[Title 40 CFR ]
[Code of Federal Regulations (annual edition) - July 1, 2009 Edition]
[From the U.S. Government Printing Office]



[[Page i]]

          

          40


          Parts 790 to 999

                         Revised as of July 1, 2009


          Protection of Environment
          



________________________

          Containing a codification of documents of general 
          applicability and future effect

          As of July 1, 2009
          With Ancillaries
                    Published by
                    Office of the Federal Register
                    National Archives and Records
                    Administration
                    A Special Edition of the Federal Register

[[Page ii]]

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[[Page iii]]







As of July 1, 2009

Title 40, Part 790 to End

Revised as of July 1, 2008

Is Replaced By

Tilte 40, Parts 790 to 999

and

Title 40, Part 1000 to End



[[Page v]]





                            Table of Contents



                                                                    Page
  Explanation.................................................     vii

  Title 40:
          Chapter I--Environmental Protection Agency 
          (Continued)                                                3
  Finding Aids:
      Table of CFR Titles and Chapters........................     479
      Alphabetical List of Agencies Appearing in the CFR......     499
      List of CFR Sections Affected...........................     509

[[Page vi]]





                     ----------------------------

                     Cite this Code: CFR
                     To cite the regulations in 
                       this volume use title, 
                       part and section number. 
                       Thus, 40 CFR 790.1 refers 
                       to title 40, part 790, 
                       section 1.

                     ----------------------------

[[Page vii]]



                               EXPLANATION

    The Code of Federal Regulations is a codification of the general and 
permanent rules published in the Federal Register by the Executive 
departments and agencies of the Federal Government. The Code is divided 
into 50 titles which represent broad areas subject to Federal 
regulation. Each title is divided into chapters which usually bear the 
name of the issuing agency. Each chapter is further subdivided into 
parts covering specific regulatory areas.
    Each volume of the Code is revised at least once each calendar year 
and issued on a quarterly basis approximately as follows:

Title 1 through Title 16.................................as of January 1
Title 17 through Title 27..................................as of April 1
Title 28 through Title 41...................................as of July 1
Title 42 through Title 50................................as of October 1

    The appropriate revision date is printed on the cover of each 
volume.

LEGAL STATUS

    The contents of the Federal Register are required to be judicially 
noticed (44 U.S.C. 1507). The Code of Federal Regulations is prima facie 
evidence of the text of the original documents (44 U.S.C. 1510).

HOW TO USE THE CODE OF FEDERAL REGULATIONS

    The Code of Federal Regulations is kept up to date by the individual 
issues of the Federal Register. These two publications must be used 
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    To determine whether a Code volume has been amended since its 
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Sections Affected (LSA),'' which is issued monthly, and the ``Cumulative 
List of Parts Affected,'' which appears in the Reader Aids section of 
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Register page number of the latest amendment of any given rule.

EFFECTIVE AND EXPIRATION DATES

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OMB CONTROL NUMBERS

    The Paperwork Reduction Act of 1980 (Pub. L. 96-511) requires 
Federal agencies to display an OMB control number with their information 
collection request.

[[Page viii]]

Many agencies have begun publishing numerous OMB control numbers as 
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OBSOLETE PROVISIONS

    Provisions that become obsolete before the revision date stated on 
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1963, 1964-1972, 1973-1985, or 1986-2000, published in eleven separate 
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INCORPORATION BY REFERENCE

    What is incorporation by reference? Incorporation by reference was 
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This material, like any other properly issued regulation, has the force 
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    What is a proper incorporation by reference? The Director of the 
Federal Register will approve an incorporation by reference only when 
the requirements of 1 CFR part 51 are met. Some of the elements on which 
approval is based are:
    (a) The incorporation will substantially reduce the volume of 
material published in the Federal Register.
    (b) The matter incorporated is in fact available to the extent 
necessary to afford fairness and uniformity in the administrative 
process.
    (c) The incorporating document is drafted and submitted for 
publication in accordance with 1 CFR part 51.
    What if the material incorporated by reference cannot be found? If 
you have any problem locating or obtaining a copy of material listed as 
an approved incorporation by reference, please contact the agency that 
issued the regulation containing that incorporation. If, after 
contacting the agency, you find the material is not available, please 
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Records Administration, Washington DC 20408, or call 202-741-6010.

CFR INDEXES AND TABULAR GUIDES

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This index is based on a consolidation of the ``Contents'' entries in 
the daily Federal Register.
    A List of CFR Sections Affected (LSA) is published monthly, keyed to 
the revision dates of the 50 CFR titles.




[[Page ix]]



REPUBLICATION OF MATERIAL

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    Raymond A. Mosley,
    Director,
    Office of the Federal Register.
    July 1, 2009.







[[Page xi]]



                               THIS TITLE

    Title 40--Protection of Environment is composed of thirty-two 
volumes. The parts in these volumes are arranged in the following order: 
parts 1-49, parts 50-51, part 52 (52.01-52.1018), part 52 (52.1019-end 
of part 52), parts 53-59, part 60 (60.1-end of part 60, sections), part 
60 (Appendices), parts 61-62, part 63 (63.1-63.599), part 63 (63.600-
63.1199), part 63 (63.1200-63.1439), part 63 (63.1440-63.6175), part 63 
(63.6580-63.8830), part 63 (63.8980-end of part 63) parts 64-71, parts 
72-80, parts 81-84, part 85-Sec.  86.599-99, part 86 (86.600-1-end of 
part 86), parts 87-99, parts 100-135, parts 136-149, parts 150-189, 
parts 190-259, parts 260-265, parts 266-299, parts 300-399, parts 400-
424, parts 425-699, parts 700-789, parts 790-999, and part 1000 to end. 
The contents of these volumes represent all current regulations codified 
under this title of the CFR as of July 1, 2009.

    Chapter I--Environmental Protection Agency appears in all thirty-two 
volumes. Regulations issued by the Council on Environmental Quality, 
including an Index to Parts 1500 through 1508, appear in the volume 
containing part 1000 to end. The OMB control numbers for title 40 appear 
in Sec.  9.1 of this chapter.

    For this volume, Michele Bugenhagen was Chief Editor. The Code of 
Federal Regulations publication program is under the direction of 
Michael L. White, assisted by Ann Worley.


[[Page 1]]



                   TITLE 40--PROTECTION OF ENVIRONMENT




                  (This book contains parts 790 to 999)

  --------------------------------------------------------------------
                                                                    Part

chapter i--Environmental Protection Agency (Continued)......         790

[[Page 3]]



         CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)




  --------------------------------------------------------------------


  Editorial Note: Nomenclature changes to chapter I appear at 65 FR 
47324, 47325, Aug. 2, 2000, and 66 FR 34375, 34376, June 28, 2001.

         SUBCHAPTER R--TOXIC SUBSTANCES CONTROL ACT (CONTINUED)
Part                                                                Page
790             Procedures governing testing consent 
                    agreements and test rules...............           5
791             Data reimbursement..........................          26
792             Good laboratory practice standards..........          33
795             Provisional test guidelines.................          46
796             Chemical fate testing guidelines............          80
797             Environmental effects testing guidelines....         101
798             Health effects testing guidelines...........         141
799             Identification of specific chemical 
                    substance and mixture testing 
                    requirements............................         226
                       SUBCHAPTERS S-T [RESERVED]

[[Page 5]]



          SUBCHAPTER R_TOXIC SUBSTANCES CONTROL ACT (CONTINUED)



PART 790_PROCEDURES GOVERNING TESTING CONSENT AGREEMENTS AND TEST RULES--Table 

of Contents




                      Subpart A_General Provisions

Sec.
790.1 Scope, purpose, and authority.
790.2 Applicability.
790.3 Definitions.
790.5 Submission of information.
790.7 Confidentiality.

  Subpart B_Procedures for Developing Consent Agreements and Test Rules

790.20 Recommendation and designation of testing candidates by the ITC.
790.22 Procedures for gathering information and negotiating consent 
          agreements on chemicals which the ITC has recommended for 
          testing with an intent to designate.
790.24 Criteria for determining whether a consensus exists concerning 
          the provisions of a draft consent agreement.
790.26 Initiation and completion of rulemaking proceedings on ITC-
          designated chemicals.
790.28 Procedures for developing consent agreements and/or test rules 
          for chemicals that have not been designated or recommended 
          with intent to designate by the ITC.

  Subpart C_Implementation, Enforcement, and Modification of Test Rules

790.40 Promulgation of test rules.
790.42 Persons subject to a test rule.
790.45 Submission of letter of intent to conduct testing or exemption 
          application.
790.48 Procedure if no one submits a letter of intent to conduct 
          testing.
790.50 Submission of study plans.
790.52 Phase II test rule.
790.55 Modification of test standards or schedules during conduct of 
          test.
790.59 Failure to comply with a test rule.

   Subpart D_Implementation, Enforcement and Modification of Consent 
                               Agreements

790.60 Contents of consent agreements.
790.62 Submission of study plans and conduct of testing.
790.65 Failure to comply with a consent agreement.
790.68 Modification of consent agreements.

                  Subpart E_Exemptions From Test Rules

790.80 Submission of exemption applications.
790.82 Content of exemption application.
790.85 Submission of equivalence data.
790.87 Approval of exemption applications.
790.88 Denial of exemption application.
790.90 Appeal of denial of exemption application.
790.93 Termination of conditional exemption.
790.97 Hearing procedures.
790.99 Statement of financial responsibility.

Appendix A to Subpart E--Schedule for Developing Consent Agreements and 
          Test Rules

    Authority: 15 U.S.C. 2603.



                      Subpart A_General Provisions



Sec. 790.1  Scope, purpose, and authority.

    (a) This part establishes procedures for gathering information, 
conducting negotiations, and developing and implementing test rules or 
consent agreements on chemical substances and mixtures under section 4 
of TSCA.
    (b) Section 4 of the Act authorizes EPA to require manufacturers and 
processors of chemical substances and mixtures to test these chemicals 
to determine whether they have adverse health or environmental effects. 
Section 4 (a) empowers the Agency to promulgate rules which require such 
testing. In addition, EPA has implied authority to enter into 
enforceable consent agreements requiring testing where they provide 
procedural safeguards equivalent to those that apply where testing is 
conducted by rule.
    (c) EPA intends to use enforceable consent agreements to accomplish 
testing where a consensus exists among EPA, affected manufacturers and/
or processors, and interested members of the public concerning the need 
for and scope of testing. If such a consensus does not exist and the 
Agency believes that it can make the findings specified in section 4(a), 
EPA will initiate proceedings to promulgate test rules which will be 
codified in part 799 of this chapter.

[[Page 6]]

    (d) Appendix A to this part presents timetables for various steps in 
the evaluation of chemicals under consideration for testing, the 
initiation and completion of negotiations to develop consent agreements, 
and the proposal and promulgation of test rules. All deadlines which are 
imposed by the Act are binding on EPA and will be observed by the 
Agency. The remaining deadlines represent target dates that EPA intends 
to meet.

[51 FR 23712, June 30, 1986]



Sec. 790.2  Applicability.

    This part is applicable to manufacturers and processors of chemical 
substances or mixtures who are subject to the testing requirements of a 
consent agreement or a rule under section 4(a) of the Act. The 
procedures for test rules are applicable to each test rule in part 799 
or this chapter unless otherwise stated in specific test rules in part 
799 of this chapter.

[51 FR 23712, June 30, 1986]



Sec. 790.3  Definitions.

    Terms defined in the Act and not explicitly defined herein are used 
with the meaning given in the Act. For the purpose of this part:
    Act means the Toxic Substances Control Act, 15 U.S.C. 2601 et seq.
    Additive means a chemical substance that is intentionally added to 
another chemical substance to improve its stability or impart some other 
desirable quality.
    Chemical means a chemical substance or mixture.
    Consortium means an association of manufacturers and/or processors 
who have made an agreement to jointly sponsor testing.
    EPA means the U.S. Environmental Protection Agency.
    Equivalence data means chemical data or biological test data 
intended to show that two substances or mixtures are equivalent.
    Equivalent means that a chemical substance or mixture is able to 
represent or substitute for another in a test or series of tests, and 
that the data from one substance can be used to make scientific and 
regulatory decisions concerning the other substance.
    Exemption means an exemption from a testing requirement of a test 
rule promulgated under section 4 of the Act and part 799 of this 
chapter.
    Impurity means a chemical substance which is uninitentionally 
present with another chemical substance.
    Joint sponsor means a person who sponsors testing pursuant to 
section 4(b)(3)(A) of the Act.
    Joint sponsorship means the sponsorship of testing by two or more 
persons in accordance with section

4(b)(3)(A) of the Act.
    Person means an individual, partnership, corporation, association, 
scientific or academic establishment, or organizational unit thereof, 
and any other legal entity.
    Principal sponsor means an individual sponsor or the joint sponsor 
who assumes primary responsibility for the direction of a study and for 
oral and written communication with EPA.
    Protocol means the plan and procedures which are to be followed in 
conducting a test.
    Reimbursement period refers to a period that begins when the data 
from the last non-duplicative test to be completed under a test rule are 
submitted to EPA and ends after an amount of time equal to that which 
had been required to develop data or after five years, whichever is 
later.
    Sponsor means the person or persons who design, direct and finance 
the testing of a substance or mixture.
    Test substance means the form of chemical substance or mixture that 
is specified for use in testing.

[49 FR 39782, Oct. 10, 1984, as amended at 51 FR 23712, June 30, 1986]



Sec. 790.5  Submission of information.

    (a) All submissions to EPA under this part must bear the Code of 
Federal Regulations (CFR) section number of the subject chemical test 
rule, or indicate the identity of the consent agreement. For all 
submissions under this part, six copies must be provided to EPA.
    (b) Submissions containing both confidential business information or 
non-confidential business information must be addressed to the Document 
Control Office (DCO) (7407M), Office of Pollution Prevention and Toxics 
(OPPT),

[[Page 7]]

Environmental Protection Agency, 1200 Pennsylvania Ave., NW., 
Washington, DC 20460-0001, ATTN: TSCA Section 4.

[50 FR 20656, May 17, 1985, as amended at 51 FR 23712, June 30, 1986; 58 
FR 34205, June 23, 1993; 60 FR 31922, June 19, 1995; 60 FR 34466, July 
3, 1995; 71 FR 33642, June 12, 2006]



Sec. 790.7  Confidentiality.

    (a) Any person subject to the requirements of a consent agreement or 
a test rule under section 4 of the Act may assert a claim of 
confidentiality for certain information submitted to EPA in response to 
the consent agreement or the test rule. Any information claimed as 
confidential will be treated in accordance with the procedures in part 2 
of this title and section 14 of the Act. Failure to assert a claim of 
confidentiality at the time the information is submitted will result in 
the information being made available to the public without further 
notice to the submitter.
    (b) A claim of confidentiality must be asserted by circling or 
otherwise marking the specific information claimed as confidential and 
designating it with the words ``confidential business information,'' 
``trade secret,'' or another appropriate phrase indicating its 
confidential character.
    (c) If a person asserts a claim of confidentiality for study plan 
information described in Sec. Sec. 790.50(c)(1)(iii)(D), (iv), (v), and 
(vi) and 790.62(b)(6), (7), (8), (9), and (10), the person must provide 
a detailed written substantiation of the claim by answering the 
questions in this paragraph. Failure to provide written substantiation 
at the time the study plan information is submitted will be considered a 
waiver of the claim of confidentiality, and the study plan information 
will be disclosed to the public without further notice.
    (1) Would disclosure of the study plan information disclose 
processes used in the manufacture or processing of a chemical substance 
or mixture? Describe how this would occur.
    (2) Would disclosure of the study plan information disclose the 
portion of a mixture comprised by any of the substances in the mixture? 
Describe how this would occur.
    (3) What harmful effects to your competitive position, if any, do 
you think would result from disclosure of this information? How would a 
competitor use such information? How substantial would the harmful 
effects be? What is the causal relationship between disclosure and the 
harmful effects?
    (4) For what period of time should confidential treatment be given? 
Until a specific date, the occurrence of a specific event, or 
permanently? Why?
    (5) What measures have you taken to guard against disclosure of this 
information to others?
    (6) To what extent has this information been disclosed to others? 
What precautions have been taken in connection with such disclosures?
    (7) Has this information been disclosed to the public in any forms? 
Describe the circumstances.
    (8) Has the information been disclosed in a patent?
    (9) Has EPA, another Federal agency, or any Federal court made any 
pertinent confidentiality determination regarding this information? If 
so, copies of such determinations must be included in the 
substantiation.
    (d) If the substantiation provided under paragraph (c) of this 
section contains information which the submitter considers confidential, 
the submitter must assert a separate claim of confidentiality for that 
information at the time of submission in accordance with paragraph (b) 
of this section.

[49 FR 39782, Oct. 10, 1984, as amended at 51 FR 23713, June 30, 1986]



  Subpart B_Procedures for Developing Consent Agreements and Test Rules

    Source: 51 FR 23713, June 30, 1986, unless otherwise noted.



Sec. 790.20  Recommendation and designation of testing candidates by the ITC.

    (a) Recommendations with intent to designate. The ITC has advised 
EPA that it will discharge its responsibilities under section 4(e) of 
the Act in the following manner:
    (1) When the ITC identifies a chemical substance or mixture that it 
believes should receive expedited consideration by EPA for testing, the 
ITC

[[Page 8]]

may add the substance or mixture to its list of chemicals recommended 
for testing and include a statement that the ITC intends to designate 
the substance or mixture for action by EPA in accordance with section 
4(e)(1)(B) of the Act.
    (2) Chemical substances or mixtures selected for expedited review 
under paragraph (a)(1) of this section may, at a later time, be 
designated for EPA action within 12 months of such designation. The 
ITC's subsequent decision would be based on the ITC's review of TSCA 
sections 8(a) and 8(d) data and other relevant information.
    (3) Where the ITC concludes that a substance or mixture warrants 
testing consideration but that expedited EPA review of testing needs is 
not justified, the ITC will add the substance or mixture to its list of 
testing recommendations without expressing an intent to designate the 
substance or mixture for EPA action in accordance with section 
4(e)(1)(B) of the Act.
    (4) The ITC reserves its right to designate any chemical that it 
determines the Agency should, within 12 months of the date first 
designated, initiate a proceeding under section 4(a) of the Act.
    (b) EPA consideration of ITC recommendations. (1) Where a substance 
or mixture is designated for EPA action under section 4(e)(1)(B) of the 
Act, the Agency will take either one of the following actions within 12 
months after receiving the ITC designation:
    (i) Initiate rulemaking proceedings under section 4(a) of the Act.
    (ii) Publish a Federal Register notice explaining the Agency's 
reasons for not initiating such rulemaking proceedings. EPA may conclude 
that rulemaking proceedings under section 4(a) of the Act are 
unnecessary if it determines that the findings specified in section 4(a) 
of the Act cannot be made or if the Agency has entered into a consent 
agreement requiring testing in accordance with the provisions of this 
subpart.
    (2) Where a substance or mixture has been recommended for testing by 
the ITC without an intent to designate, EPA will use its best efforts to 
act on the ITC's recommendations as rapidly as possible consistent with 
its other priorities and responsiblities. EPA may respond to the ITC's 
recommendations either by:
    (i) Initiating rulemaking proceedings under section 4(a) of the Act.
    (ii) Publishing a Federal Register notice explaining the Agency's 
reasons for concluding that testing is unnecessary.
    (iii) Entering into a consent agreement in accordance with this 
subpart.



Sec. 790.22  Procedures for gathering information and negotiating consent 

agreements on chemicals which the ITC has recommended for testing with an 

intent to designate.

    (a) Preliminary EPA evaluation. Following receipt of an ITC report 
containing a recommendation with an intent to designate, EPA will use 
the following procedure for completing a preliminary evaluation of 
testing needs. Appendix A \1\ to this part presents the schedule that 
EPA intends to follow for this purpose.
---------------------------------------------------------------------------

    \1\ Editorial Note: Appendix A appears at the end of subpart E.
---------------------------------------------------------------------------

    (1) EPA will publish the ITC report in the Federal Register and 
announce that interested persons have 30 days to submit comments on the 
ITC's testing recommendations.
    (2) EPA will publish a Federal Register notice adding all ITC-
recommended chemicals to the automatic reporting provisions of its rules 
under sections 8(a) and 8(d) of the Act (40 CFR parts 712 and 716).
    (3) EPA will hold a public ``focus meeting'' to discuss the ITC's 
testing recommendations and obtain comments and information from 
interested parties.
    (4) EPA will evaluate submissions received under the sections 8(a) 
and 8(d) reporting requirements, comments filed on the ITC's 
recommendations, and other information and data compiled by the Agency.
    (5) EPA will make a preliminary staff determination of the need for 
testing and, where testing appears warranted, will tentatively select 
the studies to be performed.
    (6) EPA will hold a public meeting to announce its preliminary 
testing determinations.

[[Page 9]]

    (b) Negotiation procedures for consent agreements. Where EPA 
believes that testing is necessary, the Agency will explore whether a 
consent agreement can be negotiated that satisfies the testing needs 
identified by the Agency. EPA will use the following procedures for 
negotiating, formulating and accepting consent agreements. Appendix A 
\1\ to this part presents the schedule that EPA intends to follow for 
this purpose.
    (1) In the Federal Register notice described in paragraph (a)(1) of 
this section, EPA will explain its procedures and timetable for 
negotiating consent agreements and invite persons interested in 
participating in or monitoring negotiations to contact the Agency in 
writing.
    (2) Persons who respond to EPA's notice by the announced date of the 
Agency's course-setting meeting will be deemed ``interested parties'' 
for purposes of any negotiations that EPA conducts.
    (3) Following the course-setting meeting announcing EPA's 
preliminary testing determinations, the Agency will meet with 
manufacturers, processors and other interested parties for the purpose 
of attempting to negotiate a consent agreement. To facilitate attendance 
at these meetings, EPA will contact all interested parties who have 
expressed a desire to participate in or monitor negotiations under 
paragraph (b)(2) of this section and advise them of meeting dates.
    (4) All negotiating meetings will be open to members of the public. 
The minutes of each meeting will be prepared by EPA. Meeting minutes, 
testing proposals, background documents and other materials exchanged at 
or prepared for negotiating meetings will be included in the public file 
established by EPA on each ITC-recommended chemical. Materials in this 
file will be made available for inspection in the OPPTS Reading Room 
during EPA working hours.
    (5) While negotiations are underway, EPA will promptly circulate 
meeting minutes, testing proposals, correspondence and other relevant 
materials to interested parties who expressed a desire to participate in 
or monitor negotiations pursuant to paragraph (b)(2) of this section.
    (6) As negotiations progress, EPA will make a tentative decision 
either to proceed with formulation of a consent agreement or to initiate 
rulemaking. EPA will terminate negotiations after 10 weeks and proceed 
with rulemaking unless negotiations are likely to result in a draft 
consent agreement within 4 additional weeks. By the end of this 4-week 
period, EPA either will have prepared a draft consent agreement 
reflecting the apparent consensus of the parties or will terminate 
negotiations and proceed with rulemaking. If EPA decides to proceed with 
rulemaking, no further opportunity for negotiations will be provided. 
EPA will promptly send written notice to all interested parties of the 
termination of negotiations.
    (7) Where EPA prepares a draft consent agreement, it will be 
circulated for comment to all interested parties who expressed a desire 
to participate in or monitor negotiations under paragraph (b)(2) of this 
section. A period of 4 weeks will be provided for submitting comments or 
written objections under Sec. 790.24(a).
    (8) If necessary, EPA will hold a public meeting to discuss comments 
on the draft consent agreement and to determine whether revisions in the 
agreement are appropriate.
    (9) Where a consensus exists concerning the contents of a draft 
consent agreement, it will be circulated to EPA management and 
interested parties for final approval and signature.
    (10) Upon final approval of a consent agreement, EPA will publish a 
Federal Register notice that summarizes the agreement, describes the ITC 
recommendations for the test substance, outlines the chemical's use and 
exposure characteristics, and explains the background, objectives and 
rationale of the testing to be conducted, and codifies in subpart C of 
part 799 the name of the substance(s) to be tested and the citation to 
the Federal Register notice of the agreement.

[[Page 10]]



Sec. 790.24  Criteria for determining whether a consensus exists concerning 

the provisions of a draft consent agreement.

    (a) EPA will enter into consent agreements only where there is a 
consensus among the Agency, one or more manufacturers and/or processors 
who agree to conduct or sponsor the testing, and all other interested 
parties who identify themselves in accordance with Sec. 790.22(b)(2). 
EPA will not enter into a consent agreement in either of the following 
circumstances:
    (1) EPA and affected manufacturers and/or processors cannot reach a 
consensus on the testing requirements or other provisions to be included 
in the consent agreement.
    (2) A draft consent agreement is considered inadequate by other 
interested parties who, pursuant to Sec. 790.22(b)(2), have asked to 
participate in or monitor negotiations; and these parties have submitted 
timely written objections to the draft consent agreement which provide a 
specific explanation of the grounds on which the draft agreement is 
objectionable.
    (b) EPA may reject objections described in paragraph (a)(2) of this 
section only where the Agency concludes the objections are either:
    (1) Not made in good faith.
    (2) Untimely.
    (3) Do not involve the adequacy of the proposed testing program or 
other features of the agreement that may affect EPA's ability to fulfill 
the goals and purposes of the Act.
    (4) Not accompanied by a specific explanation of the grounds on 
which the draft agreement is considered objectionable.
    (c) The unwillingness of some manufacturers and/or processors of a 
prospective test chemical to sign the draft consent agreement does not, 
in itself, establish a lack of consensus if EPA concludes that those 
manufacturers and/or processors who are prepared to sign the agreement 
are capable of accomplishing the testing to be required and that the 
draft agreement will achieve the purposes of the Act in all other 
respects.



Sec. 790.26  Initiation and completion of rulemaking proceedings on ITC-

designated chemicals.

    (a) Where EPA concludes that a consensus does not exist concerning 
the provisions of a draft consent agreement and that the findings 
specified by section 4(a) can be made, the Agency will proceed with 
rulemaking under section 4(a) of TSCA.
    (b) When EPA decides to proceed with rulemaking under paragraph (a) 
of this section, the Agency intends to publish a rulemaking proposal and 
a final rule or a notice terminating the rulemaking proceeding in 
accordance with the schedule specified in Appendix A \1\ to this part.
---------------------------------------------------------------------------

    \1\ Editorial Note: Appendix A appears at the end of subpart E.
---------------------------------------------------------------------------

    (c) Where the testing recommendations of the ITC raise unusually 
complex and novel issues that require additional Agency review and 
opportunity for public comment, the Agency may publish an Advance Notice 
of Proposed Rulemaking (ANPR). The schedule that EPA intends to follow 
for rulemaking proceedings initiated by publication of an ANPR is 
presented in appendix A \1\ to this part.



Sec. 790.28  Procedures for developing consent agreements and/or test rules 

for chemicals that have not been designated or recommended with intent to 

designate by the ITC.

    (a) Where EPA believes that testing is needed, it may also develop 
consent agreements and/or test rules on chemical substances or mixtures 
that either:
    (1) Have been recommended but not ``recommended with intent to 
designate'' by the ITC.
    (2) Have been selected for testing consideration by EPA on its own 
initiative.
    (b) When EPA wishes to initiate negotiations concerning chemicals 
described in paragraph (a) of this section, it will publish a Federal 
Register notice describing its tentative evaluation of testing needs, 
announcing a date for a public course-setting meeting, and inviting 
persons interested in participating in or monitoring negotiations to

[[Page 11]]

contact the Agency in writing. Any negotiations that EPA conducts will 
conform to the procedures specified in Sec. 790.22(b) and, to the 
extent feasible, will follow the schedules presented in appendix A \1\ 
to this part.
    (c) EPA will enter into consent agreements on chemicals described in 
paragraph (a) of this section only if there is a consensus among EPA, 
affected manufacturers and/or processors, and any other persons who have 
asked to participate in or monitor negotiations. In determining whether 
such a consensus exists, EPA will employ the criteria specified in Sec. 
790.24. In the absence of consensus, EPA will initiate rulemaking if it 
concludes that the findings specified in section 4(a) of the Act can be 
made. The schedule for initiating and completing such rulemaking 
proceedings will, to the extent feasible, follow the schedule specified 
in appendix A \1\ to this part.
---------------------------------------------------------------------------

    \1\ Editorial Note: Appendix A appears at the end of subpart E.
---------------------------------------------------------------------------



  Subpart C_Implementation, Enforcement, and Modification of Test Rules

    Source: 50 FR 20657, May 17, 1985, unless otherwise noted. 
Redesignated at 51 FR 23713, June 30, 1986.



Sec. 790.40  Promulgation of test rules.

    (a) If EPA determines that it is necessary to test a chemical 
substance or mixture by rule under section 4 of the Act, it will 
promulgate a test rule in part 799 of this chapter.
    (b) EPA will promulgate specific test rules in part 799 of this 
chapter either by a single-phase rulemaking procedure or by a two-phase 
rulemaking procedure.
    (1) Under single-phase test rule development, EPA will promulgate a 
test rule in part 799 of this chapter through a notice and comment 
rulemaking which specifies the following:
    (i) Identification of the chemical for which testing is required 
under the rule.
    (ii) The health or environmental effect or effects or other 
characteristics for which testing is being required.
    (iii) Which test substance(s) must be tested.
    (iv) Standards for the development of test data.
    (v) The EPA Good Laboratory Practice requirements for the required 
testing.
    (vi) Schedule for submission of interim reports and/or final reports 
to EPA.
    (vii) Who must submit either letters of intent to conduct testing or 
exemption applications.
    (viii) What types of data EPA will examine in determining 
equivalence if more than one test substance is to be tested.
    (2) Under two-phase test rule development, EPA will promulgate a 
Phase I test rule in part 799 of this chapter through a notice and 
comment rulemaking which specifies the following:
    (i) Identification of the chemical for which testing is required 
under the rule.
    (ii) The health or environmental effect or effects or other 
characteristics for which testing is being required.
    (iii) Which test substance(s) must be tested.
    (iv) A reference to appropriate guidelines for the development of 
test data.
    (v) The EPA Good Laboratory Practice requirements for the required 
testing.
    (vi) Who must submit either letters of intent to conduct testing and 
study plans, or exemption applications.
    (vii) What types of data EPA will examine in determining equivalence 
if more than one test substance is to be tested.
    (3) Under two-phase test rule development, test standards and 
schedules will be developed in a second phase of rulemaking as described 
in Sec. Sec. 790.50 and 790.52.

[50 FR 20657, May 17, 1985. Redesignated and amended at 51 FR 23713, 
June 30, 1986; 54 FR 36313, Sept. 1, 1989]



Sec. 790.42  Persons subject to a test rule.

    (a) Each test rule described in Sec. 790.40 will specify whether 
manufacturers, processors, or both are subject to the requirement for 
testing of the subject chemical under section 4(b)(3)(B) of the

[[Page 12]]

Act and will indicate who will be required to submit letters of intent 
to conduct testing.
    (1) If testing is being required to allow evaluation of risks:
    (i) Primarily associated with manufacture of the chemical, or
    (ii) Associated with both manufacturer and processing of the 
chemical, or
    (iii) Associated with distribution in commerce, use, and/or disposal 
activities concerning the chemical, each manufacturer of the chemical 
will be subject and must comply with the requirements of the test rule.
    (2) While legally subject to the test rule in circumstances 
described in paragraphs (a)(1) (ii) and (iii) of this section, 
processors of the chemical must comply with the requirements of the test 
rule only if processors are directed to do so in a subsequent notice as 
set forth in Sec. 790.48(b).
    (3) If testing is being required to allow evaluation of risks 
associated solely with processing of the chemical, processors will be 
subject and must comply with the requirements of the test rule.
    (4) While legally subject to the test rule in circumstances 
described in paragraph (a)(1) of this section, persons who manufacture 
less than 500 kg (1,100 lb) of the chemical annually during the period 
from the effective date of the test rule to the end of the reimbursement 
period, must comply with the requirements of the test rule only if such 
manufacturers are directed to do so in a subsequent notice as set forth 
in Sec. 790.48, or if directed to do so in a particular test rule.
    (5) While legally subject to the test rule in circumstances 
described in paragraph (a)(1) of this section, persons who manufacture 
small quantities of the chemical solely for research and development 
(meaning quantities that are not greater than those necessary for 
purposes of scientific experimentation or analysis or chemical research 
on, or analysis of, such chemical or another chemical, including such 
research or analysis for development of a product) from the effective 
date of the test rule to the end of the reimbursement period, must 
comply with the requirements of the test rule only if such manufacturers 
are directed to do so in subsequent notice set forth in Sec. 790.48, or 
if directed to do so in a particular test rule.
    (6) If testing is being required to allow evaluation of risks 
associated primarily with manufacture of a chemical for research and 
development (R & D) purposes, manufacturers of the chemical for R & D 
will be subject and must comply with the requirements of the test rule.
    (b) [Reserved]

[50 FR 20657, May 17, 1985. Redesignated at 51 FR 23713, June 30, 1986, 
and amended at 55 FR 18884, May 7, 1990]



Sec. 790.45  Submission of letter of intent to conduct testing or exemption 

application.

    (a) No later than 30 days after the effective date of a test rule 
described in Sec. 790.40, each person subject to that rule and required 
to comply with the requirements of that rule as provided in Sec. 
790.42(a) must, for each test required, either notify EPA by letter of 
his or her intent to conduct testing or submit to EPA an application for 
an exemption from testing requirements for the test.
    (b) EPA will consider letters of intent to test as commitments to 
sponsor the tests for which they are submitted unless EPA agrees to the 
substitution of an exemption application in instances where more than 
one person indicates an intent to sponsor equivalent tests.
    (c) Each letter of intent to conduct testing must include:
    (1) Identification of test rule.
    (2) Name, address, and telephone number of the firm(s) which will be 
sponsoring the tests.
    (3) Name, address, and telephone number of the appropriate 
individual to contact for further information.
    (4) For sponsors participating in a testing consortium--a list of 
all members of the consortium, the signature of an authorized 
representative of each member, and a designation of who is to serve as 
principal sponsor.
    (5) A list of the testing requirements for which the sponsor(s) 
intends to conduct tests.

[[Page 13]]

    (6) If EPA is requiring testing of more than one representative 
substance--which test substance the sponsor(s) intends to use in each of 
the tests.
    (d)(1) Any person not manufacturing or processing the subject 
chemical as of the effective date of the test rule describing in Sec. 
790.40 or by 30 days after the effective date of the rule who, before 
the end of the reimbursement period, manufacturers or processes the test 
chemical and who is subject to and required to comply with the 
requirements of the test rule must submit the letter of intent to test 
or an exemption application required by paragraph (a) of this section by 
the date manufacture or processing begins, or
    (2) When both manufacturers and processors are subject to the rule, 
any person not processing the subject chemical as of the effective date 
of the test rule described in Sec. 790.40 or by 30 days after 
publication of the Federal Register notice described in Sec. 
790.48(b)(2) who, before the end of the reimbursement period, processes 
the test chemical and who is required to comply with the requirements of 
the rule must submit the letter of intent to test or an exemption 
application required by Sec. 790.48(b)(3) of the date processing 
begins.
    (e) Manufacturers subject to a test rule described in Sec. 790.40 
who do not submit to EPA either a letter of their intent to conduct 
tests or a request for an exemption from testing for each test for which 
testing is required in the test rule will be considered in violation of 
that rule beginning on the 31st day after the effective date of the test 
rule described in Sec. 790.40 or on the date manufacture begins as 
described in paragraph (d) of this section.
    (f) Processors subject to a test rule described in Sec. 790.40 and 
required to comply with the requirements of test rule pursuant to Sec. 
790.42(a)(2) or a Federal Register notice as described in Sec. 
790.48(b)(2) who do not submit to EPA either a letter of their intent to 
conduct tests or a request for an exemption for each test for which 
testing is required in the test rule will be considered in violation of 
that rule beginning on the 31st day after the effective date of the test 
rule described in Sec. 790.40 or 31 days after publication of the 
Federal Register notice described in Sec. 790.48(b)(2) or on the date 
processing begins as described in paragraph (d) of this section, as 
appropriate.



Sec. 790.48  Procedure if no one submits a letter of intent to conduct 

testing.

    (a) If only manufacturers are subject to the rule. (1) This 
paragraph applies if testing is being required solely to allow 
evaluation of risks associated with manufacturing and the test rule 
described in Sec. 790.40 states that manufacturers only are responsible 
for testing.
    (2) If no manufacturer subject to the test rule has notified EPA of 
its intent to conduct one or more of the required tests within 30 days 
after the effective date of the test rule described in Sec. 790.40, EPA 
will notify all manufacturers, including those described in Sec. 
790.42(a)(4) and (a)(5), by certified mail or by publishing a notice of 
this fact in the Federal Register specifying the tests for which no 
letter of intent has been submitted and will give such manufacturers an 
opportunity to take corrective action.
    (3) If no manufacturer submits a letter of intent to conduct one or 
more of the required tests within 30 days after receipt of the certified 
letter or publication of the Federal Register notice described in 
paragraph (a)(2) of this section, all manufacturers subject to the rule 
will be in violation of the test rule from the 31st day after receipt of 
the certified letter or publication of the Federal Register notice 
described in this paragraph.
    (b) If manufacturers and processors are subject to the rule. (1) 
This paragraph applies if testing is being required to allow evaluation 
of risks associated with manufacturing and processing or with 
distribution in commerce, use, or disposal of the chemical and the test 
rule described in Sec. 790.40 states that manufacturers and processors 
are responsible for testing.
    (2) If no manufacturer subject to the rule has notified EPA of its 
intent to conduct testing for one or more of the required tests within 
30 days after the effective date of the test rule described in Sec. 
790.40, EPA will publish a notice in the Federal Register of this fact

[[Page 14]]

specifying the tests for which no letter of intent has been submitted.
    (3) No later than 30 days after the date of publication of the 
Federal Register notice described in paragraph (b)(2) of this section, 
each person described in Sec. 790.40(a)(4) and (5) and each person 
processing the subject chemical as of the effective date of the test 
rule described in Sec. 790.40 or by 30 days after the date of 
publication of the Federal Register notice described in paragraph (b)(2) 
of this section must, for each test specified in the Federal Register 
notice, either notify EPA by letter of his or her intent to conduct 
testing or submit to EPA an application for an exemption from testing 
requirements for the test.
    (4) If no manufacturer or processor of the test chemical has 
submitted a letter of intent to conduct one or more of the required 
tests within 30 days after the date of publication of the Federal 
Register notice described in paragraph (b)(2) of this section, EPA will 
notify all manufacturers and processors by certified letter or publish a 
Federal Register notice of this fact specifying the tests for which no 
letter of intent has been submitted. This letter or Federal Register 
notice will give the manufacturers and processors an opportunity to take 
corrective action.
    (5) If no manufacturer or processor submits a letter of intent to 
conduct one or more of the required tests within 30 days after receipt 
of the certified letter or publication of the Federal Register notice 
described in paragraph (b)(4) of this section, all manufacturers and 
processors subject to the rule will be in violation of the test rule 
from the 31st day after receipt of the certified letter or publication 
of the Federal Register notice described in paragraph (b)(4) of this 
section.
    (c) Only processors are subject to the rule. (1) This paragraph 
applies if testing is being required solely to allow evaluation of risks 
associated with processing and the test rule described in Sec. 790.40 
states that only processors are responsible for testing.
    (2) If no processor subject to the rule has notified EPA of its 
intent to conduct one or more of the required tests within 30 days after 
the effective date of the test rule described in Sec. 790.40, EPA will 
notify all the processors by certified mail or publish a notice in the 
Federal Register of this fact, specifying the tests for which no letter 
of intent has been submitted and give the processors an opportunity to 
take corrective action.
    (3) If no processor submits a letter of intent to conduct one or 
more of the required tests within 30 days after receipt of the certified 
letter or publication of the Federal Register notice described in 
paragraph (c)(2) of this section, all processors subject to the rule 
will be in violation of the test rule from the 31st day after receipt of 
the certified letter or publication of the Federal Register notice 
described in this paragraph.

[50 FR 20657, May 17, 1985. Redesignated at 51 FR 23713, June 30, 1986, 
and amended at 55 FR 18884, May 7, 1990]



Sec. 790.50  Submission of study plans.

    (a) Who must submit study plans. (1) Persons who notify EPA of their 
intent to conduct tests in compliance with the requirements of a single 
phase test rule as described in Sec. 790.40(b)(1) must submit study 
plans for those tests prior to the initiation of each of these tests, 
unless directed by a particular test rule or consent agreement to submit 
study plans at a specific time.
    (2) Persons who notify EPA of their intent to conduct tests in 
compliance with the requirements of a Phase I test rule as described in 
Sec. 790.40(b)(2) must submit the proposed study plans for those tests 
on or before 90 days after the effective date of the Phase I rule; or, 
for processors complying with the notice described in Sec. 
790.48(b)(2), 90 days after the publication date of that notice; or 60 
days after the date manufacture or processing begins as described in 
Sec. 790.45(d), as appropriate, to the address in Sec. 790.5(b).
    (3) Study plans must be prepared according to the requirements of 
this subpart B and part 792 of this chapter. Only one set of study plans 
should be prepared and submitted by persons who are jointly sponsoring 
testing.
    (4) Any person subject to a test rule may submit a study plan for 
any test

[[Page 15]]

required by the rule at any time, regardless of whether the person 
previously submitted an application for exemption from testing for that 
test.
    (5) Unless EPA has granted an extension of time for submission of 
proposed study plans, manufacturers who notify EPA that they intend to 
conduct testing in compliance with the requirements of a Phase I test 
rule as described in Sec. 790.40(b)(2) and who do not submit proposed 
study plans for those tests on or before 90 days after the effective 
date of the Phase I test rule or 60 days after the date manufacture 
begins as described in Sec. 790.45(d) will be considered in violation 
of the test rule as if no letter of intent to test had been submitted.
    (6) Unless EPA has granted an extension of time for submission of 
proposed study plans, processors who notify EPA that they intend to 
conduct testing in compliance with the requirements of a Phase I test 
rule as described in Sec. 790.40(b)(2) and who do not submit proposed 
study plans for those tests on or before 90 days after the effective 
date of the Phase I test rule or 90 days after the publication date of 
the notice described in Sec. 790.48(b)(2), or 60 days after the date 
processing begins as described in Sec. 790.45(d), as appropriate, will 
be considered in violation of the test rule as if no letter of intent to 
test had been submitted.
    (b) Extensions of time for submission of study plans. (1) EPA may 
grant requests for additional time for the development of study plans on 
a case-by-case basis. Requests for additional time for study plan 
development must be made in writing to EPA at the address in Sec. 
790.5(b). Each extension request must state why EPA should grant the 
extension.
    (2) Under two-phase rulemaking, extension requests must be submitted 
to EPA within 60 days after the effective date of the Phase I test rule 
as described in Sec. 790.40(b)(2); or for processors complying with the 
notice described in Sec. 790.48(b)(2), 60 days after the publication 
date of that notice; or 30 days after the date manufacture or processing 
begins as described in Sec. 790.45(d), as appropriate.
    (3) EPA will notify the submitter by certified mail of EPA's 
decision to grant or deny an extension request.
    (4) Persons who have been granted an extension of time for 
submission of study plans as described in paragraph (b)(1) of this 
section and who do not submit proposed study plans in compliance with 
the requirements of a Phase I test rule in accordance with the new 
deadline granted by EPA will be considered in violation of the test rule 
as if no letter of intent to test had been submitted as described in 
Sec. 790.45(e) and (f).
    (c) Content of study plans. (1) All study plans are required to 
contain the following information:
    (i) Identity of the test rule.
    (ii) The specific test requirements of that rule to be covered by 
the study plan.
    (iii)(A) The names and addresses of the test sponsors.
    (B) The names, addresses, and telephone numbers of the responsible 
administrative officials and project manager(s) in the principal 
sponsor's organization.
    (C) The name, address, and telephone number of the appropriate 
individual to contact for oral and written communications with EPA.
    (D)(1) The names and addresses of the testing facilities and the 
names, addresses, and telephone numbers of the testing facilities' 
administrative officials and project manager(s) responsible for the 
testing.
    (2) Brief summaries of the training and experience of each 
professional involved in the study, including study director, 
veterinarian(s), toxicologist(s), pathologist(s), chemist(s), 
microbiologist(s), and laboratory assistants.
    (iv) Identity and data on the chemical substance(s) being tested, 
including physical constants, spectral data, chemical analysis, and 
stability under test and storage conditions, as appropriate.
    (v) Study protocol, including the rationale for any combination of 
test protocols; the rationale for species/strain selection; dose 
selection (and supporting data); route(s) or method(s) of exposure; 
description of diet to be used and its source; including nutrients

[[Page 16]]

and contaminants and their concentrations; for in vitro test systems, a 
description of culture medium and its source; and a summary of expected 
spontaneous chronic diseases (including tumors), genealogy, and life 
span.
    (vi) Schedule for initiation and completion of each short-term test 
and of each major phase of long-term tests; dates for submission of 
interim progress and final reports to EPA that are within the reporting 
deadlines specified by EPA In the final test rule.
    (2) Information required in paragraph (c)(1)(iii)(D) of this section 
is not required in proposed study plans submitted in compliance with the 
requirements of a Phase I test rule if the information is not available 
at the time of study plan submission; however, the information must be 
submitted before the initiation of testing.
    (d) Incomplete study plans. (1) Upon receipt of a study plan, EPA 
will review the study plan to determine whether it complies with 
paragraph (c) of this section. If EPA determines that the study plan 
does not comply with paragraph (c) of this section, EPA will notify the 
submitter that the submission is incomplete and will identify the 
deficiencies and the steps necessary to complete the submission.
    (2) The submitter will have 15 days after the day it receives this 
notice to submit appropriate information to make the study plan 
complete.
    (3) If the submitter fails to provide appropriate information to 
complete a proposed study plan submitted in compliance with the 
requirements of a Phase I test rule on or before 15 days after receipt 
of the notice, the submitter will be considered in violation of the test 
rule as if no letter of intent to conduct the test had been submitted as 
described in Sec. 790.45(e) and (f).
    (e) Amendments to study plans. Test sponsors shall submit all 
amendments to study plans to the Director, Office of Compliance 
Monitoring at the address in Sec. 790.5(d).

[50 FR 20657, May 17, 1985. Redesignated and amended at 51 FR 23713, 
June 30, 1986; 52 FR 36569, Sept. 30, 1987; 54 FR 36313, Sept. 1, 1989; 
55 FR 18884, May 7, 1990; 58 FR 34205, June 23, 1993; 60 FR 34466, July 
3, 1995]



Sec. 790.52  Phase II test rule.

    (a) If EPA determines that the proposed study plan described in 
Sec. 790.50(a)(2) complies with Sec. 790.50(c), EPA will publish a 
proposed Phase II test rule in the Federal Register requesting comments 
on the ability of the proposed study plan to ensure that data from the 
test will be reliable and adequate.
    (b) EPA will provide a 45-day comment period and will provide an 
opportunity for an oral presentation upon the request of any person. EPA 
may extend the comment period if it appears from the nature of the 
issues raised by EPA's review or from public comments that further 
comment is warranted.
    (c) After receiving and considering public comments on the study 
plan, EPA will adopt, as proposed or as modified in response to EPA 
review and public comments, the study protocol section of the study 
plan, as defined by Sec. 790.50(c)(1)(v) of this chapter, as the test 
standard for the required testing, and the schedule section of the study 
plan, as defined by Sec. 790.50(c)(1)(vi) of this chapter, as the 
schedule for the required testing in a final Phase II test rule.

[50 FR 20657, May 17, 1985. Redesignated at 51 FR 23713, June 30, 1986, 
and amended at 52 FR 36569, Sept. 30, 1987]



Sec. 790.55  Modification of test standards or schedules during conduct of 

test.

    (a) Application. Any test sponsor who wishes to modify the test 
schedule for the mandatory testing conditions or requirements (i.e., 
``shall statements'') in the test standard for any test required by a 
test rule must submit an application in accordance with this paragraph. 
Application for modification must be made in writing to EPA at the 
address in Sec. 790.5(b), or by phone with written confirmation to 
follow within 10 working days. Applications must include an appropriate 
explanation and rationale for the modification. Where a test sponsor 
requests EPA to provide guidance or to clarify a non-mandatory testing 
requirement (i.e., ``should statements'') in a test standard, the test 
sponsor should submit these requests to EPA at the address in Sec. 
790.5(b).

[[Page 17]]

    (b) Adoption. (1) Where EPA concludes that the requested 
modification of a test standard or schedule for a test required under a 
test rule is appropriate, EPA will proceed in accordance with this 
paragraph (b).
    (2) Where, in EPA's judgment, the requested modification of the test 
standard or schedule would not alter the scope of the test or 
significantly change the schedule for completing the test, EPA will not 
ask for public comment before approving the modification. EPA will 
notify the test sponsor by letter of EPA's approval. EPA will place 
copies of each application and EPA approval letter in the rulemaking 
record for the test rule in question. EPA will publish a notice annually 
in the Federal Register indicating the test standards or schedules for 
tests required in test rules which have been modified under this 
paragraph (b)(2) and describing the nature of the modifications. Until 
the Federal Register notice is published, any modification approved by 
EPA under this paragraph (b)(2) shall apply only to the test sponsor who 
applied for the modification under this paragraph (a) of this section.
    (3) Where, in EPA's judgment, the requested modification of a test 
standard or schedule would significantly alter the scope of the test or 
significantly change the schedule for completing the test, EPA will 
publish a notice in the Federal Register requesting comment on the 
proposed modification. However, EPA will approve a requested 
modification of a test standard under paragraph (b)(3) of this section 
without first seeking public comment if EPA believes that an immediate 
modification to the test standard is necessary to preserve the accuracy 
or validity of an ongoing test. EPA may also modify a testing 
requirement or test condition in a test standard if EPA determines that 
the completion or achievement of this requirement or condition is not 
technically feasible. EPA may approve a test schedule extension under 
paragraph (b)(3) of this section without first seeking public comment if 
EPA determines, on a case-by-case basis, that a delay of over 12 months 
is not the fault of the test sponsor and is the result of unforeseen 
circumstances such as a lack of laboratory availability, lack of 
availability of suitable test substance (e.g., 14-C labelled test 
substance), lack of availability of healthy test organisms, or the 
unexpected failure of a long-term test. EPA will publish an annual 
notice in the Federal Register announcing the approval of any test 
standard modifications and test schedule extensions under paragraph 
(b)(3) of this section and provide a brief rationale of why the 
modification was granted.
    (4) For purposes of this paragraph (b), a requested modification of 
a test standard or schedule for a test required under a test rule would 
alter the scope of the test or significantly change the schedule for 
completing the test if the modification would:
    (i) Change the test species.
    (ii) Change the route of administration of the test chemical.
    (iii) Change the period of time during which the test species is 
exposed to the test chemical.
    (iv) Except as provided in paragraph (b)(3) of this section, extend 
the final reporting deadline more than 12 months from the date specified 
in the final rule.
    (c) Disapproval. Where EPA concludes that the requested modification 
of a test standard or schedule for a test required under a test rule is 
not appropriate, EPA will so notify the test sponsor in writing.
    (d) Timing. (1) Test sponsors should submit all applications for 
test schedule modifications at least 60 days before the reporting 
deadline for the test in question.
    (2) EPA will not normally approve any test schedule extensions 
submitted less than 30 days before the reporting deadline for the test 
in question.
    (3) Except as provided in paragraph (b)(3) of this section, EPA may 
grant extensions for up to 1 year but will normally limit extensions to 
a period of time equal to the in-life portion of the test plus 60 days.
    (4) EPA will normally approve only one deadline extension for each 
test.
    (5) Test sponsors should submit requests for test standard 
modifications as soon as they determine that the test

[[Page 18]]

cannot be successfully completed according to the test standard 
specified in the rule.

[50 FR 20657, May 17, 1985. Redesignated at 51 FR 23713, June 30, 1986, 
and amended at 52 FR 36571, Sept. 30, 1987; 54 FR 36314, Sept. 1, 1989; 
60 FR 34466, July 3, 1995]



Sec. 790.59  Failure to comply with a test rule.

    (a) Persons who notified EPA of their intent to conduct a test 
required in a test rule in part 799 of this chapter and who fail to 
conduct the test in accordance with the test standards and schedules 
adopted in the test rule, or as modified in accordance with Sec. 
790.55, will be in violation of the rule.
    (b) Any person who fails or refuses to comply with any aspect of 
this part or a test rule under part 799 of this chapter is in violation 
of section 15 of the Act. EPA will treat violations of the Good 
Laboratory Practice standards as indicated in Sec. 792.17 of this 
chapter.



   Subpart D_Implementation, Enforcement and Modification of Consent 
                               Agreements

    Source: 51 FR 23715, June 30, 1986, unless otherwise noted.



Sec. 790.60  Contents of consent agreements.

    (a) Standard provisions. All consent agreements will contain the 
following provisions:
    (1) Identification of the chemical(s) to be tested.
    (2) The health effects, environmental effects and/or other 
characteristics for which testing will be required.
    (3) The names and addresses of each manufacturer and/or processor 
who will sign the agreement.
    (4) The name and address of the manufacturer, processor or other 
entity who has agreed to act as the principal test sponsor.
    (5) The technical or commercial grade, level of purity or other 
characteristics of the test substances(s) or mixture(s).
    (6) Standards for the development of test data.
    (7) A requirement that testing will be conducted in accordance with 
the EPA Good Laboratory Practice (GLP) regulations (40 CFR part 792).
    (8) Schedules with reasonable deadlines for submitting interim 
progress and/or final reports to EPA.
    (9) A requirement that the principal sponsor will submit a study 
plan to EPA in accordance with Sec. 790.62.
    (10) A statement that the results of testing conducted pursuant to 
the consent agreement will be announced to the public in accordance with 
the procedures specified in section 4(d) of the Act and that the 
disclosure of data generated by such testing will be governed by section 
14(b) of the Act.
    (11) A requirement that the manufacturers and/or processors signing 
the consent agreement will comply with the notification requirements of 
section 12(b)(1) of the Act and part 707 of this chapter if they export 
or intend to export the substance or mixture for which the submission of 
data is required under the agreement and a statement that any other 
person who exports or intends to export such substance or mixture is 
subject to the above cited export notification requirements.
    (12) A requirement that, in the event EPA promulgates a significant 
new use rule applicable to the test chemical under section 5(a)(2), the 
consent agreement will have the status of a test rule for purposes of 
section 5(b)(1)(A) and manufacturers and/or processors signing the 
agreement will comply with the data submission requirements imposed by 
that provision.
    (13) A statement that each manufacturer and/or processor signing the 
agreement agrees that violation of its requirements will constitute a 
``prohibited act'' under section 15(1) of the Act and will trigger all 
provisions of TSCA applicable to a violation of section 15.
    (14) A statement that, in the event one or more provisions of the 
agreement are determined to be unenforceable by a court, the remainder 
of the agreement would not be presumed to be valid and EPA will then 
either initiate a rulemaking proceeding or publish in the Federal 
Register the Administrator's reason for not initiating such a 
proceeding.

[[Page 19]]

    (15) A statement that the Agency may conduct laboratory inspections 
and/or study audits of the testing being conducted pursuant to the 
consent agreement in accordance with the authority and procedures 
contained in section 11 of the Act.
    (16) A statement that EPA acceptance of a consent agreement 
constitutes ``final agency action'' for purposes of 5 U.S.C. 704.
    (17) Any other requirements that the parties agree are necessary to 
achieve the purposes of the Act.
    (b) Contents of standards for the development of data. The standards 
for the development of the data included in consent agreements will be 
based on the TSCA test guidelines in 40 CFR parts 796, 797, and 798, the 
Organization for Economic Cooperation and Development (OECD) test 
guidelines, the EPA pesticide assessment guidelines published by The 
National Technical Information Service (NTIS), or other suitable test 
methodologies. During the negotiation of consent agreements, EPA will 
initially propose suitable test guidelines as the required test 
standards; manufacturers and processors or other interested parties may 
then suggest alternative methodologies or modifications to the Agency's 
proposed guidelines. These alternative methodologies or modifications 
will be adopted only where, in the judgment of EPA, they will develop at 
least equally reliable and adequate data on the chemical substance or 
mixture subject to the agreement.
    (c) Statement of rationale for consent agreement. EPA will prepare a 
written explanation of the basis for each consent agreement. This 
document will summarize the agreement, describe any ITC testing 
recommendations for the chemical involved, outline the chemical's use 
and exposure characteristics, and explain the objectives of the testing 
to be conducted and the rationale for the specific studies selected. 
This document will be published in the Federal Register and, for ITC-
designated chemicals, will constitute the statement of EPA's reasons for 
not initiating rulemaking required by section 4(e)(1)(B) of the Act.

[51 FR 23715, June 30, 1986, as amended at 54 FR 36314, Sept. 1, 1989]



Sec. 790.62  Submission of study plans and conduct of testing.

    (a) Timing of submission. The principal sponsor of testing conducted 
pursuant to a consent agreement shall submit a study plan no later than 
45 days prior to the initiation of testing.
    (b) Content of study plans. All study plans are required to contain 
the following information:
    (1) Identity of the consent agreement under which testing will be 
performed.
    (2) The specific test requirements to be covered by the study plan.
    (3) The name and address of the principal test sponsor.
    (4) The names, addresses, and telephone numbers of the responsible 
administrative official[s] and project manager[s] in the principal 
sponsor's organization.
    (5) The names, addresses, and telephone numbers of the technical 
contacts at each manufacturer and/or processor subject to the agreement.
    (6) The names and addresses of the testing facilities responsible 
for the testing and the names, addresses, and telephone numbers of the 
administrative officials[s] and project manager[s] assigned to oversee 
the testing program at these facilities.
    (7) Brief summaries of the training and experience of each 
professional involved in the study, including study director, 
veterinarian[s], toxicologist[s], pathologist[s], chemist[s], 
microbiologist[s], and laboratory assistants.
    (8) Identity and supporting data on the chemical substance[s] being 
tested, including physical constants, spectral data, chemical analysis, 
and stability under test and storage conditions, as appropriate.
    (9) Study protocol, including the rationale for any combination of 
test protocols; the rationale for species/strain selection; dose 
selection (and supporting data); route(s) or method(s) of exposure; 
description of diet to be used and its source, including nutrients and 
contaminants and their concentrations; for in vitro test systems, a 
description of culture medium and its source; and a summary of expected 
spontaneous chronic diseases (including tumors), genealogy, and life 
span.

[[Page 20]]

    (10) A schedule, with reasonable timeables and deadlines, for 
initiation and completion of each short-term test and of each major 
phases of long-term tests, and submission of interim progress and/or 
final reports to EPA.
    (c) Review and modification. (1) Upon receipt of a study plan, EPA 
will review it to determine whether it complies with paragraph (b) of 
this section. If EPA determines that the study plan does not comply with 
paragraph (b) of this section, EPA will notify the submitter that the 
plan is incomplete and will identify the deficiencies and the steps 
necessary to complete the plan. It is the responsibility of the test 
sponsor to review the study protocols to determine if they comply with 
all the mandatory testing conditions and requirements in the test 
standards (i.e., ``shall statements'').
    (2) The submitter will have 15 days after the day it receives a 
notice under paragraph (c)(1) of this section to submit appropriate 
information to make the study plan complete.
    (3) If the submitter fails to provide appropriate information to 
complete a study plan within 15 days after having received a notice 
under paragraph (c)(1) of this section, the submitter will be considered 
to be in violation of the consent agreement and subject to enforcement 
proceedings pursuant to Sec. 790.65 (c) and (d).
    (4) The test sponsor shall submit any amendments to study plans to 
EPA at the address specified in Sec. 790.5(b).
    (d) Functions of the principal test sponsor. When testing is being 
conducted pursuant to a consent agreement, the principal test sponsor 
will be responsible for submitting interim progress and final reports to 
EPA, informing the Agency of any proposed changes in standards for the 
development of data, study plans or testing schedules, and communicating 
with the Agency about laboratory inspections and other matters affecting 
the progress of testing.

[51 FR 23715, June 30, 1986, as amended at 54 FR 36314, Sept. 1, 1989; 
60 FR 34466, July 3, 1995]



Sec. 790.65  Failure to comply with a consent agreement.

    (a) Manufacturers and/or processors who have signed a consent 
agreement and who fail to comply with the test requirements, test 
standards, GLP regulations, schedules, or other provisions contained in 
the consent agreement, or in modifications to the agreement adopted 
pursuant to Sec. 790.68, will be in violation of the consent agreement.
    (b) The Agency considers failure to comply with any aspect of a 
consent agreement to be a ``prohibited act'' under section 15 of TSCA, 
subject to all of the provisions of the Act applicable to violations of 
section 15. Section 15(1) of TSCA makes it unlawful for any person to 
fail or refuse to comply with any rule or order issued under section 4. 
Consent agreements adopted pursuant to this part are ``orders issued 
under section 4'' for purposes of section 15(1) of TSCA.
    (c) Manufacturers and/or processors who violate consent agreements 
are subject to criminal and/or civil liability. Under the penalty 
provisions of section 16 of TSCA, such firms could be subject to a civil 
penalty of up to $25,000 per violation with each day in violation 
constituting a separate violation of section 15. Intentional violations 
could lead to the imposition of criminal penalties of up to $25,000 for 
each day of violation and imprisonment for up to one year. In addition, 
EPA could invoke the remedies available under section 17 of TSCA, 
including seeking an injunction to compel adherence to the requirements 
of the consent agreement.
    (d) Noncompliance with a consent agreement will constitute conduct 
``in violation of this Act'' under section 20(a)(1) of TSCA. Thus, 
failure to comply with the requirements of a consent agreement could 
result in a citizens' civil action under section 20(a)(1) of TSCA.



Sec. 790.68  Modification of consent agreements.

    (a) Changes in the scope of testing. (1) Manufacturers or processors 
subject to a consent agreement, other persons or EPA may seek 
modifications in the scope of testing performed under the consent 
agreement. If, upon receiving a request for modification, EPA determines 
that new issues have been raised that warrant reconsideration of the 
scope of testing, or if EPA determines

[[Page 21]]

on its own that such reconsideration is appropriate, EPA will publish a 
Federal Register notice describing the proposed modification and 
soliciting public comment. If, based on the comments received, EPA 
concludes that differences of opinion may exist about the proposed 
modification, EPA will establish a schedule for conducting negotiations 
and invite parties who wish to participate in or monitor these 
negotiations to contact the Agency in writing. Any negotiations that EPA 
conducts will conform to the procedures specified in Sec. 790.22(b).
    (2) The scope of testing required by a consent agreement will be 
modified only where there is a consensus concerning the modified testing 
requirements among EPA, affected manufacturers and/or processors, and 
other persons who have asked to participate in or monitor negotiations 
under paragraph (a)(1) of this section. In determining whether a 
consensus exists, EPA will employ the criteria specified in Sec. 
790.24. In the absence of consensus, EPA may initiate rulemaking under 
section 4(a) of the Act if it concludes that any testing beyond that 
required by the consent agreement is necessary and that the other 
statutory findings required by section 4(a) can be made. While such 
rulemaking proceedings are underway, the consent agreement will remain 
in effect unless EPA finds that the testing required by the agreement is 
or may be unnecessary in view of the testing requirements included in 
EPA's proposed rule.
    (b) Changes in test standards or schedules. (1) Any test sponsor who 
wishes to modify the test schedule for any test required under a consent 
order must submit an application in accordance with this paragraph. 
Application for modification must be made in writing to EPA at the 
address in Sec. 790.5(b), or by phone with written confirmation to 
follow within 10 working days. Applications must include an appropriate 
explanation and rationale for the modification. EPA will consider only 
those applications that request modifications to mandatory testing 
conditions or requirements (``shall statements'' in the consent order). 
Where a test sponsor requests EPA to provide guidance or to clarify a 
non-mandatory testing requirement (i.e., ``should statements''), the 
test sponsor should submit these requests to EPA at the address in 
section 790.5(b).
    (2)(i) Where EPA concludes that the requested modification of a test 
standard or schedule for a test required under a consent agreement is 
appropriate, EPA will proceed in accordance with this paragraph (b)(2).
    (ii) Where, in EPA's judgment, the requested modification of a test 
standard or schedule would not alter the scope of the test or 
significantly change the schedule for completing the test, EPA will not 
ask for public comment before approving the modification. EPA will 
notify the test sponsor, and any other persons who have signed the 
consent agreement, by letter of EPA's approval. EPA will place copies of 
each application and EPA approval letter in the administrative record 
maintained for the consent agreement in question. EPA will publish a 
notice annually in the Federal Register indicating the test standards or 
schedules for test required in consent agreements which have been 
modified under this paragraph (b)(2)(ii) and describing the nature of 
the modifications.
    (iii) Where, in EPA's judgment, the requested modification of a test 
standard or schedule would significantly alter the scope of the test or 
significantly change the schedule for completing the test, EPA will 
publish a notice in the Federal Register requesting comment on the 
proposed modification. However, EPA will approve a requested 
modification of a test standard under paragraph (b)(2)(iii) of this 
section without first seeking public comment if EPA believes that an 
immediate modification to the test standard is necessary to preserve the 
accuracy or validity of an ongoing test. EPA also may modify a testing 
requirement or test condition in a test standard if EPA determines that 
the completion or achievement of this requirement or condition is not 
technically feasible. EPA may approve a requested modification of a test 
schedule under paragraph (b)(2)(iii) of this section without first 
seeking public comment if EPA determines, on a case-by-case basis, that 
a delay of over 12 months is not the fault of the test sponsor and is 
due

[[Page 22]]

to unforeseen circumstances such as a lack of laboratory availability, 
lack of availability of suitable test substance (e.g., 14-C labelled 
test substance), lack of availability of healthy test organisms, or the 
unexpected failure of a long-term test. EPA will publish an annual 
notice in the Federal Register announcing the approval of any test 
standard modifications and test scheduled extensions under paragraph 
(b)(2)(iii) of this section, and provide a brief rationale of why the 
modification was granted.
    (iv) For purposes of this paragraph (b)(2), a requested modification 
of a test standard of schedule for a test required under a consent 
agreement would alter the scope of the test or significantly change the 
schedule for completing the test if the modification would:
    (A) Change the test species.
    (B) Change the route of administration of the test chemical.
    (C) Change the period of time during which the test species is 
exposed to the test chemical.
    (D) Except as provided in paragraph (b)(2)(iii) of this section, 
extend the final reporting deadline more than 12 months from the date 
specified in the consent order.
    (3) Where EPA concludes that the requested modification of a test 
standard or schedule for a test requirement under a consent agreement is 
not appropriate, EPA will so notify the test sponsor in writing.
    (c) Timing. (1) Test sponsors should submit all applications for 
test schedule modifications at least 60 days before the reporting 
deadline for the test in question.
    (2) EPA will not normally approve any test schedule extensions 
submitted less than 30 days before the reporting deadline for the test 
in question.
    (3) Except as provided in paragraph (b)(2)(iii) of this section, EPA 
may grant extensions as shown necessary for up to 1 year but will 
normally limit extensions to a period of time equal to the in-life 
portion of the test plus 60 days.
    (4) EPA will normally approve only one deadline extension for each 
test.
    (5) Test sponsors should submit requests for test standard 
modifications as soon as they determine that the test cannot be 
successfully completed according to the test standard specified in the 
consent order.

[51 FR 23715, June 30, 1986, as amended at 52 FR 36571, Sept. 30, 1987; 
54 FR 36314, Sept. 1, 1989; 60 FR 34466, July 3, 1995]



                  Subpart E_Exemptions From Test Rules

    Source: 50 FR 20660, May 17, 1985, unless otherwise noted.



Sec. 790.80  Submission of exemption applications.

    (a) Who should file applications. (1) Any manufacturer or processor 
subject to a test rule in part 799 of this chapter may submit an 
application to EPA for an exemption from performing any or all of the 
tests required under the test rule.
    (2) Processors will not be required to apply for an exemption or 
conduct testing unless EPA so specifies in a test rule or in a special 
Federal Register notice as described in Sec. 790.48(b)(2) under the 
following circumstances:
    (i) If testing is being required to allow evaluation of risks 
associated with manufacturing and processing or with distribution in 
commerce, use, or disposal of the chemical and manufacturers do not 
submit notice(s) of intent to conduct the required testing; or
    (ii) If testing is being required solely to allow evaluation of 
risks associated with processing of the chemical.
    (b) When applications must be filed. (1) Exemption applications must 
be filed within 30 days after the effective date of the test rule 
described in Sec. 790.40 or, if being submitted in compliance with the 
Federal Register notice described in Sec. 790.48(b)(2), within 30 days 
after the publication of that notice.
    (2) Exemption applications must be filed by the date manufacture or 
processing begins by any person not manufacturing or processing the 
subject chemical as of the effective date of the test rule described in 
Sec. 790.40 or by 30 days after the effective date of the test rule 
described in Sec. 790.40, who, before the end of the reimbursement 
period, manufactures or processes the test substance and who is subject 
to the requirement to submit either a letter of

[[Page 23]]

intent to test or an exemption application.
    (3) When both manufacturers and processors are subject to the rule, 
exemption applications must be filed by the date processing begins by 
any person not processing as of the effective date of the test rule 
described in Sec. 790.40 or by 30 days after publication of the Federal 
Register notice described in Sec. 790.48(b)(2) who, before the end of 
the reimbursement period, processes the test substance and who is 
subject to the requirement to submit either a letter of intent to test 
or an exemption application.
    (c) Scope of application. A person may apply for an exemption from 
all, or one or more, specific testing requirements in a test rule in 
part 799 of this chapter.

[50 FR 20660, May 17, 1985, as amended at 58 FR 34205, June 23, 1993]



Sec. 790.82  Content of exemption application.

    The exemption application must contain:
    (a) The identity of the test rule, the chemical identity, and the 
CAS No. of the test substance on which the application is based.
    (b) The specific testing requirement(s) from which an exemption is 
sought and the basis for the exemption request.
    (c) Name, address, and telephone number of applicant.
    (d) Name, address, and telephone number of appropriate individual to 
contact for further information.
    (e)(1) If required in the test rule to establish equivalence:
    (i) The chemical identity of the test substance on which the 
application is based.
    (ii) Equivalence data specified in Sec. 790.85.
    (2) If a test rule requires testing of a single representative 
substance, EPA will consider all forms of the chemical subject to that 
rule to be equivalent and will not require the submission of equivalence 
data as described in Sec. 790.85.

[50 FR 20660, May 17, 1985, as amended at 54 FR 36315, Sept. 1, 1989]



Sec. 790.85  Submission of equivalence data.

    If EPA requires in a test rule promulgated under section 4 of the 
Act the testing of two or more test substances which are forms of the 
same chemical, each exemption applicant must submit the following data:
    (a) The chemical identity of each technical-grade chemical substance 
or mixture manufactured and/or processed by the applicant for which the 
exemption is sought. The exact type of identifying data required will be 
specified in the test rule, but may include all characteristics and 
properties of the applicant's substance or mixture, such as boiling 
point, melting point, chemical analysis (including identification and 
amount of impurities), additives, spectral data, and other physical or 
chemical information that may be relevant in determining whether the 
applicant's substance or mixture is equivalent to the specific test 
substance.
    (b) The basis for the applicant's belief that the substance or 
mixture is equivalent to the test substance or mixture.
    (c) Any other data which exemption applicants are directed to submit 
in the test rule which may bear on a determination of equivalence. This 
may include a description of the process by which each technical-grade 
chemical substance or mixture for which an exemption is sought is 
manufactured or processed prior to use or distribution in commerce by 
the applicant.



Sec. 790.87  Approval of exemption applications.

    (a) EPA will conditionally approve exemption applications if:
    (1)(i) For single-phase test rules, EPA has received a letter of 
intent to conduct the testing from which exemption is sought;
    (ii) For two-phase test rules, EPA has received a complete proposed 
study plan for the testing from which exemption is sought and has 
adopted the study plan, as proposed or modified, as test standards and 
schedules in a final Phase II test rule; and
    (2) The chemical substance or mixture with respect to which the 
application was submitted is equivalent to a test substance or mixture 
for which the

[[Page 24]]

required data have been or are being submitted in accordance with a test 
rule; and
    (3) Submission of the required test data concerning that chemical 
substance or mixture would be duplicative of data which have been or are 
being submitted to EPA in accordance with a test rule.
    (b)(1) If a single representative substance is to be tested under a 
test rule, EPA will consider all forms of the chemical subject to that 
rule to be equivalent and will contact the exemption applicant only if 
information is missing or unclear.
    (2) If two or more representative substances are to be tested under 
a test rule, EPA will evaluate equivalence claims made in each exemption 
application according to the criteria discussed in the test rule.
    (i) If EPA finds an equivalence claim to be in error or inadequately 
supported, the applicant will be notified by certified mail. The 
applicant will be given 15 days to provide clarifying information.
    (ii) Exemption applicants will be notified that equivalence has been 
accepted or rejected.
    (c) The final Phase II test rule which adopts the study plans in 
two-phase rulemaking, a separate Federal Register notice in single-phase 
rulemaking, or a letter by certified mail will give exemption applicants 
final notice that they have received a conditional exemption. All 
conditional exemptions thus granted are contingent upon the test 
sponsors' successful completion of testing according to the 
specifications in the test rule.



Sec. 790.88  Denial of exemption application.

    (a) EPA may deny any exemption application if:
    (1) EPA determines that the applicant has failed to demonstrate that 
the applicant's chemical is equivalent to the test substance; or
    (2) The exemption applicant fails to submit any of the information 
specified in Sec. 790.82; or
    (3) The exemption applicant fails to submit any of the information 
specified in Sec. 790.85 if required in the test rule; or
    (4)(i) For single-phase test rules, EPA has not received a letter of 
intent to conduct the test for which exemption is sought; or
    (ii) For two-phase test rules, EPA has not received an adequate 
study plan for the test for which exemption is sought; or
    (5) The study sponsor(s) fails to initiate the required testing by 
the deadlines adopted in the test rule; or
    (6) The study sponsor(s) fails to submit data as required in the 
test standard and deadlines for submission of test data as adopted in 
the test rule or as modified in accordance with Sec. 790.55.
    (b) EPA will notify the exemption applicant by certified mail or 
Federal Register notice of EPA's determination that the exemption 
application is denied.



Sec. 790.90  Appeal of denial of exemption application.

    (a) Within 30 days after receipt of notification that EPA has denied 
an application for exemption, the applicant may file an appeal with EPA.
    (b) The appeal shall indicate the basis for the applicant's request 
for reconsideration.
    (c)(1) The applicant may also include a request for a hearing. 
Hearings will be held according to the procedures described in Sec. 
790.97.
    (2) Hearing requests must be in writing and must be received by EPA 
within 30 days of receipt of the letter or publication of the Federal 
Register notice described in Sec. 790.88(b). Hearing requests must 
provide reasons why a hearing is necessary.
    (d) If EPA determines that there are material issues of fact, then 
the request for a hearing will be granted. If EPA denies a hearing 
request, EPA will base its decision on the written submission.
    (e) EPA will notify the applicant of its decision within 60 days 
after EPA receives the appeal described in paragraph (a) of this section 
or within 60 days after completion of a hearing described in paragraph 
(c) of this section.
    (f) The filing of an appeal from the denial of an exemption shall 
not act to stay the applicant's legal obligations under a test rule 
promulgated under section 4 of the Act.

[[Page 25]]



Sec. 790.93  Termination of conditional exemption.

    (a) EPA shall terminate a conditional exemption if it determines 
that:
    (1) The test which provided the basis for approval of the exemption 
application has not been started by the deadlines for initiation of 
testing adopted in the test rule or modified in accordance with Sec. 
790.55; or
    (2) Data required by the test rule have not been generated in 
accordance with the test standards or submitted in accordance with the 
deadlines for submission of test data that were adopted in the test rule 
or modified in accordance with Sec. 790.55; or
    (3) The testing has not been conducted or the data have not been 
generated in accordance with the Good Laboratory Practice requirements 
in part 792 of this chapter.
    (b) If EPA determines that one or more of the criteria listed in 
paragraph (a) of this section has been met, EPA will notify each holder 
of an affected conditional exemption by certified mail or Federal 
Register notice of EPA's intent to terminate that conditional exemption.
    (c) Within 30 days after receipt of a letter of notification or 
publication of a notice in the Federal Register that EPA intends to 
terminate a conditional exemption, the exemption holder may submit 
information to rebut EPA's preliminary decision or notify EPA by letter 
of its intent to conduct the required test pursuant to the test standard 
established in the final test rule. Such a letter of intent shall 
contain all of the information required by Sec. 790.45(c).
    (d)(1) The exemption holder may also include a request for a 
hearing. Hearings will be held in accordance with the procedures set 
forth in Sec. 790.97.
    (2) Hearing requests must be in writing and must be received by EPA 
within 30 days after receipt of the letter or publication in the Federal 
Register notice described in paragraph (b) of this section.
    (e) EPA will notify the exemption holder by certified letter or by 
Federal Register notice of EPA's final decision concerning termination 
of conditional exemptions and will give instructions as to what actions 
the former exemption holder must take to avoid being found in violation 
of the test rule.



Sec. 790.97  Hearing procedures.

    (a) Hearing requests must be in writing to EPA and must include the 
applicant's basis for appealing EPA's decision.
    (b) If more than one applicant has requested a hearing on similar 
grounds, all of those appeals will be considered at the same hearing 
unless confidentiality claims preclude a joint hearing.
    (c) EPA will notify each applicant of EPA's decision within 60 days 
after the hearing.



Sec. 790.99  Statement of financial responsibility.

    Each applicant for an exemption shall submit the following sworn 
statement with his or her application:

    I understand that if this application is granted before the 
reimbursement period described in section 4(c)(3)(B) of TSCA expires, I 
must pay fair and equitable reimbursement to the person or persons who 
incurred or shared in the costs of complying with the requirement to 
submit data and upon whose data the granting of my application was 
based.



   Sec. Appendix A to Subpart E of Part 790--Schedule for Developing 
                    Consent Agreements and Test Rules

    EPA intends to follow the schedule set forth in this Appendix to 
evaluate testing candidates, conduct negotiations, develop consent 
agreements where appropriate, and propose and promulate test rules in 
those instances where testing can be required under section 4(a) of TSCA 
but agreement cannot be reached in timely manner on a consent agreement. 
Where deadlines are imposed by the statute, they are binding on EPA and 
will be observed by the Agency. The remaining dates represent targets 
that EPA intends to meet.
    This schedule is based on what EPA currently believes are reasonable 
target dates. As EPA gains experience with the process and determines 
the feasibility of these schedules, it may adjust the schedule 
accordingly. EPA will solicit public comment before implementing any 
changes in the schedule.

------------------------------------------------------------------------
           Week \1\                               Event
------------------------------------------------------------------------
0.............................  Receive ITC report, recommendation.

[[Page 26]]

 
2.............................  Publish ITC report, 8(a) and 8(d)
                                 notices, and invitation for public
                                 participation in negotiations.
3-6...........................  Comment period on ITC report.
6.............................  Public focus meeting.
7-14..........................  8(a) and 8(d) reporting period.
22............................  Public meeting on course-setting
                                 decision and deadline for requests to
                                 participate in negotiations.
22-30.........................  Negotiations.
32............................  EPA decision point: consent agreement or
                                 test rule.
------------------------------------------------------------------------
\1\ The dates contained in the left-hand column are calculated from the
  date EPA receives the ITC report recommending a chemical for testing.


------------------------------------------------------------------------
      Week          Consent Agreement      Week          Test Rule
------------------------------------------------------------------------
36-40..........  Comment period on          32-60  Rule preparation,
                  consent agreement.                agency review and
                                                    sign-off.
42.............  Comment resolution            62  Publish proposed rule
                  meeting if necessary.             in Federal
                                                    Register.\1\
48.............  Sign-off consent          70-106  Agency reviews
                  agreement and Federal             comments;
                  Register notice.                  preparation of final
                                                    rule or no-test
                                                    decision, agency
                                                    review and sign-off.
                                                    \1\
50.............  Publish Federal              108  Publish final rule or
                  Register notice.                  no-test decision in
                                                    Federal Register.\1\
------------------------------------------------------------------------
\1\ As stated in Sec.  790.26, EPA may publish an Advance Notice of
  Proposed Rulemaking (ANPR) where the testing recommendations of the
  ITC raise unusually novel and complex issues that require additional
  Agency review and opportunity for public comment. EPA intends to
  publish such ANPRs by Week 62 following receipt of the initial ITC
  report; to publish a proposed rule or decision-not-to-test by Week
  108; and to publish a final rule or notice terminating the rulemaking
  process by Week 154.


[51 FR 23717, June 30, 1986]



PART 791_DATA REIMBURSEMENT--Table of Contents




                      Subpart A_General Provisions

Sec.
791.1 Scope and authority.
791.2 Applicability.
791.3 Definitions.

                      Subpart B_Hearing Procedures

791.20 Initiation of reimbursement proceeding.
791.22 Consolidation of hearings.
791.27 Pre-hearing preparation.
791.29 Appointment of hearing officer.
791.30 Hearing procedures.
791.31 Expedited procedures.
791.34 Serving of notice.
791.37 The award.
791.39 Fees and expenses.

                   Subpart C_Basis for Proposed Order

791.40 Basis for the proposed order.
791.45 Processors.
791.48 Production volume.
791.50 Costs.
791.52 Multiple tests.

                            Subpart D_Review

791.60 Review.

                          Subpart E_Final Order

791.85 Availability of final Agency order.

                        Subpart F_Prohibited Acts

791.105 Prohibited acts.

    Authority: 15 U.S.C. 2603 and 2607.

    Source: 48 FR 31791, July 11, 1983, unless otherwise noted.



                      Subpart A_General Provisions



Sec. 791.1  Scope and authority.

    (a) This part establishes procedures and criteria to be used in 
determining fair amounts of reimbursement for testing costs incurred 
under section 4(a) of the Toxic Substances Control Act (TSCA) (15 U.S.C. 
2603(a)).
    (b) Section 4(c) of TSCA requires EPA to develop rules for the 
determination of fair and equitable reimbursement (15 U.S.C. 2603 (c)).



Sec. 791.2  Applicability.

    (a) This rule is potentially applicable to all manufacturers, 
importers and processors who may be required by a specific test rule 
promulgated under section 4(a) of TSCA to conduct tests and submit data, 
and who seek the assistance of the Administrator in determining the 
amount or method of reimbursement. Persons subject to a test rule have 
an obligation from the date the test rule becomes effective until the 
end of the reimbursement period, either to test or to obtain an 
exemption and pay reimbursement.
    (b) The provisions of this rule will take effect only when private 
efforts to resolve a dispute have failed and a manufacturer or processor 
requests EPA's assistance.

[[Page 27]]



Sec. 791.3  Definitions.

    Terms defined in the Act, and not explicitly defined herein, are 
used with the meanings given in the Act.
    (a) The Act refers to the Toxic Substances Control Act (TSCA) (15 
U.S.C. 2601 et seq.).
    (b) The Agency or EPA refers to the Environmental Protection Agency.
    (c) Byproduct refers to a chemical substance produced without a 
separate commercial intent during the manufacture, processing, use or 
disposal of another chemical substance or mixture.
    (d) Dispute refers to a present controversy between parties subject 
to a test rule over the amount or method of reimbursement for the cost 
of developing health and environmental data on the test chemical.
    (e) Exemption holder refers to a manufacturer or processor, subject 
to a test rule, that has received an exemption under sections 4(c)(1) or 
4(c)(2) of TSCA from the requirement to conduct a test and submit data.
    (f) Impurity refers to a chemical substance unintentionally present 
with another chemical substance or mixture.
    (g) A party refers to a person subject to a section 4 test rule, 
who:
    (1) Seeks reimbursement from another person under these rules, or
    (2) From whom reimbursement is sought under these rules.
    (h) Reimbursement period refers to a period that begins when the 
data from the last non-duplicative test to be completed under a test 
rule is submitted to EPA and ends after an amount of time equal to that 
which had been required to develop that data or after 5 years, whichever 
is later.
    (i) Small business refers to a manufacturer or importer whose annual 
sales, when combined with those of its parent company (if any) are less 
than $30 million.
    (j) Test rule refers to a regulation ordering the development of 
data on health or environmental effects or chemical fate for a chemical 
substance or mixture pursuant to TSCA section 4(a).



                      Subpart B_Hearing Procedures



Sec. 791.20  Initiation of reimbursement proceeding.

    (a) When persons subject to a test rule are unable to reach an 
agreement on the amount or method of reimbursement for test data 
development as described in TSCA section 4(c)(3)(A), any of them may 
initiate a proceeding by filing two signed copies of a request for a 
hearing with a regional office of the American Arbitration Association 
and mailing a copy of the request to EPA, and to each person from whom 
they seek reimbursement, or who seeks reimbursement from them.
    (b) The request for hearing must contain the following:
    (1) The names and addresses of the filing party and its counsel, if 
any.
    (2) Identification of the test rule under which the dispute arose.
    (3) A list of the parties from whom reimbursement is sought or who 
are seeking reimbursement, a brief description of the attempts to reach 
agreement and a concise explanation of the issues on which the parties 
are unable to agree.
    (c) The request for a hearing shall be accompanied by the 
appropriate administrative fee, as provided in a current Fee Schedule of 
the American Arbitration Association.



Sec. 791.22  Consolidation of hearings.

    (a) Promptly upon receipt of the request for a hearing, the 
Administrator will publish a notice in the Federal Register, advising 
those subject to the test rule that a request for a hearing has been 
made.
    (b) Any other person wishing to participate in the hearing shall so 
notify EPA within 45 days of the Federal Register notice. EPA will 
promptly inform the regional office of the American Arbitration 
Association where the request has been filed of the additional parties.



Sec. 791.27  Pre-hearing preparation.

    (a) Responses to requests for hearings. After filing of the request 
for hearing, if any other party desires to file an answer it shall be 
made in writing and filed with the American Arbitration Association, and 
a copy thereof shall

[[Page 28]]

be mailed to the other parties within a period of fourteen days from the 
date of receiving the complete list of parties. After the hearing 
officer is appointed, however, no new or different claim may be 
submitted except with the hearing officer's consent.
    (b) Pre-hearing conference. At the request of the parties or at the 
discretion of the American Arbitration Association, a pre-hearing 
conference with a representative of the American Arbitration Association 
and the parties or their counsel will be scheduled in appropriate cases 
to arrange for an exchange of information and the stipulation of 
uncontested facts so as to expedite the proceedings.
    (c) Fixing of locale. The parties may mutually agree on the locale 
where the hearing is to be held. If the locale is not designated within 
45 days from the time the complete list of parties is received, the 
American Arbitration Association shall have power to determine the 
locale. Its decision shall be final and binding. If any party requests, 
and informs the other parties of its request, that the hearing be held 
in a specific locale and the other parties file no objection thereto 
within 14 days of the request, the locale shall be the one requested.
    (d) Time and place. The hearing officer shall fix the time and place 
for each hearing. The American Arbitration Association will mail notice 
to each party at least 14 days in advance.



Sec. 791.29  Appointment of hearing officer.

    (a) Qualifications of hearing officer. All hearing officers shall be 
neutral, subject to disqualification for the reasons specified in 
paragraph (f) of this section.
    (b) Appointment from panel. Promptly after receiving the complete 
list of parties at the close of the notice period described in Sec. 
791.22, the American Arbitration Association shall submit simultaneously 
to each party to the dispute an identical list of names. Each party to 
the dispute shall have thirty days from the mailing date in which to 
cross off any names objected to, number the remaining names to indicate 
the order of preference, and return the list to the American Arbitration 
Association. If a party does not return the list within the time 
specified, all persons named therein shall be deemed acceptable to that 
party. From among the persons who have been approved on all lists, and 
in accordance with the designated order of mutual preference, the 
American Arbitration Association shall invite the acceptance of a 
hearing officer to serve. If the parties fail to agree upon any of the 
persons named, or if acceptable hearing officers are unable to act, or 
if for any other reason the appointment cannot be made from the 
submitted lists, the American Arbitration Association shall have the 
power to make the appointment without the submission of any additional 
list.
    (c) Nationality of hearing officer in international dispute. If one 
of the parties is a national or resident of a country other than the 
United States, the hearing officer shall upon the request of any party, 
be appointed from among the nationals of a country other than that of 
the parties.
    (d) Number of hearing officers. The dispute shall be heard and 
determined by one hearing officer unless the American Arbitration 
Association, in its discretion, directs that a greater number of hearing 
officers be appointed.
    (e) Notice of appointment. Notice of the appointment of the hearing 
officer, together with a copy of these rules, and the signed acceptance 
of the hearing officer shall be filed prior to the opening of the first 
hearing.
    (f) Disclosure and challenge procedure. A person appointed as 
hearing officer shall disclose to the American Arbitration Association 
any circumstances likely to affect impartiality, including any bias or 
any financial or personal interest in the result of the hearing or any 
past or present relationship with the parties or their counsel. Upon 
receipt of such information from such hearing officer or other source, 
the American Arbitration Association shall communicate such information 
to the parties, and, if it deems it appropriate to do so, to the hearing 
officer and others. Thereafter, the American Arbitration Association 
shall determine whether the hearing officer should be disqualified and 
shall inform the parties of its decision, which shall be conclusive.

[[Page 29]]

    (g) Vacancies. If any hearing officer should resign, die, withdraw, 
refuse, be disqualified or be unable to perform the duties of the 
office, the American Arbitration Association may, on proof satisfactory 
to it, declare the office vacant. Vacancies shall be filled in 
accordance with the applicable provisions of these rules and the matter 
shall be reheard unless the parties shall agree otherwise.



Sec. 791.30  Hearing procedures.

    (a) Representation by counsel. Any party may be represented by 
counsel. A party intending to be so represented shall notify the other 
parties and the American Arbitration Association of the name and address 
of counsel at least 5 days prior to the date set for the hearing at 
which counsel is first to appear. When a hearing is initiated by 
counsel, or where an attorney replies for the other party, such notice 
is deemed to have been given.
    (b) Stenographic record. The American Arbitration Association shall 
make the necessary arrangements for the taking of a stenographic record. 
The parties shall share the cost of such record.
    (c) Attendance at hearings. The hearing officer shall have the power 
to require the exclusion of anyone, including a party or other essential 
person, during the testimony of any witness to protect confidential 
business information. It shall be discretionary with the hearing officer 
to determine the propriety of the attendance of any other person.
    (d) Oaths. Hearing officers shall swear or affirm their neutrality 
and their dedication to a fair and equitable resolution. Witnesses shall 
swear or affirm that they are telling the truth.
    (e) Order of proceedings. (1) A hearing shall be opened by the 
filing of the oath of the hearing officer and by the recording of the 
place, time and date of the hearing, the presence of the hearing officer 
and parties, and counsel, if any, and by the receipt by the hearing 
officer of the request for hearing and answer, if any.
    (2) The hearing officer may, at the beginning of the hearing, ask 
for statements clarifying the issues involved.
    (3) The party or parties seeking reimbursement shall then present a 
claim and proofs and witnesses, who shall submit to questions or other 
examination. The party or parties from whom reimbursement is sought 
shall then present a defense and proofs and witnesses, who shall submit 
to questions or other examination. The hearing officer has discretion to 
vary this procedure but shall afford full and equal opportunity to all 
parties for the presentation of any material or relevant proofs.
    (4) Exhibits, when offered by any party, shall be received in 
evidence by the hearing officer. The names and addresses of all 
witnesses and exhibits in order received shall be made a part of the 
record.
    (f) Hearing in the absence of a party. A hearing may proceed in the 
absence of any party which, after due notice, fails to be present or 
fails to obtain an adjournment. An award shall not be made solely on the 
default of a party. The hearing officer shall require the parties who 
are present to submit such evidence as the hearing officer may require 
for the making of an award.
    (g) Evidence. (1) The parties may offer such evidence as they desire 
and shall produce such additional evidence as the hearing officer may 
deem necessary to an understanding and determination of the dispute. The 
hearing officer shall be the judge of the relevancy and materiality of 
the evidence offered and conformity to legal rules of evidence shall not 
be necessary. All evidence shall be taken in the presence of all the 
hearing officers and of all the parties, except where any of the parties 
is absent in default, has waived the right to be present, or has been 
excluded by the hearing officer to protect confidential business 
information.
    (2) All documents not filed with the hearing officer at the hearing, 
but arranged for by agreement of the parties, shall be filed with the 
American Arbitration Association for transmission to the hearing 
officer, according to the agreed-upon schedule. All parties shall be 
afforded opportunity to examine such documents.
    (h) Evidence by affidavit and filing of documents. The hearing 
officer shall receive and consider the evidence of witnesses by 
affidavit, but shall give it only such weight as the hearing officer

[[Page 30]]

deems it entitled to after consideration of any objections made to its 
admission.
    (i) Closing of hearings. The hearing officer shall specifically 
inquire of all parties whether they have any further proofs to offer or 
witnesses to be heard. Upon receiving negative replies, the hearing 
officer shall declare the hearings closed and record the time of closing 
of the hearing. If briefs are to be filed, the hearings shall be 
declared closed as of the final date set by the hearing officer for the 
receipt of briefs. If documents are to be filed as provided for in 
paragraph (g)(2) of this section and the date set for their receipt is 
later than that set for the receipt of briefs, the later date shall be 
the date of closing the hearings.
    (j) Reopening of hearings. The hearings may be reopened on the 
hearing officer's own motion, or upon application of a party at any time 
before the award is made. If the reopening of the hearings would prevent 
the making of the award within the specified time the matter may not be 
reopened, unless the parties agree upon the extension of the time limit.
    (k) Waiver of oral hearings. The parties may provide, by written 
agreement, for the waiver of oral hearings. If the parties are unable to 
agree as to the procedure, the American Arbitration Association shall 
specify a fair and equitable procedure.
    (l) Waiver of rules. Any party who proceeds with the hearing after 
knowledge that any provision or requirement of these rules has not been 
complied with and who fails to state objection thereto in writing, shall 
be deemed to have waived the right to object.
    (m) Extensions of time. The parties may modify any period of time by 
mutual agreement. The American Arbitration Association for good cause 
may extend any period of time established by these rules, except the 
time for making the award. (Sec. 791.37(a)) The American Arbitration 
Association shall notify the parties of any such extension of time and 
its reason therefor.
    (n) Communication with hearing officer. There shall be no direct 
communication between the parties and a hearing officer other than at 
oral hearings. Any other oral or written communications from the parties 
to the hearing officer shall be directed to the American Arbitration 
Association for transmittal to the hearing officer.



Sec. 791.31  Expedited procedures.

    Unless the American Arbitration Association in its discretion 
determines otherwise, the Expedited Procedures described in this section 
shall be applied in any case where the total claim of any party does not 
exceed $5,000, exclusive of interest and hearing costs, and may be 
applied in other cases if the parties agree.
    (a) Application of rules. The expedited hearings will be conducted 
according to the same procedures as the regular ones, except for those 
specifically changed by the expedited rules in this section, Sec. 
791.31.
    (b) Notice by telephone. The parties shall accept all notices from 
the American Arbitration Association by telephone. Such notices by the 
American Arbitration Association shall subsequently be confirmed in 
writing to the parties. Notwithstanding the failure to confirm in 
writing any notice or objection hereunder, the proceeding shall 
nonetheless be valid if notice or obligation has, in fact, been given by 
telephone.
    (c) Appointment and qualifications of hearing officers. The American 
Arbitration Association shall submit simultaneously to each party to the 
dispute an identical list of five persons from which one hearing officer 
shall be appointed. Each party shall have the right to strike two names 
from the list on a peremptory basis. The list is returnable to the 
American Arbitration Association within 10 days from the date of 
mailing. If for any reasons the appointment cannot be made from the 
list, the American Arbitration Association shall have the authority to 
make the appointment without the submission of additional lists. Such 
appointment shall be subject to disqualification for the reasons 
specified in Sec. 791.29(f). The parties shall be given notice by 
telephone by the American Arbitration Association of the appointment of 
the hearing officer. The parties shall notify the American Arbitration 
Association, by telephone, within 7 days of any objections to the 
hearing

[[Page 31]]

officer(s) appointed. Any objection by a party to such hearing officer 
shall be confirmed in writing to the American Arbitration Association 
with a copy to the other parties.
    (d) Time and place of hearing. The hearing officer shall fix the 
date, time and place of the hearing. The American Arbitration 
Association will notify the parties by telephone, 7 days in advance of 
the hearing date. Formal notice of hearing will be sent by the American 
Arbitration Association to the parties.
    (e) The hearing. Generally, the hearing shall be completed within 1 
day. The hearing officer, for good cause shown, may schedule an 
additional hearing to be held within 5 days.
    (f) Time of award. Unless otherwise agreed to by the parties, the 
Award shall be rendered not later than 15 business days from the date of 
the closing of the hearing.



Sec. 791.34  Serving of notice.

    (a) Each party shall be deemed to have consented that any papers, 
notices or process necessary or proper for the initiation or 
continuation of a hearing under these rules and for any appeal to EPA or 
any court action in connection therewith may be served upon such party 
by mail addressed to such party or its attorney at its last known 
address or by personal service, within or without the state wherein the 
arbitration is to be held (whether such party be within or without the 
United States of America), provided that reasonable opportunity to be 
heard with regard thereto has been granted such party.
    (b) The American Arbitration Association shall, upon the written 
request of a party, furnish to such party, at its expense, certified 
facsimiles of any papers in the American Arbitration Association's 
possession that may be required in appeal to EPA or judicial proceedings 
relating to the hearing.



Sec. 791.37  The award.

    (a) Time of award. The award shall be made promptly by the hearing 
officer and, unless otherwise agreed by the parties, no later than 30 
days from the date of closing the hearings, or if oral hearings have 
been waived, from the date of transmitting the final statements and 
proofs to the hearing officer.
    (b) Form of award. The award shall be in writing and shall be signed 
either by the sole hearing officer or by at least a majority if there is 
more than one. It shall contain a concise statement of its basis and 
rationale, and a timetable for payment of any ordered reimbursement.
    (c) Delivery of award to parties. Parties shall accept as legal 
delivery of the award the delivery of the award or a true copy thereof 
by certified mail to the party at its last known address or to its 
attorney, or by personal service.



Sec. 791.39  Fees and expenses.

    (a) Administrative fees. (1) As a not-for-profit organization, the 
American Arbitration Association shall prescribe an Administrative Fee 
Schedule and a Refund Schedule to compensate it for the cost of 
providing administrative services. The schedule in effect at the time of 
filing or the time of refund shall be applicable.
    (2) The administrative fees shall be advanced by the initiating 
party or parties, subject to final apportionment by the hearing officer 
in the award. The administrative fee is increased by 10 percent of the 
original for each additional party.
    (3) Fees and expenses in excess of the limit contained in section 
26(b) of TSCA ($2,500 per person, or $100 per small business) will be 
paid by EPA.
    (b) Expenses. Subject to paragraph (a)(3) of this section, all 
expenses of the hearing, including the cost of recording (though not 
transcribing) the hearing and required traveling and other expenses of 
the hearing officer and of American Arbitration Association 
representatives, and the expenses of any witness or the cost of any 
proofs produced at the direct request of the hearing officer, shall be 
borne equally by the parties, unless they agree otherwise, or unless the 
hearing officer, in the award, assesses such expenses or any part 
thereof against any specified party or parties.
    (c) Hearing officer's fee. Hearing officers will normally serve 
without a fee. In prolonged or special cases the American Arbitration 
Association in consultation with the Administrator may

[[Page 32]]

determine that payment of a fee by the parties is appropriate and may 
establish a reasonable amount, taking into account the extent of service 
by the hearing officer and other relevant circumstances of the case. Any 
arrangements for compensation shall be made through the American 
Arbitration Association and not directly between the parties and the 
hearing officer.



                   Subpart C_Basis for Proposed Order



Sec. 791.40  Basis for the proposed order.

    (a) The hearing officer shall propose a fair and equitable amount of 
reimbursement. The formula in paragraph (b) of this section shall be 
presumed to be fair and equitable as applied to all persons subject to a 
test rule. However, the hearing officer has the discretion to modify the 
formula, or to use some other basis for allocation if necessary. 
Additional factors that may be taken into account include, but are not 
limited to, relative amounts of exposure attributable to each person and 
the effect of the reimbursement share on competitive position.
    (b) In general, each person's share of the test cost shall be in 
proportion to its share of the total production volume of the test 
chemical:
[GRAPHIC] [TIFF OMITTED] TC15NO91.044

Where:

R=the reimbursement share owed by company X.
C=the total cost of the testing required by the test rule.
Vx=the volume of the test chemical produced or imported by 
company X over the period defined by Sec. 791.48.
Vt=the total volume of the test chemical produced or imported 
over the period defined by Sec. 791.48.

    (c) The burden of proposing modifications to the formula shall lie 
with the party requesting the modification.



Sec. 791.45  Processors.

    (a) Generally, processors will be deemed to have fulfilled their 
testing and reimbursement responsibilities indirectly, through higher 
prices passed on by those directly responsible, the manufacturers. There 
are three circumstances in which processors will have a responsibility 
to provide reimbursement directly to those paying for the testing:
    (1) When a test rule or subsequent Federal Register notice 
pertaining to a test rule expressly obligates processors as well as 
manufacturers to assume direct testing and data reimbursement 
responsibilities.
    (2) When one or more manufacturers demonstrate to the hearing 
officer that it is necessary to include processors in order to provide 
fair and equitable reimbursement in a specific case.
    (3) When one or more processors voluntarily agree to reimburse 
manufacturers for a portion of test costs. Only those processors who 
volunteer will incur the obligation.
    (b) A hearing including processors shall be initiated in the same 
way as those including only manufacturers. Voluntary negotiations must 
be attempted in good faith first, and the request for a hearing must 
contain the names of the parties and a description of the unsuccessful 
negotiations.
    (c) When processors as well as manufacturers are required to provide 
reimbursement, the hearing officer will decide for each case how the 
reimbursement should be allocated among the participating parties. When 
a test rule is applicable solely to processors, the hearing officer will 
apply the formula to the amount of the test chemical purchased or 
processed.



Sec. 791.48  Production volume.

    (a) Production volume will be measured over a period that begins one 
calendar year before publication of the final test rule in the Federal 
Register and continues up to the latest data available upon resolution 
of a dispute.
    (b) For the purpose of determining fair reimbursement shares, 
production volume shall include amounts of the test chemical imported in 
bulk form and mixtures, and the total domestic production of the 
chemical including that produced as a byproduct. Impurities will not be 
included unless the test rule specifically includes them.

[[Page 33]]

    (c) Amounts of the test chemical manufactured for export will not be 
included unless covered by a finding under TSCA section 12(a)(2).
    (d) Chemicals excluded from the jurisdiction of TSCA by section 
3(2)(B) need not be included in the computation of production volume. 
(Chemicals used as intermediates to produce pesticides are covered by 
TSCA.)
    (e) The burden of establishing the fact that particular amounts of 
the test chemical are produced for exempt purposes lies with the party 
seeking to exclude those amounts from the calculation of his production 
volume.



Sec. 791.50  Costs.

    (a) All costs reasonable and necessary to comply with the test rule, 
taking into account the practices of other laboratories in conducting 
similar tests, are eligible for reimbursement. Necessary costs include:
    (1) Direct and indirect costs of planning, conducting, analyzing and 
submitting the test results to EPA.
    (2) A reasonable profit, and a reasonable rate of interest and 
depreciation on the tester's initial capital investment.
    (3) The cost of repeating or repairing tests where failure was 
demonstrably due to some cause other than negligence of the tester.
    (b) Costs attributable to tests beyond those specified by EPA shall 
not be eligible for reimbursement under this rule.



Sec. 791.52  Multiple tests.

    When more than one of a particular kind of test required by the test 
rule is performed, the additional costs will be shared among all those 
holding exemptions. The costs of all the tests will be added together 
and each exemption holder shall be responsible for a share of the total 
which is equal to its share of the total production of the test 
chemical. The exemption holders shall divide their shares between test 
sponsors in proportion to the costs of their respective tests. Those 
sponsoring a particular test do not have to obtain exemptions for that 
test and therefore do not have reimbursement responsibilities for the 
same tests done by others.



                            Subpart D_Review



Sec. 791.60  Review.

    (a) The hearing officer's proposed order shall become the final 
Agency order 30 days after issuance unless within the 30-day period one 
of the parties requests Agency review or the Administrator of his own 
initiative decides to review the proposed order.
    (b) The proposed order may be reviewed upon the record of the 
hearing and the petitions for review. If necesary, the Administrator may 
order the transcription of the stenographic record of the hearing, 
written briefs, oral arguments or any other reasonable aids to making an 
equitable decision.
    (c) The final Agency order may be reviewed in federal court as 
provided by 26 U.S.C. 2603(c).



                          Subpart E_Final Order



Sec. 791.85  Availablity of final Agency order.

    The final Agency order shall be available to the public for 
inspection and copying pursuant to 5 U.S.C. 552(a)(2), subject to 
necessary confidentiality restrictions.



                        Subpart F_Prohibited Acts



Sec. 791.105  Prohibited acts.

    Failure to provide information required by the Agency or to pay the 
amounts awarded under this rule within time alloted in the final order 
shall constitute a violation of 15 U.S.C. 2614(1) or 2614(3).



PART 792_GOOD LABORATORY PRACTICE STANDARDS--Table of Contents




                      Subpart A_General Provisions

Sec.
792.1 Scope.
792.3 Definitions.
792.10 Applicability to studies performed under grants and contracts.
792.12 Statement of compliance or non-compliance.
792.15 Inspection of a testing facility.
792.17 Effects of non-compliance.

                  Subpart B_Organization and Personnel

792.29 Personnel.

[[Page 34]]

792.31 Testing facility management.
792.33 Study director.
792.35 Quality assurance unit.

                          Subpart C_Facilities

792.41 General.
792.43 Test system care facilities.
792.45 Test system supply facilities.
792.47 Facilities for handling test, control, and reference substances.
792.49 Laboratory operation areas.
792.51 Specimen and data storage facilities.

                           Subpart D_Equipment

792.61 Equipment design.
792.63 Maintenance and calibration of equipment.

                 Subpart E_Testing Facilities Operation

792.81 Standard operating procedures.
792.83 Reagents and solutions.
792.90 Animal and other test system care.

            Subpart F_Test, Control, and Reference Substances

792.105 Test, control, and reference substance characterization.
792.107 Test, control, and reference substance handling.
792.113 Mixtures of substances with carriers.

              Subpart G_Protocol for and Conduct of A Study

792.120 Protocol.
792.130 Conduct of a study.
792.135 Physical and chemical characterization studies.

Subparts H-I [Reserved]

                      Subpart J_Records and Reports

792.185 Reporting of study results.
792.190 Storage and retrieval of records and data.
792.195 Retention of records.

    Authority: 15 U.S.C. 2603.

    Source: 54 FR 34043, Aug. 17, 1989, unless otherwise noted.



                      Subpart A_General Provisions



Sec. 792.1  Scope.

    (a) This part prescribes good laboratory practices for conducting 
studies relating to health effects, environmental effects, and chemical 
fate testing. This part is intended to ensure the quality and integrity 
of data submitted pursuant to testing consent agreements and test rules 
issued under section 4 of the Toxic Substances Control Act (TSCA) (Pub. 
L. 94-469, 90 Stat. 2006, 15 U.S.C. 2603 et seq.).
    (b) This part applies to any study described by paragraph (a) of 
this section which any person conducts, initiates, or supports on or 
after September 18, 1989.
    (c) It is EPA's policy that all data developed under section 5 of 
TSCA be in accordance with provisions of this part. If data are not 
developed in accordance with the provisions of this part, EPA will 
consider such data insufficient to evaluate the health and environmental 
effects of the chemical substances unless the submitter provides 
additional information demonstrating that the data are reliable and 
adequate.



Sec. 792.3  Definitions.

    As used in this part the following terms shall have the meanings 
specified:
    Batch means a specific quantity or lot of a test, control, or 
reference substance that has been characterized according to Sec. 
792.105(a).
    Carrier means any material, including but not limited to, feed, 
water, soil, and nutrient media, with which the test substance is 
combined for administration to a test system.
    Control substance means any chemical substance or mixture, or any 
other material other than a test substance, feed, or water, that is 
administered to the test system in the course of a study for the purpose 
of establishing a basis for comparison with the test substance for 
chemical or biologicaI measurements.
    EPA means the U.S. Environmental Protection Agency.
    Experimental start date means the first date the test substance is 
applied to the test system.
    Experimental termination date means the last date on which data are 
collected directly from the study.
    FDA means the U.S. Food and Drug Administration.
    Person includes an individual, partnership, corporation, 
association, scientific or academic establishment, government agency, or 
organizational unit thereof, and any other legal entity.
    Quality assurance unit means any person or organizational element, 
except

[[Page 35]]

the study director, designated by testing facility management to perform 
the duties relating to quality assurance of the studies.
    Raw data means any laboratory worksheets, records, memoranda, notes, 
or exact copies thereof, that are the result of original observations 
and activities of a study and are necessary for the reconstruction and 
evaluation of the report of that study. In the event that exact 
transcripts of raw data have been prepared (e.g., tapes which have been 
transcribed verbatim, dated, and verified accurate by signature), the 
exact copy or exact transcript may be substituted for the original 
source as raw data. ``Raw data'' may include photographs, microfilm or 
microfiche copies, computer printouts, magnetic media, including 
dictated observations, and recorded data from automated instruments.
    Reference substance means any chemical substance or mixture, or 
analytical standard, or material other than a test substance, feed, or 
water, that is administered to or used in analyzing the test system in 
the course of a study for the purposes of establishing a basis for 
comparison with the test substance for known chemical or biological 
measurements.
    Specimen means any material derived from a test system for 
examination or analysis.
    Sponsor means:
    (1) A person who initiates and supports, by provision of financial 
or other resources, a study;
    (2) A person who submits a study to the EPA in response to a TSCA 
section 4(a) test rule and/or a person who submits a study under a TSCA 
section 4 testing consent agreement or a TSCA section 5 rule or order to 
the extent the agreement, rule or order references this part; or
    (3) A testing facility, if it both initiates and actually conducts 
the study.
    Study means any experiment at one or more test sites, in which a 
test substance is studied in a test system under laboratory conditions 
or in the environment to determine or help predict its effects, 
metabolism, environmental and chemical fate, persistence, or other 
characteristics in humans, other living organisms, or media. The term 
``study'' does not include basic exploratory studies carried out to 
determine whether a test substance or a test method has any potential 
utility.
    Study completion date means the date the final report is signed by 
the study director.
    Study director means the individual responsible for the overall 
conduct of a study.
    Study initiation date means the date the protocol is signed by the 
study director.
    Test substance means a substance or mixture administered or added to 
a test system in a study, which substance or mixture is used to develop 
data to meet the requirements of a TSCA section 4(a) test rule and/or is 
developed under a TSCA section 4 testing consent agreement or section 5 
rule or order to the extent the agreement, rule or order references this 
part.
    Test system means any animal, plant, microorganism, chemical or 
physical matrix, including but not limited to, soil or water, or 
components thereof, to which the test, control, or reference substance 
is administered or added for study. ``Test system'' also includes 
appropriate groups or components of the system not treated with the 
test, control, or reference substance.
    Testing facility means a person who actually conducts a study, i.e., 
actually uses the test substance in a test system. ``Testing facility'' 
encompasses only those operational units that are being or have been 
used to conduct studies.
    TSCA means the Toxic Substances Control Act (15 U.S.C, 2601 et seq.)
    Vehicle means any agent which facilitates the mixture, dispersion, 
or solubilization of a test substance with a carrier.



Sec. 792.10  Applicability to studies performed under grants and contracts.

    When a sponsor or other person utilizes the services of a consulting 
laboratory, contractor, or grantee to perform all or a part of a study 
to which this part applies, it shall notify the consulting laboratory, 
contractor, or grantee that the service is, or is part of, a study that 
must be conducted in compliance with the provisions of this part.

[[Page 36]]



Sec. 792.12  Statement of compliance or non-compliance.

    Any person who submits to EPA a test required by a testing consent 
agreement or a test rule issued under section 4 of TSCA shall include in 
the submission a true and correct statement, signed by the sponsor and 
the study director, of one of the following types:
    (a) A statement that the study was conducted in accordance with this 
part; or
    (b) A statement describing in detail all differences between the 
practices used in the study and those required by this part; or
    (c) A statement that the person was not a sponsor of the study, did 
not conduct the study, and does not know whether the study was conducted 
in accordance with this part.



Sec. 792.15  Inspection of a testing facility.

    (a) A testing facility shall permit an authorized employee or duly 
designated representative of EPA or FDA, at reasonable times and in a 
reasonable manner, to inspect the facility and to inspect (and in the 
case of records also to copy) all records and specimens required to be 
maintained regarding studies to which this part applies. The records 
inspection and copying requirements shall not apply to quality assurance 
unit records of findings and problems, or to actions recommended and 
taken, except the EPA may seek production of these records in litigation 
or formal adjudicatory hearings.
    (b) EPA will not consider reliable for purposes of showing that a 
chemical substance or mixture does not present a risk of injury to 
health or the environment any data developed by a testing facility or 
sponsor that refuses to permit inspection in accordance with this part. 
The determination that a study will not be considered reliable does not, 
however, relieve the sponsor of a required test of any obligation under 
any applicable statute or regulation to submit the results of the study 
to EPA.
    (c) Since a testing facility is a place where chemicals are stored 
or held, it is subject to inspection under section 11 of TSCA.



Sec. 792.17  Effects of non-compliance.

    (a) The sponsor or any other person who is conducting or has 
conducted a test to fulfill the requirements of a testing consent 
agreement or a test rule issued under section 4 of TSCA will be in 
violation of section 15 of TSCA if:
    (1) The test is not being or was not conducted in accordance with 
any requirement of this part;
    (2) Data or information submitted to EPA under this part (including 
the statement required by Sec. 792.12) include information or data that 
are false or misleading, contain significant omissions, or otherwise do 
not fulfill the requirements of this part; or
    (3) Entry in accordance with Sec. 792.15 for the purpose of 
auditing test data or inspecting test facilities is denied. Persons who 
violate the provisions of this part may be subject to civil or criminal 
penalties under section 16 of TSCA, legal action in United States 
district court under section 17 of TSCA, or criminal prosecution under 
18 U.S.C. 2 or 1001.
    (b) EPA, at its discretion, may not consider reliable for purposes 
of showing that a chemical substance or mixture does not present a risk 
of injury to health or the environment any study which was not conducted 
in accordance with this part. EPA, at its discretion, may rely upon such 
studies for purposes of showing adverse effects. The determination that 
a study will not be considered reliable does not, however, relieve the 
sponsor of a required test of the obligation under any applicable 
statute or regulation to submit the results of the study to EPA.
    (c) If data submitted to fulfill a requirement of a testing consent 
agreement or a test rule issued under section 4 of TSCA are not 
developed in accordance with this part, EPA may determine that the 
sponsor has not fulfilled its obligations under section 4 of TSCA and 
may require the sponsor to develop data in accordance with the 
requirements of this part in order to satisfy such obligations.

[[Page 37]]



                  Subpart B_Organization and Personnel



Sec. 792.29  Personnel.

    (a) Each individual engaged in the conduct of or responsible for the 
supervision of a study shall have education, training, and experience, 
or combination thereof, to enable that individual to perform the 
assigned functions.
    (b) Each testing facility shall maintain a current summary of 
training and experience and job description for each individual engaged 
in or supervising the conduct of a study.
    (c) There shall be a sufficient number of personnel for the timely 
and proper conduct of the study according to the protocol.
    (d) Personnel shall take necessary personal sanitation and health 
precautions designed to avoid contamination of test, control, and 
reference substances and test systems.
    (e) Personnel engaged in a study shall wear clothing appropriate for 
the duties they perform. Such clothing shall be changed as often as 
necessary to prevent microbiological, radiological, or chemical 
contamination of test systems and test, control, and reference 
substances.
    (f) Any individual found at any time to have an illness that may 
adversely affect the quality and integrity of the study shall be 
excluded from direct contact with test systems, test, control, and 
reference substances and any other operation or function that may 
adversely affect the study until the condition is corrected. All 
personnel shall be instructed to report to their immediate supervisors 
any health or medical conditions that may reasonably be considered to 
have an adverse effect on a study.



Sec. 792.31  Testing facility management.

    For each study, testing facility management shall:
    (a) Designate a study director as described in Sec. 792.33 before 
the study is initiated.
    (b) Replace the study director promptly if it becomes necessary to 
do so during the conduct of a study.
    (c) Assure that there is a quality assurance unit as described in 
Sec. 792.35.
    (d) Assure that test, control, and reference substances or mixtures 
have been appropriately tested for identity, strength, purity, 
stability, and uniformity, as applicable.
    (e) Assure that personnel, resources, facilities, equipment, 
materials and methodologies are available as scheduled.
    (f) Assure that personnel clearly understand the functions they are 
to perform.
    (g) Assure that any deviations from these regulations reported by 
the quality assurance unit are communicated to the study director and 
corrective actions are taken and documented.



Sec. 792.33  Study director.

    For each study, a scientist or other professional of appropriate 
education, training, and experience, or combination thereof, shall be 
identified as the study director. The study director has overall 
responsibility for the technical conduct of the study, as well as for 
the interpretation, analysis, documentation, and reporting of results, 
and represents the single point of study control. The study director 
shall assure that:
    (a) The protocol, including any change, is approved as provided by 
Sec. 792.120 and is followed.
    (b) All experimental data, including observations of unanticipated 
responses of the test system are accurately recorded and verified.
    (c) Unforeseen circumstances that may affect the quality and 
integrity of the study are noted when they occur, and corrective action 
is taken and documented.
    (d) Test systems are as specified in the protocol.
    (e) All applicable good laboratory practice regulations are 
followed.
    (f) All raw data, documentation, protocols, specimens, and final 
reports are transferred to the archives during or at the close of the 
study.



Sec. 792.35  Quality assurance unit.

    (a) A testing facility shall have a quality assurance unit which 
shall be responsible for monitoring each study to assure management that 
the facilities, equipment, personnel, methods, practices, records, and 
controls are in

[[Page 38]]

conformance with the regulations in this part. For any given study, the 
quality assurance unit shall be entirely separate from and independent 
of the personnel engaged in the direction and conduct of that study. The 
quality assurance unit shall conduct inspections and maintain records 
appropriate to the study.
    (b) The quality assurance unit shall:
    (1) Maintain a copy of a master schedule sheet of all studies 
conducted at the testing facility indexed by test substance and 
containing the test system, nature of study, date study was initiated, 
current status of each study, identity of the sponsor, and name of the 
study director.
    (2) Maintain copies of all protocols pertaining to all studies for 
which the unit is responsible.
    (3) Inspect each study at intervals adequate to ensure the integrity 
of the study and maintain written and properly signed records of each 
periodic inspection showing the date of the inspection, the study 
inspected, the phase or segment of the study inspected, the person 
performing the inspection, findings and problems, action recommended and 
taken to resolve existing problems, and any scheduled date for re-
inspection. Any problems which are likely to affect study integrity 
found during the course of an inspection shall be brought to the 
attention of the study director and management immediately.
    (4) Periodically submit to management and the study director written 
status reports on each study, noting any problems and the corrective 
actions taken.
    (5) Determine that no deviations from approved protocols or standard 
operating procedures were made without proper authorization and 
documentation.
    (6) Review the final study report to assure that such report 
accurately describes the methods and standard operating procedures, and 
that the reported results accurately reflect the raw data of the study.
    (7) Prepare and sign a statement to be included with the final study 
report which shall specify the dates inspections were made and findings 
reported to management and to the study director.
    (c) The responsibilities and procedures applicable to the quality 
assurance unit, the records maintained by the quality assurance unit, 
and the method of indexing such records shall be in writing and shall be 
maintained. These items including inspection dates, the study inspected, 
the phase or segment of the study inspected, and the name of the 
individual performing the inspection shall be made available for 
inspection to authorized employees or duly designated representatives of 
EPA or FDA.
    (d) An authorized employee or a duly designated representative of 
EPA or FDA shall have access to the written procedures established for 
the inspection and may request testing facility management to certify 
that inspections are being implemented, performed, documented, and 
followed up in accordance with this paragraph.



                          Subpart C_Facilities



Sec. 792.41  General.

    Each testing facility shall be of suitable size and construction to 
facilitate the proper conduct of studies. Testing facilities which are 
not located within an indoor controlled environment shall be of suitable 
location to facilitate the proper conduct of studies. Testing facilities 
shall be designed so that there is a degree of separation that will 
prevent any function or activity from having an adverse effect on the 
study.



Sec. 792.43  Test system care facilities.

    (a) A testing facility shall have a sufficient number of animal 
rooms or other test system areas, as needed, to ensure: proper 
separation of species or test systems, isolation of individual projects, 
quarantine or isolation of animals or other test systems, and routine or 
specialized housing of animals or other test systems.
    (1) In tests with plants or aquatic animals, proper separation of 
species can be accomplished within a room or area by housing them 
separately in different chambers or aquaria. Separation of species is 
unnecessary where the protocol specifies the simultaneous exposure of 
two or more species in the

[[Page 39]]

same chamber, aquarium, or housing unit.
    (2) Aquatic toxicity tests for individual projects shall be isolated 
to the extent necessary to prevent cross-contamination of different 
chemicals used in different tests.
    (b) A testing facility shall have a number of animal rooms or other 
test system areas separate from those described in paragraph (a) of this 
section to ensure isolation of studies being done with test systems or 
test, control, and reference substances known to be biohazardous, 
including volatile substances, aerosols, radioactive materials, and 
infectious agents.
    (c) Separate areas shall be provided, as appropriate, for the 
diagnosis, treatment, and control of laboratory test system diseases. 
These areas shall provide effective isolation for the housing of test 
systems either known or suspected of being diseased, or of being 
carriers of disease, from other test systems.
    (d) Facilities shall have proper provisions for collection and 
disposal of contaminated water, soil, or other spent materials. When 
animals are housed, facilities shall exist for the collection and 
disposal of all animal waste and refuse or for safe sanitary storage of 
waste before removal from the testing facility. Disposal facilities 
shall be so provided and operated as to minimize vermin infestation, 
odors, disease hazards, and environmental contamination.
    (e) Facilities shall have provisions to regulate environmental 
conditions (e.g., temperature, humidity, photoperiod) as specified in 
the protocol.
    (f) For marine test organisms, an adequate supply of clean sea water 
or artificial sea water (prepared from deionized or distilled water and 
sea salt mixture) shall be available. The ranges of composition shall be 
as specified in the protocol.
    (g) For freshwater organisms, an adequate supply of clean water of 
the appropriate hardness, pH, and temperature, and which is free of 
contaminants capable of interfering with the study shall be available as 
specified in the protocol.
    (h) For plants, an adequate supply of soil of the appropriate 
composition, as specified in the protocol, shall be available as needed.



Sec. 792.45  Test system supply facilities.

    (a) There shall be storage areas, as needed, for feed, nutrients, 
soils, bedding, supplies, and equipment. Storage areas for feed, 
nutrients, soils, and bedding shall be separated from areas where the 
test systems are located and shall be protected against infestation or 
contamination. Perishable supplies shall be preserved by appropriate 
means.
    (b) When appropriate, plant supply facilities shall be provided. 
These include:
    (1) Facilities, as specified in the protocol, for holding, 
culturing, and maintaining algae and aquatic plants.
    (2) Facilities, as specified in the protocol, for plant growth, 
including but not limited to, greenhouses, growth chambers, light banks, 
and fields.
    (c) When appropriate, facilities for aquatic animal tests shall be 
provided. These include but are not limited to aquaria, holding tanks, 
ponds, and ancillary equipment, as specified in the protocol.



Sec. 792.47  Facilities for handling test, control, and reference substances.

    (a) As necessary to prevent contamination or mixups, there shall be 
separate areas for:
    (1) Receipt and storage of the test, control, and reference 
substances.
    (2) Mixing of the test, control, and reference substances with a 
carrier, e.g., feed.
    (3) Storage of the test, control, and reference substance mixtures.
    (b) Storage areas for test, control, and/or reference substance and 
for test, control, and/or reference mixtures shall be separate from 
areas housing the test systems and shall be adequate to preserve the 
identity, strength, purity, and stability of the substances and 
mixtures.



Sec. 792.49  Laboratory operation areas.

    Separate laboratory space and other space shall be provided, as 
needed, for the performance of the routine and specialized procedures 
required by studies.

[[Page 40]]



Sec. 792.51  Specimen and data storage facilities.

    Space shall be provided for archives, limited to access by 
authorized personnel only, for the storage and retrieval of all raw data 
and specimens from completed studies.



                           Subpart D_Equipment



Sec. 792.61  Equipment design.

    Equipment used in the generation, measurement, or assessment of data 
and equipment used for facility environmental control shall be of 
appropriate design and adequate capacity to function according to the 
protocol and shall be suitably located for operation, inspection, 
cleaning, and maintenance.



Sec. 792.63  Maintenance and calibration of equipment.

    (a) Equipment shall be adequately inspected, cleaned, and 
maintained. Equipment used for the generation, measurement, or 
assessment of data shall be adequately tested, calibrated, and/or 
standardized.
    (b) The written standard operating procedures required under Sec. 
792.81(b)(11) shall set forth in sufficient detail the methods, 
materials, and schedules to be used in the routine inspection, cleaning, 
maintenance, testing, calibration, and/or standardization of equipment, 
and shall specify, when appropriate, remedial action to be taken in the 
event of failure or malfunction of equipment. The written standard 
operating procedures shall designate the person responsible for the 
performance of each operation.
    (c) Written records shall be maintained of all inspection, 
maintenance, testing, calibrating, and/or standardizing operations. 
These records, containing the date of the operation, shall describe 
whether the maintenance operations were routine and followed the written 
standard operating procedures. Written records shall be kept of 
nonroutine repairs performed on equipment as a result of failure and 
malfunction. Such records shall document the nature of the defect, how 
and when the defect was discovered, and any remedial action taken in 
response to the defect.



                 Subpart E_Testing Facilities Operation



Sec. 792.81  Standard operating procedures.

    (a) A testing facility shall have standard operating procedures in 
writing, setting forth study methods that management is satisfied are 
adequate to insure the quality and integrity of the data generated in 
the course of a study. All deviations in a study from standard operating 
procedures shall be authorized by the study director and shall be 
documented in the raw data. Significant changes in established standard 
operating procedures shall be properly authorized in writing by 
management.
    (b) Standard operating procedures shall be established for, but not 
limited to, the following:
    (1) Test system room preparation.
    (2) Test system care.
    (3) Receipt, identification, storage, handling, mixing, and method 
of sampling of the test, control, and reference substances.
    (4) Test system observations.
    (5) Laboratory or other tests.
    (6) Handling of test systems found moribund or dead during study.
    (7) Necropsy of test systems or postmortem examination of test 
systems.
    (8) Collection and identification of specimens.
    (9) Histopathology.
    (10) Data handling, storage and retrieval.
    (11) Maintenance and calibration of equipment.
    (12) Transfer, proper placement, and identification of test systems.
    (c) Each laboratory or other study area shall have immediately 
available manuals and standard operating procedures relative to the 
laboratory or field procedures being performed. Published literature may 
be used as a supplement to standard operating procedures.
    (d) A historical file of standard operating procedures, and all 
revisions thereof, including the dates of such revisions, shall be 
maintained.



Sec. 792.83  Reagents and solutions.

    All reagents and solutions in the laboratory areas shall be labeled 
to indicate identity, titer or concentration,

[[Page 41]]

storage requirements, and expiration date. Deteriorated or outdated 
reagents and solutions shall not be used.



Sec. 792.90  Animal and other test system care.

    (a) There shall be standard operating procedures for the housing, 
feeding, handling, and care of animals and other test systems.
    (b) All newly received test systems from outside sources shall be 
isolated and their health status or appropriateness for the study shall 
be evaluated. This evaluation shall be in accordance with acceptable 
veterinary medical practice or scientific methods.
    (c) At the initiation of a study, test systems shall be free of any 
disease or condition that might interfere with the purpose or conduct of 
the study. If during the course of the study, the test systems contract 
such a disease or condition, the diseased test systems should be 
isolated, if necessary. These test systems may be treated for disease or 
signs of disease provided that such treatment does not interfere with 
the study. The diagnosis, authorization of treatment, description of 
treatment, and each date of treatment shall be documented and shall be 
retained.
    (d) Warm-blooded animals, adult reptiles, and adult terrestrial 
amphibians used in laboratory procedures that require manipulations and 
observations over an extended period of time, or in studies that require 
these test systems to be removed from and returned to their test system-
housing units for any reason (e.g., cage cleaning, treatment, etc.), 
shall receive appropriate identification (e.g., tattoo, color code, ear 
tag, ear punch, etc.). All information needed to specifically identify 
each test system within the test system-housing unit shall appear on the 
outside of that unit. Suckling mammals and juvenile birds are excluded 
from the requirement of individual identification unless otherwise 
specified in the protocol.
    (e) Except as specified in paragraph (e)(1) of this section, test 
systems of different species shall be housed in separate rooms when 
necessary. Test systems of the same species, but used in different 
studies, should not ordinarily be housed in the same room when 
inadvertent exposure to test, control, or reference substances or test 
system mixup could affect the outcome of either study. If such mixed 
housing is necessary, adequate differentiation by space and 
identification shall be made.
    (1) Plants, invertebrate animals, aquatic vertebrate animals, and 
organisms that may be used in multispecies tests need not be housed in 
separate rooms, provided that they are adequately segregated to avoid 
mixup and cross contamination.
    (2) [Reserved]
    (f) Cages, racks, pens, enclosures, aquaria, holding tanks, ponds, 
growth chambers, and other holding, rearing, and breeding areas, and 
accessory equipment, shall be cleaned and sanitized at appropriate 
intervals.
    (g) Feed, soil, and water used for the test systems shall be 
analyzed periodically to ensure that contaminants known to be capable of 
interfering with the study and reasonably expected to be present in such 
feed, soil, or water are not present at levels above those specified in 
the protocol. Documentation of such analyses shall be maintained as raw 
data.
    (h) Bedding used in animal cages or pens shall not interfere with 
the purpose or conduct of the study and shall be changed as often as 
necessary to keep the animals dry and clean.
    (i) If any pest control materials are used, the use shall be 
documented. Cleaning and pest control materials that interfere with the 
study shall not be used.
    (j) All plant and animal test systems shall be acclimatized to the 
environmental conditions of the test, prior to their use in a study.



            Subpart F_Test, Control, and Reference Substances



Sec. 792.105  Test, control, and reference substance characterization.

    (a) The identity, strength, purity, and composition, or other 
characteristics which will appropriately define the test, control, or 
reference substance shall be determined for each batch and shall be 
documented before its use in a study. Methods of synthesis, fabrication, 
or derivation of the test, control,

[[Page 42]]

or reference substance shall be documented by the sponsor or the testing 
facility, and such location of documentation shall be specified.
    (b) When relevant to the conduct of the study the solubility of each 
test, control, or reference substance shall be determined by the testing 
facility or the sponsor before the experimental start date. The 
stability of the test, control or reference substance shall be 
determined before the experimental start date or concomitantly according 
to written standard operating procedures, which provide for periodic 
analysis of each batch.
    (c) Each storage container for a test, control, or reference 
substance shall be labeled by name, chemical abstracts service number 
(CAS) or code number, batch number, expiration date, if any, and, where 
appropriate, storage conditions necessary to maintain the identity, 
strength, purity, and composition of the test, control, or reference 
substance. Storage containers shall be assigned to a particular test 
substance for the duration of the study.
    (d) For studies of more than 4 weeks experimental duration, reserve 
samples from each batch of test, control, and reference substances shall 
be retained for the period of time provided by Sec. 792.195.
    (e) The stability of test, control, and reference substances under 
storage conditions at the test site shall be known for all studies.



Sec. 792.107  Test, control, and reference substance handling.

    Procedures shall be established for a system for the handling of the 
test, control, and reference substances to ensure that:
    (a) There is proper storage.
    (b) Distribution is made in a manner designed to preclude the 
possibility of contamination, deterioration, or damage.
    (c) Proper identification is maintained throughout the distribution 
process.
    (d) The receipt and distribution of each batch is documented. Such 
documentation shall include the date and quantity of each batch 
distributed or returned.



Sec. 792.113  Mixtures of substances with carriers.

    (a) For each test, control, or reference substance that is mixed 
with a carrier, tests by appropriate analytical methods shall be 
conducted:
    (1) To determine the uniformity of the mixture and to determine, 
periodically, the concentration of the test, control, or reference 
substance in the mixture.
    (2) When relevant to the conduct of the experiment, to determine the 
solubility of each test, control, or reference substance in the mixture 
by the testing facility or the sponsor before the experimental start 
date.
    (3) To determine the stability of the test, control or reference 
substance in the mixture before the experimental start date or 
concomitantly according to written standard operating procedures, which 
provide for periodic analysis of each batch.
    (b) Where any of the components of the test, control, or reference 
substance carrier mixture has an expiration date, that date shall be 
clearly shown on the container. If more than one component has an 
expiration date, the earliest date shall be shown.
    (c) If a vehicle is used to facilitate the mixing of a test 
substance with a carrier, assurance shall be provided that the vehicle 
does not interfere with the integrity of the test.



              Subpart G_Protocol for and Conduct of A Study



Sec. 792.120  Protocol.

    (a) Each study shall have an approved written protocol that clearly 
indicates the objectives and all methods for the conduct of the study. 
The protocol shall contain but shall not necessarily be limited to the 
following information:
    (1) A descriptive title and statement of the purpose of the study.
    (2) Identification of the test, control, and reference substance by 
name, chemical abstracts service (CAS) number or code number.
    (3) The name and address of the sponsor and the name and address of 
the testing facility at which the study is being conducted.

[[Page 43]]

    (4) The proposed experimental start and termination dates.
    (5) Justification for selection of the test system.
    (6) Where applicable, the number, body weight, sex, source of 
supply, species, strain, substrain, and age of the test system.
    (7) The procedure for identification of the test system.
    (8) A description of the experimental design, including methods for 
the control of bias.
    (9) Where applicable, a description and/or identification of the 
diet used in the study as well as solvents, emulsifiers and/or other 
materials used to solubilize or suspend the test, control, or reference 
substances before mixing with the carrier. The description shall include 
specifications for acceptable levels of contaminants that are reasonably 
expected to be present in the dietary materials and are known to be 
capable of interfering with the purpose or conduct of the study if 
present at levels greater than established by the specifications.
    (10) The route of administration and the reason for its choice.
    (11) Each dosage level, expressed in milligrams per kilogram of body 
or test system weight or other appropriate units, of the test, control, 
or reference substance to be administered and the method and frequency 
of administration.
    (12) The type and frequency of tests, analyses, and measurements to 
be made.
    (13) The records to be maintained.
    (14) The date of approval of the protocol by the sponsor and the 
dated signature of the study director.
    (15) A statement of the proposed statistical method.
    (b) All changes in or revisions of an approved protocol and the 
reasons therefor shall be documented, signed by the study director, 
dated, and maintained with the protocol.



Sec. 792.130  Conduct of a study.

    (a) The study shall be conducted in accordance with the protocol.
    (b) The test systems shall be monitored in conformity with the 
protocol.
    (c) Specimens shall be identified by test system, study, nature, and 
date of collection. This information shall be located on the specimen 
container or shall accompany the specimen in a manner that precludes 
error in the recording and storage of data.
    (d) In animal studies where histopathology is required, records of 
gross findings for a specimen from postmortem observations shall be 
available to a pathologist when examining that specimen 
histopathologically.
    (e) All data generated during the conduct of a study, except those 
that are generated by automated data collection systems, shall be 
recorded directly, promptly, and legibly in ink. All data entries shall 
be dated on the day of entry and signed or initialed by the person 
entering the data. Any change in entries shall be made so as not to 
obscure the original entry, shall indicate the reason for such change, 
and shall be dated and signed or identified at the time of the change. 
In automated data collection systems, the individual responsible for 
direct data input shall be identified at the time of data input. Any 
change in automated data entries shall be made so as not to obscure the 
original entry, shall indicate the reason for change, shall be dated, 
and the responsible individual shall be identified.



Sec. 792.135  Physical and chemical characterization studies.

    (a) All provisions of the GLPs shall apply to physical and chemical 
characterization studies designed to determine stability, solubility, 
octanol water partition coefficient, volatility, and persistence (such 
as biodegradation, photodegradation, and chemical degradation studies).
    (b) The following GLP standards shall not apply to studies designed 
to determine physical and chemical characteristics of a test, control, 
or reference substance:

Section 792.31 (c), (d), and (g)
Section 792.35 (b) and (c)
Section 792.43
Section 792.45
Section 792.47
Section 792.49
Section 792.81(b) (1), (2), (6) through (9), and (12)
Section 792.90
Section 792.105 (a) through (d)

[[Page 44]]

Section 792.113
Section 792.120(a) (5) through (12), and (15)
Section 792.185(a) (5) through (8), (10), (12), and (14)
Section 792.195 (c) and (d)

Subparts H-I [Reserved]



                      Subpart J_Records and Reports



Sec. 792.185  Reporting of study results.

    (a) A final report shall be prepared for each study and shall 
include, but not necessarily be limited to, the following:
    (1) Name and address of the facility performing the study and the 
dates on which the study was initiated and was completed, terminated, or 
discontinued.
    (2) Objectives and procedures stated in the approved protocol, 
including any changes in the original protocol.
    (3) Statistical methods employed for analyzing the data.
    (4) The test, control, and reference substances identified by name, 
chemical abstracts service (CAS) number or code number, strength, 
purity, and composition, or other appropriate characteristics.
    (5) Stability, and when relevant to the conduct of the study, the 
solubility of the test, control, and reference substances under the 
conditions of administration.
    (6) A description of the methods used.
    (7) A description of the test system used. Where applicable, the 
final report shall include the number of animals or other test organisms 
used, sex, body weight range, source of supply, species, strain and 
substrain, age, and procedure used for identification.
    (8) A description of the dosage, dosage regimen, route of 
administration, and duration.
    (9) A description of all circumstances that may have affected the 
quality or integrity of the data.
    (10) The name of the study director, the names of other scientists 
or professionals and the names of all supervisory personnel, involved in 
the study.
    (11) A description of the transformations, calculations, or 
operations performed on the data, a summary and analysis of the data, 
and a statement of the conclusions drawn from the analysis.
    (12) The signed and dated reports of each of the individual 
scientists or other professionals involved in the study, including each 
person who, at the request or direction of the testing facility or 
sponsor, conducted an analysis or evaluation of data or specimens from 
the study after data generation was completed.
    (13) The locations where all specimens, raw data, and the final 
report are to be stored.
    (14) The statement prepared and signed by the quality assurance unit 
as described in Sec. 792.35(b)(7).
    (b) The final report shall be signed and dated by the study 
director.
    (c) Corrections or additions to a final report shall be in the form 
of an amendment by the study director. The amendment shall clearly 
identify that part of the final report that is being added to or 
corrected and the reasons for the correction or addition, and shall be 
signed and dated by the person responsible. Modification of a final 
report to comply with the submission requirements of EPA does not 
constitute a correction, addition, or amendment to a final report.
    (d) A copy of the final report and of any amendment to it shall be 
maintained by the sponsor and the test facility.



Sec. 792.190  Storage and retrieval of records and data.

    (a) All raw data, documentation, records, protocols, specimens, and 
final reports generated as a result of a study shall be retained. 
Specimens obtained from mutagenicity tests, specimens of soil, water, 
and plants, and wet specimens of blood, urine, feces, and biological 
fluids, do not need to be retained after quality assurance verification. 
Correspondence and other documents relating to interpretation and 
evaluation of data, other than those documents contained in the final 
report, also shall be retained.
    (b) There shall be archives for orderly storage and expedient 
retrieval of all raw data, documentation, protocols, specimens, and 
interim and final reports. Conditions of storage shall minimize 
deterioration of the documents or

[[Page 45]]

specimens in accordance with the requirements for the time period of 
their retention and the nature of the documents of specimens. A testing 
facility may contract with commercial archives to provide a repository 
for all material to be retained. Raw data and specimens may be retained 
elsewhere provided that the archives have specific reference to those 
other locations.
    (c) An individual shall be identified as responsible for the 
archives.
    (d) Only authorized personnel shall enter the archives.
    (e) Material retained or referred to in the archives shall be 
indexed to permit expedient retrieval.



Sec. 792.195  Retention of records.

    (a) Record retention requirements set forth in this section do not 
supersede the record retention requirements of any other regulations in 
this subchapter.
    (b)(1) Except as provided in paragraph (c) of this section, 
documentation records, raw data, and specimens pertaining to a study and 
required to be retained by this part shall be retained in the archive(s) 
for a period of at least ten years following the effective date of the 
applicable final test rule.
    (2) In the case of negotiated testing agreements, each agreement 
will contain a provision that, except as provided in paragraph (c) of 
this section, documentation records, raw data, and specimens pertaining 
to a study and required to be retained by this part shall be retained in 
the archive(s) for a period of at least ten years following the 
publication date of the acceptance of a negotiated test agreement.
    (3) In the case of testing submitted under section 5, except for 
those items listed in paragraph (c) of this section, documentation 
records, raw data, and specimens pertaining to a study and required to 
be retained by this part shall be retained in the archive(s) for a 
period of at least five years following the date on which the results of 
the study are submitted to the agency.
    (c) Wet specimens, samples of test, control, or reference 
substances, and specially prepared material which are relatively fragile 
and differ markedly in stability and quality during storage, shall be 
retained only as long as the quality of the preparation affords 
evaluation. Specimens obtained from mutagenicity tests, specimens of 
soil, water, and plants, and wet specimens of blood, urine, feces, 
biological fluids, do not need to be retained after quality assurance 
verification. In no case shall retention be required for longer periods 
than those set forth in paragraph (b) of this section.
    (d) The master schedule sheet, copies of protocols, and records of 
quality assurance inspections, as required by Sec. 792.35(c) shall be 
maintained by the quality assurance unit as an easily accessible system 
of records for the period of time specified in paragraph (b) of this 
section.
    (e) Summaries of training and experience and job descriptions 
required to be maintained by Sec. 792.29(b) may be retained along with 
all other testing facility employment records for the length of time 
specified in paragraph (b) of this section.
    (f) Records and reports of the maintenance and calibration and 
inspection of equipment, as required by Sec. 792.63 (b) and (c), shall 
be retained for the length of time specified in paragraph (b) of this 
section.
    (g) If a facility conducting testing or an archive contracting 
facility goes out of business, all raw data, documentation, and other 
material specified in this section shall be transferred to the archives 
of the sponsor of the study. The EPA shall be notified in writing of 
such a transfer.
    (h) Specimens, samples, or other non-documentary materials need not 
be retained after EPA has notified in writing the sponsor or testing 
facility holding the materials that retention is no longer required by 
EPA. Such notification normally will be furnished upon request after EPA 
or FDA has completed an audit of the particular study to which the 
materials relate and EPA has concluded that the study was conducted in 
accordance with this part.
    (i) Records required by this part may be retained either as original 
records or as true copies such as photocopies, microfilm, microfiche, or 
other accurate reproductions of the original records.

[[Page 46]]



PART 795_PROVISIONAL TEST GUIDELINES--Table of Contents




Subpart A [Reserved]

             Subpart B_Provisional Chemical Fate Guidelines

Sec.
795.70 Indirect photolysis screening test: Sunlight photolysis in waters 
          containing dissolved humic substances.

         Subpart C_Provisional Environmental Effects Guidelines

795.120 Gammarid acute toxicity test.

             Subpart D_Provisional Health Effects Guidelines

795.225 Dermal pharmacokinetics of DGBE and DGBA.
795.228 Oral/dermal pharmacokinetics.
795.231 Pharmacokinetics of isopropanal.
795.232 Inhalation and dermal pharmacokinetics of commercial hexane.
795.250 Developmental neurotoxicity screen.

    Authority: 15 U.S.C. 2603.

Subpart A [Reserved]



             Subpart B_Provisional Chemical Fate Guidelines



Sec. 795.70  Indirect photolysis screening test: Sunlight photolysis in waters 

containing dissolved humic substances.

    (a) Introduction. (1) Chemicals dissolved in natural waters are 
subject to two types of photoreaction. In the first case, the chemical 
of interest absorbs sunlight directly and is transformed to products 
when unstable excited states of the molecule decompose. In the second 
case, reaction of dissolved chemical is the result of chemical or 
electronic excitation transfer from light-absorbing humic species in the 
natural water. In contrast to direct photolysis, this photoreaction is 
governed initially by the spectroscopic properties of the natural water.
    (2) In general, both indirect and direct processes can proceed 
simultaneously. Under favorable conditions the measurement of a 
photoreaction rate constant in sunlight (KpE) in a natural 
water body will yield a net value that is the sum of two first-order 
reaction rate constants for the direct (kDE) and indirect 
(kIE) pathways which can be expressed by the relationship

                               Equation 1

kpE=kDE+kIE.


This relationship is obtained when the reaction volume is optically thin 
so that a negligible fraction of the incident light is absorbed and is 
sufficiently dilute in test chemical; thus the direct and indirect 
photoreaction processes become first-order.
    (3) In pure water only, direct photoreaction is possible, although 
hydrolysis, biotransformation, sorption, and volatilization also can 
decrease the concentraton of a test chemical. By measuring 
kpE in a natural water and kDE in pure water, 
kIE can be calculated.
    (4) Two protocols have been written that measure kDE in 
sunlight or predict kDE in sunlight from laboratory 
measurements with monochromatic light (USEPA (1984) under paragraph 
(f)(14) and (15) of this section; Mill et al. (1981) under paragraph 
(f)(9) of this section; Mill et al. (1982) under paragraph (f)(10) of 
this section; Mill et al. (1983) under paragraphs (f)(11) of this 
section). As a preface to the use of the present protocol, it is not 
necessary to know kDE; it will be determined under conditions 
that definitively establish whether kIE is significant with 
respect to kDE.
    (5) This protocol provides a cost effective test method for 
measuring kIE for test chemicals in a natural water 
(synthetic humic water, SHW) derived from commercial humic material. It 
describes the preparation and standardization of SHW. To implement the 
method, a test chemical is exposed to sunlight in round tubes containing 
SHW and tubes containing pure water for defined periods of time based on 
a screening test.
    (6) To correct for variations in solar irradiance during the 
reaction period, an actinometer is simultaneously insolated. From these 
data, an indirect photoreaction rate constant is calculated that is 
applicable to clear-sky, near-surface, conditions in fresh water bodies.

[[Page 47]]

    (7) In contrast to kDE, which, once measured, can be 
calculated for different seasons and latitudes, kIE only 
applies to the season and latitude for which it is determined. This 
condition exists because the solar action spectrum for indirect 
photoreaction in humic-containing waters is not generally known and 
would be expected to change for different test chemicals. For this 
reason, kpE, which contains kIE, is likewise valid 
only for the experimental data and latitude.
    (8) The value of kpE represents an atypical quantity 
because kIE will change somewhat from water body to water 
body as the amount and quality of dissolved aquatic humic substances 
change. Studies have shown, however, that for optically-matched natural 
waters, these differences are usually within a factor of two (Zepp et 
al. (1981) under paragraph (f)(17) of this section).
    (9) This protocol consists of three separate phases that should be 
completed in the following order: In Phase 1, SHW is prepared and 
adjusted; in Phase 2, the test chemical is irradiated in SHW and pure 
water (PW) to obtain approximate sunlight photoreaction rate constants 
and to determine whether direct and indirect photoprocesses are 
important; in Phase 3, the test chemical is again irradiated in PW and 
SHW. To correct for photobleaching of SHW and also solar irradiance 
variations, tubes containing SHW and actinometer solutions are exposed 
simultaneously. From these data kpE is calculated that is the 
sum of kIE and kDE (Equation 1) (Winterle and Mill 
(1985) under paragraph (f)(12) of this section).
    (b) Phase 1--Preparation and standardization of synthetic natural 
water--(1) Approach. (i) Recent studies have demonstrated that natural 
waters can promote the indirect (or sensitized) photoreaction of 
dissolved organic chemicals. This reactivity is imparted by dissolved 
organic material (DOM) in the form of humic substances. These materials 
absorb sunlight and produce reactive intermediates that include singlet 
oxygen (\1\02) (Zepp et al. (1977) under paragraph (f)(20) of 
this section, Zepp et al. (1981) under paragraph (f)(17) of this 
section, Zepp et al. (1981) under paragraph (f)(18) of this section, 
Wolff et al. (1981) under paragraph (f)(16) of this section, Haag et al. 
(1984) under paragraph (f)(6) of this section, Haag et al. (1984) under 
paragraph (f)(7) of this section); peroxy radicals (RO2-) 
(Mill et al. (1981) under paragraph (f)(9) of this section; Mill et al. 
(1983) under paragraph (f)(8) of this section); hydroxyl radicals (HO-) 
(Mill et al. (1981) under paragraph (f)(9) of this section, Draper and 
Crosby (1981, 1984) under paragraphs (f)(3) and (4) of this section); 
superoxide anion (02--) and hydroperoxy radicals 
(HO-). (Cooper and Zika (1983) under paragraph (f)(1) of this section, 
Draper and Crosby (1983) under paragraph (f)(2) of this section); and 
triplet excited states of the humic substances (Zepp et al. (1981) under 
paragraph (f)(17) of this section, Zepp et al. (1985) under paragraph 
(f)(21) of this section). Synthetic humic waters, prepared by extracting 
commercial humic or fulvic materials with water, photoreact similarly to 
natural waters when optically matched (Zepp et al. (1981) under 
paragraphs (f)(17) and (18) of this section).
    (ii) The indirect photoreactivity of a chemical in a natural water 
will depend on its response to these reactive intermediates, and 
possibly others yet unknown, as well as the ability of the water to 
generate such species. This latter feature will vary from water-to-water 
in an unpredictable way, judged by the complexity of the situation.
    (iii) The approach to standardizing a test for indirect 
photoreactivity is to use a synthetic humic water (SHW) prepared by 
water-extracting commercial humic material. This material is 
inexpensive, and available to any laboratory, in contrast to a specific 
natural water. The SHW can be diluted to a dissolved organic carbon 
(DOC) content and uv-visible absorbance typical of most surface fresh 
waters.
    (iv) In recent studies it has been found that the reactivity of SHW 
mixtures depends on pH, and also the history of sunlight exposure (Mill 
et al. (1983) under paragraph (f)(11) of this section). The SHW 
solutions initially photobleach with a time-dependent rate constant. As 
such, an SHW test system has been designed that is buffered to maintain 
pH and is pre-aged in sunlight to produce, subsequently, a predictable 
bleaching behavior.

[[Page 48]]

    (v) The purpose of Phase 1 is to prepare, pre-age, and dilute SHW to 
a standard mixture under defined, reproducible conditions.
    (2) Procedure. (i) Twenty grams of Aldrich humic acid are added to a 
clean 2-liter Pyrex Erlenmeyer flask. The flask is filled with 2 liters 
of 0.1 percent NaOH solution. A stir bar is added to the flask, the 
flask is capped, and the solution is stirred for 1 hour at room 
temperature. At the end of this time the dark brown supernatant is 
decanted off and either filtered through coarse filter paper or 
centrifuged and then filtered through 0.4 )m microfilter. The pH is 
adjusted to 7.0 with dilute H2SO4 and filter 
sterilized through a 0.2 )m filter into a rigorously cleaned 2-liter 
Erlenmeyer flask. This mixture contains roughly 60 ppm DOC and the 
absorbance (in a 1 cm path length cell) is approximately 1.7 at 313 nm 
and 0.7 at 370 nm.
    (ii) Pre-aging is accomplished by exposing the concentrated solution 
in the 2-liter flask to direct sunlight for 4 days in early spring or 
late fall; 3 days in late spring, summer, or early fall. At this time 
the absorbance of the solution is measured at 370 nm, and a dilution 
factor is calculated to decrease the absorbance to 0.50 in a 1 cm path 
length cell. If necessary, the pH is re-adjusted to 7.0. Finally, the 
mixture is brought to exact dilution with a precalculated volume of 
reagent-grade water to give a final absorbance of 0.500 in a 1-cm path 
length cell at 370 nm. It is tightly capped and refrigerated.
    (iii) This mixture is SHW stock solution. Before use it is diluted 
10-fold with 0.010 M phosphate buffer to produce a pH 7.0 mixture with 
an absorbance of 5.00x10-2 at 370 nm, and a dissolved organic 
carbon of about 5 ppm. Such values are characteristic of many surface 
fresh waters.
    (3) Rationale. The foregoing procedure is designed to produce a 
standard humic-containing solution that is pH controlled, and 
sufficiently aged that its photobleaching first-order rate constant is 
not time dependent. It has been demonstrated that after 7 days of winter 
sunlight exposure, SHW solutions photobleached with a nearly constant 
rate constant (Mill et al. (1983) under paragraph (f)(11) of this 
section).
    (c) Phase 2--Screening test--(1) Introduction and purpose. (i) Phase 
2 measurements provide approximate solar photolysis rate constants and 
half-lives of test chemicals in PW and SHW. If the photoreaction rate in 
SHW is significantly larger than in PW (factor of  2X) then 
the test chemical is subject to indirect photoreaction and Phase 3 is 
necessary. Phase 2 data are needed for more accurate Phase 3 
measurements, which require parallel solar irradiation of actinometer 
and test chemical solutions. The actinometer composition is adjusted 
according to the results of Phase 2 for each chemical, to equalize as 
much as possible photoreaction rate constants of chemical in SHW and 
actinometer.
    (ii) In Phase 2, sunlight photoreaction rate constants are measured 
in round tubes containing SHW and then mathematically corrected to a 
flat water surface geometry. These rate constants are not corrected to 
clear-sky conditions.
    (2) Procedure. (i) Solutions of test chemicals should be prepared 
using sterile, air-saturated, 0.010 M, pH 7.0 phosphate buffer and 
reagent-grade (or purer) chemicals. \1\ Reaction mixtures should be 
prepared with chemicals at concentrations at less than one-half their 
solubility in pure water and at concentrations such that, at any 
wavelengths above 290 nm, the absorbance in a standard quartz sample 
cell with a 1-cm path length is less than 0.05. If the chemicals are too 
insoluble in water to permit reasonable handling or analytical 
procedures, 1-volume percent acetonitrile may be added to the buffer as 
a cosolvent.
---------------------------------------------------------------------------

    \1\ The water should be ASTM Type IIA, or an equivalent grade.
---------------------------------------------------------------------------

    (ii) This solution should be mixed 9.00:1.00 by volume with PW or 
SHW stock solution to provide working solutions. In the case of SHW, it 
gives a ten-fold dilution of SHW stock solution. Six mL aliquots of each 
working solution should then be transferred to separate 12 x 100 mm 
quartz tubes with screw tops and tightly sealed with Mininert valves. 
\2\ Twenty four tubes are required for each chemical solution

[[Page 49]]

(12 samples and 12 dark controls), to give a total of 48 tubes.
---------------------------------------------------------------------------

    \2\ Mininert Teflon sampling vials are available from Alltech 
Associates, Inc., 202 Campus Dr., Arlington Heights, IL 60004.
---------------------------------------------------------------------------

    (iii) The sample tubes are mounted in a photolysis rack with the 
tops facing geographically north and inclined 30[deg] from the 
horizontal. The rack should be placed outdoors over a black background 
in a location free of shadows and excessive reflection.
    (iv) Reaction progress should be measured with an analytical 
technique that provides a precision of at least 5 
percent. High pressure liquid chromatography (HPLC) or gas chromatograph 
(GC) have proven to be the most general and precise analytical 
techniques.
    (v) Sample and control solution concentrations are calculated by 
averaging analytical measurements for each solution. Control solutions 
should be analyzed at least twice at zero time and at other times to 
determine whether any loss of chemical in controls or samples has 
occurred by some adventitious process during the experiment.
    (vi) Whenever possible the following procedures should be completed 
in clear, warm, weather so that solutions will photolyze more quickly 
and not freeze.
    (A) Starting at noon on day zero, expose to sunlight 24 sample tubes 
mounted on the rack described above. Tape 24 foil-wrapped controls to 
the bottom of the rack.
    (B) Analyze two sample tubes and two unexposed controls in PW and 
SHW for chemical at 24 hours. Calculate the round tube photolysis rate 
constants (kp)SHW and (kp)W 
if the percent conversions are J 20 percent but F 80 percent. The rate 
constants (kp)SHW and (kp)W 
are calculated, respectively, from Equations 2 and 3:

                               Equation 2

(kp)SHW=(1/t)Pn(Co/
    Ct)SHW (in d-1)

                               Equation 3

(kp)W=(1/t)Pn(Co/
    Ct)W (in d-1),


where the subscript identifies a reaction in SHW or PW; t is the 
photolysis time in calendar days; Co is the initial molar 
concentration; and Ct is the molar concentration in the 
irradiated tube at t. In this case t=1 day.

    (C) If less than 20 percent conversion occurs in SHW in 1 day, 
repeat the procedure for SHW and PW at 2 days, 4 days, 8 days, or 16 
days, or until 20 percent conversion is reached. Do not extend the 
experiment past 16 days. If less than 20 percent photoreaction occurs in 
SHW at the end of 16 days the chemical is ``photoinert''. Phase 3 is not 
applicable.
    (D) If more than 80 percent photoreaction occurs at the end of day 1 
in SHW, repeat the experiment with eight each of the remaining foil-
wrapped PW and SHW controls. Divide these sets into four sample tubes 
each, leaving four foil-wrapped controls taped to the bottom of the 
rack.
    (1) Expose tubes of chemical in SHW and PW to sunlight starting at 
0900 hours and remove one tube and one control at 1, 2, 4, and 8 hours. 
Analyze all tubes the next day.
    (2) Extimate (kp)SHW for the first tube in 
which photoreaction is J 20 percent but F 80 percent. If more than 80 
percent conversion occurs in the first SHW tube, report: ``The half-life 
is less than one hour'' and end all testing. The chemical is 
``photolabile.'' Phase 3 is not applicable.
    (3) The rate constants (kp)SHW and 
(kp)W are calculated from equations 2 and 3 but 
the time of irradiation must be adjusted to reflect the fact that day-
averaged rate constants are approximately one-third of rate constants 
averaged over only 8 daylight hours. For 1 hour of insolation enter 
t=0.125 day into equation 2. For reaction times of 2, 4, and 8 hours 
enter 0.25, 0.50 and 1.0 days, respectively. Proceed to Phase 3 testing.
    (4) Once (kp)SHW and 
(kp)W are measured, determine the ratio R from 
equation 4:

                               Equation 4

R=(kp)SHW/(kp)W.


The coefficient R, defined by Equation 4, is equal to 
[(kI+kD)/kD]. If R is in the range 0 to 
1, the photoreaction is inhibited by the synthetic humic water and Phase 
3 does not apply. If R is in the range 1 to 2, the test chemical is 
marginally susceptable to indirect photolysis. In this case, Phase 3 
studies are optional. If R is greater than 2,

[[Page 50]]

Phase 3 measurements are necessary to measure kpE and to 
evaluate kIE.
    (vii) Since the rate of photolysis in tubes is faster than the rate 
in natural water bodies, values of near-surface photolysis rate 
constants in natural and pure water bodies, kpE and 
kDE, respectively, can be obtained from 
(kp)SHW and (kp)W from 
Equations 5 and 6:

                               Equation 5

kpE=0.45(kp)SHW

                               Equation 6

kDE=0.45(kp)W.


The factor 0.45 is an approximate geometric correction for scattered 
light in tubes versus horizontal surfaces. A rough value of 
kIE, the rate constant for indirect photolysis in natural 
waters or SHW, can be estimated from the difference between 
kpE and kDE using Equation 7:

                               Equation 7

kIE=kpE-kDE.

    (3) Criteria for Phase 2. (i) If no loss of chemical is found in 
dark control solutions compared with the analysis in tubes at zero time 
(within experimental error), any loss of chemical in sunlight is assumed 
to be due to photolysis, and the procedure provides a valid estimate of 
kpE and kDE. Any loss of chemical in the dark-
control solutions may indicate the intervention of some other loss 
process such as hydrolysis, microbial degradation, or volatilization. In 
this case, more detailed experiments are needed to trace the problem and 
if possible eliminate or minimize the source of loss.
    (ii) Rate constants determined by the Phase 2 protocol depend upon 
latitude, season, and weather conditions. Note that 
(kp)SHW and kD values apply to round 
tubes and kpE and kDE values apply to a natural 
water body. Because both (kp)SHW and kD 
are measured under the same conditions the ratio 
((kp)SHW/kD) is a valid measure of the 
susceptibility of a chemical to indirect photolysis. However, since SHW 
is subject to photobleaching, (kp)SHW will 
decrease with time because the indirect rate will diminish. Therefore, R 
2 is considered to be a conservative limit because 
(kp)SHW will become systematically smaller with 
time.
    (4) Rationale. The Phase 2 protocol is a simple procedure for 
evaluating direct and indirect sunlight photolysis rate constants of a 
chemical at a specific time of year and latitude. It provides a rough 
rate constant for the chemical in SHW that is necessary for Phase 3 
testing. By comparison with the direct photoreaction rate constant, it 
can be seen whether the chemical is subject to indirect photoreaction 
and whether Phase 3 tests are necessary.
    (5) Scope and limitations. (i) Phase 2 testing separates test 
chemicals into three convenient categories: ``Photolabile'', 
``photoinert'', and those chemicals having sunlight half-lives in round 
tubes in the range of 1 hour to 50 days. Chemicals in the first two 
categories fall outside the practical limits of the test, and cannot be 
used in Phase 3. All other chemicals are suitable for Phase 3 testing.
    (ii) The test procedure is simple and inexpensive, but does require 
that the chemical dissolve in water at sufficient concentrations to be 
measured by some analytical technique but not have appreciable 
absorbance in the range 290 to 825 nm. Phase 2 tests should be done 
during a clear-sky period to obtain the best results. Testing will be 
less accurate for chemicals with half-lives of less than 1 day because 
dramatic fluctuations in sunlight intensity can arise from transient 
weather conditions and the difficulty of assigning equivalent reaction 
times. Normal diurnal variations also affect the photolysis rate 
constant. Phase 3 tests should be started as soon as possible after the 
Phase 2 tests to ensure that the (kp)SHW estimate 
remains valid.
    (6) Illustrative Example. (i) Chemical A was dissolved in 0.010 M pH 
7.0 buffer. The solution was filtered through a 0.2 )m filter, air 
saturated, and analyzed. It contained 1.7x10 -5 M A, five-
fold less than its water solubility of 8.5x10 -5 M at 25 
[deg]C. A uv spectrum (1-cm path length) versus buffer blank showed no 
absorbance greater than 0.05 in the wavelength interval 290 to 825 nm, a 
condition required for the Phase 2 protocol. The 180 mL mixture was 
diluted

[[Page 51]]

by the addition of 20 mL of SHW stock solution.
    (ii) The SHW solution of A was photolyzed in sealed quartz tubes 
(12x100 mm) in the fall season starting on October 1. At the end of 1 
and 2 days, respectively, the concentration of A was found to be 1.13x10 
-5 M and 0.92x10 -5 M compared to unchanged dark 
controls (1.53x10 -5 M).
    (iii) The tube photolysis rate constant of chemical A was calculated 
from Equation 2 under paragraph (c)(2)(vi)(B) of this section. The first 
time point at day 1 was used because the fraction of A remaining was in 
the range 20 to 80 percent:

(kp)SHW=(1/1d)Pn(1.53x10 -5/1.13 x10 
    -5) (kp)SHW=0.30 d-1.

    (iv) From this value, kpE was found to be 0.14 d-\1\ 
using equation 5 under paragraph (c)(2)(vii) of this section:

kpE=0.45(0.30 d-1)=0.14d-1.

    (v) From measurements in pure water, kD for chemical A 
was found to be 0.085 d-1. Because the ratio of 
(kp)SHW/kD(=3.5) is greater than 2, 
Phase 3 experiments were started.
    (d) Phase 3--Indirect photoreaction with actinometer: Calculation of 
kIE and kpE--(1) Introduction and purpose.
    (i) The purpose of Phase 3 is to measure kIo, the 
indirect photolysis rate constant in tubes, and then to calculate 
kpE for the test chemical in a natural water. If the 
approximate (kp)SHW determined in Phase 2 is not 
significantly greater than kD measured for the experiment 
date of Phase 2, then Phase 3 is unnecessary because the test chemical 
is not subject to indirect photoreaction.
    (ii) In the case (kp)SHW is significantly 
larger than kD, Phase 3 is necessary. The rate constant 
(kp)SHW is used to choose an actinometer 
composition that matches the actinometer rate to the test chemical rate. 
Test chemical solutions in SHW and in pure water buffer are then 
irradiated in sunlight in parallel with actinometer solutions, all in 
tubes.
    (iii) The actinometer used is the p-nitroacetophenone-pyridine 
(PNAP/PYR) system developed by Dulin and Mill (1982) under paragraph 
(f)(5) of this section and is used in two EPA test guidelines (USEPA 
(1984) under paragraphs (f) (14) and (15) of this section). By varying 
the pyridine concentration, the PNAP photolysis half-life can be 
adjusted over a range of several hours to several weeks. The starting 
PNAP concentration is held constant.
    (iv) SHW is subject to photobleaching that decreases its ability to 
promote indirect photolysis based on its ability to absorb sunlight. 
This effect will be significant when the test period exceeds a few days. 
To correct for photobleaching, tubes containing SHW are irradiated in 
action to the other tubes above.
    (v) At any time, the loss of test chemical is given by Equation 8 
assuming actinometric correction to constant light flux:

                               Equation 8

-(d[C]/dt)=kI[C]+kD[C].

    (vi) The indirect photolysis rate constant, kI, is 
actually time dependent because SHW photobleaches; the rate constant 
kI, after pre-aging, obeys the formula:

                               Equation 9

kI=kIo exp(-kt),


in which kIo is the initial indirect photoreaction rate 
constant and k is the SHW photobleaching rate constant. After 
substituting equation 9 for kI in Equation 8 under paragraph 
(d)(1)(v) of this section, and rearranging, one obtains

-(d[C]/[C]=kIo[exp(-kt)]dt+kD dt.


This expression is integrated to give Equation 10:

                               Equation 10

Pn(Co/C)SHW=(kIo/k)[1-exp(-
    kt)]+kD t.


The term (kIo/k) can now be evaluated. Since in pure water, 
Pn(Co/C)W=kD t, then subtracting this 
equation from Equation 10 gives

                               Equation 11

Pn(Co/C)SHW-Pn(co/
    C)W=(kIo/k)[1-exp(-kt)].


The photobleaching fraction, [1-exp(-kt)], is equivalent to the 
expression [1-

[[Page 52]]

(A370/A[deg]370)], where A[deg]370 and 
A370 are the absorbances at 370 nm, and are proportional to 
humic sensitizer content at times zero and t. Therefore, 
(kIo/k) is derived from the slope of a linear regression 
using [Pn(Co/C)SHW-Pn(Co/
C)W] as the dependent variable and [1-(A370/
A[deg]370)SHW] as the independent variable.
    (vii) To evaluate kIo, the parameter k has to be 
evaluated under standard sunlight conditions. Therefore, the photolysis 
rate constant for the PNAP/PYR actinometer (kA) is used to 
evaluate k by linear regression on Equation 12:

                               Equation 12

Pn(A[deg]370/A370)=(k/
    kA)Pn(Co/C)PNAP,


where the slope is (k/kA) and the value of kA is 
calculated from the concentration of pyridine and the absorption of 
light by PNAP: kA=2.2(0.0169)[PYR]ka. Values of 
ka are listed in the following Table 1.

 Table 1--Day Averaged Rate Constant (ka) \1\ for Sunlight Absorption by
          PNAP as a Function of Season and Decadic Latitude \2\
------------------------------------------------------------------------
                                                       Season
                 Latitude                 ------------------------------
                                           Spring  Summer   Fall  Winter
------------------------------------------------------------------------
20[deg]N.................................     515     551    409     327
30[deg]N.................................     483     551    333     232
40[deg]N.................................     431     532    245     139
50[deg]N.................................     362     496    154      64
------------------------------------------------------------------------
\1\ ka=@ ega Lg in the units of day \-1\, (Mill et al. (1982) under
  paragraph (f)(10) of this section).
\2\ For use in Equation 15 under paragraph (d)(2)(i) of this section.


The value of kIo is then given by Equation 13:

                               Equation 13

kIo=(kIo/k)(k/kA)kA.

    (viii) To obtain kD, determine the ratio (kD/
kA) from a linear regression of Pn(Co/
C)W versus Pn(Co/C)PNAP according to 
Equation 13a:

                              Equation 13a

Pn(Co/C)W=(kD/
    kA)Pn(Co/C)PNAP.


The slope is (kD/kA), and kD is 
obtained by multiplication of this slope with the known value of 
kA: i.e., kD=(kD/
kA)kA.
    (ix) Then, (kp)SHW values in SHW are 
determined by summing kD and KIo as follows:

                               Equation 14

(kp)SHW=kIo+kD.

    (x) Finally, kpE is calculated from the precise 
relationship, Equation 5a:

                               Equation 5a

kpE=0.455(kp)SHW.

    (2) Procedure. (i) Using the test chemical photoreaction rate 
constant in round tubes, (kp) SHW' determined in 
Phase 2 under paragraph (c) of this section, and the absorption rate 
constant, k[alpha] found in Table 1, under paragraph (d)(1)(vii) of this 
section, calculate the molar pyridine concentration required by the 
PNAP/PYR actinometer using Equation 15:

                               Equation 15

[PYR]/M=26.9[(kp) SHW/ka].


This pyridine concentration makes the actinometer rate constant match 
the test chemical rate constant.
    (A) The variable ka (= @ e ga Lg) 
is equal to the day-averaged rate constant for sunlight absorption by 
PNAP (USEPA (1984) under paragraph (f)(14) of this section; Mill et al. 
(1982) under paragraph (f)(10) of this section, Zepp and Cline (1977) 
under paragraph (f)(19) of this section) which changes with season and 
latitude.
    (B) The variable ka is selected from Table 1 under 
paragraph (d)(1)(vii) of this section for the season nearest the mid-
experiment date of Phase 2 studies and the decadic latitude nearest the 
experimental site.
    (ii) Once [PYR] is determined, an actinometer solution is prepared 
by adding 1.00 mL of 1.0x10-2 M (0.165 gms/100 mL) PNAP stock 
solution (in CH3 CN solvent) and the required volume, V, of 
PYR to a 1 liter volumetric flask. The flask is then filled with 
distilled water to give 1 liter of solution. The volume V can be 
calculated from Equation 16:

                               Equation 16

V/mL=[PYR]/0.0124.


[[Page 53]]



The PNAP/PYR solutions should be wrapped with aluminum foil and kept out 
of bright light after preparation.
    (iii) The following solutions should be prepared and individually 
added in 6.00 mL aliquots to 12/100 mm quartz sample tubes; 8 tubes 
should be filled with each solution:
    (A) PNAP/PYR actinometer solution.
    (B) Test chemical in pH 7.0, 0.010 M phosphate buffer.
    (C) Test chemcial in pH 7.0, 0.010 M phosphate buffer/SHW.
    (D) pH 7.0, 0.010 M phosphate buffer/SHW. Four tubes of each set are 
wrapped in foil and used as controls.
    (iv) The tubes are placed in the photolysis rack (Phase 2, 
Procedure) at 0900 hours on day zero, with the controls taped to the 
bottom of the rack. One tube of each composition is removed, along with 
their respective controls, according to a schedule found in Table 2, 
which categorizes sampling times on the basis of 
(kp)SHW determined in Phase 1.

    Table 2--Category and Sampling Procedure for Test and Actinometry
                                Solutions
------------------------------------------------------------------------
            Category                 kp (d-1)SHW      Sampling procedure
------------------------------------------------------------------------
A..............................  5.5 J Kp J 0.69      Sample at 0, 1, 2,
                                                       4, and 8h.
B..............................  0.69 kp   Sample at 0, 1, 2,
                                  J 0.017              4, and 8d.
C..............................  0.17 kp   Sample at 0, 4, 8,
                                  J 0.043              16, and 32d.
------------------------------------------------------------------------

    (v) The tubes containing PNAP, test chemical, and their controls are 
analyzed for residual concentrations soon after the end of the 
experiment. PNAP is conveniently analyzed by HPLC, using a 30 cm 
C18 reverse phase column and a uv detector set at 280 nm. The 
mobile phase is 2 percent acetic acid, 50 percent acetonitrile and 48 
percent water (2 mL/min flow rate). Tubes containing only SHW (solution 
D) should be analyzed by absorption spectroscopy at 370 nm after storage 
at 4 [deg]C in the dark. The absorbance range to be measured is 0.05 to 
0.01 AU (1 cm).
    (vi) If controls are well-behaved and show no significant loss of 
chemical or absorbance change, then kI can be calculated. In 
tabular form (see Table 4 under paragraph (d)(6)(iii)(A) of this 
section) arrange the quantities Pn(Co/Ct) 
SHW, Pn(Co/Ct)SHW, [1-
(A370/A\o\370)], Pn(A\o\370/
A370), and Pn(Co/C)PNAP in order of 
increasing time. According to Equation 11 under paragraph (d)(1)(vi) of 
this section in the form of Equation 17,

                               Equation 17

Pn(Co/C)SHW-Pn(Co/
    C)W=(kIo/k)[1-(A370/
    A\o\370)],


plot the quantities [Pn(Co/Ct)SHW-
Pn(Co/Ct)W] versus the independent 
variable [1-(A370/A\o\370)]. Obtain the slope (S1) 
by least square linear regression. Under the assumptions of the 
protocol, S1=(kIo/k).
    (vii) According to Equation 12 under paragraph (d)(1)(vii) of this 
section, plot the quantities Pn(A\o\370/A370) 
versus the independent variable Pn(Co/
Ct)PNAP. Obtain the slope (S2) by least squares 
linear regression on Equation 12 under paragraph (d)(1)(vii) of this 
section. Under the assumptions of the protocol, S2=(k/kA).
    (viii) Then, using Equation 13a under paragraph (d)(1)(vii) of this 
section, determine the slope (S3) by least squares linear regression. 
Under the assumptions of the protocol, S3 is equal to (kD/
kA).
    (ix) From Equation 18

                               Equation 18

kA=0.0372[PYR]ka,


calculate kA using ka values found in Table 1 
under paragraph (d)(1)(vii) of this section. The value of ka 
chosen must correspond to the date closest to the mid-experiment date 
and latitude closest to that of the experimental site.
    (x) The indirect photoreaction rate constant, kIo, is 
determined using Equation 19,

                               Equation 19

kIo=(S1)(kA)(S2),


by incorporating the quantities kA, S1, and S2 determined as 
described in paragraphs (d)(2) (ix), (vi), and (vii) of this section, 
respectively.
    (xi) The rate constant kD is calculated from Equation 20,

                               Equation 20

kD=(S3)(kA),


[[Page 54]]



using the quantities S3 and kA determined as described above.
    (xii) Then, (kp)SHW is obtained by summing 
kD and kIo, as described by Equation 14 in 
paragraph (d)(1)(ix) of this section:

                               Equation 14

(kp)SHW=kIo+kD.

    (xiii) Finally, kpE is obtained by multiplying 
(kp) SNW by the factor 0.455, as described by 
Equation 5a in paragraph (d)(1)(x) of this section:

                               Equation 5a

kpE=0.455 (kp)SHW


As determined, kpE is the net environmental photoreaction 
rate constant. It applies to clear sky conditions and is valid for 
predicting surface photoreaction rates in an average humic containing 
freshwater body. It is strictly valid only for the experimental latitude 
and season.
    (3) Criteria for Phase 3. As in Phase 2, Phase 3 tests are assumed 
valid if the dark controls are well behaved and show no significant loss 
of chemical. In such a case, loss of test chemical in irradiated samples 
is due to photoreaction.
    (4) Rationale. Simultaneous irradiation of a test chemical and 
actinometer provide a means of evaluating sunlight intensities during 
the reaction period. Parallel irradiation of SHW solutions allows 
evaluation of the extent of photobleaching and loss of sensitizing 
ability of the natural water.
    (5) Scope and limitations of Phase 3 protocol. Test chemicals that 
are classified as having half-lives in SHW in the range of 1 hour to 50 
days in Phase 2 listing are suitable for use in Phase 3 testing. Such 
chemicals have photoreaction half-lives in a range accommodated by the 
PNAP/PYR actinometry in sunlight and also accommodate the persistence of 
SHW in sunlight.
    (6) Illustrative example. (i) From Phase 2 testing, under paragraph 
(c)(6)(iii) of this section, chemical A was found to have a photolysis 
rate constant, (kp)SHW' of 0.30 d-1 in 
fall in round tubes at latitude 33[deg] N. Using Table 1 under paragraph 
(d)(1)(vii) of this section for 30[deg] N, the nearest decadic latitude, 
a fall value of ka equal to 333 d-1 is found for 
PNAP. Substitution of (kp)SHW and ka 
into Equation 15 under paragraph (d)(2)(i) of this section gives [PYR] = 
0.0242 M. This is the concentration of pyridine that gives an 
actinometer rate constant of 0.30 d-1 in round tubes in fall 
at this latitude.
    (ii) The actinometer solution was made up by adding a volume of 
pyridine (1.95 mL) calculated from equation 16 under paragraph 
(d)(2)(ii) of this section to a 1 liter volumetric flask containing 1.00 
mL of 1.00 x 10-2 M PNAP in acetonitrile. The flask was 
filled to the mark with distilled water to give final concentrations of 
[PYR]=0.0242 M and [PNAP]=1.00x10-5 M. Ten tubes of each of 
the following solutions were placed in the photolysis rack at 1,200 
hours on day zero:
    (A) Chemical A (1.53x10-5 M) in standard SHW (0.010 M, pH 
7 phosphate buffer).
    (B) Chemical A (1.53x10-5), in 0.010 M, pH 7 phosphate 
buffer.
    (C) SHW standard solution diluted with water 0.90 to 1.00 to match 
solution A.
    (D) PNAP/PYR actinometer solution. Ten additional foil-wrapped 
controls of each mixture were taped to the bottom of the rack.
    (iii) The test chemical had been placed in category B, Table 2 under 
the paragraph (d)(2)(iv) of this section, on the basis of its Phase 2 
rate constant under paragraph (c) of this section. Accordingly, two 
tubes of each irradiated solution and two tubes of each blank solution 
were removed at 0, 1, 2, 4, and 8 days at 1,200 hours. The averaged 
analytical results obtained at the end of the experiment are shown in 
the following Table 3.

                     Table 3--Chemical Analytical Results for Illustrative Example, Phase 3
----------------------------------------------------------------------------------------------------------------
                                                  10\5\[C]\SHW\,                                   10\5\ [PNAP],
                       Day                               M        10\5\[C]\W\, M     A\SHW\370           M
----------------------------------------------------------------------------------------------------------------
0...............................................           1.53            1.53           0.0500           1.00
1...............................................           1.03            1.40           0.0470           0.810

[[Page 55]]

 
2...............................................           0.760           1.30           0.0440           0.690
4...............................................           0.300           1.01           0.0370           0.380
8...............................................           0.130           0.800          0.0320           0.220
----------------------------------------------------------------------------------------------------------------


Data for solutions A through D are given in column 2 through 5, 
respectively. No significant chemical loss was found in the dark 
controls.
    (A) From these items the functions Pn(Co/C) 
SNW' Pn(Co/C)W' [1--(A370/
A\o\370)SNW], Pn(A\o\370/
A370), and Pn(Co/C)PNAP were 
calculated, as shown in the following Table 4 which was derived from 
Table 3 under paragraph (d)(6)(iii) of this section:

            Table 4--Photoreaction Function for Illustrative Examples, Phase 3, Derived From Table 3
----------------------------------------------------------------------------------------------------------------
                                                                    1-(A 370 /     Pn(A\o\370 /
               Day                  Pn(Co/C)SHW      Pn(Co/C)W       A\o\370)          A370)      Pn(Co /C) PNAP
----------------------------------------------------------------------------------------------------------------
0...............................           0              0               0               0                0
1...............................           0.396          0.0888          0.0600          0.0618           0.211
2...............................           0.700          0.163           0.120           0.128            0.371
4...............................           1.629          0.415           0.260           0.301            0.968
8...............................           2.465          0.648           0.360           0.446            1.514
----------------------------------------------------------------------------------------------------------------

    (B) Slope S1=(kIo/k) was calculated according to Equation 
17 under paragraph (d)(2)(vi) of this section and was found to be 4.96 
by a least squares regression with a correlation coefficient equal to 
0.9980. The following Figure 1 shows a plot of Equation 17 under 
paragraph (d)(2)(vi) of this section and its best-fit line.
[GRAPHIC] [TIFF OMITTED] TC01AP92.034

   Figure 1--Graphic determination of S1=(kIo/k) based on 
         Equation 17 under paragraph (d)(2)(vi) of this section.
    (C) Slope S2=(k/ka) was also derived from Table 4 under 
paragraph (d)(6)(iii)(A) of this section by a fit of 
Pn(A\o\370 /A370) SHW and 
Pn(Co /C)PNAP to Equation 12 under paragraph 
(d)(l)(vii) of this section. This plot is displayed in the following 
Figure 2; the slope S2 was found to be 0.295 and the correlation 
coefficient was equal to 0.9986.

[[Page 56]]

[GRAPHIC] [TIFF OMITTED] TC01AP92.035

    Figure 2--Graphic determination of S2=(k/kA) based on 
        Equation 12 under paragraph (d)(1)(vii) of this section.
    (D) Using the data in columns 3 and 6 in Table 4 under paragraph 
(d)(6)(iii)(A) of this section, slope S3 was calculated by regression 
from Equation 13a under paragraph (d)(1)(viii) of this section and was 
found to be 0.428 with correlation coefficient equal to 0.99997.
    (E) Using Equation 18 under paragraph (d)(2)(ix) of this section, 
kA was found to be =0.300 d-1.
    (F) The values of S1, S2, and kA were then combined in 
Equation 19 under paragraph (d)(2)(x) of this section to give 
kIo as follows:

                               Equation 19

kIo=(4.96)(0.300)(0.295)=0.439 d-1.

    (G) The rate constant kD was calculated from the product 
of S3 and kA as expressed in Equation 20 under paragraph 
(d)(2)(xi) of this section as follows:

                               Equation 20

kD=(0.428)(0.300)=0.128d-1.

    (H) The sum of kD and kIo was multiplied by 
0.455 to obtain kpE as follows:

                               Equation 21

kpE=(0.455)(0.439+0.128)d-1=0.258 d-1.

    (I) Since kpE is a first-order rate constant, the half-
life, t1/2E, is given by Equation 22:

                               Equation 22

t1/2E=0.693/kpE.

Substituting the value of kpE from Equation 21 under 
paragraph (d)(6)(iii)(H) of this section in Equation 22 yielded

                               Equation 23

t1/2E=0.693/0.258d-1=2.7 d.

    (e) Data and reporting--(1) Test conditions--(i) Specific analytical 
and recovery procedures. (A) Provide a detailed description or reference 
for the analytical procedures used, including the calibration data and 
precision.
    (B) If extraction methods were used to separate the solute from the 
aqueous solution, provide a description of the extraction method as well 
as the recovery data.
    (ii) Other test conditions. (A) Report the site and latitude where 
the photolysis experiments were carried out.
    (B) Report the dates of photolysis, weather conditions, times of 
exposure, and the duration of exposure.
    (C) If acetonitrile was used to solubilize the test chemical, report 
the volume percent.
    (D) If a significant loss of test chemical occurred in the control 
solutions for pure water and SHW, indicate the causes and how they were 
eliminated or minimized.
    (2) Test data report--(i) Phase 2 Screening Test under paragraph (c) 
of this section. (A) Report the initial molar concentration of test 
chemical, Co, in pure water and SHW for each replicate and 
the mean value.
    (B) Report the molar concentration of test chemical, Ct, 
in pure water and SHW for each replicate and the mean value for each 
time point t.
    (C) Report the molar concentration of test chemical for each 
replicate control sample and the mean value for each time point.
    (D) Report the values of (kp)SHW and 
(kp)W for the time point t in which the fraction 
of test chemical photoreacted is in the range 20 to 80 percent.
    (E) If small losses of test chemical were observed in SHW and pure 
water, report a first-order rate constant loss, 
(kp)loss. Calculate and report 
(kp)obs for SHW and/or pure water. Calculate and 
report the corrected first-order rate

[[Page 57]]

constant for SHW and/or pure water using the relationship expressed in 
Equation 24:

                               Equation 24

kp=(kp)obs-
    (kp)loss.

    (F) Report the value of R calculated from Equation 4 under paragraph 
(c)(2)(vi)(D)(4) of this section.
    (G) Report the values of kpE and kDE obtained 
from Equations 5 and 6, respectively under paragraph (c)(2)(vii) of this 
section; report the corresponding half-life calculated from Equation 22 
under paragraph (d)(6)(iii)(I) of this section.
    (ii) Phase 3--Indirect photoreaction with actinometer. (A) Report 
the initial molar concentration of test chemical, Co, in pure 
water and in SHW for each replicate and the mean value.
    (B) Report the initial absorbance A\o\370 of the SNW 
solution.
    (C) Report the initial molar concentration of PNAP of each replicate 
and the mean value in the actinometer. Report the concentration of 
pyridine used in the actinometer which was obtained from Equation 15 
under paragraph (d)(2)(i) of this section.
    (D) Report the time and date the photolysis experiments were 
started, the time and date the experiments were completed, and the 
elapsed photolysis time in days.
    (E) For each time point t, report the separate values of the 
absorbance of the SHW solution, and the mean values.
    (F) For each time point for the controls, report the separate values 
of the molar concentrations of test chemical in pure water and SHW, and 
the absorbance of the SHW solution, and the mean values.
    (G) Tabulate and report the following data: t, [C]\SHW\, [C]\W\, 
A\SNW\370, [PNAP].
    (H) From the data in (G), tabulate and report the following data: t, 
Pn(Co/C)SNW, Pn(Co/C)W, [1-
(A370/A\o\370)SNW], Pn(A\o\370/
A370), Pn(Co/C)PNAP.
    (I) From the linear regression analysis of the appropriate data in 
step (H) in Equation 17 under paragraph (d)(2)(vi) of this section, 
report the slope S1 and the correlation coefficient.
    (J) From the linear regression analysis of the appropriate data in 
step (H) in Equation 12 under paragraph (d)(1)(vii) of this section, 
report the slope S2 and the correlation coefficient.
    (K) From the linear regression analysis of the appropriate data in 
step (H) in Equation 13a under paragraph (d)(1)(viii) of this section, 
report the slope S3 and the correlation coefficient.
    (L) If loss of chemical was observed during photolysis in pure water 
and SHW, then report the data Pn(Co/C)corr, 
Pn(Co/C)obs, Pn(Co/C)loss as 
described in paragraph (e)(2)(E) of this section. Repeat steps (H), (I), 
(J), (K) where applicable and report S1, S2, S3 and the corresponding 
correlation coefficients.
    (M) Report the value of the actinometer rate constant obtained from 
Equation 18 under paragraph (d)(2)(ix) of this section.
    (N) Report the value of kIo obtained from Equation 19 
under paragraph (d)(2)(x) of this section.
    (O) Report the value of kD obtained from Equation 20 
under paragraph (d)(2)(xi) of this section.
    (P) Report the value of (kpE)SHW, obtained 
from Equation 14 under paragraph (d)(1)(ix) of this section, and the 
value of kpE obtained from Equation 5a under paragraph 
(d)(1)(x) of this section.
    (Q) Report the half-life, t1/2E, obtained from Equation 
22 under paragraph (d)(6)(iii)(I) of this section.
    (f) References. For additional background information on this test 
guideline the following references should be consulted.
    (1) Cooper W.J., Zika R.G. ``Photochemical formation of hydrogen 
peroxide in surface and ground waters exposed to sunlight.'' Science, 
220:711. (1983).
    (2) Draper W.M., Crosby D.G. ``The photochemical generation of 
hydrogen peroxide in natural waters.'' Archives of Environmental 
Contamination and Toxicology, 12:121. (1983).
    (3) Draper, W.M. and Crosby D.G. ``Solar photooxidation of 
pesticides in dilute hydrogen peroxide.'' Journal of Agricultural and 
Food Chemistry, 32:231. (1984).
    (4) Draper W.M., Crosby D.G. ``Hydrogen peroxide and hydroxyl 
radical:

[[Page 58]]

Intermediates in indirect photolysis reactions in water.'' Journal of 
Agricultural and Food Chemistry, 29:699. (1981).
    (5) Dulin D., Mill T. ``Development and evaluation of sunlight 
actinometers.'' Environmental Science and Technology, 6:815. (1982).
    (6) Haag H.R., Hoigne J., Gassman E., Braun A.M. ``Singlet oxygen in 
surface waters--Part I; Furfuryl alcohol as a trapping agent.'' 
Chemosphere, 13:631. (1984).
    (7) Haag W.R., Hoigne J., Gassman E., Braun A.M. ``Singlet oxygen in 
surface waters--Part II: Quantum yields of its production by some 
natural humic materials as a function of wavelength.'' Chemosphere, 
13:641. (1984).
    (8) Mill T., Winterle J.S., Fischer A., Tse D., Mabey W.R., Drossman 
H., Liu A., Davenport J.E. Toxic substances process data generation and 
protocol development. Work assignment 12, test standard development. 
``Section 3. Indirect photolysis.'' Draft final report. EPA Contract No. 
68-03-2981. Environmental Research Laboratory, Office of Research and 
Development, EPA, Athens, GA, and Office of Pollution Prevention and 
Toxics, EPA, Washington, DC. (1984).
    (9) Mill T., Mabey W.R., Bomberger D.C., Chou T.W., Hendry D.G., 
Smith J.H. ``Laboratory protocols for evaluating the fate of organic 
chemicals in air and water. Chapter 3. Photolysis in water. Chapter 4. 
Oxidation in water.'' EPA 600/3-82-022. Environmental Research 
Laboratory, Office of Research and Development, EPA, Athens, GA. (1981).
    (10) Mill T., Mabey W.R., Winterle J.S., Davenport J.E., Barich 
V.P., Dulin D.E., Tse D.S., Lee G. ``Design and validation of screening 
and detailed methods for environmental processes. Apendix C. Lower-tier 
direct photolysis protocol.'' Draft final report. EPA Contract No. 68-
01-6325. Office of Pollution Prevention and Toxics, EPA, Washington, DC. 
(1982).
    (11) Mill T., Davenport J.E., Winterle J.S., Mabey W.R., Dossman H., 
Tse D., Liu A. Toxic substances process data generation and protocol 
development. Work assignment 12. ``Appendix B. Upper-tier protocol for 
direct photolysis in water.'' Draft final report. EPA Contract No. 68-
03-2981. Environmental Research Laboratory, Office of Research and 
Development, EPA, Athens, GA, and Office of Pollution Prevention and 
Toxics, EPA, Washington, DC. (July 1983).
    (12) Winterle J.S., Mill T. Toxic substances process data generation 
and protocol development. Work assignment 18. ``Indirect photoreaction 
protocol.'' Draft EPA special report. EPA Contract No. 68-03-2981. 
Environmental Research Laboratory, Office of Research and Development, 
EPA, Athens, GA and Office of Pollution Prevention and Toxics, EPA, 
Washington, DC. (1985).
    (13) Mill T., Hendry D.G., Richardson H. ``Free radical oxidants in 
natural waters.'' Science, 207:886. (1980).
    (14) U.S. Environmental Protection Agency (USEPA), Office of 
Pollution Prevention and Toxics (OPPT). ``Chemical fate test guidelines. 
Test guideline (CG, CS-6000). Photolysis in aqueous solution.'' EPA-560/
6-84-003. NTIS publication PB-84-233287. (1984).
    (15) USEPA, OPPT. ``Chemical fate test guidelines. Test guildeline 
(CG, CS-6010). Laboratory determination of the direct photolysis 
reaction quantum yield in aqueous solution and sunlight photolysis.'' 
EPA-560/6-84-003. NTIS publication PB-84-233287. (1984).
    (16) Wolff C.J.M., Halmans M.T.H., Van der Heijde H.B. ``The 
formation of singlet oxygen in surface waters.'' Chemosphere, 10:59. 
(1981).
    (17) Zepp R.G., Baughman G.L., Schlotzhauer P.F. ``Comparison of 
photochemical behavior of various humic substances in water: I. Sunlight 
induced reactions of aquatic pollutants photosensitized by humic 
substances.'' Chemosphere, 10:109. (1981).
    (18) Zepp R.G., Baughman G.L., Schlozhauer P.F. ``Comparison of 
photochemical behavior of various humic substances in water: II. 
Photosensitized oxygenations.'' Chemosphere, 10:119. (1981).
    (19) Zepp R.G., Cline D.M. ``Rates of direct photolysis in aquatic 
environments.'' Environmental Science and Technology, 11:359. (1977).
    (20) Zepp, R.G., Wolfe N.L., Baughman G.L., Hollis R.C. ``Singlet 
oxygen in natural waters.'' Nature, 267:421. (1977).

[[Page 59]]

    (21) Zepp R.G., Schlotzhauer P.F., Merritt S.R. ``Photosensitized 
transformations involving electronic energy transfer in natural waters: 
role of humic substances.'' Environmental Science and Technology, 19:74. 
(1985).

[53 FR 34522, Sept. 7, 1988; 53 FR 37393, Sept. 26, 1988]



         Subpart C_Provisional Environmental Effects Guidelines



Sec. 795.120  Gammarid acute toxicity test.

    (a) Purpose. This guideline is intended for use in developing data 
on the acute toxicity of chemical substances and mixtures subject to 
environmental effects test regulations under the Toxic Substances 
Control Act (TSCA) (Pub. L. 94-469, 90 Stat. 2003 (15 U.S.C. 2601 et 
seq.)). This guideline describes a test to develop data on the acute 
toxicity of chemicals to gammarids. The United States Environmental 
Protection Agency (EPA) will use data from this test in assessing the 
hazard of a chemical to aquatic organisms.
    (b) Definitions. The definitions in section 3 of TSCA and in part 
792 of this chapter, Good Laboratory Practice Standards, apply to this 
test guideline. The following definitions also apply to this guideline:
    Death means the lack of reaction of a test organism to gentle 
prodding.
    Flow-through means a continuous or an intermittent passage of test 
solution or dilution water through a test chamber or a holding or 
acclimation tank, with no recycling.
    LC50 means the median lethal concentration, i.e., that concentration 
of a chemical in air or water killing 50 percent of the test batch of 
organisms within a particular period of exposure (which shall be 
stated).
    Loading means the ratio of the biomass of gammarids (grams, wet 
weight) to the volume (liters) of test solution in either a test chamber 
or passing through it in a 24-hour period.
    Solvent means a substance (e.g., acetone) which is combined with the 
test substance to facilitate introduction of the test substance into the 
dilution water.
    Static system means a test chamber in which the test solution is not 
renewed during the period of the test.
    (c) Test procedures--(1) Summary of the test. In preparation for the 
test, test chambers are filled with appropriate volumes of dilution 
water. If a flow-through test is performed, the flow of dilution water 
through each chamber is adjusted to the rate desired. In a static test, 
the test substance is introduced into each test chamber. In a flow-
through test, the rate in which the test substance is added is adjusted 
to establish and maintain the desired concentration of test substance in 
each test chamber. The test is started by randomly introducing 
gammarids, which have been acclimated to the test conditions, into the 
test chambers. Gammarids in the test chambers are observed periodically 
during the test; the dead gammarids are removed and the findings 
recorded. Dissolved oxygen concentration, pH, temperature, and the 
concentration of test substance in test chambers are measured at 
specified intervals. Data collected during the test are used to develop 
concentration--response curves and LC50 values for the test substance.
    (2) [Reserved]
    (3) Range-finding test. (i) A range-finding test should be conducted 
to establish test substance concentrations to be used for the definitive 
test.
    (ii) The gammarids shall be exposed to a wide-range of 
concentrations of the test substance (e.g., 1, 10, 100 mg/1, etc.), 
usually under static conditions.
    (iii) A minimum of five gammarids should be exposed to each 
concentration of test substance for a period of 96 hours. The exposure 
period may be shortened if data suitable for determining concentrations 
in the definitive test can be obtained in less time. Nominal 
concentrations of the test substance may be acceptable.
    (4) Definitive test. (i) The purpose of the definitive test is to 
determine the 24, 48, 72, and 96--hour LC50 values and the 
concentration-response curves.
    (ii) A minimum of 20 gammarids per concentration shall be exposed to 
five or more concentrations of the test substance chosen in a geometric 
series in which the ratio is between 1.5 and 2.0 (e.g., 2, 4, 8, 16, 32, 
64 mg/L). The range

[[Page 60]]

and number of concentrations to which the organisms are exposed shall be 
such that in 96 hours there is at least one concentration resulting in 
mortality greater than 50 and less than 100 percent, and one 
concentration causing greater than zero and less than 50 percent 
mortality. An equal number of gammarids may be placed in two or more 
replicate test chambers. Solvents should be avoided, if possible. If 
solvents have to be used, a solvent control, as well as a dilution 
control, shall be tested at the highest solvent concentration employed 
in the treatments. The solvent should not be toxic or have an effect on 
the toxicity of the test substance. The concentration of solvent should 
not exceed 0.1 ml/L.
    (iii) Every test shall include a concurrent control using gammarids 
from the same population or culture container. The control group shall 
be exposed to the same dilution water, conditions and procedures, except 
that none of the test substance shall be is added to the chamber.
    (iv) The dissolved oxygen concentration, temperature and pH of the 
test solution shall be measured at the beginning of the test and at 24, 
48, 72 and 96 hours in at least one replicate each of the control, and 
the highest, lowest and middle test concentrations.
    (v) The test duration is 96 hours. The test is unacceptable if more 
than 10 percent of the control organisms die during the test.
    (vi) In addition to death, any abnormal behavior or appearance shall 
also be reported.
    (vii) Gammarids shall be randomly assigned to the test chambers. 
Test chambers shall be positioned within the testing area in a random 
manner or in a way in which appropriate statistical analyses can be used 
to determine whether there is any variation due to placement.
    (viii) Gammarids shall be introduced into the test chambers after 
the test substance has been added.
    (ix) Observations on compound solubility shall be recorded. The 
investigator should record the appearance of surface slicks, 
precipitates, or material adhering to the sides of the test chambers.
    (5) [Reserved]
    (6) Analytical measurements--(i) Water quality analysis. The 
hardness, acidity, alkalinity, pH, conductivity, TOC or COD, and 
particulate matter of the dilution water shall be measured at the 
beginning of each definitive test.
    (ii) Collection of samples for measurement of test substance. Each 
sample to be analyzed for the test substance concentrations shall be 
taken at a location midway between the top, bottom, and sides of the 
test chamber. Samples should not include any surface scum or material 
dislodged from the bottom or sides. Samples shall be analyzed 
immediately or handled and stored in a manner which minimizes loss of 
test substance through microbial degradation, photogradation, chemical 
reaction, volatilization, or sorption.
    (iii) Measurement of test substance. (A) For static tests, the 
concentration of dissolved test substance (that which passes through a 
0.45 micron filter) shall be measured in each test chamber at least at 
the beginning (zero-hour, before gammarids are added) and at the end of 
the test. During flow-through tests, the concentration of dissolved test 
substance shall be measured in each test chamber at least at 0 and 96-
hours and in at least one chamber whenever a malfunction of the test 
substance delivery system is observed.
    (B) The analytical methods used to measure the amount of test 
substance in a sample shall be validated before beginning the test. This 
involves adding a known amount of the test substance to each of three 
water samples taken from a chamber containing dilution water and the 
same number of gammarids as are placed in each test chamber. The nominal 
concentrations of the test substance in these samples should span the 
concentration range to be used in the test. Validation of the analytical 
method should be performed on at least two separate days prior to 
starting the test.
    (C) An analytical method is not acceptable if likely degradation 
products of the test substance give positive or negative interferences, 
unless it is shown that such degradation products are not present in the 
test chambers during the test.
    (D) Among replicate test chambers, the measured concentrations shall 
not

[[Page 61]]

vary more than 20 percent. The measured concentration of the test 
substance in any chamber during the test shall not vary more than plus 
or minus 30 percent from the measured concentration in that chamber at 
zero time.
    (E) The mean measured concentration of dissolved test substance 
shall be used to calculate all LC50's and to plot all concentration-
response curves.
    (d) Test conditions for definitive test--(1) Test species--(i) 
Selection. (A) The amphipods, Gammarus fasciatus, G. pseudolimnaeus, and 
G. lacustris are specified for this test.
    (B) Gammarids can be cultured in the laboratory or collected from 
natural sources. If collected, they must be held in the laboratory for 
at least 14 days prior to testing.
    (C) Gammarids used in a particular test shall be of similar age and/
or size and from the same source or culture population.
    (ii) Acclimation. If the holding water is from the same source as 
the dilution water, acclimation to the dilution water shall be done 
gradually over a 48-hour period. The gammarids then shall be held at 
least 7 days in the dilution water prior to testing. Any changes in 
water temperature should not exceed 2 [deg]C per day. Gammarids should 
be held for a minimum of 7 days at the test temperature prior to 
testing.
    (iii) Care and handling. Gammarids shall be cultured in dilution 
water under similar environmental conditions to those used in the test. 
Organisms shall be handled as little as possible. When handling is 
necessary it should be done as gently, carefully and quickly as 
possible. During culturing and acclimation, gammarids shall be observed 
carefully for signs of stress and mortality. Dead and abnormal 
individuals shall be discarded.
    (iv) Feeding. The organisms shall not be fed during testing. During 
culturing, holding, and acclimation, a sufficient quantity of deciduous 
leaves, such as maple, aspen, or birch, should be placed in the culture 
and holding containers to cover the bottom with several layers. These 
leaves should be aged for at least 30 days in a flow-through system 
before putting them in aquaria. As these leaves are eaten, more aged 
leaves should be added. Pelleted fish food may also be added.
    (2) Facilities--(i) Apparatus--(A) Facilities needed to perform this 
test include:
    (1) Containers for culturing, acclimating and testing gammarids;
    (2) Containers for aging leaves under flow-through conditions;
    (3) A mechanism for controlling and maintaining the water 
temperature during the culturing, acclimation and test periods;
    (4) Apparatus for straining particulate matter, removing gas 
bubbles, or aerating the dilution water, as necessary; and
    (5) An apparatus for providing a 16-hour light and 8-hour dark 
photoperiod with a 15- to 30-minute transition period.
    (B) Facilities should be well ventilated and free of fumes and 
disturbances that may affect the test organism.
    (C) Test chambers shall be covered loosely to reduce the loss of 
test solution or dilution water due to evaporation and to minimize the 
entry of dust or other particulates into the solutions.
    (ii) Construction materials. Construction materials and equipment 
that may contact the stock solution, test solution or dilution water 
should not contain substances that can be leached or dissolved into 
aqueous solutions in quantities that can alter the test results. 
Materials and equipment that contact stock or test solutions should be 
chosen to minimize sorption of test substances. Glass, stainless steel, 
and perfluorocarbon plastic should be used wherever possible. Concrete, 
fiberglass, or plastic (e.g., PVC) may be used for holding tanks, 
acclimation tanks, and water supply systems, but they should be aged 
prior to use. Rubber, coopper, brass, galvanized metal, and lead should 
not come in contact with the dilution water, stock solution, or test 
solution.
    (iii) Test substance delivery system. In flow-through tests, 
diluters, metering pump systems or other suitable devices shall be used 
to deliver the test substance to the test chambers. The system used 
shall be calibrated before each test. The general operation of the

[[Page 62]]

test substance delivery system shall be checked twice daily during a 
test. The 24-hour flow shall be equal to at least five times the volume 
of the test chamber. During a test, the flow rates should not vary more 
than 10 percent from one test chamber to another.
    (iv) Test chambers. Test chambers shall contain at least one liter 
of test solution. Test chambers made of stainless steel should be 
welded, not soldered. Test chambers made of glass should be glued using 
clear silicone adhesive. As little adhesive as possible should be left 
exposed in the interior of the chamber. A substrate, such as a bent 
piece of stainless steel screen, should be placed on the bottom of each 
test chamber to provide cover for the gammarids.
    (v) Cleaning of test system. Test substance delivery systems and 
test chambers should be cleaned before each test. They should be washed 
with detergent and then rinsed sequentially with clean water, pesticide-
free acetone, clean water, and 5-percent nitric acid, followed by two or 
more changes of dilution water.
    (vi) Dilution water. (A) Clean surface or ground water, 
reconstituted water, or dechlorinated tap water is acceptable as 
dilution water if gammarids will survive in it for the duration of the 
culturing, acclimating, and testing periods without showing signs of 
strees. The quality of the dilution water should be constant enough that 
the month-to-month variation in hardness, acidity, alkalinity, 
conductivity, TOC or COD, and particulate matter is not more than 10 
percent. The pH should be constant within 0.4 unit. In addition, the 
dilution water should meet the following specifications measured at 
least twice a year:

------------------------------------------------------------------------
                 Substance                      Maximum concentration
------------------------------------------------------------------------
Particulate matter.........................  20 mg/L
Total organic carbon (TOC) or..............  2 mg/L
  chemical oxygen demand (COD).............  5 mg/L
Boron, fluoride............................  100 ug/L
Un-ionized ammonia.........................  1 ug/L
Aluminum, arsenic, chromium, cobalt,         1 ug/L
 copper, iron, lead, nickel, zinc.
Residual chlorine..........................  3 ug/L
Cadmium, mercury, silver...................  100 ng/L
Total organophosphorus pesticides..........  50 ng/L
Total organochlorine pesticides plus:
  polychlorinated biphenyls (PCBs) or......  50 ng/L
  organic chlorine.........................  25 ng/L
------------------------------------------------------------------------

    (B) If the dilution water is from a ground or surface water source, 
conductivity and total organic carbon (TOC) or chemical oxygen demand 
(COD) shall be measured. Reconstituted water can be made by adding 
specific amounts of reagent-grade chemicals to deionized or distilled 
water. Glass-distilled or carbon-filtered deionized water with a 
conductivity less than 1 micromho/cm is acceptable as the diluent for 
making reconstituted water.
    (C) The concentration of dissolved oxygen in the dilution water 
shall be between 90 and 100 percent saturation. If necessary, the 
dilution water can be aerated before the addition of the test substance. 
All reconstituted water should be aerated before use.
    (3) Test parameters. Environmental parameters during the test shall 
be maintained as specified below:
    (i) Water temperature of 18 1 [deg]C.
    (ii) Dissolved oxygen concentration between 60 and 105 percent 
saturation.
    (iii) The number of gammarids placed in a test chamber shall not be 
so great as to affect the results of the test. Ten gammarids per liter 
is the recommended level of loading for the static test. Loading 
requirements for the flow-through test will vary depending on the flow 
rate of dilution water. The loading should not cause the dissolved 
oxygen concentration to fall below the recommended levels.
    (iv) Photoperiod of 16 hours light and 8 hours darkness.
    (e) Reporting. The sponsor shall submit to the EPA all data 
developed by the test that are suggestive or predictive of toxicity. In 
addition, the test report shall include, but not necessarily be limited 
to, the following information:
    (1) Name and address of the facility performing the study and the 
dates on which the study was initiated and completed.
    (2) Objectives and procedures stated in the approved protocol, 
including any changes in the original protocol.
    (3) Statistical methods employed for analyzing the data.
    (4) The test substance identified by name, Chemical Abstracts (CAS) 
number or code number, source, lot or batch number, strength, purity, 
and

[[Page 63]]

composition, or other appropriate characteristics.
    (5) Stability of the test substance under the conditions of the 
test.
    (6) A description of the methods used, including:
    (i) The source of the dilution water, its chemical characteristics 
(e.g., hardness, pH, etc.) and a description of any pretreatment.
    (ii) A description of the test substance delivery system, test 
chambers, the depth and volume of solution in the chamber, the way the 
test was begun (e.g., test substance addition), the loading, the 
lighting, and the flow rate.
    (iii) Frequency and methods of measurements and observations.
    (7) The scientific name, weight, length, source, and history of the 
organisms used, and the acclimation procedures and food used.
    (8) The concentrations tested, the number of gammarids and 
replicates per test concentration. The reported results should include:
    (i) The results of dissolved oxygen, pH and temperature 
measurements.
    (ii) If solvents are used, the name and source of the solvent, the 
nominal concentration of the test substance in the stock solution, the 
highest solvent concentration in the test solution and a description of 
the solubility determination in water and solvents.
    (iii) The measured concentration of the test substance in each test 
chamber just before the start of the test and at all subsequent sampling 
periods.
    (iv) In each test chamber at each observation period, the number of 
dead and live test organisms, the percentage of organisms that died, and 
the number of test organisms that showed any abnormal effects in each 
test chamber at each observation period.
    (v) The 48, 72 and 96-hour LC50's and their 95 percent confidence 
limits. When sufficient data have been generated, the 24-hour LC50 value 
also. These calculations should be made using the mean measured test 
substance concentrations.
    (vi) The observed no-effect concentration (the highest concentration 
tested at which there were no mortalities or abnormal behavioral or 
physiological effects), if any.
    (vii) Methods and data for all chemical analyses of water quality 
and test substance concentrations, including method validations and 
reagent blanks.
    (9) A description of all circumstances that may have affected the 
quality or integrity of the data.
    (10) The names of the sponsor, study director, principal 
investigator, names of other scientists or professionals, and the names 
of all supervisory personnel involved in the study.
    (11) A description of the transformations, calculations, or 
operations performed on the data, a summary and analysis of the data, 
and a statement of the conclusions drawn from the analysis. Results of 
the analysis of data should include the calculated LC50 value, 95 
percent confidence limits, slope of the transformed concentration-
response line, and the results of a goodness-of-fit test (e.g., chi-
square test).
    (12) The signed and dated reports prepared by any individual 
scientist or other professional involved in the study, including each 
person who, at the request or direction of the testing facility or 
sponsor, conducted an analysis or evaluation of data or specimens from 
the study after data generation was completed.
    (13) The locations where all specimens, raw data, and the final 
report are stored.
    (14) The statement prepared and signed by the quality assurance 
unit.

[52 FR 24462, July 1, 1987]



             Subpart D_Provisional Health Effects Guidelines



Sec. 795.225  Dermal pharmacokinetics of DGBE and DGBA.

    (a) Purpose. The purpose of these studies is to determine:
    (1) The absorption of diethylene glycol butyl ether (DGBE) after 
administration by the dermal route.
    (2) The biotransformation of DGBE administered dermally.
    (3) The dermal absorption of DGBE and diethylene glycol butyl ether 
acetate (DGBA).
    (b) Test procedures--(1) Animal selection--(i) Species. The species 
utilized for investigating DGBE and DGBA shall be the rat, a species for 
which historical

[[Page 64]]

data on the toxicity and carcinogenicity of many compounds are available 
and which is used extensively in percutaneous absorption studies.
    (ii) Animals. Adult female Sprague Dawley rats shall be used. The 
rats shall be 7 to 8 weeks old and weigh 180 to 220 grams. Prior to 
testing, the animals shall be selected at random for each group. Animals 
showing signs of ill health shall not be used.
    (iii) Animal care. (A) The animals should be housed in 
environmentally controlled rooms with 10 to 15 air changes per hour. The 
rooms should be maintained at a temperature of 25 2 [deg]C and humidity of 50 10 
percent with a 12-hour light/dark cycle per day. The rats should be 
isolated for at least 7 days prior to use.
    (B) During the acclimatization period, the rats should be housed in 
cages on hardwood chip bedding. All animals shall be provided with 
conventional laboratory diets and water ad libitum.
    (2) Administration of DGBE and DGBA--(i) Test substances. These 
studies require the use of \14\C-labeled DGBE and DGBA. The use of 
\14\C-DGBE and \14\C-DGBA is required for the determinations in 
paragraphs (a) (1), (2), and (3) of this section because they will 
facilitate the work and improve the reliability of quantitative 
determinations.
    (ii) Dosage and treatment. (A) Two doses of DGBA shall be used in 
the study, a ``low'' dose and a ``high'' dose. Three doses of DGBE shall 
be used in the study, a neat ``low'' dose, an aqueous ``low'' dose, and 
neat ``high'' dose. When administered dermally, the ``high'' dose level 
should ideally induce some overt toxicity such as weight loss. The 
``low'' dose level should correspond to a no observed effect level.
    (B) For dermal treatment, the doses shall be applied in a volume 
adequate to deliver the prescribed doses. The backs of the rats should 
be lightly shaved with an electric clipper shortly before treatment. The 
dose shall be applied with a micropipette on a specific area (for 
example, 2 cm\2\) on the freshly shaven skin.
    (iii) Washing efficiency study. Before initiation of the dermal 
absorption studies described in paragraph (b)(2)(iv)(A) of this section, 
an initial washing efficiency experiment shall be performed to assess 
the extent of removal of the applied DGBE and DGBA by washing with soap 
and water. Groups of four rats should be lightly anesthetized with 
sodium pentobarbital. These animals shall then be treated with dermal 
doses of test substance at the low dose level. Soon after application (5 
to 10 minutes) the treated animals shall be washed with soap and water 
then housed in individual metabolism cages for excreta collection. Urine 
and feces shall be collected at 8, 24, and 48 hours following dosing. 
Collection of excreta shall continue every 24 hours if a significant 
amounts of DGBE, DGBA, or metabolites continue to be eliminated.
    (iv) Determination of absorption, biotransformation, and excretion. 
(A) Eight animals shall be dosed once dermally with the low dose of 
\14\C-DGBE.
    (B) Eight animals shall be dosed once dermally with the high dose of 
\14\C-DGBE.
    (C) Eight animals shall be dosed once dermally with the low dose of 
\14\C-DGBA.
    (D) Eight animals shall be dosed once dermally with the high dose of 
\14\C-DGBA.
    (E) The high and low doses of \14\C-DGBE and \14\C-DGBA shall be 
kept on the skin for 24 hours. After application, the animals shall be 
placed in metabolism cages for excreta collection. After 24 hours, any 
test material remaining on the skin will be washed off and the 
containment cell removed. Radiolabeled material in the wash will be 
accounted for in the total recovery. Urine and feces shall be collected 
at 8, 24, 48, 72, and 96 hours after dosing, and if necessary, daily 
thereafter until at least 90 percent of the dose has been excreted or 
until 7 days after dosing, whichever occurs first.
    (3) Observation of animals--(i) Urinary and fecal excretion. The 
quantities of total \14\C excreted in urine and feces by rats dosed as 
specified in paragraph (b)(2)(iv) of this section shall be determined at 
8, 24, 48, 72 and 96 hours after dosing, and if necessary, daily 
thereafter until at least 90 percent of the dose has been excreted or 
until 7 days after dosing (whichever occurs first). Four animals from 
each group shall be used for this purpose.

[[Page 65]]

    (ii) Biotransformation after dermal dosing. Appropriate qualitative 
and quantitative methods shall be used to assay urine specimens 
collected from rats dosed with DGBE as specified in paragraph (b)(2)(iv) 
of this section. Any metabolite which comprises greater than 10 percent 
of the dose shall be identified.
    (c) Data and reporting--(1) Treatment of results. Data shall be 
summarized in tabular form.
    (2) Evaluation of results. All observed results, quantitative or 
incidental, shall be evaluated by an appropriate statistical method.
    (3) Test report. In addition to the reporting requirements as 
specified in the TSCA Good Laboratory Practice Standards, in part 792, 
subpart J of this chapter, the following specific information shall be 
reported:
    (i) Species, strain, and supplier of laboratory animals.
    (ii) Information on the degree (i.e., specific activity for a 
radiolabel) and sites of labeling of the test substances.
    (iii) A full description of the sensitivity and precision of all 
procedures used to produce the data.
    (iv) Relative percent absorption by the dermal route for rats 
administered low and high doses of \14\C-DGBE and \14\C-DGBA.
    (v) Quantity of isotope, together with percent recovery of the 
administered dose, in feces and urine.
    (vi) Biotransformation pathways and quantities of DGBE and 
metabolites in urine collected after administering single high and low 
dermal doses to rats.

[53 FR 5946, Feb. 26, 1988, as amended at 54 FR 41834, Oct. 12, 1989]



Sec. 795.228  Oral/dermal pharmacokinetics.

    (a) Purpose. The purposes of these studies are to:
    (1) Ascertain whether the pharmacokinetics and metabolism of a 
chemical substance or mixture (``test substance'') are similar after 
oral and dermal administration.
    (2) Determine bioavailability of a test substance after oral and 
dermal administration.
    (3) Examine the effects of repeated dosing on the pharmacokinetics 
and metabolism of the test substance.
    (b) Definitions. (1) Bioavailability refers to the rate and relative 
amount of administered test substance which reaches the systemic 
circulation.
    (2) Metabolism means the study of the sum of the processes by which 
a particular substance is handled in the body and includes absorption, 
tissue distribution, biotransformation, and excretion.
    (3) Percent absorption means 100 times the ratio between total 
excretion of radioactivity following oral or dermal administration and 
total excretion following intravenous administration of test substance.
    (4) Pharmacokinetics means the study of the rates of absorption, 
tissue distribution, biotransformation, and excretion.
    (c) Test procedures--(1) Animal selection--(i) Species. The rat 
shall be used for pharmacokinetics testing because it has been used 
extensively for metabolic and toxicological studies. For dermal 
bioavailability studies, the rat and the mini-pig shall be used.
    (ii) Test animals. For pharmacokinetics testing and dermal studies, 
adult male and female Sprague-Dawley rats, 7 to 9 weeks of age, shall be 
used. For dermal studies, young adult mini-pigs shall also be used. The 
animals should be purchased from a reputable dealer and shall be 
identified upon arrival at the testing laboratory. The animals shall be 
selected at random for the test groups and any animal showing signs of 
ill health shall not be used. In all studies, unless otherwise 
specified, each test group shall contain at least 4 animals of each sex 
for a total of at least 8 animals.
    (iii) Animal care. (A) The animals shall be housed in 
environmentally controlled rooms with at least 10 air changes per hour. 
The rooms shall be maintained at a temperature of 24 2 [deg]C and humidity of 50 20 
percent with a 12-hour light/dark cycle per day. The animals shall be 
kept in a quarantine facility for at least 7 days prior to use and shall 
be acclimated to the experimental environment for a minimum of 48 hours 
prior to administration of the test substance.
    (B) During the acclimatization period, the animals shall be housed 
in suitable cages. All animals shall be

[[Page 66]]

provided with certified feed and tap water ad libitum. The mini-pig diet 
shall be supplemented with adequate amounts of ascorbic acid in the 
drinking water.
    (2) Administration of test substance--(i) Test substance. The use of 
a radioactive test substance is required for all studies. Ideally, the 
purity, radioactive and nonradioactive, is greater than 99 percent. The 
radioactive and nonradioactive test substances shall be chromatographed 
separately and together to establish purity and identity. If the purity 
is less than 99 percent or if the chromatograms differ significantly, 
EPA should be consulted.
    (ii) Dosage and treatment--(A) Intravenous. The low dose of test 
substance, in an appropriate vehicle, shall be administered 
intravenously to groups of rats and mini-pigs of each sex. If feasible, 
the same low dose should be used for intravenous, oral, and dermal 
studies.
    (B) Oral. Two doses of text substance shall be used in the oral 
study, a low dose and a high dose. The high dose should ideally induce 
some overt toxicity, such as weight loss. The low dose should correspond 
to a no-observed effect level. The oral dosing shall be accomplished by 
gavage or by administering the encapsulated test substance. If feasible, 
the same high and low doses should be used for oral and dermal studies.
    (C) Dermal. (1) Dermal treatment. For dermal treatment, two doses, 
comparable to the low and high oral doses, shall be dissolved in a 
suitable vehicle and applied in volumes adequate to deliver comparable 
doses. The backs of the animals should be lightly shaved with an 
electric clipper 24 hours before treatment. The test substance shall be 
applied to the intact shaven skin (approximately 2 cm\2\ for rats, 5 
cm\2\ for mini-pigs). The dosed areas shall be protected with a suitable 
porous covering which is secured in place, and the animals shall be 
housed separately.
    (2) Washing efficacy study. Before initiation of the dermal 
absorption studies, an initial washing efficacy experiment shall be 
conducted to assess the removal of the applied low dose of the test 
substance by washing the exposed skin area with soap and water and an 
appropriate organic solvent. The low dose shall be applied to 4 rats and 
4 mini-pigs in accordance with paragraph (c)(2)(ii)(C)(1) of this 
section. After application (5 to 10 minutes), the treated areas of 2 
rats and 2 mini-pigs shall be washed with soap and water and the treated 
areas of the remaining rats and pigs shall be washed with an appropriate 
solvent. The amounts of test substance recovered in the washings shall 
be determined to assess efficacy of its removal by washing.
    (iii) Dosing and sampling schedule--(A) Rat studies. After 
administration of the test substance, each rat shall be placed in a 
metabolic unit to facilitate collection of excreta. For the dermal 
studies, excreta from the rats shall also be collected during the 6 hour 
exposure periods. At the end of each collection period, the metabolic 
units shall be cleaned to recover any excreta that might adhere to them. 
All studies, except the repeated dosing study, shall be terminated at 7 
days or after at least 90 percent of the radioactivity has been 
recovered in the excreta, whichever occurs first.
    (1) Intravenous study. Group A shall be dosed once intravenously at 
the low dose of test substance.
    (2) Oral study. (i) Group B shall be dosed once per os with the low 
dose of test substance.
    (ii) Group C shall be dosed once per os with the high dose of test 
substance.
    (3) Dermal studies. Unless precluded by corrosivity, the test 
substance shall be applied and kept on the skin for a minimum of 6 
hours. At the time of removal of the porous covering, the treated area 
shall be washed with an appropriate solvent to remove any test substance 
that may be on the skin surface. Both the covering and the washing shall 
be assayed to recover residual radioactivity. At the termination of the 
studies, each animal shall be sacrificed and the exposed skin area 
removed. An appropriate section of the skin shall be solubilized and 
assayed for radio-activity to ascertain if the skin acts as a reservoir 
for the test substance. Studies on the dermal absorption of corrosive 
test substances should be discussed with EPA prior to initiation.

[[Page 67]]

    (i) Group D shall be dosed once dermally with the low dose of test 
compound.
    (ii) Group E shall be dosed once dermally with the high dose of the 
test substance.
    (4) Repeated dosing study. Group F shall receive a series of single 
daily oral low doses of nonradioactive test substance over a period of 
at least 7 days. Twenty-four hours after the last nonradioactive dose, a 
single oral low dose of radioactive test substance shall be 
administered. Following dosing with the radioactive substance, the rats 
shall be placed in individual metabolic units as described in paragraph 
(c)(2)(iii) of this section. The study shall be terminated at 7 days 
after the last dose, or after at least 90 percent of the radioactivity 
has been recovered in the excreta, whichever occurs first.
    (B) Mini-Pig studies. For all mini-pig studies, the test groups 
shall consist of four young adult animals. After administration of the 
test substance, each mini-pig shall be kept in a metabolic unit to 
facilitate collection of excreta. At the end of each collection period, 
the metabolic units are to be cleaned to recover any excreta that might 
adhere to them. All studies shall be terminated at 7 days, or after at 
least 90 percent of the radio-activity has been recovered in the 
excreta, whichever occurs first.
    (1) Intravenous study. Group G is to be dosed once intravenously at 
the low dose of the test substance.
    (2) Dermal studies. Following the experimental guidance described in 
(c)(2)(iii)(A)(3) of this section:
    (i) Group H shall be dosed once dermally with the low dose of test 
substance.
    (ii) Group I shall be dosed once dermally with the high dose of the 
test substance.
    (3) Types of studies--(i) Pharmacokinetics studies--(A) Rat studies. 
Groups A through F shall be used to determine the kinetics of absorption 
of the test substance. In the group administered the test substance by 
intravenous routes, (i.e., Group A), the concentration of radioactivity 
in blood and excreta shall be measured following administration. In 
groups administered the test substance by the oral and dermal route 
(i.e., Groups B, C, D, E and F), the concentration of radioactivity in 
blood and excreta shall be measured at selected time intervals during 
and following the exposure period.
    (B) Mini-Pig studies. Groups G, H, and I shall be used to determine 
the extent of dermal absorption of the test substance. The amount of 
radioactivity in excreta shall be determined at selected time intervals.
    (ii) Metabolism studies--Rat studies. Groups A through F shall be 
used to determine the metabolism of the test substance. Urine, feces, 
and expired air shall be collected for identification and quantification 
of the test substance and metabolites.
    (4) Measurements--(i) Pharmacokinetics. Four animals from each group 
shall be used for these purposes.
    (A) Rat studies--(1) Bioavailability. The levels of radioactivity 
shall be determined in whole blood, blood plasma or blood serum at 15 
and 30 minutes and at 1, 2, 8, 24, 48, and 96 hours after initiation of 
dosing.
    (2) Extent of absorption. The total quantities of radioactivity 
shall be determined for excerta collected daily for 7 days or until at 
least 90 percent of the radioactivity has been recovered in the excreta.
    (3) Excretion. The quantities of radioactivity eliminated in the 
urine, feces, and expired air shall be determined separately at 
appropriate time intervals. The collection of carbon dioxide may be 
discontinued when less than one percent of the dose is found to be 
exhaled as radioactive carbon dioxide in 24 hours.
    (4) Tissue distribution. At the termination of each study, the 
quantities of radioactivity in blood and in various tissues, including 
bone, brain, fat, gastrointestinal tract, gonads, heart, kidney, liver, 
lungs, muscle, skin, and residual carcass of each animal shall be 
determined.
    (5) Changes in pharmacokinetics. Results of pharmacokinetics 
measurements (i.e., bioavailability and extent of absorption, tissue 
distribution, and excretion) obtained in rats receiving

[[Page 68]]

the single low oral dose of the test substance (Groups B and C) shall be 
compared to the corresponding results obtained in rats receiving 
repeated oral doses of the test substance (Group F).
    (B) Mini-Pig studies--Extent of absorption. The total quantities of 
radioactivity shall be determined for excreta daily for 7 days or until 
at least 90 percent of the test substance has been excreted.
    (ii) Metabolism. Four animals from each group shall be used for 
these purposes.
    (A) Rat studies--(1) Biotransformation. Appropriate qualitative and 
quantitative methods shall be used to assay urine, feces, and expired 
air collected from rats. Efforts shall be made to identify any 
metabolite which comprises 5 percent or more of the administered dose 
and the major radioactive components of blood.
    (2) Changes in biotransformation. Appropriate qualitative and 
quantitative assay methodology shall be used to compare the composition 
of radioactive compounds in excreta from rats receiving a single oral 
dose (Groups B and C) with those in the excreta from rats receiving 
repeated oral doses (Group H).
    (d) Data and reporting. The final test report shall include the 
following:
    (1) Presentation of results. Numerical data shall be summarized in 
tabular form. Pharmacokinetic data shall also be presented in graphical 
form. Qualitative observations shall also be reported.
    (2) Evaluation of results. All quantitative results shall be 
evaluated by an appropriate statistical method.
    (3) Reporting results. In addition to the reporting requirements as 
specified in 40 CFR part 792, the following specific information shall 
be reported:
    (i) Species and strains of laboratory animals.
    (ii) Chemical characterization of the test substance, including:
    (A) For the radioactive test substances, information on the site(s) 
and degree of radiolabeling, including type of label, specific activity, 
chemical purity, and radiochemical purity.
    (B) For the nonradioactive compound, information on chemical purity.
    (C) Results of chromatography.
    (iii) A full description of the sensitivity, precision, and accuracy 
of all procedures used to generate the data.
    (iv) Percent of absorption of test substance after oral and dermal 
exposures to rats and dermal exposure to mini-pigs.
    (v) Quantity and percent recovery of radioactivity in feces, urine, 
expired air, and blood. In dermal studies on rats and mini-pigs, include 
recovery data for skin, skin washings, and residual radioactivity in the 
covering as well as results of the washing efficacy study.
    (vi) Tissue distribution reported as quantity of radioactivity in 
blood and in various tissues, including bone, brain, fat, 
gastrointestinal tract, gonads, heart, kidney, liver, lung, muscle, skin 
and in residual carcass of rats.
    (vii) Materials balance developed from each study involving the 
assay of body tissues and excreta.
    (viii) Biotransformation pathways and quantities of test substance 
and metabolites in excreta collected after administering single high and 
low doses to rats.
    (ix) Biotransformation pathways and quantities of the test substance 
and metabolites in excreta collected after administering repeated low 
doses to rats.
    (x) Pharmacokinetics model(s) developed from the experimental data.

[54 FR 33411, Aug. 14, 1989; 54 FR 49844, Dec. 1, 1989; 55 FR 25392, 
June 21, 1990]



Sec. 795.231  Pharmacokinetics of isopropanal.

    (a) Purpose. The purposes of these studies are to:
    (1) Ascertain whether the pharmacokinetics and metabolism of the 
``test substance'' are similar after oral and inhalation administration.
    (2) Determine bioavailability of the test substance after oral and 
inhalation administration.
    (3) Examine the effects of repeated dosing on the pharmacokinetics 
and metabolism of the test substance.
    (b) Definitions. (1) ``Bioavailability'' refers to the rate and 
relative amount of administered test substance which reaches the 
systemic circulation.
    (2) ``Metabolism'' means the study of the sum of the processes by 
which a

[[Page 69]]

particular substance is handled in the body, and includes absorption, 
tissue distribution, biotransformation, and excretion.
    (3) ``Pharmacokinetics'' means the study of the rates of absorption, 
tissue distribution, biotransformation, and excretion.
    (c) Test procedures--(1) Animal selection--(i) Species. The rat 
shall be used because it has been used extensively for metabolic and 
toxicological studies.
    (ii) Test animals. For pharmacokinetics testing, adult male and 
female rats (Fischer 344 or strain used for major toxicity testing), 7 
to 9 weeks of age, shall be used. The animals should be purchased from a 
reputable dealer and shall be identified upon arrival at the testing 
laboratory. The animals shall be selected at random for the testing 
groups and any animal showing signs of ill health shall not be used. In 
all studies, unless otherwise specified, each test group shall contain 
at least four animals of each sex for a total of at least eight animals.
    (iii) Animal care. (A) Animal care and housing should be in 
accordance with DHEW Publication No. (NIH)-85-23, 1985, entitled 
``Guidelines for the Care and Use of Laboratory Animals.''
    (B) The animals should be housed in environmentally controlled rooms 
with at least 10 air changes per hour. The rooms shall be maintained at 
a temperature of 22 2 [deg]C and humidity of 50 
20 percent with a 12-hour light/dark cycle per 
day. The animals shall be kept in a quarantine facility for at least 7 
days prior to use and shall be acclimated to the experimental 
environment for a minimum of 48 hours prior to treatment.
    (C) During the acclimatization period, the animals should be housed 
in suitable cages. All animals shall be provided with certified feed and 
tap water ad libitum.
    (2) Administration of test substance--(i) Test substance. The use of 
radioactive test substance is required for all materials balance and 
metabolite identification requirements of the study. Ideally, the purity 
of both radioactive and nonradioactive test substance should be greater 
than 99 percent. The radioactive and nonradioactive substances shall be 
chromatographed separately and together to establish purity and 
identity. If the purity is less than 99 percent or if the chromatograms 
differ significantly, EPA should be consulted.
    (ii) Dosage and treatment--(A) Intravenous. The low dose of test 
substance, in an appropriate vehicle, shall be administered 
intravenously to four rats of each sex.
    (B) Oral. Two doses of test substance shall be used in the oral 
portion of the study, a low dose and a high dose. The high dose should 
ideally induce some overt toxicity, such as weight loss. The low dose 
level should correspond to a no-observed effect level. The oral dosing 
shall be accomplished by gavage or by administering an encapsulated test 
substance. If feasible, the same high and low doses should be used for 
oral and dermal studies.
    (C) Inhalation. Two concentrations of the test substance shall be 
used in this portion of the study, a low concentration and a high 
concentration. The high concentration should ideally induce some overt 
toxicity, while the low concentration should correspond to a no observed 
level. Inhalation treatment should be conducted using a ``nose-cone'' or 
``head only'' apparatus to prevent ingestion of the test substance 
through ``grooming''.
    (iii) Dosing and sampling schedule. After administration of the test 
substance, each rat shall be placed in a separate metabolic unit to 
facilitate collection of excreta. For the inhalation studies, excreta 
from the rats shall also be collected during the exposure periods. At 
the end of each collection period, the metabolic units shall be cleaned 
to recover any excreta that might adhere to the cages. All studies, 
except the repeated dose study, shall be terminated at 7 days, or after 
at least 90 percent of the radioactivity has been recovered in the 
excreta, whichever occurs first.
    (A) Intravenous study. Group A shall be dosed once intravenousely at 
the low dose of test substance.
    (B) Oral studies. (1) Group B shall be dosed once per os with the 
low dose of the test substance.
    (2) Group C shall be dosed once per os with the high dose of the 
test substance.

[[Page 70]]

    (C) Inhalation studies. A single 6-hour exposure period shall be 
used for each group.
    (1) Group D shall be exposed to a mixture of the test substance in 
air at the low concentration.
    (2) Group E shall be exposed to a mixture of test substance in air 
at the high concentration.
    (D) Repeated dosing study. Group F shall receive a series of single 
daily oral low doses of nonradioactive test substance over a period of 
at least 7 consecutive days. Twenty four hours after the last 
nonradioactive dose, a single oral low dose of radioactive test 
substance shall be administered. Following dosing with radioactive 
substance, the rats shall be placed in individual metabolic units as 
described in paragraph (c)(2)(iii) of this section. The study shall be 
terminated 7 days after the last dose, or after at least 90 percent of 
the radioactivity has been recovered in the excreta, whichever occurs 
first.
    (3) Types of studies--(i) Pharmacokinetics studies. Groups A through 
F shall be used to determine the kinetics of absorption of the test 
substance. In groups administered the substance by intravenous or oral 
routes, (i.e., Groups A, B, C, F), the concentration of radioactivity in 
blood and excreta including expired air shall be measured following 
administration. In groups administered the substance by the inhalation 
route (i.e., Groups D and E), the concentration of radioactivity in 
blood shall be measured at selected time intervals during and following 
the exposure period. In the groups administered the substance by 
inhalation (i.e., Groups D and E), the concentration of radioactivity in 
excreta (including expired air) shall be measured at selected time 
intervals following the exposure period. In addition, in the groups 
administered the substance by inhalation, the concentration of test 
substance in inspired air shall be measured at selected time intervals 
during the exposure period.
    (ii) Metabolism studies. Groups A through F shall be used to 
determine the metabolism of the test substance. Excreta (urine, feces, 
and expired air) shall be collected for identification and 
quantification of test substance and metabolites.
    (4) Measurements--(i) Pharmacokinetics. Four animals from each group 
shall be used for these purposes.
    (A) Bioavailability. The levels of radioactivity shall be determined 
in whole blood, blood plasma or blood serum at 15 minutes, 30 minutes, 
1, 2, 3, 6, 9, and 18 hours after dosing; and at 30 minutes, 3, 6, 6.5, 
7, 8, 9, 12, and 18 hours after initation of inhalation exposure.
    (B) Extent of absorption. The total quantities of radioactivity 
shall be determined for excreta collected daily for 7 days, or after at 
least 90 percent of the radioactivity has been recovered in the excreta, 
whichever occurs first.
    (C) Excretion. The quantities of radioactivity eliminated in the 
urine, feces, and expired air shall be determined separately at 
appropriate time intervals. The collection of the intact test substance 
or its metabolites, including carbon dioxide, may be discontinued when 
less than 1 percent of the administered dose is found to be exhaled as 
radioactive carbon dioxide in 24 hours.
    (D) Tissue distribution. At the termination of each study, the 
quantities of radioactivity in blood and in various tissues, including 
bone, brain, fat, gastrointestinal tract, gonads, heart, kidney, liver, 
lungs, muscle, skin, spleen, and residual carcass of each animal shall 
be determined.
    (E) Changes in pharmacokinetics. Results of pharmacokinetics 
measurements (i.e., biotransformation, extent of absorption, tissue 
distribution, and excretion) obtained in rats receiving the single low 
oral dose of test substance (Group B) shall be compared to the 
corresponding results obtained in rats receiving repeated oral doses of 
test substance (Group F).
    (F) Biotransformation. Appropriate qualitative and quantitative 
methods shall be used to assay urine, feces, and expired air collected 
from rats. Efforts shall be made to identify any metabolite which 
comprises 5 percent or more of the dose eliminated.
    (G) Changes in biotransformation. Appropriate qualitative and 
quantitative assay methodology shall be used to compare the composition 
of radioactive substances in excreta from the rats receiving a single 
oral dose

[[Page 71]]

(Groups B and C) with those in the excreta from rats receiving repeated 
oral doses (Group F).
    (ii) [Reserved]
    (d) Data and reporting. The final test report shall include the 
following:
    (1) Presentation of results. Numerical data shall be summarized in 
tabular form. Pharmacokinetics data shall also be presented in graphical 
form. Qualitative observations shall also be reported.
    (2) Evaluation of results. All quantitative results shall be 
evaluated by an appropriate statistical method.
    (3) Reporting results. In addition to the reporting requirements as 
specified in the EPA Good Laboratory Practice Standards (40 CFR 
792.185), the following specific information shall be reported:
    (i) Species and strains of laboratory animals.
    (ii) Chemical characterization of the test substance, including:
    (A) For the radioactive test substance, information on the site(s) 
and degree of radiolabeling, including type of label, specific activity, 
chemical purity, and radiochemical purity.
    (B) For the nonradioactive substance, information on chemical 
purity.
    (C) Results of chromatography.
    (iii) A full description of the sensitivity, precision, and accuracy 
of all procedures used to generate the data.
    (iv) Extent of absorption of the test substance as indicated by: 
percent absorption of the administered oral dose; and total body burden 
after inhalation exposure.
    (v) Quantity and percent recovery of radioactivity in feces, urine, 
expired air, and blood.
    (vi) Tissue distribution reported as quantity of radioactivity in 
blood and in various tissues, including bone, brain, fat, 
gastrointestinal tract, gonads, heart, kidney, liver, lung, muscle, 
skin, spleen and in residual carcass of each rat.
    (vii) Biotransformation pathways and quantities of the test 
substance and metabolites in excreta collected after administering 
single high and low doses to rats.
    (viii) Biotransformation pathways and quantities of the test 
substance and metabolites in excreta collected after administering 
repeated low doses to rats.
    (ix) Pharmacokinetics model(s) developed from the experimental data.

[54 FR 43261, Oct. 23, 1989]



Sec. 795.232  Inhalation and dermal pharmacokinetics of commercial hexane.

    (a) Purposes. The purposes of these studies are to:
    (1) Determine the bioavailability of the test substances after 
dermal and inhalation administration.
    (2) Compare the pharmacokinetics and metabolism of the test 
substances after intravenous, dermal, and inhalation administration.
    (3) Examine the effects of repeated doses on the pharmacokinetics 
and metabolism of the test substances.
    (b) Definitions. (1) Bioavailability refers to the relative amount 
of administered test substance which reaches the systemic circulation 
and the rate at which this process occurs.
    (2) Metabolism means the sum of the enzymatic and nonenzymatic 
processes by which a particular substance is handled in the body.
    (3) Pharmacokinetics means the study of the rates of absorption, 
tissue distribution, biotransformation, and excretion.
    (4) Low dose should correspond to 1 /10 of the high dose.
    (5) High dose shall not exceed the lower explosive limit (LEL) and 
ideally should induce minimal toxicity.
    (6) Test substance refers to the unlabeled and both radiolabeled 
mixtures (\14\C-n-hexane and \14\C-methylcyclopentane) of commercial 
hexane used in the testing.
    (c) Test procedures--(1) Animal selection--(i) Species. The rat 
shall be used for pharmacokinetics testing because it has been used 
extensively for metabolic and toxicological studies.
    (ii) Test animals. Adult male and female rats shall be used for 
testing. The rats shall be 7 to 9 weeks old and their weight range 
should be comparable from group to group. The animals shall be purchased 
from a reputable dealer and shall be permanently identified upon 
arrival. The animals shall be selected at random for the testing groups, 
and any animal showing signs of ill health shall not be used.

[[Page 72]]

    (iii) Animal care. (A) Animal care and housing shall be in 
accordance with DHHS/PHS NIH Publication No. 86-23, 1985, ``Guidelines 
for the Care and Use of Laboratory Animals.''
    (B) The animals shall be housed in environmentally controlled rooms 
with at least 10 air changes per hour. The rooms shall be maintained at 
a temperature of 18 to 26 degrees centigrade and humidity of 40 to 70 
percent with a 12-hour light/dark cycle per day. The animal subjects 
shall be kept in a quarantine facility for at least 7 days prior to use, 
and shall be acclimated to the experimental environment for a minimum of 
48 hours prior to treatment.
    (C) During the acclimatization period, the rats shall be housed in 
suitable cages. All animals shall be provided with certified feed and 
tap water ad libitum.
    (2) Administration of test substances--(i) Test substances. The 
study will require the use of both radiolabeled and unlabeled test 
substances. All unlabeled commercial hexane shall be from the same lot 
number. Two kinds of radiolabeled test substances will be tested. \14\C-
n-hexane shall be the only radiolabeled component of one, and \14\C-MCP 
shall be the only radiolabeled component of the other test substance. 
The use of both radiolabeled test substances is required for all 
pharmacokinetics and metabolism studies described in this rule, except 
for the bioavailability measurements required in (c)(4)(i)(A) of this 
section. The bioavailability measurements need only be conducted with 
the test substance containing \14\C-n-hexane or an unlabeled test 
substance may be used if it can be demonstrated that the analytical 
sensitivity of the method used with the unlabeled test substance is 
equal to or greater than the sensitivity which could be obtained with 
the radiolabeled test substance. If an unlabeled test substance is used 
for bioavailability measurements, these measurements shall be extended 
to include relevant metabolites of n-hexane. These test substances shall 
contain at least 40 liquid volume percent but no more than 55 liquid 
volume percent n-hexane and no less than 10 liquid volume percent 
methylcyclopentane (MCP) and otherwise conform to the specifications 
prescribed in the American Society for Testing and Materials Designation 
D 1836-83 (ASTM D 1836), ``Standard Specification for Commercial 
Hexanes'', published in the 1986 Annual Book of ASTM Standards: 
Petroleum Products and Lubricants, ASTM D 1836-83, pp. 966-967, 1986, 
which is incorporated by reference in accordance with 5 U.S.C. 552(a). 
ASTM D 1863-83 is available for public inspection at the National 
Archives and Records Administration (NARA). For information on the 
availability of this material at NARA, call 202-741-6030, or go to: 
http://www.archives.gov/federal--register/code--of--federal--
regulations/ibr--locations.html. Copies may be obtained from the Non-
Confidential Information Center (NCIC) (7407), Office of Pollution 
Prevention and Toxics, U.S. Environmental Protection Agency, Room B-607 
NEM, 401 M Street, SW., Washington, DC 20460, between the hours of 12 
p.m. and 4 p.m. weekdays excluding legal holidays. This incorporation by 
reference was approved by the Director of the Office of the Federal 
Register in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. This 
material is incorporated as it exists on the date of approval, and a 
notice of any change in this material will be published in the Federal 
Register.
    (ii) Dosage and treatment--(A) Intravenous. An appropriate dose of 
the test substance shall be administered intravenously. The intravenous 
data obtained in this portion of the study shall be suitable for the 
determination of absorption, distribution, and excretion parameters of 
the test substance. Factors that should be considered in the selection 
of the intravenous doses are: The acute toxicity of the test substance, 
the availability of a suitable vehicle (if saline is unsuitable) and the 
solubility of the test substance in the vehicle.
    (B) Inhalation. Two concentrations of each test substance shall be 
used in this portion of the study, a low concentration and a high 
concentration. The high concentration should induce minimal toxicity, 
but shall not exceed the lower explosive limit (LEL). The low 
concentration shall correspond to 1/10 of the high concentration. 
Inhalation treatment shall be conducted

[[Page 73]]

using a ``nose-cone'' or ``head only'' apparatus to reduce ingestion of 
the test substance through ``grooming'' or dermal absorption.
    (C) Dermal. Dermal absorption studies should be conducted by the 
methodology of Susten, A.S., Dames, B.L. and Niemeier, R.W., ``In vivo 
percutaneous absorption studies of volatile solvents in hairless mice. 
I. Description of a skin depot'', In: Journal of Applied Toxicology 
6:43-46, (1986), or by some other suitable method because the test 
substances have significant volatility. The high and low doses shall be 
tested in rats.
    (iii) Dosing and sampling schedule. Each experimental group shall 
contain at least four animals of each sex. After administration of the 
test substance, each rat shall be placed in an individual metabolic unit 
for collection of urine, feces, and expired air. For the dermal studies, 
excreta from the rats shall also be collected during the exposure 
periods. At the end of each collection period, the metabolic units shall 
be cleaned to recover any excreta that might adhere to the units. All 
studies, except the repeated dose studies, shall be terminated at 7 
days, or after at least 90 percent of the administered radioactivity has 
been recovered in the excreta, whichever occurs first. All studies 
described below shall be conducted separately with each radiolabeled 
test substance.
    (A) Intravenous study. Group A shall be given a single intravenous 
dose of the radiolabeled test substance to result in a level of 
commercial hexane in the blood that approximates the level from the 
other routes of exposure so that the data can be used to determine 
absorption and excretion parameters.
    (B) Inhalation studies. A single 6-hour exposure period shall be 
used for each group.
    (1) Group B shall be exposed to a mixture of the radiolabeled test 
substance in air at the low concentration.
    (2) Group C shall be exposed to a mixture of the radiolabeled test 
substance in air at the high concentration.
    (C) Dermal studies. The test substance shall be applied and kept on 
the skin for a minimum of 6 hours. The covering apparatus components 
shall be assayed to recover residual radioactivity. At the termination 
of the studies, each animal shall be sacrificed and the exposed skin 
area removed. An appropriate section of the skin shall be solubilized 
and assayed for radioactivity to ascertain whether the skin acts as a 
reservoir for the test substance.
    (1) Group D shall be given one dermal, low dose of the radiolabeled 
test substance.
    (2) Group E shall be given one dermal, high dose of the radiolabeled 
test substance.
    (D) Repeated dosing study. Group F shall receive a series of single 
daily 6-hour inhalation exposures to unlabeled test substance at the low 
dose over a period of at least 7 days. A single 6-hour inhalation 
exposure to the radiolabeled test substance at the low dose shall be 
administered 24 hours after the last unlabeled exposure. Following 
administration of the radiolabeled substance, the rats shall be placed 
in individual metabolic units and excreta collected. The study shall be 
terminated 7 days after the last exposure, or after at least 90 percent 
of the radioactivity has been recovered in the excreta, whichever occurs 
first.
    (3) Types of studies--(i) Pharmacokinetics studies. Groups A through 
F shall be used to determine the kinetics of absorption of the test 
substance. In animal subjects administered the test substance 
intravenously (i.e., Group A), the concentration of test substance in 
blood and excreta shall be measured following administration. In animal 
subjects administered the test substance by the inhalation and dermal 
routes (i.e., Groups B through F), the concentration of test substance 
in blood shall be measured at selected time intervals during and 
following the exposure period. In animal subjects administered the test 
substance by the inhalation route (i.e., Groups B, C, and F) the 
concentration of test substance in excreta shall be measured following 
exposure. In animal subjects administered the test substance by the 
dermal route (i.e., Groups D and E) the concentration of test substance 
in excreta shall be measured during and following exposure. These 
measurements allow calculation of uptake, half lives, and clearance. In 
addition, in the groups

[[Page 74]]

administered the test substance by inhalation (i.e., Groups B, C, and 
F), the concentration of test substance in the exposure chamber air 
shall be measured at selected time intervals during the exposure period.
    (ii) Metabolism studies. Groups A through F shall be used to 
determine the metabolism of the test substance. Excreta (urine, feces, 
and expired air) shall be collected for identification and measurement 
of the quantities of test substance and metabolites.
    (4) Measurements--(i) Pharmacokinetics. At least four animals from 
each group shall be used for these purposes.
    (A) Bioavailability. The levels of test substance and relevant 
metabolites, as appropriate, shall be determined in whole blood, blood 
plasma or blood serum at appropriate intervals after initiation of 
intravenous, dermal, and inhalation exposure. The sampling intervals 
should be compatible with the exposure route under study. The 
determinations need only be done on animals administered the test 
substance containing \14\C-n-hexane or, if the analytical sensitivity is 
equal or greater, unlabeled test substance may be used.
    (B) Extent of absorption. The total quantities of radioactivity 
shall be determined for excreta collected daily for 7 days, or until at 
least 90 percent of theradioactivity has been recovered in the excreta, 
whichever occurs first.
    (C) Excretion. The quantities of radioactivity eliminated in the 
urine, feces, and expired air shall be determined separately at time 
intervals that provide accurate measurement of clearance and excretory 
rates. The collection of carbon dioxide may be discontinued when less 
than one percent of the dose is found to be exhaled as radioactive 
carbon dioxide in 24 hours.
    (D) Tissue distribution. At the termination of each study, the 
quantities of radioactivity shall be determined in blood and in various 
tissues, including bone, brain, fat, gastrointestinal tract, gonads, 
heart, kidney, liver, lungs, muscle, skin, spleen, thymus, and residual 
carcass of each animal.
    (E) Change in pharmacokinetics. Results of pharmacokinetics 
measurements (i.e., biotransformation, extent of absorption, tissue 
distribution, and excretion) obtained in rats receiving the single 
inhalation exposure to the low dose of the test substance (Group B) 
shall be compared to the corresponding results obtained in rats 
receiving repeated inhalation exposures to the low dose of the test 
substance (Group F).
    (ii) Metabolism. At least four animals from each group shall be used 
for these purposes.
    (A) Biotransformation. Appropriate qualitative and quantitative 
methods shall be used to assay urine, feces, and expired air collected 
from rats. Efforts shall be made to identify any metabolite which 
comprises 5 percent or more of the dose administered.
    (B) Changes in biotransformation. Appropriate qualitative and 
quantitative assay methods shall be used to compare the composition of 
radioactive compounds in excreta from rats receiving a single inhalation 
exposure (Groups B and C) with that from rats receiving repeated 
inhalation exposures (Group F).
    (d) Data and reporting. The final test report shall include the 
following:
    (1) Presentation of results. Numerical data shall be summarized in 
tabular form. Pharmacokinetics data shall also be presented in graphical 
form. Qualitative observations shall also be reported.
    (2) Evaluation of results. All data shall be evaluated by 
appropriate statistical methods.
    (3) Reporting results. In addition to the reporting requirements as 
specified in 40 CFR part 792, the following information shall be 
reported.
    (i) Strain of laboratory animals.
    (ii) Chemical characterization of the test substances, including:
    (A) For the radiolabeled test substances, information on the sites 
and degree of radiolabeling, including type of label, specific activity, 
chemical purity prior to mixing with the unlabeled hexane mixture, and 
radiochemical purity.
    (B) For the unlabeled test substance, information on lot number and 
the percentage of MCP and n-hexane.
    (C) Results of chromatography.
    (iii) A full description of the sensitivity, precision, and accuracy 
of all procedures used to obtain the data.

[[Page 75]]

    (iv) Percent and rate of absorption of the test substance after 
inhalation and dermal exposures.
    (v) Quantity and percent recovery of radioactivity in feces, urine, 
expired air, and blood. For dermal studies, include recovery data for 
skin and residual radioactivity in the covering apparatus.
    (vi) Tissue distribution reported as quantity of radioactivity in 
blood, in various tissues including bone, brain, fat, gastrointestinal 
tract, gonads, heart, kidney, liver, lung, muscle, skin, spleen, thymus, 
and in residual carcass.
    (vii) Biotransformation pathways, to the extent possible, and 
quantities of the test substances and metabolites in excreta collected 
after administering single high and low doses.
    (viii) Biotransformation pathways, to the extent possible, and 
quantities of test substances and metabolites in excreta collected after 
administering repeated low doses.
    (ix) Pharmacokinetics models to the extent they can be developed 
from the experimental data.

[55 FR 632, Jan. 8, 1990, as amended at 58 FR 34205, June 23, 1993; 60 
FR 34466, July 3, 1995; 69 FR 18803, Apr. 9, 2004]



Sec. 795.250  Developmental neurotoxicity screen.

    (a) Purpose. In the assessment and evaluation of the toxic 
characteristics of a chemical, it is important to determine when 
acceptable exposures in the adult may not be acceptable to a developing 
organism. This test is designed to provide information on the potential 
functional and morphologic hazards to the nervous system which may arise 
in the offspring from exposure of the mother during pregnancy and 
lactation.
    (b) Principle of the test method. The test substance is administered 
to several groups of pregnant animals during gestation and lactation, 
one dose level being used per group. Offspring are randomly selected 
from within litters for neurotoxicity evaluation. The evaluation 
includes observation to detect gross neurological and behavioral 
abnormalities, determination of motor activity, neuropathological 
evaluation, and brain weights. Measurements are carried out periodically 
during both postnatal development and adulthood.
    (c) Test procedures--(1) Animal selection--(i) Species and strain. 
Testing should be performed in the Sprague Dawley rat.
    (ii) Age. Young adult animals (nulliparous females) shall be used.
    (iii) Sex. Pregnant females shall be used at each dose level.
    (iv) Number of animals. The objective is for a sufficient number of 
pregnant rats to be exposed to ensure that an adequate number of 
offspring are produced for neurotoxicity evaluation. At least 20 litters 
are recommended at each dose level. This number assumes a coefficient of 
variation of 20 to 25 percent for most behavioral tests. If, based upon 
experience with historical control data or data for positive controls in 
a given laboratory, the coefficient of variation for a given task is 
higher than 20 to 25 percent, then calculation of appropriate sample 
sizes to detect a 20 percent change from control values with 80 percent 
power would need to be done. For most designs, calculations can be made 
according to Dixon and Massey (1957) under paragraph (e)(5) of this 
section, Neter and Wasserman (1974) under paragraph (e)(10) of this 
section, Sokal and Rohlf (1969) under paragraph (e)(11) of this section, 
or Jensen (1972) under paragraph (e)(8) of this section.
    (A) On day 4 after birth, the size of each litter should be adjusted 
by eliminating extra pups by random selection to yield, as nearly as 
possible, 4 males and 4 females per litter. Whenever the number of male 
or female pups prevents having 4 of each sex per litter, partial 
adjustment (for example, 5 males and 3 females) is permitted. 
Adjustments are not appropriate for litters of less than 8 pups. 
Elimination of runts only is not appropriate. Individual pups should be 
identified uniquely after standardization of litters. A method that may 
be used can be found in Adams et al. (1985) under paragraph (e)(1) of 
this section.
    (B) After standardization of litters, males and females shall be 
randomly assigned to one of each of three behavioral tasks. 
Alternatively, more than one of the behavioral tasks may be conducted in 
the same animal. In the

[[Page 76]]

latter case, a minimum of 1 to 2 days should separate the tests when 
conducted at about the same age.
    (C) One male and one female shall be randomly selected from each 
litter for sacrifice at weaning as specified in paragraph (c)(8) of this 
section.
    (2) Control group. A concurrent control group shall be used. This 
group shall be a sham treated group, or, if a vehicle is used in 
administering the test substance, a vehicle control group. Animals in 
the control groups shall be handled in an identical manner to test group 
animals. The vehicle shall neither be developmentally toxic nor have 
effects on reproduction.
    (3) Dose levels and dose selection. (i) At least 3 dose levels plus 
a control (vehicle control, if a vehicle is used) shall be used.
    (ii) If the substance has been shown to be developmentally toxic 
either in a standard developmental toxicity study or a pilot study, the 
highest dose level shall be the maximum dose which will not induce in 
utero or neonatal deaths or malformations sufficient to preclude a 
meaningful evaluation of neurotoxicity.
    (iii) In the absence of standard developmental toxicity, unless 
limited by the physicochemical nature or biologicial properties of the 
substance, the highest dose level shall induce some overt maternal 
toxicity but shall not result in a reduction in weight gain exceeding 20 
percent during gestation and lactation.
    (iv) The lowest dose should not produce any grossly observable 
evidence of either maternal or developmental neurotoxicity.
    (v) The intermediate dose(s) shall be equally spaced between the 
highest and lowest dose.
    (4) Dosing period. Day 0 in the test is the day on which a vaginal 
plug and/or sperm are observed. The dose period shall cover the period 
from day 6 of gestation through weaning (21 days postnatally).
    (5) Administration of test substance. The test substance or vehicle 
should be administered orally by intubation. The test substance shall be 
administered at the same time each day. The animals shall be weighed 
periodically and the dosage based on the most recent weight 
determination.
    (6) Observation of dams. (i) A gross examination of the dams shall 
be made at least once each day, before daily treatment. The animals 
shall be observed by trained technicians who are blind with respect to 
the animal's treatment, using standardized procedures to maximize inter-
observer reliability. Where possible, it is advisable that the same 
observer be used to evaluate the animals in a given study. If this is 
not possible, some demonstration of inter-observer reliability is 
required.
    (ii) During the treatment and observation periods, cage-side 
observations shall include:
    (A) Any responses with respect to body position, activity level, 
coordination of movement, and gait.
    (B) Any unusual or bizarre behavior including, but not limited to 
headflicking, head searching, compulsive biting or licking, self-
mutilation, circling, and walking backwards.
    (C) The presence of:
    (1) Convulsions.
    (2) Tremors.
    (3) Increased levels of lacrimation and/or red-colored tears.
    (4) Increased levels of salivation.
    (5) Piloerection.
    (6) Pupillary dilation or constriction.
    (7) Unusual respiration (shallow, labored, dyspneic, gasping, and 
retching) and/or mouth breathing.
    (8) Diarrhea.
    (9) Excessive or diminished urination.
    (10) Vocalization.
    (iii) Signs of toxicity shall be recorded as they are observed, 
including the time of onset, the degree and duration.
    (iv) Animals shall be weighed at least weekly.
    (v) The day of delivery of litters shall be recorded.
    (7) Study conduct--(i) Observation of offspring. (A) All offspring 
shall be examined cage-side daily for gross signs of mortality and 
morbidity.
    (B) All offspring shall be examined outside the cage for gross signs 
of toxicity whenever they are weighed or removed from their cages for 
behavioral testing. The offspring shall be observed by trained 
technicians, who are blind with respect to the animal's treatment

[[Page 77]]

using standardized procedures to maximize inter-observer reliability. 
Where possible, it is advisable that the same observer be used to 
evaluate the animals in a given study. If this is not possible, some 
demonstration of inter-observer reliability is required. At a minimum, 
the end points outlined in paragraph (c)(6)(ii) of this section shall be 
monitored as appropriate for the developmental stage being observed.
    (C) Any gross signs of toxicity in the offspring shall be recorded 
as they are observed, including the time of onset, the degree, and 
duration.
    (ii) Developmental landmarks. Live pups should be counted and 
litters weighed by weighing each individual pup at birth, or soon 
thereafter, and on days 4, 7, 13, 17, and 21, and biweekly thereafter. 
The age of the pups at the time of the appearance of the following 
developmental landmarks shall be determined:
    (A) Vaginal opening. General procedure for this determination may be 
found in Adams et al. (1985) under paragraph (e)(1) of this section.
    (B) Testes descent. General procedure for this determination may be 
found in Adams et al. (1985) under paragraph (e)(1) of this section.
    (iii) Motor activity. (A) Motor activity shall be monitored 
specifically on days 13, 17, 21, 45 (2 days), and 
60 (2 days). Motor activity shall be monitored by 
an automated activity recording apparatus. The device used shall be 
capable of detecting both increases and decreases in activity, i.e., 
baseline activity as measured by the device shall not be so low as to 
preclude decreases nor so high as to preclude increases. Each device 
shall be tested by standard procedures to ensure, to the extent 
possible, reliability of operation across devices and testing of animals 
within dose groups shall be balanced across devices.
    (B) Each animal shall be tested individually. The test session shall 
be long enough to demonstrate habituation of motor activity in control 
animals, i.e., to approach asymptotic levels by the last 20 percent of 
the session. Animals' activity counts shall be collected in equal time 
periods of no greater than 10 minutes duration. All sessions shall have 
the same duration. Treatment groups shall be counter-balanced across 
test times.
    (C) Efforts shall be made to ensure that variations in the test 
conditions are minimal and are not systematically related to treatment. 
Among the variables which can affect motor activity are sound level, 
size, and shape of the test cage, temperature, relative humidity, 
lighting conditions, odors, use of home cage or novel test cage, and 
environmental distractions.
    (D) Additional information on the conduct of a motor activity study 
may be obtained in the TSCA motor activity guideline, in Sec. 798.6200 
of this chapter.
    (iv) Auditory startle test. An auditory startle habituation test 
shall be performed on the offspring on days 22 and 60. Details on the 
conduct of this testing may be obtained in Adams et al. (1985) under 
paragraph (e)(1) of this section. In performing the auditory startle 
task, the mean response amplitude on each block of 10 trials (5 blocks 
of 10 trials per session on each day of testing) shall be made. While 
use of pre-pulse inhibition is not a requirement, it may be used at the 
discretion of the investigator. Details on the conduct of this testing 
may be obtained from Ison (1984) under paragraph (e)(7) of this section.
    (v) Active avoidance test. Active avoidance testing shall be 
conducted beginning at 60 to 61 days of age. Details on the apparatus 
may be obtained in Brush and Knaff (1959) and on the conduct of testing 
from Brush (1962), under paragraphs (e)(2) and (e)(4) of this section, 
respectively; reviews on active avoidance conditioning by Brush (1971) 
and McAllister and McAllister (1971) can be found under paragraphs 
(e)(3) and (e)(9) of this section, respectively. In performing the 
active avoidance task, the following measures should be made:
    (A) Mean number of shuttles during the adaptation period preceding 
each daily session.
    (B) Mean number and latency of avoidances per session, presented in 
blocks of 10 trials (2 blocks of 10 trials per session across 5 
sessions).
    (C) Mean number and latency of escapes per session, presented in 
blocks of 10 trials as above.

[[Page 78]]

    (D) Mean duration of shocks per session, presented in blocks of 10 
trials as above.
    (E) Mean number of shuttles during the inter-trial intervals.
    (8) Post-mortem evaluation--(i) Age of animals. One male and one 
female per litter shall be sacrificed at weaning and the remainder 
following the last behavioral measures. Neuropathology and brain weight 
determinations shall be made on animals sacrificed at weaning and after 
the last behavioral measures.
    (ii) Neuropathology. Details for the conduct of neuropathology 
evaluation may be obtained in the TSCA neuropathology guideline, in 
Sec. 798.6400 of this chapter. At least 6 offspring per dose group 
shall be randomly selected from each sacrificed group (weaning and 
adulthood) for neuropathologic evaluation. These animals shall be 
balanced across litters, and equal numbers of males and females shall be 
used. The remaining sacrificed animals shall be used to determine brain 
weight. Animals shall be perfused in situ by a generally recognized 
technique. After perfusion, the brain and spinal cord shall be removed 
and gross abnormalities noted. Cross-sections of the following areas 
shall be examined: The forebrain, the center of the cerebrum and 
midbrain, the cerebellum and pons, and the medulla oblongata; the spinal 
cord at cervical and lumbar swelling; Gasserian ganglia, dorsal root 
ganglia, dorsal and ventral root fibers, proximal sciatic nerve (mid-
thigh and sciatic notch), sural nerve (at knee), and tibial nerve (at 
knee). Tissue samples from both the central and peripheral nervous 
system shall be further immersion-fixed and stored in appropriate 
fixative for further examination. After dehydration, tissue specimens 
shall be cleared with xylene and embedded in paraffin or paraplast 
except for the sural nerve which should be embedded in plastic. A method 
for plastic embedding is described by Spencer et al. under paragraph 
(e)(12) of this section. Tissue sections shall be prepared from the 
tissue blocks. The following general testing sequence is recommended for 
gathering histopathological data:
    (A) General staining. A general staining procedure shall be 
performed on all tissue specimens in the highest treatment group. 
Hematoxylin and eosin (H&E) shall be used for this purpose. The staining 
shall be differentiated properly to achieve bluish nuclei with pinkish 
background.
    (B) Special stains. Based on the results of the general staining, 
selected sites and cellular components shall be further evaluated by use 
of specific techniques. If H&E screening does not provide such 
information, a battery of stains shall be used to assess the following 
components in all appropriate required samples: Neuronal body (e.g., 
Einarson's gallocyanin), axon (e.g., Kluver's Luxol Fast Blue), and 
neurofibrils (e.g., Bielchosky). In addition, nerve fiber teasing shall 
be used. A section of normal tissue shall be included in each staining 
to assure that adequate staining has occurred. Any changes shall be 
noted and representative photographs shall be taken. If lesions are 
observed, the special techniques shall be repeated in the next lower 
treatment group until no further lesions are detectable.
    (C) Alternative technique. If the anatomical locus of expected 
neuropathology is well-defined, epoxy-embedded sections stained with 
toluidine blue may be used for small sized tissue samples. This 
technique obviates the need for special stains.
    (iii) Brain weight. At least 10 animals that are not sacrificed for 
histopathology shall be used to determine brain weight. The animals 
shall be decapitated and the brains carefully removed, blotted, chilled, 
and weighed. The following dissection shall be performed on an ice-
cooled glass plate: First, the rhombencephalon is separated by a 
transverse section from the rest of the brain and dissected into the 
cerebellum and the medulla oblongata/pons. A transverse section is made 
at the level of the ``optic chiasma'' which delimits the anterior part 
of the hypothalamus and passes through the anterior commissure. The 
cortex is peeled from the posterior section and added to the anterior 
section. This divides the brain into four sections, the telencephalon, 
the diencephalon/mid-brain, the medulla oblongata/pons, and the 
cerebellum. Sections shall be

[[Page 79]]

weighed as soon as possible after dissection to avoid drying. Detailed 
methodology is available in Glowinski and Iversen (1966) under paragraph 
(e)(6) of this section.
    (d) Data reporting and evaluation. In addition to the reporting 
requirements specified in part 792, subpart J of this chapter, the final 
test report shall include the following information.
    (1) Description of system and test methods. (i) A detailed 
description of the procedures used to standardize observation and 
operational definitions for scoring observations.
    (ii) Positive control data from the laboratory performing the test 
that demonstrate the sensitivity of the procedures being used. These 
data do not have to be from studies using prenatal exposures. However, 
the laboratory must demonstrate competence in testing neonatal animals 
perinatally exposed to chemicals and establish test norms for the 
appropriate age group.
    (iii) Procedures for calibrating and assuring the equivalence of 
devices and balancing treatment groups.
    (iv) A short justification explaining any decisions where 
professional judgement is involved such as fixation technique and choice 
of stains.
    (2) Results. The following information shall be arranged by test 
group dose level.
    (i) In tabular form, data for each animal shall be provided showing:
    (A) Its identification number and litter from which it came.
    (B) Its body weight and score on each developmental landmark at each 
observation time; total session activity counts and intrasession 
subtotals on each day measured; auditory startle response magnitude 
session counts and intrasession subtotals on each day measured; 
avoidance session counts and intrasession counts on each day measured; 
time and cause of death (if appropriate); locations, nature or 
frequency, and severity of the lesions; total brain weight; absolute 
weight of each of the four sections; and weight of each section as a 
percentage of total brain weight. A commonly used scale such as 1+, 2+, 
3+, and 4+ for degree of severity of lesions ranging from very slight to 
extensive may be used for morphologic evaluation. Any diagnoses derived 
from neurologic signs and lesions, including naturally occurring 
diseases or conditions, shall also be recorded.
    (ii) Summary data for each group shall include:
    (A) The number of animals at the start of the test.
    (B) Body weights of the dams during gestation and lactation.
    (C) Litter size and mean weight at birth.
    (D) The number of animals showing each observation score at each 
observation time.
    (E) The percentage of animals showing each abnormal sign at each 
observation time.
    (F) The mean and standard deviation for each continuous end point at 
each observation time. These will include body weight, motor activity 
counts, acoustic startle responses, performance in active avoidance 
tests, and brain weights (both absolute and relative).
    (G) The number of animals in which any lesion was found.
    (H) The number of animals affected by each different type of lesion, 
the average grade of each type of lesion, and the frequency of each 
different type and/or location of lesions.
    (3) Evaluation of data. An evaluation of the test results shall be 
made. The evaluation shall include the relationship between the doses of 
the test substance and the presence or absence, incidence, and severity 
of any neurotoxic effect. The evaluation shall include appropriate 
statistical analyses. The choice of analyses shall consider tests 
appropriate to the experimental design and needed adjustments for 
multiple comparisons.
    (e) References. For additional background information on this test 
guideline, the following references should be consulted:
    (1) Adams, J., Buelke-Sam, J., Kimmel, C.A., Nelson, C.J., Reiter, 
L.W., Sobotka, T.J., Tilson, H.A., and Nelson, B.K. ``Collaborative 
behavioral teratology study: Protocol design and testing procedure.'' 
Neurobehavioral Toxicology and Teratology. 7: 579-586. (1985).
    (2) Brush, F.R. ``The effects of inter-trial interval on avoidance 
learning in the rat.'' Journal of Comparative Physiology and Psychology. 
55: 888-892. (1962).

[[Page 80]]

    (3) Brush, F.R. ``Retention of aversively motivated behavior.'' In: 
``Adverse Conditioning and Learning.'' Brush, F.R., ed., New York: 
Academic Press. (1971).
    (4) Brush, F.R. and Knaff, P.R. ``A device for detecting and 
controlling automatic programming of avoidance-conditioning in a 
shuttle-box.'' American Journal of Psychology. 72: 275-278 (1959).
    (5) Dixon, W.J. and Massey, E.J. ``Introduction to Statistical 
Analysis.'' 2nd ed. New York: McGraw-Hill. (1957).
    (6) Glowinski, J. and Iversen, L.L. ``Regional studies of 
catecholamines in the rat brain-I.'' Journal of Neurochemistry. 13: 655-
669. (1966).
    (7) Ison, J.R. ``Reflex modification as an objective test for 
sensory processing following toxicant exposure.'' Neurobehavioral 
Toxicology and Teratology. 6: 437-445. (1984).
    (8) Jensen, D.R. ``Some simultaneous multivariate procedures using 
Hotelling's T2 Statistics.'' Biometrics. 28: 39-53. (1972).
    (9) McAllister, W.R. and McAllister, D.E. ``Behavioral measurement 
of conditioned fear.'' In: ``Adverse Conditioning and Learning.'' Brush, 
F.R., ed., New York: Academic Press (1971).
    (10) Neter, J. and Wasserman, W. ``Applied Linear Statistical 
Models.'' Homewood: Richard D. Irwin, Inc. (1974).
    (11) Sokal, R.P. and Rohlf, E.J. ``Biometry.'' San Francisco: W.H. 
Freeman and Co. (1969).
    (12) Spencer, P.S., Bischoff, M.C., and Schaumburg, H.H., 
``Neuropathological methods for the detection of neurotoxic disease.'' 
In: ``Experimental and Clinical Neurotoxicology.'' Spencer, P.S. and 
Schaumburg, H.H., eds., Baltimore, MD: Williams & Wilkins, pp. 743-757. 
(1980).

[53 FR 5957, Feb. 26, 1988]



PART 796_CHEMICAL FATE TESTING GUIDELINES--Table of Contents




Subpart A [Reserved]

               Subpart B_Physical and Chemical Properties

Sec.
796.1050 Absorption in aqueous solution: Ultraviolet/visible spectra.
796.1950 Vapor pressure.

                      Subpart C_Transport Processes

796.2750 Sediment and soil adsorption isotherm.

                   Subpart D_Transformation Processes

796.3100 Aerobic aquatic biodegradation.
796.3500 Hydrolysis as a function of pH at 25 [deg]C.

    Authority: 15 U.S.C. 2603.

Subpart A [Reserved]



               Subpart B_Physical and Chemical Properties



Sec. 796.1050  Absorption in aqueous solution: Ultraviolet/visible spectra.

    (a) Introductory information--(1) Guidance information. (i) 
Molecular formula.
    (ii) Structural formula.
    (2) Standard documents. The spectrophotometric method is based on 
national standards and consensus methods which are applied to measure 
the absorption spectra.
    (b) Method--(1)(i) Introduction, purpose, scope, relevance, 
application and limits of test. (A) The primary environmental purpose in 
determining the ultraviolet-visible (UV-VIS) absorption spectrum of a 
chemical compound is to have some indication of the wavelengths at which 
the compounds may be susceptible to photochemical degradation. Since 
photochemical degradation is likely to occur in both the atmosphere and 
the aquatic environment, spectra appropriate to these media will be 
informative concerning the need for further persistence testing.
    (B) Degradation will depend upon the total energy absorbed in 
specific wavelength regions. Such energy absorption is characterized by 
both molar absorption coefficient (molar extinction coefficient) and 
band width. However, the absence of measurable absorption does not 
preclude the possibility of photodegradation.
    (ii) Definitions and units. The UV-VIS absorption spectrum of a 
solution is a function of the concentration, c1, expressed in 
mol/L, of all absorbing species present; the path length, d, of the 
spectrophotometer cell, expressed in cm; and the molar absorption 
(extinction) coefficient,[egr]i, of each species. The

[[Page 81]]

absorbance (optical density) A of the solution is then given by:
[GRAPHIC] [TIFF OMITTED] TC15NO91.045


For a resolvable absorbance peak, the band width [lambda] is the 
wavelength range, expressed in nm=10-9 m, of the peak at half 
the absorbance maximum.
    (iii) Reference substances. (A) The reference substances need not be 
employed in all cases when investigating a new substance. They are 
provided primarily so that calibration of the method may be performed 
from time to time and to offer the chance to compare the results when 
another method is applied.
    (B) Reference compounds appropriate for the calibration of the 
system are:
    (1) Potassium dichromate (in 0.005 mol/L, 
H2SO4 solution) from J.A.A. Ketelaar, paragraph 
(d)(2) of this section:

log [egr]...................................   3.56   3.63   3.16   3.50
[lambda] in nm..............................    235    257    313    350
 

    (2) Fluoranthene (in methanol) from C.R.C. Atlas of Spectral Data, 
paragraph (d)(3) of this section:

log [egr]............................   4.75   4.18   4.73   3.91   3.92
[lambda] in nm.......................    237    236    288    339    357
 

    (3) 4-nitrophenol (in methanol) from C.R.C. Atlas of Spectral Data, 
paragraph (d)(3) of this section:

log [egr]...............................................    3.88    4.04
[lambda] in nm..........................................     288     311
 


See also paragraph (d)(1) of this section.
    (iv) Principle of the test method. This method utilizes a double-
beam spectrophotometer which records only the absorption differences 
between the blank and test solutions to give the spectrum of the 
chemical being tested.
    (v) Quality criteria--Reproducibility and sensitivity. (A) 
Reproducibility and sensitivity, need not be measured directly. Instead, 
the accuracy of the system in measuring the spectra of reference 
compounds will be defined so as to assure appropriate reproducibility 
and sensitivity. It is preferable to use a recording double-beam 
spectrophotometer to obtain the UV-VIS spectrum of the test compound. 
Such an instrument should have a photometric accuracy of 0.02 units over the absorbance range of 0 to 2 units. It 
should be capable of recording absorbances at wavelengths of 200 to 750 
nanometers nm with a wavelength accuracy of 0.5 
nm. The cells employed with the instrument must necessarily be 
transparent over this wavelength range and must have a path length 
determined to within 1 percent. To ensure that the instrument is 
performing satisfactorily, spectra for test solutions of 
K2Cr2O7 (for absorbance accuracy) and 
holmium glass (for wavelength accuracy) should be run periodically.
    (B) In the event that a recording double-beam instrument is not 
available, it will be necessary to determine the absorbance of the test 
solution in a single-beam instrument at 5-nm intervals over the entire 
wavelength range and at 1-nm intervals where there are indicated 
absorbance maxima. Wavelength and absorbance tests should be done as 
with the double-beam instrument.
    (2) Description of the test procedure--(i) Preparation--(A) 
Preparation of test solutions. (1) Solutions should be prepared by 
accurately weighing an appropriate amount of the purest form of the test 
substance available. This should be made up in a concentration which 
will result in at least one absorbance maximum in the range 0.5 to 1.5 
units.
    (2) The absorption of a compound is due to its particular chemical 
form. It is often the case that different forms are present, depending 
on whether the medium is acidic, basic, or neutral. Consequently, 
spectra under all three conditions are required where solubility and 
concentration allow. Where it is not possible to obtain sufficient 
concentrations in any of the aqueous media, a suitable organic solvent 
should be used (methanol preferred).
    (3) The acid medium should have a pH of less than 2, and the basic 
medium should be at least pH 10. The solvent for the neutral solution, 
and for preparing the acidic and basic ones, should be distilled water, 
transparent to ultraviolet radiation down to 200 nm. If methanol must be 
used, acidic and basic solutions can be prepared by adding 10 percent by 
volume of HCl or NaOH in aqueous solution ([HCl], [NaOH]=1 mol/L).
    (4) In theory, all chemical species other than that being tested are

[[Page 82]]

present in both beams and would therefore not appear in the recorded 
spectrum of a double-beam instrument. In practice, because the solvent 
is usually present in great excess, there is a threshold value of 
wavelength below which it is not possible to record the spectrum of the 
test chemical. Such a wavelength will be a property of the solvent or of 
the test medium. In general, distilled water is useful from 200 nm 
(dissolved ions will often increase this), methanol from 210 nm, hexane 
from 210 nm, acetonitrile from 215 nm and dichloromethane from 235 nm.
    (B) Blank solutions. A blank must be prepared which contains the 
solvent and all chemical species other than the test chemical. The 
absorption spectrum of this solution should be recorded in a manner 
identical to that of the test solution and preferably on the same chart. 
This ``baseline'' spectrum should never record an absorbance reading 
varying more than 0.05 from the nominal zero 
value.
    (C) Cells. Cell pathlengths are usually between 0.1 cm and 10 cm. 
Cell lengths should be selected to permit recording of at least one 
maximum in the absorbance range of 0.5 to 1.5 units. Which set of cells 
should be used will be governed by the concentration and the absorbance 
of the test solution as indicated by the Beer-Lambert Law. The cells 
should be transparent over the range of the spectrum being recorded, and 
the path-lengths should be known to an accuracy of at least 1 per cent. 
Cells should be thoroughly cleaned in an appropriate manner (chromic 
acid is useful for quartz cells) and rinsed several times with the test 
or blank solutions.
    (ii) Performance of the test. Both cells to be employed should be 
rinsed with the blank solution and then filled with same. The instrument 
should be set to scan at a rate appropriate for the required wavelength 
resolution and the spectrum of the blank recorded. The sample cell 
should then be rinsed and filled with the test solution and the scanning 
repeated, preferably on the same spectrum chart, to display the 
baseline. The test should be carried out at 25 [deg]C.
    (c) Data and reporting--(1) Treatment of results. (i) The molar 
absorption coefficient [egr] should be calculated for all absorbance 
maxima of the test substance. The formula for this calculation is:
[GRAPHIC] [TIFF OMITTED] TC15NO91.046


where the quantities are as defined above (see Definitions and units).

    (ii) For each peak which is capable of being resolved, either as 
recorded or by extrapolated symmetrical peaks, the bandwidth should be 
recorded.
    (2) Test report. (i) The report should contain a copy of each of the 
three spectra (3 pH conditions). If neither water nor methanol solutions 
are feasible, there will be only one spectrum. Spectra should include a 
readable wave-length scale. Each spectrum should be clearly marked with 
the test conditions.
    (ii) For each maximum in each spectrum, the [egr] value and 
bandwidth (when applicable) should be calculated and reported, along 
with the wavelength of the maximum. This should be presented in tabular 
form.
    (iii) The various test conditions should be included, such as scan 
speed, the name and model of the spectrophotom-eter, the slit width 
(where available), cell type and path length, the concentrations of the 
test substance, and the nature and acidity of the solvent medium. A 
recent test spectrum on appropriate reference materials for photometric 
and wavelength accuracy should also be submitted (see Reproducibility 
and sensitivity).
    (d) Literature references. For additional background information on 
this test guideline, the following references should be consulted:
    (1) Milazzo, G., Caroli, S., Palumbo-Doretti, M., Violante, N., 
Analytical Chemistry, 49: 711 (1977).
    (2) Katelaar, J.A.A., Photoelectric Spectrometry Group Bulletin, 8, 
(Cambridge, 1955).
    (3) Chemical Rubber Company, Atlas of Spectral Data, (Cliffland, 
Ohio).

[50 FR 39472, Sept. 27, 1985]



Sec. 796.1950  Vapor pressure.

    (a) Introduction--(1) Background and purpose. (i) Volatilization, 
the evaporative loss of a chemical, depends upon

[[Page 83]]

the vapor pressure of chemical and on environmental conditions which 
influence diffusion from a surface. Volatilization is an important 
source of material for airborne transport and may lead to the 
distribution of a chemical over wide areas and into bodies of water far 
from the site of release. Vapor pressure values provide indications of 
the tendency of pure substances to vaporize in an unperturbed situation, 
and thus provide a method for ranking the relative volatilities of 
chemicals. Vapor pressure data combined with water solubility data 
permit the calculation of Henry's law constant, a parameter essential to 
the calculation of volatility from water.
    (ii) Chemicals with relatively low vapor pressures, high 
adsorptivity onto solids, or high solubility in water are less likely to 
vaporize and become airborne than chemicals with high vapor pressures or 
with low water solubility or low adsorptivity to solids and sediments. 
In addition, chemicals that are likely to be gases at ambient 
temperatures and which have low water solubility and low adsorptive 
tendencies are less likely to transport and persist in soils and water. 
Such chemicals are less likely to biodegrade or hydrolyze and are prime 
candidates for atmospheric oxidation and photolysis (e.g., smog 
formation or stratospheric alterations). On the other hand, nonvolatile 
chemicals are less frequently involved in atmosphere transport, so that 
concerns regarding them should focus on soils and water.
    (iii) Vapor pressure data are an important consideration in the 
design of other chemical fate and effects tests; for example, in 
preventing or accounting for the loss of violatile chemicals during the 
course of the test.
    (2) Definitions and units. (i) ``Desorption efficiency'' of a 
particular compound applied to a sorbent and subsequently extracted with 
a solvent is the weight of the compound which can be recovered from the 
sorbent divided by the weight of the compound originally sorbed.
    (ii) ``Pascal'' (Pa) is the standard international unit of vapor 
pressure and is defined as newtons per square meter (N/m\2\). A newton 
is the force necessary to give acceleration of one meter per second 
squared to one kilogram of mass.
    (iii) The ``torr'' is a unit of pressure which equals 133.3 pascals 
or 1 mm Hg at 0 [deg]C.
    (iv) ``Vapor pressure'' is the pressure at which a liquid or solid 
is in equilibrium with its vapor at a given temperature.
    (v) ``Volatilization'' is the loss of a substance to the air from a 
surface or from solution by evaporation.
    (3) Principle of the test methods. (i) The isoteniscope procedure 
uses a standardized technique [ASTM 1978] that was developed to measure 
the vapor pressure of certain liquid hydrocarbons. The sample is 
purified within the equipment by removing dissolved and entrained gases 
until the measured vapor pressure is constant, a process called 
``degassing.'' Impurities more volatile than the sample will tend to 
increase the observed vapor pressure and thus must be minimized or 
removed. Results are subject to only slight error for samples containing 
nonvolatile impurities.
    (ii) Gas saturation (or transpiration) procedures use a current of 
inert gas passed through or over the test material slowly enough to 
ensure saturation and subsequent analysis of either the loss of material 
or the amount (and sometimes kind) of vapor generated. Gas saturation 
procedures have been described by Spencer and Cliath (1969) under 
paragraph (d)(2) of this section. Results are easy to obtain and can be 
quite precise. The same procedures also can be used to study 
volatilization from laboratory scale environmental simulations. Vapor 
pressure is computed on the assumption that the total pressure of a 
mixture of gases is equal to the sum of the pressures of the separate or 
component gases and that the ideal gas law is obeyed. The partial 
pressure of the vapor under study can be calculated from the total gas 
volume and the weight of the material vaporized. If v is the volume 
which contains w grams of the vaporized material having a molecular 
weight M, and if p is the pressure of the vapor in equilibrium at 
temperature T (K), then the vapor pressure, p, of the sample is 
calculated by

p=(w/M)(RT/v),


[[Page 84]]



where R is the gas constant (8.31 Pa m\2\ mol-1 
K-1) when the pressure is in pascals (Pa) and the volume is 
in cubic meters. As noted by Spencer and Cliath (1970) under paragraph 
(d)(3) of this section, direct vapor pressure measurements by gas 
saturation techniques are more directly related to the volatilization of 
chemicals than are other techniques.

    (iii) In an effort to improve upon the procedure described by 
Spencer and Cliath (1969) under paragraph (d)(2) of this section, and to 
determine the applicability of the gas saturation method to a wide 
variety of chemical types and structures, EPA has sponsored research and 
development work at SRI International (EPA 1982) under paragraph (d)(1) 
of this section. The procedures described in this Test Guideline are 
those developed under that contract and have been evaluated with a wide 
variety of chemicals of differing structure and vapor pressures.
    (4) Applicability and specificity. (i) A procedure for measuring the 
vapor pressure of materials released to the environment ideally would 
cover a wide range of vapor pressure values, at ambient temperatures. No 
single procedure can cover this range, so two different procedures are 
described in this section, each suited for a different part of the 
range. The isoteniscope procedure is for pure liquids with vapor 
pressures from 0.1 to 100 kPa. For vapor pressures of 10-5 to 
10 \3\ Pa, a gas saturation procedure is to be used.
    (ii) With respect to the isoteniscope method, if compounds that boil 
close to or form azeotropes with the test material are present, it is 
necessary to remove the interfering compounds and use pure test 
material. Impurities more volatile than the sample will tend to increase 
the observed vapor pressure above its true value but the purification 
steps will tend to remove these impurities. Soluble, nonvolatile 
impurities will decrease the apparent vapor pressure. However, because 
the isoteniscope procedure is a static, fixed-volume method in which an 
insignificant fraction of the liquid sample is vaporized, it is subject 
to only slight error for samples containing nonvolatile impurities. That 
is, the nonvolatile impurities will not be concentrated due to 
vaporization of the sample.
    (iii) The gas saturation method is applicable to solid or liquid 
chemicals. Since the vapor pressure measurements are made at ambient 
temperatures, the need to extrapolate data from high temperatures is not 
necessary and high temperature extrapolation, which can often cause 
serious errors, is avoided. The method is most reliable for vapor 
pressures below 10 \3\ Pa. Above this limit, the vapor pressures are 
generally overestimated, probably due to aerosol formation. Finally, the 
gas saturation method is applicable to the determination of the vapor 
pressure of impure materials.
    (b) Test procedures--(1) Test conditions. (i) The apparatus in the 
isoteniscope method is described in paragraph (b)(2)(i) of this section.
    (ii) The apparatus used in the gas saturation method is described in 
paragraph (b)(2)(ii) of this section.
    (2) Performance of the tests--(i) Isoteniscope Procedure. The 
isoteniscope procedure described as ANSI/ASTM Method D 2879-86 is 
applicable for the measurement of vapor pressures of liquids with vapor 
pressures of 0.1 to 100 kilopascals (kPa) (0.75 to 750 torr). ASTM D 
2879-86 is available for inspection at the National Archives and Records 
Administration (NARA). For information on the availability of this 
material at NARA, call 202-741-6030, or go to: http://www.archives.gov/
federal--register/code--of--federal--regulations/ibr--locations.html. 
This incorporation by reference was approved by the Director of the 
Office of the Federal Register. This material is incorporated as it 
exists on the date of approval and a notice of any change in this 
material will be published in the Federal Register. Copies of the 
incorporated material may be obtained from the Non-Confidential 
Information Center (NCIC) (7407), Office of Pollution Prevention and 
Toxics, U.S. Environmental Protection Agency, Room B-607 NEM, 401 M St., 
SW., Washington, DC 20460, between the hours of 12 p.m. and 4 p.m. 
weekdays excluding legal holidays, or from the American Society for 
Testing and Materials (ASTM), 1916 Race Street, Philadelphia, PA 19103.

[[Page 85]]

The isoteniscope method involves placing liquid sample in a thermostated 
bulb (the isoteniscope) connected to a manometer and a vacuum pump. 
Dissolved and entrained gases are removed from the sample in the 
isoteniscope by degassing the sample at reduced presssure. The vapor 
pressure of the sample at selected temperatures is determined by 
balancing the pressure due to the vapor of the sample against a known 
pressure of an inert gas. The vapor pressure of the test compound is 
determined in triplicate at 25 0.5 [deg]C and at 
any other suitable temperatures (0.5[deg]). It is 
important that additional vapor pressure measurements be made at other 
temperatures, as necessary, to assure that there is no need for further 
degassing, as described in the ASTM method.
    (ii) Gas saturation procedure. (A) The test procedures require the 
use of a constant-temperature box as depicted in the following Figure 1.
[GRAPHIC] [TIFF OMITTED] TC01AP92.036

        Figure 1--Schematic Diagram of Vapor Saturation Apparatus

The insulated box, containing sample holders, may be of any suitable 
size and shape. The sketch in Figure 1 shows a box containing three 
solid sample holders and three liquid sample holders, which allows for 
the triplicate analysis of either a solid or liquid sample. The 
temperature within the box is controlled to 0.5[deg] or better. Nitrogen gas, split into six streams 
and controlled by fine needle valves (approximately 0.79 mm orifice), 
flows into the box via 3.8 mm (0.125 in.) i.d. copper tubing. After 
temperature equilibration, the gas flows through the sample and the 
sorbent trap and exits from the box. The flow rate of the effluent 
carrier gas is measured at room temperature with a bubble flow meter or 
other suitable device. The flow rate is checked frequently during the 
experiment to assure that there is an accurate value for the total 
volume of carrier gas. The flow rate is used to calculate the total 
volume (at room temperature) of gas that has passed

[[Page 86]]

through the sample and sorbent [(vol/time) x time = volume]. The vapor 
pressure of the test substance can be calculated from the total gas 
volume and the mass of sample vaporized. If v is the volume of gas that 
transported mass w of the vaporized test material having a molecular 
weight M, and if p is the equilibrium vapor pressure of the sample at 
temperature T, then p is calculated by the equation

    p=(w/M)(RT/v).


In this equation, R is the gas constant (8.31 Pa m\3\mol-1 
K-1). The pressure is expressed in pascals (Pa), the volume 
in cubic meters (m\3\), mass in grams and T in kelvins (K). T=273.15+t, 
if t is measured in degrees Celsius ([deg]C).
    (B) Solid samples are loaded into 5 mm i.d. glass tubing between 
glass wool plugs. The following Figure 2 depicts a drawing of a sample 
holder and absorber system.
[GRAPHIC] [TIFF OMITTED] TC01AP92.037

                Figure 2--Solid Compound Sampling System
    (C) Liquid samples are contained in a holder as shown in the 
following Figure 3.
[GRAPHIC] [TIFF OMITTED] TC01AP92.038

                Figure 3--Liquid Compound Sampling System

The most reproducible method for measuring the vapor pressure of liquids 
is to coat the liquid on glass beads and to pack the holder in the 
designated place with these beads.
    (D) At very low vapor pressures and sorbent loadings, adsorption of 
the chemical on the glass wool separating the sample and the sorbent and 
on the glass surfaces may be a serious problem. Therefore, very low 
loadings should be avoided whenever possible. Incoming nitrogen gas 
(containing no interfering impurities) passes through a coarse frit and 
bubbles through a 38 cm column of liquid sample. The stream passes 
through a glass wool column to trap aerosols and then through a sorbent 
tube, as described above. The pressure drop across the glass wool column 
and the sorbent tube are negligible.
    (E) With both solid and liquid samples, at the end of the sampling 
time, the front and backup sorbent sections are analyzed separately. The 
compound on each section is desorbed by adding the sorbent from that 
section to 1.0 ml of desorption solvent in a small vial and allowing the 
mixture to stand at a suitable temperature until no more test compound 
desorbs. It is extremely important that the desorption solvent contain 
no impurities which would interfere with the analytical method of 
choice. The resulting solutions are analyzed quantitatively by a 
suitable analytical method to determine the weight of sample desorbed 
from each section. The choice of the analytical method,

[[Page 87]]

sorbent, and desorption solvent is dictated by the nature of the test 
material. Commonly used sorbents include charcoal, Tenax GC, and XAD-2. 
Describe in detail the sorbent, desorption solvent, and analytical 
methods employed.
    (F) Measure the desorption efficiency for every combination of 
sample, sorbent, and solvent used. The desorption efficiency is 
determined by injecting a known mass of sample onto a sorbent and later 
desorbing it and analyzing for the mass recovered. For each combination 
of sample, sorbent, and solvent used, carry out the determination in 
triplicate at each of three concentrations. Desorption efficiency may 
vary with the concentration of the actual sample and it is important to 
measure the efficiency at or near the concentration of sample under gas 
saturation test procedure conditions.
    (G) To assure that the gas is indeed saturated with test compound 
vapor, sample each compound at three differing gas flow rates. 
Appropriate flow rates will depend on the test compound and test 
temperature. If the calculated vapor pressure shows no dependence on 
flow rate, then the gas is assumed to be saturated.
    (c) Data and reporting. (1) Report the triplicate calculated vapor 
pressures for the test material at each temperature, the average 
calculated vapor pressure at each temperature, and the standard 
deviation.
    (2) Provide a description of analytical methods used to analyze for 
the test material and all analytical results.
    (3) For the isoteniscope procedure, include the plot of p vs. the 
reciprocal of the temperature in K, developed during the degasing step 
and showing linearity in the region of 298.15 K (25 [deg]C) and any 
other required test temperatures.
    (4) For the gas saturation procedure, include the data on the 
calculation of vapor pressure at three or more gas flow rates at each 
test temperature, showing no dependence on flow rate. Include a 
description of sorbents and solvents employed and the desorption 
efficiency calculations.
    (5) Provide a description of any difficulties experienced or any 
other pertinent information.
    (d) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) U.S. Environmental Protection Agency. Evaluation of Gas 
Saturation Methods to Measure Vapor Pressures: Final Report, EPA 
Contract No. 68-01-5117 with SRI International, Menlo Park, California 
(1982).
    (2) Spencer, W.F. and Cliath, M.M. ``Vapor Density of Dieldrin,'' 
Journal of Agricultural and Food Chemistry, 3:664-670 (1969).
    (3) Spencer, W.F. and Cliath, M.M. ``Vapor Density and Apparent 
Vapor Pressure of Lindane,'' Journal of Agricultural and Food Chemistry, 
18:529-530 (1970).

[50 FR 39252, Sept. 27, 1985, as amended at 53 FR 12525, Apr. 15, 1988; 
53 FR 21641, June 9, 1988; 60 FR 34466, July 3, 1995; 69 FR 18803, Apr. 
9, 2004]



                      Subpart C_Transport Processes



Sec. 796.2750  Sediment and soil adsorption isotherm.

    (a) Introduction--(1) Background and purpose. The adsorption of 
chemicals to sediments and soils is an important process that affects a 
chemical's distribution in the environment. If a chemical is adsorbed to 
soil particles, it will remain on the soil surface and will not reach 
ground water. If a chemical is not adsorbed, it will leach through the 
soil profile and may reach ground waters and then surface waters. 
Similarly, if a chemical adsorbed to sediment, it will accumulate in the 
bed and suspended load of aquatic systems. If a chemical is not adsorbed 
to sediment, it will accumulate in the water column of aquatic systems. 
Information on the adsorption potential is needed under certain 
circumstances to assess the transport of chemicals in the environment. 
This section describes procedures that will enable sponsors to determine 
the adsorption isotherm of a chemical on sediments and soils.
    (2) Definitions and units. (i) The ``cation exchange capacity'' 
(CEC) is the sum total of exchangeable cations that a sediment or soil 
can adsorb. The CEC is expressed in milliequivalents of negative charge 
per 100 grams (meq/100g) or milliequivalents of negative

[[Page 88]]

charge per gram (meq/g) of soil or sediment.
    (ii) ``Clay mineral analysis'' is the estimation or determination of 
the kinds of clay-size minerals and the amount present in a sediment or 
soil.
    (iii) ``Organic matter'' is the organic fraction of the sediment or 
soil; it includes plant and animal residues at various stages of 
decomposition, cells and tissues of soil organisms, and substances 
synthesized by the microbial population.
    (iv) ``Particle size analysis'' is the determination of the various 
amounts of the different particle sizes in a sample (i.e., sand, silt, 
clay), usually by sedimentation, sieving, micrometry, or combinations of 
these methods. The names and diameter range commonly used in the United 
States are:

------------------------------------------------------------------------
                  Name                            Diameter range
------------------------------------------------------------------------
Very coarse sand.......................  2.0 to 1.0 mm
Coarse sand............................  1.0 to 0.5 mm
Medium sand............................  0.5 to 0.25 mm
Fine sand..............................  0.25 to 0.125 mm
Very fine sand.........................  0.125 to 0.062 mm
Silt...................................  0.062 to 0.002 mm
Clay...................................  <0.002 mm
------------------------------------------------------------------------

    (v) The ``pH'' of a sediment or soil is the negative logarithm to 
the base ten of the hydrogen ion activity of the sediment or soil 
suspension. It is usually measured by a suitable sensing electrode 
coupled with a suitable reference electrode at a 1/1 solid/solution 
ratio by weight.
    (vi) The adsorption ratio, ``Kd,'' is the amount of test 
chemical adsorbed by a sediment or soil (i.e., the solid phase) divided 
by the amount of test chemical in the solution phase, which is in 
equilibrium with the solid phase, at a fixed solid/solution ratio.
    (vii) ``Sediment'' is the unconsolidated inorganic and organic 
material that is suspended in and being transported by surface water, or 
has settled out and has deposited into beds.
    (viii) ``Soil'' is the unconsolidated mineral material on the 
immediate surface of the earth that serves as a natural medium for the 
growth of land plants. Its formation and properties are determined by 
various factors such as parent material, climate, macro- and 
microorganisms, topography, and time.
    (ix) ``Soil aggregate'' is the combination or arrangement of soil 
separates (sand, silt, clay) into secondary units. These units may be 
arranged in the soil profile in a distinctive characteristic pattern 
that can be classified according to size, shape, and degree of 
distinctness into classes, types, and grades.
    (x) ``Soil classification'' is the systematic arrangement of soils 
into groups or categories. Broad groupings are based on general soil 
characteristics while subdivisions are based on more detailed 
differences in specific properties. The soil classification system used 
in this standard and the one used today in the United States is the 7th 
Approximation-Comprehensive System. The ranking of subdivisions under 
this system is: Order, Suborder, Great group, family, and series.
    (xi) A ``soil horizon'' is a layer of soil approximately parallel to 
the land surface. Adjacent layers differ in physical, chemical, and 
biological properties such as color, structure, texture, consistency, 
kinds and numbers of organisms present, and degree of acidity or 
alkalinity.
    (xii) ``Soil Order'' is the broadest category of soil classification 
and is based on the general similarities of soil physical/chemical 
properties. The formation of soil by similar general genetic processes 
causes these similarities. The Soil Orders found in the United States 
are: Alfisol, Aridisol, Entisol, Histosol, Inceptisol, Mollisol, Oxisol, 
Spodosol, Ultisol, and Vertisol.
    (xiii) ``Soil series'' is the basic unit of soil classification and 
is a subdivision of a family. A series consists of soils that were 
developed under comparable climatic and vegetational conditions. The 
soils comprising a series are essentially alike in all major profile 
characteristics except for the texture of the ``A'' horizon (i.e., the 
surface layer of soil).
    (xiv) ``Soil texture'' is a classification of soils that is based on 
the relative proportions of the various soil separates present. The soil 
textural classes are: clay, sandy clay, silty clay, clay loam, silty 
clay loam, sandy clay loam, loam, silt loam, silt, sandy loam, loamy 
sand, and sand.
    (3) Principle of the test method. (i) The extent of adsorption of a 
chemical onto sediment or soil is measured, using this

[[Page 89]]

test guideline, by equilibrating aqueous solutions containing different, 
but environmentally realistic, concentrations of the test chemical with 
a known quantity of sediment or soil. After equilibrium is reached, the 
distribution of the chemical between the water phase and the solid phase 
is quantitatively measured by a suitable analytical method. Then, 
sorption constants are calculated by using the Freundlich equation:

                               Equation 1

x/m=Cs=KCel/n

where:

Ce=Equilibrium concentration of the chemical in the solution 
phase
Cs=Equilibrium concentration of the chemical in the solid 
phase
K=Freundlich adsorption coefficient
m=The mass of the solid in grams
l/n=Exponent where n is a constant
x=The mass in micrograms of the chemical adsorbed by m grams of solid.


Logarithmetic transformation of the Freundlich equation yields the 
following linear relationship:

                               Equation 2

log Cs=log K+(l/n) log Ce

    (ii) In order to estimate the environmental movement of the test 
chemical, the values K and l/n are compared with the values of other 
chemicals whose behavior in soil and sediment systems is well-documented 
in scientific literature.
    (iii) The adsorption isotherm (AI) test has many desirable features. 
First, adsorption results are highly reproducible. The test provides 
excellent quantitative data readily amenable to statistical analyses. 
Also, it has relatively modest requirements for chemicals, soils, 
laboratory space, and equipment. It allows solution phase organic 
chemical determinations that are relatively uncomplicated. A chemical 
extraction-mass balance procedure to elicit information on chemical 
transformations occurring at colloid interfaces can be incorporated into 
this test. The ease of performing the isotherm test and mass balance 
will depend upon the physical/chemical properties of the test chemical 
and the availability of suitable analytical techniques to measure the 
chemical.
    (iv) The papers by Aharonson and Kafkafi (1975) under paragraph 
(d)(1) of this section, Harvey (1974) under paragraph (d)(3) of this 
section, Murray (1975) under paragraph (d)(4) of this section, Saltzman 
(1972) under paragraph (d)(5) of this section, Weber (1971) under 
paragraph (d)(6) of this section, and Wu (1975) under paragraph (d)(7) 
of this section served as the basis for this section. The soil and 
colloid chemistry literature and the analytical chemistry literature 
substantiate the experimental conditions and procedures specified in 
this guideline as accepted, standard procedures.
    (4) Applicability and specificity. The AI Test Guideline can be used 
to determine the soil and sediment adsorption potential of sparingly 
water soluble to infinitely soluble chemicals. In general, a chemical 
having a water solubility of less than 0.5 ppm need not be tested with 
soil as the solid phase, since the literature indicates that these 
chemicals are, in general, immobile in soils, see Goring and Hamaker 
(1972) under paragraph (d)(2) of this section. However, this does not 
preclude future soil adsorption/transformation testing of these 
chemicals if more refined data are needed for the assessment process.
    (b) Test procedures--(1) Test conditions--(i) Special laboratory 
equipment. (A) Equilibrating solutions that contain, besides the test 
chemical, 0.01M calcium nitrate dissolved in sterilized, distilled-
deionized H2O adjusted to neutral pH 7 by boiling to remove 
CO2.
    (B) Containers shall be composed of material that (1) adsorb 
negligible amounts of test chemical, and (2) withstand high speed 
centrifugation. The volume of the container is not a major 
consideration; however, it is extremely important that the amount of 
soil or sediment and the solid/solution ratio used in the study result 
in minimal container headspace. It is also extremely important that the 
containers be sterilized before use.
    (C) A 150 micron (100 mesh) stainless-steel or brass sieve.
    (D) Drying oven, with circulating air, that can attain 100 [deg]C.

[[Page 90]]

    (E) Vortex mixer or a comparable device.
    (F) Rotary shaker or a comparable device.
    (G) High speed temperature-controlled centrifuge capable of 
sedimenting particles greater than 0.5 micron from aqueous solution.
    (ii) Temperature. The test procedure shall be performed at 23 5 [deg]C.
    (iii) Replications. Three replications of the experimental 
treatments shall be used.
    (iv) Soil pretreatment. The following soil pretreatment steps shall 
be performed under the following conditions:
    (A) Decrease the water content, air or oven-dry soils at or below 50 
[deg]C.
    (B) Reduce aggregate size before and during sieving, crush and grind 
dried soil very gently.
    (C) Eliminate microbial growth during the test period using a 
chemical or physical treatment that does not alter or minimally alters 
the soil surface properties.
    (D) Sieve soils with a 100 mesh stainless-steel or brass sieve.
    (E) Store all solutions and soils at temperatures between 0 and 5 
[deg]C.
    (v) Sediment pretreatment. The following sediment pretreatment steps 
shall be performed under the following conditions:
    (A) Decrease the H2O content by air or oven-drying 
sediments at or below 50 [deg]C. Sediments should not be dried 
completely and should remain moist at all times prior to testing and 
analysis.
    (B) Eliminate microbial growth during the test period by using a 
chemical and/or physical treatment that does not alter or minimally 
alters the colloid surface's properties.
    (C) Store at temperatures between 0 and 5[deg]C.
    (vi) Solid/solution ratio. The solid/solution ratio shall be equal 
to or greater than 1/10. If possible, the ratios should be equal to or 
greater than 1/5. The sediment or soil dry weight after drying for a 24-
hour minimum at 90 [deg]C is recommended for use as the weight of the 
solid for ratio and data calculations. If an insufficient amount of 
chemical remains in the water phase for quantification, the solid/
solution ratio should be adjusted so that measurable amounts of the test 
chemical remain in solution.
    (vii) Equilibration time. The equilibration time will depend upon 
the length of time needed for the parent chemical to attain an 
equilibrium distribution between the solid phase and the aqueous 
solution phase. The equilibration time shall be determined by the 
following procedure:
    (A) Equilibrate one solution containing a known concentration of the 
test chemical with the sediment or soil in a solid/solution ratio equal 
to or greater than \1/10\ and preferably equal to or greater than \1/5\. 
It is important that the concentration of the test chemical in the 
equilibrating solution (1) does not exceed one-half of its solubility 
and (2) should be 10 ppm or less at the end of the equilibration period.
    (B) Measure the concentration of the chemical in the solution phase 
at frequent intervals during the equilibration period.
    (C) Determine the equilibration time by plotting the measured 
concentration versus time of sampling; the equilibration time is the 
minimum period of time needed to establish a rate of change of solution 
concentration of 5 percent or less per 24 hours.
    (viii) Centrifugation time. Calculate the centrifugation time, 
tc, necessary to remove particles from solution greater than 
approximately 0.5 [micro]m (5x10-5 [micro]m) equivalent 
diameter (which represents all particles except the fine clay fraction) 
using the following equation:

                               Equation 3

tc(min)=1.41x10\9\ [log(R2/R1)]/N\2\

where:

tc=centrifuge time in minutes
R2=distance from centrifuge spindle to deposition surface of 
centrifuge
R1=distance from spindle to surface of the sample
N=number of revolutions of the centrifuge per minute.

    (ix) Storage of solutions. If the chemical analysis is delayed 
during the course of the experiment, store all solutions between 0 and 5 
[deg]C.
    (x) Solvents for extraction. It is important that the solvent used 
to extract the chemical from the sediment or soil is reagent grade or 
better. Solvents

[[Page 91]]

shall contain no impurities which could interfere with the determination 
of the test compound.
    (2) Test procedure--(i) Equilibration. Add six solutions containing 
different concentrations of the test chemical to at least one gram of 
each solid. The initial concentration of the test chemical in these 
solutions will depend on the affinity the chemical has for the sediment 
or soil. Therefore, after equilibrium is attained, it is extremely 
important that the highest concentration of the test chemical in the 
equilibrating solution does not exceed 10 ppm, is at least one order of 
magnitude greater than the lowest concentration reported, and does not 
exceed one half of its solubility.
    (A) Immediately after the solutions are added to the solids, tightly 
cap the containers and vigorously agitate them for several minutes with 
a vortex mixture or similar device.
    (B) Shake the containers throughout the equilibration period at a 
rate that suspends all solids in the solution phase.
    (ii) Centrifugation. When the equilibration time has expired, 
centrifuge the containers for tc minutes.
    (iii) Chemical extraction. (A) After centrifugation, remove the 
supernatant aqueous phase from the solid-solution mixture.
    (B) Extract the chemical adsorbed on the sediment or soil colloid 
surfaces with solvent.
    (iv) Chemical analysis. Determine the amount of parent test chemical 
in the aqueous equilibrating solution and organic solvent extractions. 
Use any method or combination of methods suitable for the identification 
and quantitative detection of the parent test chemical.
    (c) Reporting. Report the following information:
    (1) Temperature at which the test was conducted.
    (2) Detailed description of the analytical technique(s) used in the 
chemical extraction, recovery, and quantitative analysis of the parent 
chemical.
    (3) Amount of parent test chemical applied, the amount recovered, 
and the percent recovered.
    (4) Extent of adsorption by containers and the approach used to 
correct the data for adsorption by containers.
    (5) The individual observations, the mean values, and graphical 
plots of x/m as a function of Ce for each sediment or soil 
for (i) the equilibration time determination and (ii) the isotherm 
determination.
    (6) The quantities K, n, and l/n.
    (7) Soil information: Soil Order, series, texture, sampling 
location, horizon, general clay fraction mineralogy.
    (8) Sediment information: sampling location, general clay fraction 
mineralogy.
    (9) Sediment and soil physical-chemical properties: percent sand, 
silt, and clay (particle size analysis); percent organic matter; pH (1/1 
solids/H2O); and cation exchange capacity.
    (10) The procedures used to determine the physical/chemical 
properties listed under paragraphs (c) (7) through (9) of this section.
    (d) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) Aharonson, N., Kafkafi, U. ``Adsorption, mobility and 
persistence of thiabendazole and methyl 2-benzimidasole carbamate in 
soils,'' Journal of Agricultural and Food Chemistry, 23:720-724 (1975).
    (2) Goring, C.A.I., Hamaker, J.W., (eds). Organic Chemicals in the 
Soil Environment. Vol. I & II (New York: Marcel Dekker, Inc., 1972).
    (3) Harvey, R.G. et al. ``Soil adsorption and volatility of 
dinitroaniline herbicides,'' Weed Science, 22:120-124 (1974).
    (4) Murray, D.S. et al. ``Comparative adsorption, desorption, and 
mobility of dipropetryn and prometryn in soil,'' Journal of Agricultural 
and Food Chemistry, 23:578-581 (1973).
    (5) Saltzman, S.L. et al. ``Adsorption, desorption of parathion as 
affected by soil organic matter,'' Journal of Agricultural and Food 
Chemistry, 20:1224-1226 (1972).
    (6) Weber, J.B. ``Model soil system, herbicide leaching, and 
sorption,'' Weed Science, 19:145-160 (1971).

[[Page 92]]

    (7) Wu, C.H., et al. ``Napropamide adsorption, desorption, and 
movement in soils,'' Weed Science, 23:454-457 (1975).

[50 FR 39252, Sept. 27, 1985, as amended at 52 FR 19058, May 20, 1987; 
54 FR 29715, July 14, 1989]



                   Subpart D_Transformation Processes



Sec. 796.3100  Aerobic aquatic biodegradation.

    (a) Introduction--(1) Purpose. (i) This Guideline is designed to 
develop data on the rate and extent of aerobic biodegradation that might 
occur when chemical substances are released to aquatic environments. A 
high biodegradability result in this test provides evidence that the 
test substance will be biodegradable in natural aerobic freshwater 
environments.
    (ii) On the contrary, a low biodegradation result may have other 
causes than poor biodegradability of the test substance. Inhibition of 
the microbial inoculum by the test substance at the test concentration 
may be observed. In such cases, further work is needed to assess the 
aerobic aquatic biodegradability and to determine the concentrations at 
which toxic effects are evident. An estimate of the expected 
environmental concentration will help to put toxic effects into 
perspective.
    (2) Definitions. (i) ``Adaptation'' is the process by which a 
substance induces the synthesis of any degradative enzymes necessary to 
catalyze the transformation of that substance.
    (ii) ``Ultimate Biodegradability'' is the breakdown of an organic 
compound to CO2, water, the oxides or mineral salts of other 
elements and/or to products associated with normal metabolic processes 
of microorganisms.
    (iii) ``Ready Biodegradability'' is an expression used to describe 
those substances which, in certain biodegradation test procedures, 
produce positive results that are unequivocal and which lead to the 
reasonable assumption that the substance will undergo rapid and ultimate 
biodegradation in aerobic aquatic environments.
    (3) Principle of the test method. This Guideline method is based on 
the method described by William Gledhill (1975) under paragraph (d)(1) 
of this section. The method consists of a 2-week inoculum buildup period 
during which soil and sewage microorganisms are provided the opportunity 
to adapt to the test compound. This inoculum is added to a specially 
equipped Erlenmeyer flask containing a defined medium with test 
substance. A reservoir holding barium hydroxide solution is suspended in 
the test flask. After inoculation, the test flasks are sparged with 
CO2-free air, sealed, and incubated, with shaking in the 
dark. Periodically, samples of the test mixture containing water-soluble 
test substances are analyzed for dissolved organic carbon (DOC) and the 
Ba(OH)2 from the reservoirs is titrated to measure the amount 
of CO2 evolved. Differences in the extent of DOC 
disappearance and CO2 evolution between control flasks 
containing no test substance, and flasks containing test substance are 
used to estimate the degree of ultimate biodegradation.
    (4) Prerequisites. The total organic carbon (TOC) content of the 
test substance shall be calculated or, if this is not possible, 
analyzed, to enable the percent of theoretical yield of carbon dioxide 
and percent of DOC loss to be calculated.
    (5) Guideline information. (i) Information on the relative 
proportions of the major components of the test substance will be useful 
in interpreting the results obtained, particularly in those cases where 
the result lies close to a ``pass level.''
    (ii) Information on the toxicity of the chemical may be useful in 
the interpretation of low results and in the selection of appropriate 
test concentrations.
    (6) Reference substances. Where investigating a chemical substance, 
reference compounds may be useful and an inventory of suitable reference 
compounds needs to be identified. In order to check the activity of the 
inoculum the use of a reference compound is desirable. Aniline, sodium 
citrate, dextrose, phthalic acid and trimellitic acid will exhibit 
ultimate biodegradation under the conditions of this Test Guideline 
method. These reference substances must yield 60 percent of theoretical 
maximum CO2 and show a removal of 70 percent DOC within 28

[[Page 93]]

days. Otherwise the test is regarded as invalid and shall be repeated 
using an inoculum from a different source.
    (7) Reproducibility. The reproducibility of the method has not yet 
been determined; however it is believed to be appropriate for a 
screening test which has solely an acceptance but no rejective function.
    (8) Sensitivity. The sensitivity of the method is determined by the 
ability to measure the endogenous CO2 production of the 
inoculum in the blank flask and by the sensitivity limit of the 
dissolved organic carbon analysis. If the test is adapted to handle 
\14\C-labeled test substances, test substance concentrations can be much 
lower.
    (9) Possibility of standardization. This possibility exists. The 
major difficulty is to standardize the inoculum in such a way that 
interlaboratory reproducibility is ensured.
    (10) Possibility of automation. None at present, although parts of 
the analyses may be automated.
    (b) Test procedures--(1) Preparations--(i) Apparatus. The shake 
flask apparatus under the following Figure 1 contains 10 mL of 0.2N 
Ba(OH)2 in an open container suspended over 1 liter of 
culture medium in a 2-liter Erlenmeyer flask.
[GRAPHIC] [TIFF OMITTED] TC01AP92.039

        Figure 1--Shake-Flask System for Carbon Dioxide Evolution

The Ba(OH)2 container is made by placing a constriction just 
above the 10 mL mark of a 50 mL heavy-duty centrifuge tube and attaching 
the centrifuge tube to a 2 mm I.D. x 9 mm O.D. glass tube by means of 3 
glass support rods. The centrifuge tube opening is large enough to 
permit CO2 to diffuse into the Ba(OH)2, while the 
constriction permits transferal of the flask to and from the shaker 
without Ba(OH)2 spillage into the medium. For periodic 
removal and addition of base from the center well, a polypropylene 
capillary tube, attached at one end to a 10 ml disposable syringe, is 
inserted through the 9 mm O.D. glass tube into the Ba(OH)2 
reservoir. The reservoir access port is easily sealed during incubation 
with a serum bottle stopper. Two glass tubes are added for sparging, 
venting, and medium sampling. The tops of these

[[Page 94]]

tubes are connected with a short section of flexible tubing during 
incubation.
    (ii) Reagents and stock solutions. (A) Stock solutions, I, II, and 
III under the following Table 1.
    (B) Yeast extract.
    (C) Vitamin-free casamino acids.
    (D) 70 percent O2 in nitrogen or CO2-free air.
    (E) 0.2N Ba(OH)2.
    (F) 0.1 N HCl.
    (G) 20 percent H2SO4.
    (H) Phenolphthalein.
    (I) Dilution water--distilled, deionized water (DIW).
    (iii) Soil inoculum. A fresh sample of an organically rich soil is 
used as the inoculum in the ultimate biodegradation test. Soil is 
collected, prepared, and stored according to the recommendations of 
Pramer and Bartha (1972) under paragraph (d)(2) of this section. The 
soil surface is cleared of litter and a soil sample is obtained 10 to 20 
cm below the surface. The sample is screened through a sieve with 2 to 5 
mm openings and stored in a polyethylene bag at 2 to 4 [deg]C for not 
more than 30 days prior to use. The soil is never allowed to air-dry, 
and shall not be frozen during storage.

           Table 1--Medium Employed for Assay of CO2 Evolution
------------------------------------------------------------------------
                                                                 Stock
                                                                Solution
       Solution \1\                      Compound              Conc. (g/
                                                                   L)
------------------------------------------------------------------------
I                           NH4Cl............................    35
                            KNO3.............................    15
                            K2HPO4[middot]3H2O...............   750
                            NaH2PO4[middot]H2O...............    25
II \2\                      KCl..............................    10
                            MgSO4............................    20
                            FeSO4[middot]7H2O................     1
III                         CaCl2............................     5
                            ZnCl2............................     0.05
                            MnCl2[middot]4H2O................     0.5
                            CuCl2............................     0.05
                            CoCl2............................     0.001
                            H3 BO3...........................     0.001
                            MoO3.............................     0.0004
------------------------------------------------------------------------
\1\= Each liter of test medium contains 1 mL of each solution.
\2\= Final pH is adjusted to 3.0 with 0.10 N HCl.

    (iv) Acclimation Medium. Acclimation medium is prepared by adding, 
for each liter of distilled, deionized water (DIW): 1 mL each of 
solutions I, II, and III in Table 1 in paragraph (b)(1)(iii) of this 
section, 1.0 gm of soil inoculum (prepared according to paragraph 
(b)(1)(iii) of this section), 2.0 mL of aerated mixed liquor (obtained 
from an activated sludge treatment plant not more than 2 days prior to 
commencing the acclimation phase, and stored in the interim at 4 [deg]C) 
and 50 mL raw domestic influent sewage. This medium is mixed for 15 
minutes and filtered through a glass wool plug in a glass funnel. The 
filtrate is permitted to stand for 1 hour, refiltered through glass 
wool, and supplemented with 25 mg/L each of Difco vitamin-free casamino 
acids and yeast extract. Appropriate volumes are added to 2-liter 
Erlenmeyer flasks. Test compounds are added incrementally during the 
acclimation period at concentrations equivalent to 4, 8, and 8 mg/L 
carbon on days 0, 7, and 11, respectively. On day 14, the medium is 
refiltered through glass wool prior to use in the test. For evaluating 
the biodegradability of a series of functionally or structurally related 
chemicals, media from all inoculum flasks may be combined before final 
filtration.
    (2) Procedures. (i) Inoculum (100 mL of acclimation medium) is added 
to 900 mL DIW containing 1 mL each of solutions I, II, and III in Table 
1 under paragraph (b)(1)(iii) of this section in a 2-liter Erlenmeyer 
flask. Test compound equivalent to 10 mg/liter carbon is added to each 
of the replicate flasks containing the test medium. Ten mL of 0.2 N Ba 
(OH)2 are added to the suspended reservoir in each flask and 
duplicate 10 mL samples of Ba(OH)2 are also saved as 
titration blanks for analysis with test samples. Flasks are sparged with 
CO2-free air (for volatile test materials, sparging is done 
prior to addition of the chemical), sealed, and placed on a gyrotary 
shaker (approximately 125 rpm) at 20 to 25 [deg]C in the dark. For each 
set of experiments, each test, reference, inhibited, and control system 
should be analyzed at time zero and at a minimum of four other times 
from time zero through day 28. Sampling must be made with sufficient 
frequency to allow for a smooth plot of biodegradation with time. 
Sampling times should be varied by the investigator as deemed 
appropriate to match the rate of degradation of the test substance. 
Tests may be terminated when biodegradation reaches a plateau and is

[[Page 95]]

consistent (10 percent) over 3 consecutive days or 
on day 28, whichever occurs first. For chemicals which are water soluble 
at the test concentration, an adequate volume (5 to 10 mL) of medium is 
removed for DOC analysis. Each sample for DOC analysis should be 
filtered through a membrane filter of 0.45 micrometer pore diameter 
before DOC analysis. For all test and reference compounds, 
Ba(OH)2 from the center well is removed for analysis. The 
center well is rinsed with 10 mL CO2-free DIW and is refilled 
with fresh base. Rinse water is combined with the Ba(OH)2 
sample to be analyzed. Flasks are resealed and placed on the shaker. On 
the day prior to terminating the test, 3 mL of 20 percent 
H2SO4 are added to the medium to release carbonate 
bound CO2.
    (ii) For each set of experiments, each test substance shall be 
tested in triplicate.
    (iii) For each set of experiments, one or two reference compounds 
are included to assess the microbial activity of the test medium. 
Duplicate reference flasks are prepared by adding reference compound 
equivalent to 10 mg/liter carbon to each of two flasks containing the 
test medium. Reference compounds which are positive for ultimate 
biodegradability include: sodium citrate, dextrose, phthalic acid, 
trimellitic acid, and aniline.
    (iv) For each test set, triplicate controls receiving inoculated 
medium and no test compound, plus all test and reference flasks, are 
analyzed for CO2 evolution and DOC removal. Results from 
analysis of the control flasks (DOC, CO2 evolution, etc.) are 
subtracted from corresponding experimental flasks containing test 
compound in order to arrive at the net effect due to the test compound.
    (v) A test system containing a growth inhibitor should be 
established as a control for each substance tested for biodegradation by 
this method. That inhibited system must contain the same amount of 
water, mineral nutrients, inoculum, and test substance used in the 
uninhibited test systems, plus 50 mg/L mercuric chloride 
(HgCl2) to inhibit microbial activity.
    (vi) Flasks shall be incubated in the dark to minimize both 
photochemical reactions and algal growth. Appropriate sterile controls 
or controls containing a metabolic inhibitor, such as 50 mg/1 
HgCl2, are needed to correct for interferences due to 
nonbiological degradation. With volatile organic materials, sparging 
with CO2-free air is performed only once, just prior to 
addition of the test chemical. Analyses for CO2 evolution and 
DOC removal are conducted within 2 to 3 hours of sampling to minimize 
interferences which may occur in storage. All glassware should be free 
of organic carbon contaminants.
    (3) Analytical measurements. The quantity of CO2 evolved 
is measured by titration of the entire Ba(OH)2 sample (10 mL 
Ba(OH)2+10 mL rinse water) with 0.1 N HCl to the 
phenolphthalein end point. Ba(OH)2 blanks are also 
supplemented with 10 mL CO2-free DIW and titrated in a 
similar manner. Samples (5 mL) for DOC are centrifuged and/or filtered 
and supernatant or filtrate analyzed by a suitable total organic carbon 
method.
    (c) Data and reporting--(1) Treatment of results. (i) Test compound 
(10 mg carbon) is theoretically converted to 0.833 mmol CO2. 
Absorbed CO2 precipitates as BaCO3 from 
Ba(OH)2, causing a reduction in alkalinity by the equivalent 
of 16.67 mL of 0.1 N HCl for complete conversion of the test compound 
carbon to CO2. Therefore, the percent theoretical 
CO2 evolved from the test compound is calculated at any 
sampling time from the formula:

Percent CO2 evolution=[(TF-CF)/16.67] 100 (for 10 mg/L test 
    compound carbon)

where:

TF= mL 0.1 N HCl required to titrate Ba(OH)2 samples from the 
test flask
CF= mL 0.1 N HCl required to titrate Ba(OH)2 samples from the 
control flask.

    (ii) The cumulative percent CO2 evolution at any sample 
time is calculated as the summation of the percent CO2 
evolved at all sample points of the test.
    (iii) The percent DOC disappearance from the test compound is 
calculated from the following equation:

Percent DOC Removal=[1-(DTFx- DCFx)/
    (DTFo- DCFo)] 100

where:


[[Page 96]]


DTF= Dissolved organic carbon from test flask
DCF= Dissolved organic carbon from control flask
o= Day zero measurements
x= Day of measurements during test.

    (iv) The difference between the amount of 0.1 N HCl used for the 
Ba(OH)2 titration blank samples and the Ba(OH)2 
samples from the control units (no test compound) is an indication of 
the activity of the microorganisms in the test system. In general, this 
difference is approximately 1 to 3 mL of 0.1 N HCl at each sampling 
time. A finding of no difference in the titration volumes between these 
two samples indicates a poor inoculum. In this case, the validity of the 
test results is questionable and the test set shall be rerun beginning 
with the acclimation phase.
    (v) CO2 evolution in the reference flasks is also 
indicative of the activity of the microbial test system. The suggested 
reference compounds should all yield final CO2 evolution 
values of at least 60 percent of theoretical CO2. If, for any 
test set, the percent theoretical CO2 evolution value for the 
reference flasks is outside this range, the test results are considered 
invalid and the test is rerun.
    (vi) Inhibition by the test compound is indicated by lower 
CO2 evolution in the test flasks than in the control flasks. 
If inhibition is noted, the study for this compound is rerun beginning 
with the acclimation phase. During the test phase for inhibitory 
compounds, the test chemical is added incrementally according to the 
schedule: Day 0--0.5 mg/liter as organic carbon, Day 2--1 mg/liter C, 
Day 4--1.5 mg/liter C, Day 7--2 mg/liter C, Day 10--5 mg/liter C. For 
this case, the Ba(OH)2 is sampled on Day 10, and weekly 
thereafter. The total test duration remains 28 days.
    (vii) The use of \14\C-labeled chemicals is not required. If 
appropriately labeled test substance is readily available and if the 
investigator chooses to use this procedure with labeled test substance, 
this is an acceptable alternative. If this option is chosen, the 
investigator may use lower test substance concentrations if those 
concentrations are more representative of environmental levels.
    (2) Test report. (i) For each test and reference compound, the 
following data shall be reported.
    (ii) Information on the inoculum, including source, collection date, 
handling, storage and adaptation possibilities (i.e., that the inoculum 
might have been exposed to the test substance either before or after 
collection and prior to use in the test).
    (iii) Results from each test, reference, inhibited (with 
HgCl2) and control system at each sampling time, including an 
average result for the triplicate test substance systems and the 
standard deviation for that average.
    (iv) Average cumulative percent theoretical CO2 evolution 
over the test duration.
    (v) Dissolved organic carbon due to test compound at each sampling 
time (DTF-DCF).
    (vi) Average percent DOC removal at each sampling time.
    (vii) Twenty-eight day standard deviation for percent CO2 
evolution and DOC removal.
    (d) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) Gledhill, W.E. ``Screening Test for Assessment of Ultimate 
Biodegradability: Linear Alkyl Benzene Sulfonate,'' Applied 
Microbiology, 30:922-929 (1975).
    (2) Pramer, D., Bartha, R. ``Preparation and Processing of Soil 
Samples for Biodegradation Testing,'' Environmental Letters, 2:217-224 
(1972).

[50 FR 39252, Sept. 27, 1985, as amended at 52 FR 19058, May 20, 1987]



Sec. 796.3500  Hydrolysis as a function of pH at 25 [deg]C.

    (a) Introduction--(1) Background and purpose. (i) Water is one of 
the most widely distributed substances in the environment. It covers a 
large portion of the earth's surface as oceans, rivers, and lakes. The 
soil also contains water, as does the atmosphere in the form of water 
vapor. As a result of this ubiquitousness, chemicals introduced into the 
environment almost always come into contact with aqueous media. Certain 
classes of these chemicals, upon such contact, can undergo hydrolysis,

[[Page 97]]

which is one of the most common reactions controlling chemical stability 
and is, therefore, one of the main chemical degradation paths of these 
substances in the environment.
    (ii) Since hydrolysis can be such an important degradation path for 
certain classes of chemicals, it is necessary, in assessing the fate of 
these chemicals in the environment, to know whether, at what rate, and 
under what conditions a substance will hydrolyze. Some of these 
reactions can occur so rapidly that there may be greater concern about 
the products of the transformation than about the parent compounds. In 
other cases, a substance will be resistant to hydrolysis under typical 
environmental conditions, while, in still other instances, the substance 
may have an intermediate stability that can result in the necessity for 
an assessment of both the original compound and its transformation 
products. The importance of transformation of chemicals via hydrolysis 
in aqueous media in the environment can be determined quantitatively 
from data on hydrolysis rate constants. This hydrolysis Test Guideline 
represents a test to allow one to determine rates of hydrolysis at any 
pH of environmental concern at 25[deg]C.
    (2) Definitions and units. (i) ``Hydrolysis'' is defined as the 
reaction of an organic chemical with water, such that one or more bonds 
are broken and the reaction products of the transformation incorporate 
the elements of water (H2O).
    (ii) ``Elimination'' is defined in this Test Guideline to be a 
reaction of an organic chemical (RX) in water in which the X group is 
lost. These reactions generally follow the same type of rate laws that 
hydrolysis reactions follow and, thus, are also covered in this Test 
Guideline.
    (iii) A ``first-order reaction'' is defined as a reaction in which 
the rate of disappearance of the chemical substance being tested is 
directly proportional to the concentration of the chemical substance and 
is not a function of the concentrations of any other substances present 
in the reaction mixture.
    (iv) The ``half-life'' of a chemical is defined as the time required 
for the concentration of the chemical substance being tested to be 
reduced to one-half its initial value.
    (v) ``Hydrolysis'' refers to a reaction of an organic chemical with 
water such that one or more bonds are broken and the reaction products 
incorporate the elements of water (H2O). This type of 
transformation often results in the net exchange of a group X, on an 
organic chemical RX, for the OH group from water. This can be written 
as:

RX+HOH[rarr]ROH+HX.

    (A) Another result of hydrolysis can be the incorporation of both H 
and OH in a single product. An example of this is the hydrolysis of 
epoxides, which can be represented by


    (B) The hydrolysis reaction can be catalyzed by acidic or basic 
species, including OH- and H3O= 
(H=). The promotion of the reaction by 
H3O- or OH- is called specific acid or 
specific base catalysis, respectively, as contrasted with general acid 
or base catalysis encountered with other cationic or anionic species. 
Usually, the rate law for chemical RX can be written as:

                               Equation 1

-d[RX]/d= = kh[RX]=kA[H=] 
    [RX]

+kB[OH-] [RX]+k'N [H2O] 
    [RX],


where KA, kB and k'N are the second-
order rate constants for acid and base catalyzed and neutral water 
processes, respectively. In dilute solutions, such as are encountered in 
following this Test Guideline, water is present in great excess and its 
concentration is, thus, essentially constant during the course of the 
hydrolysis reaction. At fixed pH, the reaction, therefore, becomes 
pseudo first-order, and the rate constant (kh) can be written 
as:

                               Equation 2

kh=kA [H=]+kB 
    [OH-]+kN,


where kN is the first-order neutral water rate constant. 
Since this is a

[[Page 98]]

pseudo first-order process, the half-life is independent of the 
concentration and can be written as:

                               Equation 3

t1/2=0.693/kh.


At constant pH, Equation 1 can be integrated to yield the first order 
rate expression

                               Equation 4

log10C=- (kh t/
    2.303)+log10Co,


where C is the concentration of the test chemical at time t and 
Co is the initial chemical concentration (t=0).
    (C) At a given pH, Equation 2 under paragraph (a)(2)(v)(B) of this 
section contains three unknowns, kA, kB, and 
kN. Therefore, three equations (i.e., measurements at three 
different pH's at a fixed temperature) are required if one wishes to 
solve for these quantities. Making suitable approximations for 
quantities that are negligible, the expressions for kA, 
kB, and kN using values of kh measured 
at pH 3, 7, and 11 are:

                               Equation 5

kA=10\3\ [kh (3)-kh (7)+10-4 
    kh (11)]

kB=10\3\ [kh (11)-kh 
    (7)+10-4 kh (3)]

kN=kh (7)-10-4 [kh 
    (3)+kh (11)]


The calculated rate constants from equation 5 under this paragraph can 
be employed in equation 2 under paragraph (a)(2)(v)(B) of this section 
to calculate the hydrolysis rate of a chemical at any pH of 
environmental concern.
    (D) The equations under paragraph (a)(2) of this section apply 
whether the test chemical has one or more hydrolyzable groups. In the 
latter case, the rate may be written as:

                               Equation 6

-d[RX]/dt=[RX]=k2 [RX]+ . . . . +kn

[RX]=(k1+k2+ . . . . . kn) 
    [RX]=kh [RX].


Equation 6 applies to the hydrolysis rate of a molecule having n 
hydrolyzable groups, each of which follows first-order reaction 
kinetics. The measured kh is now the sum of the individual 
reaction rates and is the only rate constant required in this section.
    (3) Principle of the test method. Procedures described in this 
section enable sponsors to obtain quantitative information on hydrolysis 
rates through a determination of hydrolysis rate constants and half-
lives of chemicals at pH 3.00, 7.00, and 11.00 at 25 [deg]C. The three 
measured rate constants are used to determine the acidic, basic, and 
neutral rate constants associated with a hydrolytic reaction. The latter 
constants can then be employed in determining the hydrolysis rates of 
chemicals at any pH of environmental concern at 25 [deg]C.
    (4) Applicability and specificity. There are several different 
common classes of organic chemicals that are subject to hydrolysis 
transformation, including esters, amides, lactones, carbamates, 
organophosphates, and alkyl halides. Processes other than nucleophilic 
displacement by water can also take place. Among these are elimination 
reactions that exhibit behavior similar to hydrolysis and, therefore, 
are also covered in this section.
    (b) Test procedures--(1) Test conditions--(i) Special laboratory 
equipment. (A) A thermostatic bath that can be maintained at a 
temperature of 25 1 [deg]C.
    (B) A pH meter that can resolve differences of 0.05 pH units or 
less.
    (C) Stoppered volumetric flasks (no grease) or glass ampoules that 
can be sealed.
    (ii) Purity of water. Reagent-grade water (e.g., water meeting ASTM 
Type IIA standards or an equivalent grade) shall be used to minimize 
biodegradation. ASTM Type IIA water is described in ASTM D 1193-77 
(Reapproved 1983), ``Standard Specification for Reagent Water.'' ASTM D 
1193-77 (Reapproved 1983) is available for inspection at the National 
Archives and Records Administration (NARA). For information on the 
availability of this material at NARA, call 202-741-6030, or go to: 
http://www.archives.gov/federal--register/code--of--federal--
regulations/ibr--locations.html. This incorporation by reference was 
approved by the Director of the Office of the Federal Register. This 
material is incorporated as it exists on the date of approval and a 
notice of any change in this material

[[Page 99]]

will be published in the Federal Register. Copies of the incorporated 
material may be obtained from the Non-Confidential Information Center 
(NCIC) (7407), Office of Pollution Prevention and Toxics, U.S. 
Environmental Protection Agency, Room B-607 NEM, 401 M St., SW., 
Washington, DC 20460, between the hours of 12 p.m. and 4 p.m. weekdays 
excluding legal holidays, or from the American Society for Testing and 
Materials (ASTM), 1916 Race Street, Philadelphia, PA 19103.
    (iii) Sterilization. All glassware shall be sterilized. Aseptic 
conditions shall be used in the preparation of all solutions and in 
carrying out all hydrolysis experiments to eliminate or minimize 
biodegradation. Glassware can be sterilized in an autoclave or by any 
other suitable method.
    (iv) Precautions for volatility. If the chemical is volatile the 
reaction vessels shall be almost completely filled and sealed.
    (v) Temperature controls. All hydrolysis reactions shall be carried 
out at 25 [deg]C (1 [deg]C) and with the 
temperature controlled to 0.1 [deg]C.
    (vi) pH conditions. It is recommended that all hydrolysis 
experiments be performed at pH 3.00, 7.00, and 11.00 0.05 using the appropriate buffers described in 
paragraph (b)(2)(i)(A) of this section.
    (vii) Concentration of solutions of chemical substances. The 
concentration of the test chemical shall be less than one-half the 
chemical's solubility in water but not greater than 10-3 M.
    (viii) Effect of acidic and basic groups. Complications can arise 
upon measuring the rate of hydrolysis of chemicals that reversibly 
ionize or are protonated in the pH range 3.00 to 11.00. Therefore, for 
these chemicals, it is recommended that these hydrolysis tests be 
performed at pH 5.00, 7.00, and 900 0.05 using the 
appropriate buffers described in paragraphs (b)(2)(i) (A) and (B) of 
this section. If a test chemical reversibly ionizes or protonates in the 
pH range 5.00 to 9.00, then it is recommended that additional hydrolysis 
tests should be carried out at pH 6.00 and 8.00 0.05 using the buffers described in paragraph 
(b)(2)(i)(B) of this section.
    (ix) Buffer catalysis. For certain chemicals, buffers may catalyze 
the hydrolysis reaction. If this is suspected, hydrolysis rate 
determination shall be carried out with the appropriate buffers and the 
same experiments repeated at buffer concentrations lowered by at least a 
factor of five. If the hydrolysis reaction produces a change of greater 
than 0.05 pH units in the lower concentration buffers at the end of the 
measurement time, the test chemical concentrations also shall be lowered 
by at least a factor of five. Alternatively, test chemical 
concentrations and buffer concentrations may both be lowered 
simultaneously by a factor of five. A sufficient criterion for 
minimization of buffer catalysis is an observed equality in the 
hydrolysis rate constant for two different solutions differing in buffer 
or test chemical concentration by a factor of five.
    (x) Photosensitive chemicals. The solution absorption spectrum can 
be employed to determine whether a particular chemical is potentially 
subject to photolytic transformation upon exposure to light. For 
chemicals that absorb light of wavelengths greater than 290 nm, the 
hydrolysis experiment shall be carried out in the dark, under amber or 
red safelights, in amber or red glassware, or employing other suitable 
methods for preventing photolysis. The absorption spectrum of the 
chemical in aqueous solution can be measured under Sec. 796.1050.
    (xi) Chemical analysis of solutions. In determining the 
concentrations of the test chemicals in solution, any suitable 
analytical method may be employed, although methods which are specific 
for the compound to be tested are preferred. Chromatographic methods are 
recommended because of their compound specificity in analyzing the 
parent chemical without interferences from impurities. Whenever 
practicable, the chosen analytical method should have a precision within 
5 percent.
    (2) Preparation--(i) Reagents and solutions--(A) Buffer solutions. 
Prepare buffer solutions using reagent-grade chemicals and reagent-grade 
water as follows:
    (1) pH 3.00: use 250 mL of 0.100M potassium hydrogen phthalate; 111 
mL of 0.100M hydrochloric acid; and adjust

[[Page 100]]

volume to 500 mL with reagent-grade water.
    (2) pH 7.00: use 250 mL of 0.100M potassium dihydrogen phosphate; 
145 mL of 0.100M sodium hydroxide; and adjust volume to 500 mL with 
reagent-grade water.
    (3) pH 11.00: use 250 mL of 0.0500M sodium bicarbonate; 113 mL of 
0.100M sodium hydroxide; and adjust volume to 500 mL with reagent-grade 
water.
    (B) Additional buffer solutions. For chemicals that ionize or are 
protonated as discussed in paragraph (b)(1)(viii) of this section, 
prepare buffers using reagent-grade water and reagent-grade chemicals as 
follows:
    (1) pH 5.00: use 250 mL of 0.100M potassium hydrogen phthalate; 113 
mL of 0.100M sodium hydroxide; and adjust volume to 500 mL with reagent-
grade water.
    (2) pH 6.00: use 250 mL of 0.100M potassium dihydrogen phosphate; 28 
mL of 0.100M sodium hydroxide; and adjust volume to 500 mL with reagent-
grade water.
    (3) pH 8.00: use 250 mL of 0.100M potassium dihydrogen phosphate; 
234 mL of 0.100M sodium hydroxide; and adjust volume to 500 mL with 
reagent-grade water.
    (4) pH 9.00: use 250 mL of 0.0250M borax (Na2 
B4O7); 23 mL of 0.100M hydrochloric aid; and 
adjust volume to 500 mL with reagent-grade water.
    (C) Adjustment of buffer concentrations. (1) The concentrations of 
all the above buffer solutions are the maximum concentration to be 
employed in carrying out hydrolysis measurements. If the initial 
concentration of the test chemical is less than 10-3 M, the 
buffer concentration shall be lowered by a corresponding amount; e.g., 
if the initial test chemical concentration is 10-4 M, the 
concentration of the above buffers shall be reduced by a factor of 10. 
In addition, for those reactions in which an acid or base is not a 
reaction product, the minimum buffer concentration necessary for 
maintaining the pH within +0.05 units shall be employed.
    (2) Check the pH of all buffer solutions with a pH meter at 25 
[deg]C and adjust the pH to the proper value, if necessary.
    (D) Preparation of test solution. (1) If the test chemical is 
readily soluble in water, prepare an aqueous solution of the chemical in 
the appropriate buffer and determine the concentration of the chemical. 
Alternatively, a solution of the chemical in water may be prepared and 
added to an appropriate buffer solution and the concentration of the 
chemical then determined. In the latter case, the aliquot shall be small 
enough so that the concentration of the buffer in the final solution and 
the pH of the solution remain essentially unchanged. Do not employ heat 
in dissolving the chemical. The final concentration shall not be greater 
than one-half the chemical's solubility in water and not greater than 
10-3 M.
    (2) If the test chemical is too insoluble in pure water to permit 
reasonable handling and analytical procedures, it is recommended that 
the chemical be dissolved in reagent-grade acetonitrile and buffer 
solution and then added to an aliquot of the acetonitrile solution. Do 
not employ heat to dissolve the chemical in acetonitrile. The final 
concentration of the test chemical shall not be greater than one-half 
the chemical's solubility in water and not greater than 10-3 
M. In addition, the final concentration of the acetonitrile shall be one 
volume percent or less.
    (3) Performance of the test. Carry out all hydrolysis experiments by 
employing one of the procedures described in this paragraph. Prepare the 
test solutions as described in paragraph (b)(2)(i) of this section at pH 
3.00, 7.00, and 11.00 0.05, and determine the 
initial test chemical concentration (Co) in triplicate. 
Analyze each reaction mixture in triplicate at regular intervals, 
employing one of the following procedures:
    (i) Procedure 1. Analyze each test solution at regular intervals to 
provide a minimum of six measurements with the extent of hydrolysis 
between 20 to 70 percent. Rates should be rapid enough so that 60 to 70 
percent of the chemical is hydrolyzed in 672 hours.
    (ii) Procedure 2. If the reaction is too slow to conveniently follow 
hydrolysis to high conversion in 672 hours but still rapid enough to 
attain at least 20 percent conversion, take 15 to 20 time points at 
regular intervals after 10 percent conversion is attained.

[[Page 101]]

    (iii) Procedure 3. (A) If chemical hydrolysis is less than 20 
percent after 672 hours, determine the concentration (C) after this time 
period.
    (B) If the pH at the end of concentration measurements employing any 
of the above three procedures has changed by more than 0.05 units from 
the initial pH, repeat the experiment using a solution having a test 
chemical concentration lowered sufficiently to keep the pH variation 
within 0.05 pH units.
    (iv) Analytical methodology. Select an analytical method that is 
most applicable to the analysis of the specific chemical being tested 
under paragraph (b)(1)(xi) of this section.
    (c) Data and reporting--(1) Treatment of results. (i) If Procedure 1 
or 2 were employed in making concentration measurements, use a linear 
regression analysis with Equation 4 under paragraph (a)(2)(v)(B) of this 
section to calculate kh at 25 [deg]C for each pH employed in 
the hydrolysis experiments. Calculate the coefficient of determination 
(R\2\) for each rate constant. Use Equation 3 under paragraph 
(a)(2)(v)(B) of this section to calculate the hydrolysis half-life using 
kh.
    (ii) If Procedure 3 was employed in making rate measurements, use 
the mean initial concentration (Co) and the mean 
concentration of chemical (C) in Equation 4 under paragraph (a)(2)(v)(B) 
of this section to calculate kh for each pH used in the 
experiments. Calculate the hydrolysis half-life using kh in 
Equation 3 under paragraph (a)(2)(v)(B) of this section.
    (iii) For each set of three concentration replicates, calculate the 
mean value of C and the standard deviation.
    (iv) For test chemicals that are not ionized or protonated between 
pH 3 and 11, calculate kA, kB, and kN 
using Equation 5.
    (2) Specific analytical and recovery procedures. (i) Provide a 
detailed description or reference for the analytical procedure used, 
including the calibration data and precision.
    (ii) If extraction methods were used to separate the solute from the 
aqueous solution, provide a description of the extraction method as well 
as the recovery data.
    (3) Test data report. (i) For Procedures 1 and 2, report 
kh, the hydrolysis half-life (t1/2), and the 
coefficient of determination (R\2\) for each pH employed in the rate 
measurements. In addition, report the individual values, the mean value, 
and the standard deviation for each set of replicate concentration 
measurements. Finally, report kA, kB, and 
kN.
    (ii) For Procedure 3, report kh and the half-life for 
each pH employed in the rate measurements. In addition, report the 
individual values, the mean value, and the standard deviation for each 
set of replicate concentration measurements. Finally, report 
kA, kB, and kN.
    (iii) If, after 672 hours, the concentration (C) is the same as the 
initial concentration (Co) within experimental error, then 
kh cannot be calculated and the chemical can be reported as 
being persistent with respect to hydrolysis.

[50 FR 39252, Sept. 27, 1985, as amended at 53 FR 10391, Mar. 31, 1988; 
53 FR 12526, Apr. 15, 1988; 53 FR 22323, June 15, 1988; 60 FR 34467, 
July 3, 1995; 69 FR 18803, Apr. 9, 2004]



PART 797_ENVIRONMENTAL EFFECTS TESTING GUIDELINES--Table of Contents




Subpart A [Reserved]

                      Subpart B_Aquatic Guidelines

Sec.
797.1050 Algal acute toxicity test.
797.1300 Daphnid acute toxicity test.
797.1330 Daphnid chronic toxicity test.
797.1400 Fish acute toxicity test.
797.1600 Fish early life stage toxicity test.
797.1930 Mysid shrimp acute toxicity test.
797.1950 Mysid shrimp chronic toxicity test.

    Authority: 15 U.S.C. 2603.

    Source: 50 FR 39321, Sept. 27, 1985, unless otherwise noted.

Subpart A [Reserved]



                      Subpart B_Aquatic Guidelines



Sec. 797.1050  Algal acute toxicity test.

    (a) Purpose. The guideline in this section is intended for use in 
developing data on the acute toxicity of chemical substances and 
mixtures (``chemicals'') subject to environmental effects test 
regulations under the Toxic Substances Control Act (TSCA) (Pub. L. 94-
469, 90 Stat. 2003, 15 U.S.C. 2601 et seq.). This

[[Page 102]]

guideline prescribes test procedures and conditions using freshwater and 
marine algae to develop data on the phytotoxicity of chemicals. The 
United States Environmental Protection Agency (U.S. EPA) will use data 
from these tests in assessing the hazard of a chemical to the 
environment.
    (b) Definitions. The definitions in section 3 of the Toxic 
Substances Control Act (TSCA) and the definitions in part 792--Good 
Laboratory Practice Standards of this chapter apply to this test 
guideline. The following definitions also apply to this guideline:
    (1) Algicidal means having the property of killing algae.
    (2) Algistatic means having the property of inhibiting algal growth.
    (3) ECx means the experimentally derived chemical concentration that 
is calculated to effect X percent of the test criterion.
    (4) Growth means a relative measure of the viability of an algal 
population based on the number and/or weight of algal cells per volume 
of nutrient medium or test solution in a specified period of time.
    (5) Static system means a test container in which the test solution 
is not renewed during the period of the test.
    (c) Test procedures--(1) Summary of the test. (i) In preparation for 
the test, fill test containers with appropriate volumes of nutrient 
medium and/or test solution. Start the test by introducing algae into 
the test and control containers in the growth chambers. Environmental 
conditions within the growth chambers are established at predetermined 
limits.
    (ii) At the end of 96 hours enumerate the algal cells in all 
containers to determine inhibition or stimulation of growth in test 
containers compared to controls. Use data to define the concentration-
response curve, and calculate the EC10, EC50, and 
EC90 values.
    (2) [Reserved]
    (3) Range-finding test. (i) A range-finding test should be conducted 
to determine:
    (A) If definitive testing is necessary.
    (B) Test chemical concentrations for the definitive test.
    (ii) Algae are exposed to a widely spaced (e.g., log interval) 
chemical concentration series. The lowest value in the series, exclusive 
of controls, should be at the chemical's detection limit. The upper 
value, for water soluble compounds, should be the saturation 
concentration. No replicates are required; and nominal concentrations of 
the chemical are acceptable unless definitive testing is not required.
    (iii) The test is performed once for each of the recommended algal 
species or selected alternates. Test chambers should contain equal 
volumes of test solution and approximately 1x10\4\ Selenastrum cells/ml 
or 7.7x10\4\ Skeletonema cells/ml of test solution. The algae should be 
exposed to each concentration of test chemical for up to 96 hours. The 
exposure period may be shortened if data suitable for the purposes of 
the range-finding test can be obtained in less time.
    (iv) Definitive testing is not necessary if the highest chemical 
concentration tested (water saturation concentration or 1000 mg/l) 
results in less than a 50 percent reduction in growth or if the lowest 
concentration tested (analytical detection limit) results in greater 
than a 50 percent reduction in growth.
    (4) Definitive test. (i) The purpose of the definitive test is to 
determine the concentration response curves, the EC10's, 
EC50's, and EC90's for algal growth for each 
species tested, with a minimum amount of testing beyond the range-
finding test.
    (ii) Algae should be exposed to five or more concentrations of the 
test chemical in a geometric series in which the ratio is between 1.5 
and 2.0 (e.g., 2, 4, 8, 16, 32, and 64 mg/l). Algae shall be placed in a 
minimum of three replicate test containers for each concentration of 
test chemical and control. More than three replicates may be required to 
provide sufficient quantities of test solution for determination of test 
substance concentration at the end of the test. Each test chamber should 
contain equal volumes of test solution and approximately 1x10\4\ 
Selenastrum cells/ml or 7.7x10\4\ Skeletonema cells/ml of test solution. 
The chemical concentrations should result in greater than 90 percent of 
algal growth being inhibited or stimulated at the highest concentrations 
of test substance compared to controls.

[[Page 103]]

    (iii) Every test shall include a control consisting of the same 
nutrient medium, conditions, procedures, and algae from the same 
culture, except that none of the test substance is added. If a carrier 
is present in any of the test chambers, a separate carrier control is 
required.
    (iv) The test begins when algae from 5- to 10-day-old stock cultures 
are placed in the test chambers containing test solutions having the 
appropriate concentrations of the test substance. Algal growth in 
controls should reach the logarithmic growth phase by 96 hours. If 
logarithmic growth cannot be demonstrated, the test shall be repeated. 
At the end of 24, 48, 72, and 96 hours the algal growth response (number 
or weight of algal cells/ml) in all test containers and controls shall 
be determined by an indirect (spectrophotometry, electronic cell 
counters, dry weight, etc.) or a direct (actual microscopic cell count) 
method. Indirect methods shall be calibrated by a direct microscopic 
count. The percentage inhibition or stimulation of growth for each 
concentration, EC10, EC50, EC90 and the 
concentration-response curves are determined from these counts.
    (v) At the end of the definitive test, the following additional 
analyses of algal growth response shall be performed:
    (A) Determine whether the altered growth response between controls 
and test algae was due to a change in relative cell numbers, cell sizes 
or both. Also note any unusual cell shapes, color differences, 
flocculations, adherence of algae to test containers, or aggregation of 
algal cells.
    (B) In test concentrations where growth is maximally inhibited, 
algistatic effects may be differentiated from algicidal effects by the 
following two methods for Skeletonema and by the second method for 
Selenastrum.
    (1) Add 0.5 ml of a 0.1 percent solution (weight/volume) of Evans 
blue stain to a 1 milliliter aliquot of algae from a control container 
and to a 1 milliliter aliquot of algae from the test container having 
the lowest concentration of test chemical which completely inhibited 
algal growth (if algal growth was not completely inhibited, select an 
aliquot of algae for staining from the test container having the highest 
concentration of test chemical which inhibited algal growth). Wait 10 to 
30 minutes, examine microscopically, and determine the percent of the 
cells which stain blue (indicating cell mortality). A staining control 
shall be performed concurrently using heat-killed or formaldehyde-
preserved algal cells; 100 percent of these cells shall stain blue.
    (2) Remove 0.5 ml aliquots of test solution containing growth-
inhibited algae from each replicate test container having the 
concentration of test substance evaluated in paragraph (c)(4)(v)(B)(1) 
of this section. Combine these aliquots into a new test container and 
add a sufficient volume of fresh nutrient medium to dilute the test 
chemical to a concentration which does not affect growth. Incubate this 
subculture under the environmental conditions used in the definitive 
test for a period of up to 9 days, and observe for algal growth to 
determine if the algistatic effect noted after the 96-hour test is 
reversible. This subculture test may be discontinued as soon as growth 
occurs.
    (5) [Reserved]
    (6) Analytical measurements--(i) Chemical. (A) Glass distilled or 
deionized water shall be used in the preparation of the nutrient medium. 
The pH of the test solution shall be measured in the control and test 
containers at the beginning and at the end of the definitive test. The 
concentration of test chemical in the test containers shall be 
determined at the beginning and end of the definitive test by standard 
analytical methods which have been validated prior to the test. An 
analytical method is unacceptable if likely degradation products of the 
chemical, such as hydrolysis and oxidation products, give positive or 
negative interference.
    (B) At the end of the test and after aliquots have been removed for 
algal growth-response determinations, microscopic examination, mortal 
staining, or subculturing, the replicate test containers for each 
chemical concentration may be pooled into one sample. An aliquot of the 
pooled sample may then be taken and the concentration of test chemical 
determined. In

[[Page 104]]

addition, the concentration of test chemical associated with the algae 
alone should be determined. Separate and concentrate the algal cells 
from the test solution by centrifuging or filtering the remaining pooled 
sample and measure the test substance concentration in the algal-cell 
concentrate.
    (ii) Numerical. Algal growth response (as percent of inhibition or 
stimulation in the test solutions compared to the controls) is 
calculated at the end of the test. Mean and standard deviation should be 
calculated and plotted for each treatment and control. Appropriate 
statistical analyses should provide a goodness-of-fit determination for 
the concentration response curves. The concentration response curves are 
plotted using the mean measured test solution concentrations obtained at 
the end of the test.
    (d) Test conditions--(1) Test species. Species of algae recommended 
as test organisms for this test are the freshwater green alga, 
Selenastrum capricornutum, and the marine diatom, Skeletonema costatum. 
Algae to be used in acute toxicity tests may be initially obtained from 
commercial sources and subsequently cultured using sterile technique. 
Toxicity testing shall not be performed until algal cultures are shown 
to be actively growing (i.e., capable of logarithmic growth within the 
test period) in at least 2 subcultures lasting 7 days each prior to the 
start of the definitive test. All algae used for a particular test shall 
be from the same source and the same stock culture. Test algae shall not 
have been used in a previous test, either in a treatment or a control.
    (2) Facilities--(i) General. (A) Facilities needed to perform this 
test include: a growth chamber or a controlled environment room that can 
hold the test containers and will maintain the air temperature, lighting 
intensity and photoperiod specified in this test guideline; apparatus 
for culturing and enumerating algae; a source of distilled and/or 
deionized water; and apparatus for carrying out analyses of the test 
chemical.
    (B) Disposal facilities should be adequate to accommodate spent 
glassware, algae and test solutions at the end of the test and any bench 
covering, lab clothing, or other contaminated materials.
    (ii) Test containers. Erlenmeyer flasks should be used for test 
containers. The flasks may be of any volume between 125 and 500 ml as 
long as the same size is used throughout a test and the test solution 
volume does not exceed 50 percent of the flask volume.
    (iii) Cleaning and sterilization. New test containers may contain 
substances which inhibit growth of algae. They shall therefore be 
cleaned thoroughly and used several times to culture algae before being 
used in toxicity testing. All glassware used in algal culturing or 
testing shall be cleaned and sterilized prior to use according to 
standard good laboratory practices.
    (iv) Conditioning. Test containers should be conditioned by a rinse 
with the appropriate test solutions prior to the start of the test. 
Decant and add fresh test solutions after an appropriate conditioning 
period for the test chemical.
    (v) Nutrient medium. (A) Formulation and sterilization of nutrient 
medium used for algal culture and preparation of test solutions should 
conform to those currently recommended by the U.S. EPA for freshwater 
and marine algal bioassays. No chelating agents are to be included in 
the nutrient medium used for test solution preparation. Nutrient medium 
should be freshly prepared for algal testing and may be dispensed in 
appropriate volumes in test containers and sterilized by autoclaving or 
filtration. The pH of the nutrient medium shall be 7.5 (0.1) for Selenastrum and 8.1 (0.1) 
for Skeletonema at the start of the test and may be adjusted prior to 
test chemical addition with 0.1N NaOH or HC1.
    (B) Dilution water used for preparation of nutrient medium and test 
solutions should be filtered, deionized or glass distilled. Saltwater 
for marine algal nutrient medium and test solutions should be prepared 
by adding a commercial, synthetic, sea salt formulation or a modified 
synthetic seawater formulation to distilled/deionized water to a 
concentration of 30 parts per thousand.
    (vi) Carriers. Nutrient medium shall be used in making stock 
solutions of

[[Page 105]]

the test chemical. If a carrier other than nutrient medium is absolutely 
necessary to dissolve the chemical, the volume used shall not exceed the 
minimum volume necessary to dissolve or suspend the chemical in the test 
solution.
    (3) Test parameters. (i) The test temperature shall be 24 [deg]C for 
Selenastrum and 20 [deg]C for Skeletonema. Excursions from the test 
temperature shall be no greater than 2 [deg]C. 
Temperature should be recorded hourly during the test.
    (ii) Test chambers containing Selenastrum shall be illuminated 
continuously and those containing Skeletonema shall be provided a 14-
hour light and 10-hour dark photoperiod with a 30 minute transition 
period under fluorescent lamps providing 300 25 
uEin/m\2\ sec (approximately 400 ft-c) measured adjacent to the test 
chambers at the level of test solution.
    (iii) Stock algal cultures should be shaken twice daily by hand. 
Test containers shall be placed on a rotary shaking apparatus and 
oscillated at approximately 100 cycles/minute for Selenastrum and at 
approximately 60 cycles/minute for Skeletonema during the test. The rate 
of oscillation should be determined at least once daily during testing.
    (iv) The pH of nutrient medium in which algae are subcultured shall 
be 7.5 (0.1) for Selenastrum and 8.1 (0.1) for Skeletonema, and is not adjusted after the 
addition of the algae. The pH of all test solutions shall be measured at 
the beginning and end of the test.
    (v) Light intensity shall be monitored at least daily during the 
test at the level of the test solution.
    (e) Reporting. The sponsor shall submit to the EPA all data 
developed by the test that are suggestive or predictive of acute 
phytotoxicity. In addition to the general reporting requirements 
prescribed in part 792--Good Laboratory Practice Standards of this 
Chapter, the following shall be reported:
    (1) Detailed information about the test organisms, including the 
scientific name, method of verification, and source.
    (2) A description of the test chambers and containers, the volumes 
of solution in the containers, the way the test was begun (e.g., 
conditioning, test substance additions, etc.), the number of replicates, 
the temperature, the lighting, and method of incubation, oscillation 
rates, and type of apparatus.
    (3) The concentration of the test chemical in the control and in 
each treatment at the end of the test and the pH of the solutions.
    (4) The number of algal cells per milliliter in each treatment and 
control and the method used to derive these values at the beginning, 24, 
48, and 72 hours, and end of the test; the percentage of inhibition or 
stimulation of growth relative to controls; and other adverse effect in 
the control and in each treatment.
    (5) The 96-hour EC10, EC50, and 
EC90 values, and when sufficient data have been generated, 
the 24, 48, and 72 hour LC50's and 95 percent confidence 
limits, the methods used to derive these values, the data used to define 
the shape of the concentration-response curve and the goodness-of-fit 
determination.
    (6) Methods and data records of all chemical analyses of water 
quality and test substance concentrations, including method validations 
and reagent blanks.
    (7) The results of any optional analyses such as: Microscopic 
appearance of algae, size or color changes, percent mortality of cells 
and the fate of subcultured cells, the concentration of test substance 
associated with algae and test solution supernate or filtrate.
    (8) If the range-finding test showed that the highest concentration 
of the chemical tested (not less than 1000 mg/l or saturation 
concentration) had no effect on the algae, report the results and 
concentration and a statement that the chemical is of minimum phytotoxic 
concern.
    (9) If the range-finding test showed greater than a 50 percent 
inhibition of algal growth at a test concentration below the analytical 
detection limit, report the results, concentration, and a statement that 
the chemical is phytotoxic below the analytical detection limit.

[50 FR 39321, Sept. 27, 1985, as amended at 52 FR 19058, May 20, 1987]



Sec. 797.1300  Daphnid acute toxicity test.

    (a) Purpose. This guideline is intended for use in developing data 
on

[[Page 106]]

the acute toxicity of chemical substances and mixtures (``chemicals'') 
subject to environmental effects test regulations under the Toxic 
Substances Control Act (TSCA) (Pub. L. 94-469, 90 Stat. 2003, 15 U.S.C. 
2601 et seq.). This guideline prescribes an acute toxicity test in which 
daphnids (Daphnia magna or D. pulex) are exposed to a chemical in static 
and flow-through systems. The United States Environmental Protection 
Agency will use data from this test in assessing the hazard a chemical 
may present in the aquatic environment.
    (b) Definitions. The definitions in section 3 of the Toxic 
Substances Control Act (TSCA) and part 792--Good Laboratory Practice 
Standards of this chapter apply to this test guideline. In addition, the 
following definitions apply to this guideline:
    (1) Brood stock means the animals which are cultured to produce test 
organisms through reproduction.
    (2) EC50 means that experimentally derived concentration 
of test substance in dilution water that is calculated to affect 50 
percent of a test population during continuous exposure over a specified 
period of time. In this guideline, the effect measured is 
immobilization.
    (3) Ephippium means a resting egg which develops under the carapace 
in response to stress conditions in daphnids.
    (4) Flow-through means a continuous or an intermittent passage of 
test solution or dilution water through a test chamber or culture tank 
with no recycling.
    (5) Immobilization means the lack of movement by the test organisms 
except for minor activity of the appendages.
    (6) Loading means the ratio of daphnid biomass (grams, wet weight) 
to the volume (liters) of test solution in a test chamber at a point in 
time, or passing through the test chamber during a specific interval.
    (7) Static system means a test system in which the test solution and 
test organisms are placed in the test chamber and kept there for the 
duration of the test without renewal of the test solution.
    (c) Test procedures--(1) Summary of the test. (i) Test chambers are 
filled with appropriate volumes of dilution water. In the flow-through 
test, the flow of dilution water through each chamber is adjusted to the 
rate desired. The test chemical is introduced into each treatment 
chamber. The addition of test chemical in the flow-through system is 
conducted at a rate which is sufficient to establish and maintain the 
desired concentration in the test chamber. The test is started within 30 
minutes after the test chemical has been added and uniformly distributed 
in static test chambers or after the concentration of test chemical in 
each flow-through test chamber reaches the prescribed level and remains 
stable. At the initiation of the test, daphnids which have been cultured 
and acclimated in accordance with the test design are randomly placed 
into the test chambers. Daphnids in the test chambers are observed 
periodically during the test, the immobile daphnids removed, and the 
findings recorded.
    (ii) Dissolved oxygen concentration, pH, temperature, the 
concentration of test chemical and other water quality parameters are 
measured at specified intervals in selected test chambers. Data are 
collected during the test to develop concentration-response curves and 
determine EC50 values for the test chemical.
    (2) [Reserved]
    (3) Range-finding test. (i) A range-finding test should be conducted 
to establish test solution concentrations for the definitive test.
    (ii) The daphnids should be exposed to a series of widely spaced 
concentrations of the test chemical (e.g., 1, 10, 100 mg/1, etc.), 
usually under static conditions.
    (iii) A minimum of five daphnids should be exposed to each 
concentration of test chemical for a period of 48 hours. The exposure 
period may be shortened if data suitable for the purpose of the range-
finding test can be obtained in less time. No replicates are required 
and nominal concentrations of the chemical are acceptable.
    (4) Definitive test. (i) The purpose of the definitive test is to 
determine the concentration-response curves and the 24- and 48-hour 
EC50 values with the

[[Page 107]]

minimum amount of testing beyond the range-finding test.
    (ii) A minimum of 20 daphnids per concentration shall be exposed to 
five or more concentrations of the chemical chosen in a geometric series 
in which the ratio is between 1.5 and 2.0 (e.g., 2, 4, 8, 16, 32, and 64 
mg/l). An equal number of daphnids shall be placed in two or more 
replicates. If solvents, solubilizing agents or emulsifiers have to be 
used, they shall be commonly used carriers and shall not possess a 
synergistic or antagonistic effect on the toxicity of the test chemical. 
The concentration of solvent should not exceed 0.1 mg/l. The 
concentration ranges shall be selected to determine the concentration-
response curves and EC50 values at 24 and 48 hours. 
Concentration of test chemical in test solutions should be analyzed 
prior to use.
    (iii) Every test shall include controls consisting of the same 
dilution water, conditions, procedures and daphnids from the same 
population (culture container), except that none of the chemical is 
added.
    (iv) The dissolved oxygen concentration, temperature and pH shall be 
measured at the beginning and end of the test in each chamber.
    (v) The test duration is 48 hours. The test is unacceptable if more 
than 10 percent of the control organisms are immobilized during the 48-
hour test period. Each test chamber shall be checked for immobilized 
daphnids at 24 and 48 hours after the beginning of the test. 
Concentration-response curves and 24-hour and 48-hour EC50 
values for immobilization shall be determined along with their 95 
percent confidence limits.
    (vi) In addition to immobility, any abnormal behavior or appearance 
shall also be reported.
    (vii) Test organisms shall be impartially distributed among test 
chambers in such a manner that test results show no significant bias 
from the distributions. In addition, test chambers within the testing 
area shall be positioned in a random manner or in a way in which 
appropriate statistical analyses can be used to determine the variation 
due to placement.
    (viii) The concentration of the test chemical in the chambers should 
be measured as often as is feasible during the test. In the static test 
the concentration of test chemical shall be measured, at a minimum, at 
the beginning of the test and at the end of the test in each test 
chamber. In the flow-through test the concentration of test chemical 
shall be measured at a minimum:
    (A) In each chamber at the beginning of the test and at 48 hours 
after the start of the test;
    (B) In at least one appropriate chamber whenever a malfunction is 
detected in any part of the test substance delivery system.

Among replicate test chambers of a treatment concentration, the measured 
concentration of the test chemical shall not vary more than 20 percent.
    (5) [Reserved]
    (6) Analytical measurements. (i) Test chemical. Deionized water 
should be used in making stock solutions of the test chemical. Standard 
analytical methods should be used whenever available in performing the 
analyses. The analytical method used to measure the amount of test 
chemical in a sample shall be validated before beginning the test by 
appropriate laboratory practices. Any analytical method is not 
acceptable if likely degradation products of the test chemical, such as 
hydrolysis and oxidation products, give positive or negative 
interferences which cannot be systematically identified and corrected 
mathematically.
    (ii) Numerical. The number of immobilized daphnids shall be counted 
during each definitive test. Appropriate statistical analyses should 
provide a goodness-of-fit determination for the concentration-response 
curves. A 24- and 48-hour EC50 and corresponding 95 percent 
interval shall be calculated.
    (d) Test conditions--(1) Test species--(i) Selection. (A) The 
cladocerans, Daphnia magna or D. pulex, are the test species to be used 
in this test. Either species may be used for testing of a particular 
chemical. The species identity of the test organisms should be verified 
using appropriate systematic keys. First instar daphnids, <=24 hours 
old, are to be used to start the test.
    (B) Daphnids to be used in acute toxicity tests should be cultured 
at the

[[Page 108]]

test facility. Records should be kept regarding the source of the 
initial stock and culturing techniques. All organisms used for a 
particular test shall have originated from the same culture population.
    (C) Daphnids shall not be used for a test (1) if cultures contain 
ephippia; (2) if adults in the cultures do not produce young before day 
12; (3) if more than 20 percent of the culture stock die during the 2 
days preceding the test; (4) if adults in the culture do not produce an 
average of at least 3 young per adult per day over the 7-day period 
prior to the test and (5) if daphnids have been used in any portion of a 
previous test, either in a treatment or in a control.
    (ii) Acclimation. (A) Brood daphnids shall be maintained in 100-
percent dilution water at the test temperature for at least 48 hours 
prior to the start of the test. This is easily accomplished by culturing 
them in the dilution water at the test temperature. During production of 
neonates, daphnids should not be fed.
    (B) During culturing and acclimation to the dilution water, daphnids 
should be maintained in facilities with background colors and light 
intensities similar to those of the testing area.
    (iii) Care and handling. (A) Daphnids should be cultured in dilution 
water under similar environmental conditions to those used in the test. 
Organisms should be handled as little as possible. When handling is 
necessary it should be done as gently, carefully, and quickly as 
possible. During culturing and acclimation, daphnids should be observed 
carefully for ephippia and other signs of stress, physical damage and 
mortality. Dead and abnormal individuals shall be discarded. Organisms 
that touch dry surfaces or are dropped or injured in handling shall be 
discarded.
    (B) Smooth glass tubes (I.D. greater than 5 mm) equipped with rubber 
bulb should be used for transferring daphnids with minimal culture media 
carry-over. Care should be exercised to introduce the daphnids below the 
surface of any solution to avoid trapping air under the carapace.
    (iv) Feeding. A variety of foods (e.g., unicellular green algae) 
have been demonstrated to be adequate for daphnid culture. Daphnids 
shall not be fed during testing.
    (2) Facilities--(i) Apparatus. (A) Facilities needed to perform this 
test include: (1) Containers for culturing and acclimating daphnids; (2) 
a mechanism for controlling and maintaining the water temperature during 
the culturing, acclimation, and test periods; (3) apparatus for 
straining particulate matter, removing gas bubbles, or aerating the 
water as necessary; and (4) an apparatus for providing a 16-hour light 
and 8-hour dark photoperiod with a 15 to 30 minute transition period. In 
addition, the flow-through system shall contain appropriate test 
chambers in which to expose daphnids to the test chemical and an 
appropriate test substance delivery system.
    (B) Facilities should be well ventilated and free of fumes and 
disturbances that may affect the test organisms.
    (C) Test chambers shall be loosely covered to reduce the loss of 
test solution or dilution water due to evaporation and to minimize the 
entry of dust or other particulates into the solutions.
    (ii) Construction materials. (A) Materials and equipment that 
contact test solutions should be chosen to minimize sorption of test 
chemicals from the dilution water and should not contain substances that 
can be leached into aqueous solution in quantities that can affect the 
test results.
    (B) For static tests, daphnids can be conveniently exposed to the 
test chemical in 250 ml beakers or other suitable containers.
    (C) For flow-through tests, daphnids can be exposed in glass or 
stainless steel containers with stainless steel or nylon screen bottoms. 
The containers should be suspended in the test chamber in such a manner 
to insure that the test solution flows regularly into and out of the 
container and that the daphnids are always submerged in at least 5 
centimeters of test solution. Test chambers can be constructed using 250 
ml beakers or other suitable containers equipped with screened overflow 
holes, standpipes or V-shaped notches.
    (iii) Dilution water. (A) Surface or ground water, reconstituted 
water or

[[Page 109]]

dechlorinated tap water are acceptable as dilution water if daphnids 
will survive in it for the duration of the culturing, acclimation and 
testing periods without showing signs of stress. The quality of the 
dilution water should be constant and should meet the following 
specifications:

------------------------------------------------------------------------
                 Substance                      Maximum concentration
------------------------------------------------------------------------
Particulate matter........................  20 mg/liter.
Total organic carbon or...................  2 mg/liter.
      Chemical oxygen demand..............  5 mg/liter.
Un-ionized ammonia........................  1 [micro]g/liter.
Residual chlorine.........................  <3 [micro]g/liter.
Total organophosphorus pesticides.........  50 ng/liter.
Total organochlorine pesticides plus        50 ng/liter.
 polychlorinated biphenyls (PCBs) or.
Organic chlorine..........................  25 ng/liter.
------------------------------------------------------------------------

    (B) The above water quality parameters under paragraph 
(d)(2)(iii)(A) of this section shall be measured at least twice a year 
or whenever it is suspected that these characteristics may have changed 
significantly. If dechlorinated tap water is used, daily chlorine 
analysis shall be performed.
    (C) If the diluent water is from a ground or surface water source, 
conductivity and total organic carbon (TOC) or chemical oxygen demand 
(COD) shall be measured. Reconstituted water can be made by adding 
specific amounts of reagent-grade chemicals to deionized or distilled 
water. Glass distilled or carbon-filtered deionized water with a 
conductivity less than 1 [micro]ohm/cm is acceptable as the diluent for 
making reconstituted water.
    (iv) Cleaning. All test equipment and test chambers shall be cleaned 
before each use using standard laboratory procedures.
    (v) Test substance delivery system. In flow-through tests, 
proportional diluters, metering pump systems, or other suitable devices 
should be used to deliver test chemical to the test chambers. The system 
shall be calibrated before each test. Calibration includes determining 
the flow rate through each chamber and the concentration of the test 
chemical in each chamber. The general operation of the test substance 
delivery system should be checked twice during a test. The 24-hour flow 
through a test chamber shall be equal to at least 5 times the volume of 
the test chamber. During a test, the flow rates should not vary more 
than 10 percent from any one test chamber to another.
    (3) Test parameters. Environmental parameters of the water contained 
in test chambers shall be maintained as specified below:
    (i) The test temperature shall be 20 [deg]C. Excursions from the 
test temperature shall be no greater than 2 
[deg]C.
    (ii) Dissolved oxygen concentration between 60 and 105 percent 
saturation. Aeration, if needed to achieve this level, shall be done 
before the addition of the test chemical. All treatment and control 
chambers shall be given the same aeration treatment.
    (iii) The number of daphnids placed in a test chamber shall not 
affect test results. Loading shall not exceed 40 daphnids per liter test 
solution in the static system. In the flow-through test, loading limits 
will vary depending on the flow rate of dilution water. Loading shall 
not cause the dissolved oxygen concentration to fall below the 
recommended levels.
    (iv) Photoperiod of 16 hours light and 8 hours darkness.
    (e) Reporting. The sponsor shall submit to the U.S. EPA all data 
developed by the test that are suggestive or predictive of acute 
toxicity and all concomitant gross toxicological manifestations. In 
addition to the reporting requirements prescribed in part 792--Good 
Laboratory Practice Standards of this chapter, the reporting of test 
data shall include the following:
    (1) The name of the test, sponsor, testing laboratory, study 
director, principal investigator, and dates of testing.
    (2) A detailed description of the test chemical including its 
source, lot number, composition (identity and concentration or major 
ingredients and major impurities), known physical and chemical 
properties and any carriers or other additives used and their 
concentrations.
    (3) The source of the dilution water, its chemical characteristics 
(e.g., conductivity, hardness, pH, etc.) and a description of any 
pretreatment.
    (4) Detailed information about the daphnids used as brood stock, 
including the scientific name and method of

[[Page 110]]

verification, age, source, treatments, feeding history, acclimation 
procedures, and culture method. The age of the daphnids used in the test 
shall be reported.
    (5) A description of the test chambers, the volume of solution in 
the chambers, the way the test was begun (e.g., conditioning, test 
chemical additions), the number of test organisms per test chamber, the 
number of replicates per treatment, the lighting, the method of test 
chemical introduction or the test substance delivery system and the flow 
rate (in flow-through test) expressed as volume additions per 24 hours.
    (6) The concentration of the test chemical in each test chamber at 
times designated for static and flow-through tests.
    (7) The number and percentage of organisms that were immobilized or 
showed any adverse effects in each test chamber at each observation 
period.
    (8) Utilizing the average measured test chemical concentration, 
concentration-response curves should be fitted to immobilization data at 
24 and 48 hours. A statistical test of goodness-of-fit should be 
performed and the results reported.
    (9) The 24- and 48-hour EC50 values and their respective 
95 percent confidence limits using the mean measured test chemical 
concentration and the methods used to calculate both the EC50 
values and their confidence limits.
    (10) All chemical analyses of water quality and test chemical 
concentrations, including methods, method validations and reagent 
blanks.
    (11) The data records of the culture, acclimation and test 
temperatures.
    (12) Any deviation from this test guideline and anything unusual 
about the test, e.g., diluter failure, temperature fluctuations, etc.

[50 FR 39321, Sept. 27, 1985, as amended at 52 FR 19059, May 20, 1987]



Sec. 797.1330  Daphnid chronic toxicity test.

    (a) Purpose. This guideline is intended for use in developing data 
on the chronic toxicity of chemical substances and mixtures 
(``chemicals'') subject to environmental effects test regulations under 
the Toxic Substances Control Act (TSCA) (Pub. L. 94-469, 90 Stat. 2003, 
15 U.S.C. 2601 et seq.). This guideline prescribes a chronic toxicity 
test in which daphnids are exposed to a chemical in a renewal or a flow-
through system. The United States Environmental Protection Agency will 
use data from this test in assessing the hazard a chemical may present 
to the aquatic environment.
    (b) Definitions. The definitions in section 3 of the Toxic 
Substances Control Act (TSCA), and the definitions in part 792 Good 
Laboratory Practice Standards of this chapter apply to this test 
guideline. In addition, the following definitions apply to this 
guideline:
    (1) Brood stock means the animals which are cultured to produce test 
organisms through reproduction.
    (2) Chronic toxicity test means a method used to determine the 
concentration of a substance in water that produces an adverse effect on 
a test organism over an extended period of time. In this test guideline, 
mortality and reproduction (and optionally, growth) are the criteria of 
toxicity.
    (3) EC50 means that experimentally derived concentration 
of test substance in dilution water that is calculated to affect 50 
percent of a test population during continuous exposure over a specified 
period of time. In this guideline, the effect measured is 
immobilization.
    (4) Ephippium means a resting egg which develops under the carapace 
in response to stress conditions in daphnids.
    (5) Flow-through means a continuous or intermittent passage of test 
solution or dilution water through a test chamber or culture tank with 
no recycling.
    (6) Immobilization means the lack of movement by daphnids except for 
minor activity of the appendages.
    (7) Loading means the ratio of daphnid biomass (grams, wet weight) 
to the volume (liters) of test solution in a test chamber at a point in 
time or passing through the test chamber during a specific interval.
    (8) MATC (Maximum Acceptable Toxicant Concentration) means the 
maximum concentration at which a chemical can be present and not be 
toxic to the test organism.

[[Page 111]]

    (9) Renewal system means the technique in which test organisms are 
periodically transferred to fresh test solution of the same composition.
    (c) Test procedures--(1) Summary of the test. (i) Test chambers are 
filled with appropriate volumes of dilution water. In the flow-through 
test the flow of dilution water through each chamber is then adjusted to 
the rate desired. The test substance is introduced into each test 
chamber. The addition of test substance in the flow-through system is 
done at a rate which is sufficient to establish and maintain the desired 
concentration of test substance in the test chamber.
    (ii) The test is started within 30 minutes after the test substance 
has been added and uniformly distributed in the test chambers in the 
renewal test or after the concentration of test substance in each test 
chamber of the flow-through test system reaches the prescribed level and 
remains stable. At the initiation of the test, daphnids which have been 
cultured or acclimated in accordance with the test design, are randomly 
placed into the test chambers. Daphnids in the test chambers are 
observed periodically during the test, immobile adults and offspring 
produced are counted and removed, and the findings are recorded. 
Dissolved oxygen concentration, pH, temperature, the concentration of 
test substance, and other water quality parameters are measured at 
specified intervals in selected test chambers. Data are collected during 
the test to determine any significant differences (p<=0.05) in 
immobilization and reproduction as compared to the control.
    (2) [Reserved]
    (3) Range-finding test. (i) A range-finding test should be conducted 
to establish test solution concentrations for the definitive test.
    (ii) The daphnids should be exposed to a series of widely spaced 
concentrations of the test substance (e.g., 1, 10, 100 mg/l), usually 
under static conditions.
    (iii) A minimum of five daphnids should be exposed to each 
concentration of test substance for a period of time which allows 
estimation of appropriate chronic test concentrations. No replicates are 
required and nominal concentrations of the chemical are acceptable.
    (4) Definitive test. (i) The purpose of the definitive test is to 
determine concentration-response curves, EC50 values and 
effects of a chemical on immobilization and reproduction during chronic 
exposure.
    (ii) A minimum of 20 daphnids per concentration shall be exposed to 
five or more concentrations of the chemical chosen in a geometric series 
in which the ratio is between 1.5 and 2.0 (e.g., 2, 4, 8, 16, 32, 64 mg/
l). An equal number of daphnids shall be placed in two or more 
replicates. The concentration ranges shall be selected to determine the 
concentration-response curves, EC50 values and MATC. 
Solutions shall be analyzed for chemical concentration at designated 
times during the test.
    (iii) Every test shall include controls consisting of the same 
dilution water, conditions, procedures and daphnids from the same 
population (culture container), except that none of the chemical is 
added.
    (iv) The test duration is 21 days. The test is unacceptable if:
    (A) More than 20 percent of the control organisms appear to be 
immobilized, stressed or diseased during the test.
    (B) Each control daphnid living the full 21 days produces an average 
of less than 60 young.
    (C) Any ephippia are produced by control animals.
    (v) The number of immobilized daphnids in each chamber shall be 
recorded on day 21 of the test. After offspring are produced, they shall 
be counted and removed from the test chambers every 2 or 3 days. 
Concentration-response curves, EC50 values and associated 95 
percent confidence limits for adult immobilization shall be determined 
for day 21. An MATC shall be determined for the most sensitive test 
criteria measured (number of adult animals immobilized, number of young 
per adult, and number of immobilized young per adult).
    (vi) In addition to immobility, any abnormal behavior or appearance 
shall also be reported.
    (vii) Test organisms shall be impartially distributed among test 
chambers in such a manner that test results show

[[Page 112]]

no significant bias from the distributions. In addition, test chambers 
within the testing area shall be positioned in a random manner as in a 
way in which appropriate statistical analyses can be used to determine 
the variation due to placement.
    (5) [Reserved]
    (6) Analytical measurements. (i) Test chemical. Deionized water 
should be used in making stock solutions of the test substance. Standard 
analytical methods should be used whenever available in performing the 
analyses. The analytical method used to measure the amount of test 
substance in a sample shall be validated before beginning the test by 
appropriate laboratory practices. An analytical method is not acceptable 
if likely degradation products of the test substance, such as hydrolysis 
and oxidation products, give positive or negative interferences which 
cannot be systematically identified and corrected mathematically.
    (ii) Numerical. The number of immobilized adults, total offspring 
per adult, and immobilized offspring per adult shall be counted during 
each test. Appropriate statistical analyses should provide a goodness-
of-fit determination for the adult immobilization concentration-response 
curves calculated on day 21. A 21-day EC50 based on adult 
immobilization and corresponding 95 percent confidence intervals shall 
also be calculated. Appropriate statistical tests (e.g., analysis of 
variance, mean separation test) should be used to test for significant 
chemical effects on chronic test criteria (cumulative number of 
immobilized adults, cumulative number of offspring per adult and 
cumulative number of immobilized offspring per adult) on day 21. An MATC 
shall be calculated using these chronic test criteria.
    (d) Test conditions--(1) Test species--(i) Selection. (A) The 
cladocerans, Daphnia magna or D. pulex, are the species to be used in 
this test. Either species can be utilized for testing of a particular 
chemical. The species identity of the test organisms should be verified 
using appropriate systematic keys.
    (B) First instar daphnids, <=24 hours old, are to be used to start 
the test.
    (ii) Acquisition. (A) Daphnids to be used in chronic toxicity tests 
should be cultured at the test facility. Records should be kept 
regarding the source of the initial stock and culturing techniques. All 
organisms used for a particular test shall have originated from the same 
culture population.
    (B) Daphnids shall not be used for a test if:
    (1) Cultures contain ephippia.
    (2) Adults in the cultures do not produce young before day 12.
    (3) More than 20 percent of the culture stock die in the 2 days 
preceding the test.
    (4) Adults in the culture do not produce an average of at least 3 
young per adult per day over the 7-day period prior to the test.
    (5) Daphnids have been used in any portion of a previous test either 
in a treatment or in a control.
    (iii) Feeding. (A) During the test the daphnids shall be fed the 
same diet and with the same frequency as that used for culturing and 
acclimation. All treatments and control(s) shall receive, as near as 
reasonably possible, the same ration of food on a per-animal basis.
    (B) The food concentration depends on the type used. Food 
concentrations should be sufficient to support normal growth and 
development and to allow for asexual (parthenogenic) reproduction. For 
automatic feeding devices, a suggested rate is 5 to 7 mg food (either 
solids or algal cells, dry weight) per liter dilution water or test 
solution. For manual once-a-day feeding, a suggested rate is 15 mg food 
(dry weight) per liter dilution water or test solution.
    (iv) Loading. The number of test organisms placed in a test chamber 
shall not affect test results. Loading shall not exceed 40 daphnids per 
liter in the renewal system. In the flow-through test, loading limits 
will vary depending on the flow rate of the dilution water. Loading 
shall not cause the dissolved oxygen concentration to fall below the 
recommended level.
    (v) Care and handling of test organisms. (A) Daphnids should be 
cultured in dilution water under similar environmental conditions to 
those used in the test. A variety of foods have been demonstrated to be 
adequate for daphnid

[[Page 113]]

culture. They include algae, yeasts and a variety of mixtures.
    (B) Organisms should be handled as little as possible. When handling 
is necessary it should be done as gently, carefully, and quickly as 
possible. During culturing and acclimation, daphnids should be observed 
carefully for ephippia and other signs of stress, physical damage, and 
mortality. Dead and abnormal individuals shall be discarded. Organisms 
that touch dry surfaces or are dropped or injured during handling shall 
be discarded.
    (C) Smooth glass tubes (I.D. greater than 5mm) equipped with a 
rubber bulb can be used for transferring daphnids with minimal culture 
media carry-over.
    (D) Care should be exercised to introduce the daphnids below the 
surface of any solution so as not to trap air under the carapace.
    (vi) Acclimation. (A) Brood daphnids shall be maintained in 100 
percent dilution water at the test temperature for at least 48 hours 
prior to the start of the test. This is easily accomplished by culturing 
them in dilution water at the test temperature. During acclimation, 
daphnids shall be fed the same food as will be used for the definitive 
test.
    (B) During culturing and acclimation to the dilution water, daphnids 
should be maintained in facilities with background colors and light 
intensities similar to those of the testing area.
    (2) Facilities--(i) General. (A) Facilities needed to perform this 
test include:
    (1) Containers for culturing and acclimating daphnids.
    (2) A mechanism for controlling and maintaining the water 
temperature during the culturing, acclimation and test periods.
    (3) Apparatus for straining particulate matter, removing gas 
bubbles, or aerating the water when water supplies contain particulate 
matter, gas bubbles, or insufficient dissolved oxygen, respectively.
    (4) An apparatus for providing a 16-hour light and 8-hour dark 
photoperiod.
    (5) An apparatus to introduce food if continuous or intermittent 
feeding is used.
    (6) In addition, the flow-through test shall contain appropriate 
test chambers in which to expose daphnids to the test substance and an 
appropriate test substance delivery system.
    (B) Facilities should be well ventilated and free of fumes and other 
disturbances that may affect the test organisms.
    (ii) Test chambers. (A) Materials and equipment that contact test 
solutions should be chosen to minimize sorption of test chemicals from 
the dilution water and should not contain substances that can be leached 
into aqueous solution in quantities that can affect test results.
    (B) For renewal tests, daphnids can be conveniently exposed to the 
test solution in 250 ml beakers or other suitable containers.
    (C) For flow-through tests daphnids can be exposed in glass or 
stainless steel containers with stainless steel or nylon screen bottoms. 
Such containers shall be suspended in the test chamber in such a manner 
to ensure that the test solution flows regularly into and out of the 
container and that the daphnids are always submerged in at least 5 
centimeters of test solution. Test chambers can be constructed using 250 
ml beakers or other suitable containers equipped with screened overflow 
holes, standpipes or V-shaped notches.
    (D) Test chambers shall be loosely covered to reduce the loss of 
test solution or dilution water due to evaporation and to minimize the 
entry of dust or other particulates into the solutions.
    (iii) Test substance delivery system. (A) In the flow-through test, 
proportional diluters, metering pump systems or other suitable systems 
should be used to deliver the test substance to the test chambers.
    (B) The test substance delivery system shall be calibrated before 
each test. Calibration includes determining the flow rate through each 
chamber and the concentration of the test substance in each chamber. The 
general operation of the test substance delivery system should be 
checked twice daily during a test. The 24-hour flow rate through a test 
chamber shall be equal to at least five times the volume

[[Page 114]]

of the test chamber. During a test, the flow rates shall not vary more 
than 10 percent from any one test chamber to another. For the renewal 
test, test substance dilution water shall be completely replaced at 
least once every 3 days.
    (iv) Dilution water. (A) Surface or ground water, reconstituted 
water, or dechlorinated tap water are acceptable as dilution water if 
daphnids will survive in it for the duration of the culturing, 
acclimation, and testing periods without showing signs of stress. The 
quality of the dilution water should be constant and should meet the 
following specificiations:

------------------------------------------------------------------------
                 Substance                      Maximum concentration
------------------------------------------------------------------------
Particulate matter.........................  20 mg/l.
Total organic carbon or....................  2 mg/l.
      Chemical oxygen demand...............  5 mg/l.
Un-ionized ammonia.........................  20 [micro]g/l.
Residual chlorine..........................  <3 [micro]g/l.
Total organophosphorus pesticides..........  50 ng/l.
Total organochlorine pesticides plus         50 ng/l.
 polychlorinated biphenyls (PCBs).
      or organic chlorine..................  25 ng/l.
------------------------------------------------------------------------

    (B) The water quality characteristics listed above shall be measured 
at least twice a year or when it is suspected that these characteristics 
may have changed significantly. If dechlorinated tap water is used, 
daily chlorine analysis shall be performed.
    (C) If the diluent water is from a ground or surface water source, 
conductivity and total organic carbon (TOC) or chemical oxygen demand 
(COD) shall be measured. Reconstituted water can be made by adding 
specific amounts of reagent-grade chemicals to deionized or distilled 
water. Glass distilled or carbon filtered deionized water with a 
conductivity of less than 1 microohm/cm is acceptable as the diluent for 
making reconstituted water.
    (D) If the test substance is not soluble in water an appropriate 
carrier should be used.
    (v) Cleaning of test system. All test equipment and test chambers 
shall be cleaned before each use following standard laboratory 
procedures. Cleaning of test chambers may be necessary during the 
testing period.
    (3) Test parameters. (i) Environmental conditions of the water 
contained in test chambers should be maintained as specified in this 
paragraph:
    (A) The test temperature shall be 20 [deg]C. Excursions from the 
test temperature shall be no greater than 2 
[deg]C.
    (B) Dissolved oxygen concentration between 60 and 105 percent 
saturation. Aeration, if needed to achieve this level, shall be done 
before the addition of the test substance. All treatment and control 
chambers shall be given the same aeration treatment.
    (C) Photoperiod of 16-hours light and 8-hours darkness.
    (ii) Additional measurements include:
    (A) The concentration of the test substance in the chambers shall be 
measured during the test.
    (B) At a minimum, the concentration of test substance should be 
measured as follows:
    (1) In each chamber before the test.
    (2) In each chamber on days 7, 14, and 21 of the test.
    (3) In at least one appropriate chamber whenever a malfunction is 
detected in any part of the test substance delivery system. Equal 
aliquots of test solution may be removed from each replicate chamber and 
pooled for analysis. Among replicate test chambers of a treatment 
concentration, the measured concentration of the test substance should 
not vary more than 20 percent.
    (4) An apparatus for providing a 16-hour light and 8-hour dark 
photoperiod.
    (C) The dissolved oxygen concentration, temperature and pH shall be 
measured at the beginning of the test and on days 7, 14, and 21 in at 
least two chambers of the high, middle, low, and control test 
concentrations.
    (e) Reporting. The sponsor shall submit to the U.S. Environmental 
Protection Agency all data developed by the test that are suggestive or 
predictive of chronic toxicity and all associated toxicologic 
manifestations. In addition to the reporting requirements prescribed in 
the part 792--Good Laboratory Practice Standards of this chapter the 
reporting of test data shall include the following:
    (1) The name of the test, sponsor, testing laboratory, study 
director, principal investigator, and dates of testing.

[[Page 115]]

    (2) A detailed description of the test substance including its 
source, lot number, composition (identity and concentration of major 
ingredients and major impurities), known physical and chemical 
properties, and any carriers or other additives used and their 
concentrations.
    (3) The source of the dilution water, its chemical characteristics 
(e.g., conductivity, hardness, pH), and a description of any 
pretreatment.
    (4) Detailed information about the daphnids used as brood stock, 
including the scientific name and method of verification, age, source, 
treatments, feeding history, acclimation procedures, and culture 
methods. The age of the daphnids used in the test shall be reported.
    (5) A description of the test chambers, the volume of solution in 
the chambers, the way the test was begun (e.g., conditioning, test 
substance additions), the number of test organisms per test chamber, the 
number of replicates per treatment, the lighting, the renewal process 
and schedule for the renewal chronic test, the test substance delivery 
system and flow rate expressed as volume additions per 24 hours for the 
flow-through chronic test, and the method of feeding (manual or 
continuous) and type of food.
    (6) The concentration of the test substance in test chambers at 
times designated for renewal and flow-through tests.
    (7) The number and percentage of organisms that show any adverse 
effect in each test chamber at each observation period.
    (8) The cumulative adult and offspring immobilization values and the 
progeny produced at designated observation times, the time (days) to 
first brood and the number of offspring per adult in the control 
replicates and in each treatment replicate.
    (9) All chemical analyses of water quality and test substance 
concentrations, including methods, method validations and reagent 
blanks.
    (10) The data records of the culture, acclimation, and test 
temperatures.
    (11) Any deviation from this test guideline, and anything unusual 
about the test, (e.g., dilution failure, temperature fluctuations).
    (12) The MATC to be reported is calculated as the geometric mean 
between the lowest measured test substance concentration that had a 
significant (p<=0.05) effect and the highest measured test substance 
concentration that had no significant (p<=0.05) effect on day 21 of the 
test. The most sensitive of the test criteria (number of adult animals 
immobilized, the number of young per female and the number of 
immobilized young per female) is used to calculate the MATC. The 
criterion selected for MATC computation is the one which exhibits an 
effect (a statistically significant difference between treatment and 
control groups; p<=0.05) at the lowest test substance concentration for 
the shortest period of exposure. Appropriate statistical tests (analysis 
of variance, mean separation test) shall be used to test for significant 
test substance effects. The statistical tests employed and the results 
of these tests shall be reported.
    (13) Concentration-response curves utilizing the average measured 
test substance concentration shall be fitted to cumulative adult 
immobilization data at 21 days. A statistical test of goodness-of-fit 
shall be performed and the results reported.
    (14) An EC50 value based on adult immobilization with 
corresponding 95 percent confidence limits when sufficient data are 
present for day 21. These calculations shall be made using the average 
measured concentration of the test substance.

[50 FR 39321, Sept. 27, 1985, as amended at 52 FR 19060, May 20, 1987]



Sec. 797.1400  Fish acute toxicity test.

    (a) Purpose. This guideline may be used to develop data on the acute 
toxicity of chemical substances and mixtures (``chemicals'') subject to 
environmental effects test regulations under the Toxic Substances 
Control Act (TSCA) (Pub. L. 94-469, 90 Stat. 2003, 15 U.S.C. 2601 et 
seq.). This guideline prescribes tests to be used to develop data on the 
acute toxicity of chemicals to fish. The United States Environmental 
Protection Agency (EPA) will use data from these tests in assessing the 
hazard of a chemical to the environment.
    (b) Definitions. The definitions in section 3 of the Toxic 
Substances Control

[[Page 116]]

Act (TSCA), and the definitions in part 792--Good Laboratory Practice 
Standards of this chapter apply to this test guideline. The following 
definitions also apply to this guideline:
    (1) Acclimation means the physiological compensation by test 
organisms to new environmental conditions (e.g., temperature, hardness, 
pH).
    (2) Acute toxicity test means a method used to determine the 
concentration of a substance that produces a toxic effect on a specified 
percentage of test organisms in a short period of time (e.g., 96 hours). 
In this guideline, death is used as the measure of toxicity.
    (3) Carrier means a solvent used to dissolve a test substance prior 
to delivery to the test chamber.
    (4) Conditioning means the exposure of construction materials, test 
chambers, and testing apparatus to dilution water or to test solutions 
prior to the start of a test in order to minimize the sorption of the 
test substance onto the test facilities or the leaching of substances 
from the test facilities into the dilution water or test solution.
    (5) Death means the lack of opercular movement by a test fish.
    (6) Flow-through means a continuous or an intermittent passage of 
test solution or dilution water through a test chamber, or a holding or 
acclimation tank with no recycling.
    (7) Incipient LC50 means that test substance 
concentration, calculated from experimentally-derived mortality data, 
that is lethal to 50 percent of a test population when exposure to the 
test substance is continued until the mean increase in mortality does 
not exceed 10 percent in any concentration over a 24-hour period.
    (8) LC50 means that test substance concentration, 
calculated from experimentally-derived mortality data, that is lethal to 
50 percent of a test population during continuous exposure over a 
specified period of time.
    (9) Loading means the ratio of fish biomass (grams, wet weight) to 
the volume (liters) of test solution in a test chamber or passing 
through it in a 24-hour period.
    (10) Static means the test solution is not renewed during the period 
of the test.
    (11) Test solution means the test substance and the dilution water 
in which the test substance is dissolved or suspended.
    (c) Test procedures--(1) Summary of the test. (i) Test chambers are 
filled with appropriate volumes of dilution water. If a flow-through 
test is performed, the flow of dilution water through each chamber is 
adjusted to the rate desired.
    (ii) The test substance is introduced into each test chamber. In a 
flow-through test, the amount of test substance which is added to the 
dilution water is adjusted to establish and maintain the desired 
concentration of test substance in each test chamber.
    (iii) Test fish which have been acclimated in accordance with the 
test design are introduced into the test and control chambers by 
stratified random assignment.
    (iv) Fish in the test and control chambers are observed periodically 
during the test; dead fish are removed at least twice each day and the 
findings are recorded.
    (v) The dissolved oxygen concentration, pH, temperature and the 
concentration of test substance are measured at intervals in selected 
test chambers.
    (vi) Concentration-response curves and LC50 values for 
the test substance are developed from the mortality data collected 
during the test.
    (2) [Reserved]
    (3) Range finding test. If the toxicity of the test substance is not 
already known, a range finding test should be performed to determine the 
range of concentrations to be used in the definitive test. The highest 
concentration of test substance for use in the range finding test should 
not exceed its solubility in water or the permissible amount of the 
carrier used.
    (4) Definitive test. (i) A minimum of 20 fish should be exposed to 
each of five or more test substance concentrations. The range of 
concentrations to which the fish are exposed should be such that in 96 
hours there are at least two partial mortality exposures bracketing 50 
percent survival.
    (ii) For exposure to each concentration of a test substance, an 
equal number of test fish shall be placed in two or more replicate test 
chambers. Test fish

[[Page 117]]

shall be impartially distributed among test chambers in such a manner 
that test results show no significant bias from the distributions.
    (iii) Every test shall include a control consisting of the same 
dilution water, conditions, procedures, and fish from the same group 
used in the test, except that none of the test substance is added.
    (iv) Mortality data collected during the test are used to calculate 
a 96-hour LC50. The 24-, 48-, and 72-hour values should be 
calculated whenever there is sufficient mortality data to determine such 
values. If the 96-hour LC50 is less than 50 percent of the 
estimated 48-hour LC50 in a flow-through test, the test shall 
be continued until the mean increase in mortality at any test 
concentration does not exceed 10 percent over a 24-hour period or until 
14 days.
    (v) Test fish shall not be fed while they are being exposed to the 
test substance under static conditions or during the first 96 hours of 
flow-through testing. If the test continues past 96 hours, the fish 
should be fed a suitable food at a maintenance level every other day 
beginning on test day 5. Any excess food and the fecal material should 
be removed when observed.
    (5) Test results. (i) Death is the primary criterion used in this 
test guideline to evaluate the toxicity of the test substance.
    (ii) In addition to death, any abnormal behavior such as, but not 
limited to, erratic swimming, loss of reflex, increased excitability, 
lethargy, or any changes in appearance or physiology such as 
discoloration, excessive mucous production, hyperventilation, opaque 
eyes, curved spine, or hemorrhaging shall be recorded.
    (iii) Observations on compound solubility shall be recorded. The 
investigator shall report the appearance of surface slicks, 
precipitates, or material adhering to the sides of the test chamber.
    (iv) Each test and control chamber shall be checked for dead fish 
and observations recorded at 24, 48, 72, and 96 hours after the 
beginning of the test or within one hour of the designated times. If the 
test is continued past 96 hours, additional observations shall be made 
every 24 hours until termination.
    (v) The mortality data is used to calculate LC50's and 
their 95 percent confidence limits, and to plot concentration-response 
curves for each time interval whenever sufficient data exists. The 
methods recommended for use in calculating LC50's include 
probit, logit, binomial, and moving average angle.
    (vi) A test is unacceptable if more than 10 percent of the control 
fish die or exhibit abnormal behavior during a 96-hour test. If a flow-
through test is continued past 96 hours, the maximum allowable 
additional mortality is 10 percent.
    (6) Analytical measurements--(i) Water quality analysis. (A) The 
hardness, acidity, alkalinity, pH, conductivity, TOC or COD, and 
particulate matter of the dilution water should be measured at the 
beginning of each static test and at the beginning and end of each flow-
through test. The month to month variation of the above values should be 
less than 10 percent and the pH should vary less than 0.4 units.
    (B) During static tests, the dissolved oxygen concentration, 
temperature, and pH shall be measured in each test chamber at the 
beginning and end of the test. The test solution volume shall not be 
reduced by more than 10 percent as a result of these measurements.
    (C) During flow-through tests, dissolved oxygen, temperature and pH 
measurements shall be made in each chamber at the beginning and end of 
the test.
    (ii) Collection of samples for measurement of test substance. Test 
solution samples to be analyzed for the test substance should be taken 
midway between the top, bottom, and sides of the test chamber. These 
samples should not include any surface scum or material dislodged from 
the bottom or sides. Samples should be analyzed immediately or handled 
and stored in a manner which minimizes loss of test substance through 
microbial degradation, photodegradation, chemical reaction, 
volatilization, or sorption.
    (iii) Measurement of test substance. (A) For static tests, the 
concentration of the test substance shall be measured at a minimum in 
each test chamber at each test concentration at the beginning (0-hour, 
before fish are added) and at the end of the test. During flow-

[[Page 118]]

through tests, the concentration of test substance shall be measured as 
follows:
    (1) In at least the chamber of each test concentration at 0-hour.
    (2) In at least the chamber of each test concentration at 96-hours 
and every 4 days thereafter, as long as the test is continued.
    (3) In at least one appropriate chamber whenever a malfunction is 
detected in any part of the test substance delivery system.
    (4) Equal aliquots of test solution may be removed from each 
replicate chamber and pooled for analysis.
    (B) Filters and their holders used for determining the dissolved 
test substance concentrations should be prewashed with several volumes 
of distilled water and undergo a final rinse with test solution. Glass 
or stainless steel filter holders are best for organic test substances, 
while plastic holders are best for metals. The sample should be filtered 
within 30 minutes after it is taken from the test chamber.
    (C) The analytical methods used to measure the amount of test 
substance in a sample shall be validated before beginning the test. The 
accuracy of a method should be verified by a method such as using known 
additions. This involves adding a known amount of the test substance to 
three water samples taken from a chamber containing dilution water and 
the same number and species of fish as are used in the test. The nominal 
concentration of the test substance in those samples should span the 
concentration range to be used in the test.
    (D) An analytical method is not acceptable if likely degradation 
products of the test substance give positive or negative interferences, 
unless it is shown that such degradation products are not present in the 
test chambers during the test.
    (E) In addition to analyzing samples of test solution, at least one 
reagent blank, containing all reagents used, should also be analyzed.
    (F) If the measured concentrations of dissolved test substance are 
considerably lower (e.g., <50 percent) than the nominal concentrations, 
the total test substance concentration should be measured in the highest 
test concentration.
    (G) Among replicate test chambers, the measured concentrations shall 
not vary more than 20 percent. The measured concentration of the test 
substance in any chamber during the test should not vary more than 30 
percent from the measured concentration at time 0.
    (H) The mean measured concentration of test substance shall be used 
to calculate all LC60's and to plot all concentration-
response curves.
    (d) Test conditions--(1) Test species--(i) Selection. The test 
species for this test are the rainbow trout (Salmo gairdneri), bluegill 
(Lepomis macrochirus) and fathead minnow (Pimephales promelas). The 
particular species of fish to be used will be prescribed in the test 
rule.
    (ii) Age and condition of fish. (A) Juvenile fish shall be used. 
Fish used in a particular test shall be the same age and be of normal 
size and appearance for their age. The longest fish shall not be more 
than twice the length of the shortest.
    (B) All newly acquired fish should be quarantined and observed for 
at least 14 days prior to use in a test.
    (C) Fish shall not be used for a test if they appear stressed or if 
more than five percent die during the 48 hours immediately prior to the 
test.
    (iii) Acclimation of test fish. (A) If the holding water is not from 
the same source as the test dilution water, acclimation to the dilution 
water should be done gradually over a 48-hour period. The fish should 
then be held an additional 14 days in the dilution water prior to 
testing. Any changes in water temperature should not exceed 3 [deg]C per 
day. Fish should be held for a minimum of 7 days at the test temperature 
prior to testing.
    (B) During the final 48-hours of acclimation, fish should be 
maintained in facilities with background colors and light intensities 
similar to those of the testing area and should not be fed.
    (2) Facilities--(i) General. Facilities needed to perform this test 
include:
    (A) Flow-through tanks for holding and acclimating fish.
    (B) A mechanism for controlling and maintaining the water 
temperature

[[Page 119]]

during the holding, acclimation and test periods.
    (C) Apparatus for straining particulate matter, removing gas 
bubbles, or insufficient dissolved oxygen, respectively.
    (D) Apparatus for providing a 16-hour light and 8-hour dark 
photoperiod with a 15- to 30-minute transition period.
    (E) Chambers for exposing test fish to the test substance.
    (F) A test substance delivery system for flow-through tests.
    (ii) Construction materials. Construction materials and commercially 
purchased equipment that may contact the stock solution, test solution, 
or dilution water should not contain substances that can be leached or 
dissolved into aqueous solutions in quantities that can alter the test 
results. Materials and equipment that contact stock or test solutions 
should be chosen to minimize sorption of test chemicals. Glass, 
stainless steel, and perfluorocarbon plastic should be used whenever 
possible. Concrete, fiberglass, or plastic (e.g., PVC) may be used for 
holding tanks, acclimation tanks, and water supply systems, but they 
should be used to remove rust particles. Rubber, copper, brass, 
galvanized metal, epoxy glues, and lead should not come in contact with 
the dilution water, stock solution, or test solution.
    (iii) Test substance delivery system. In flow-through tests, 
diluters, metering pump systems, or other suitable devices should be 
used to deliver the test substance to the test chambers. The system used 
should be calibrated before each test. Calibration includes determining 
the flow rate through each chamber and the concentration of the test 
substance delivered to each chamber. The general operation of the test 
substance delivery system should be checked twice daily during a test. 
The 24-hour flow rate through a test chamber should be a minimum of 6 
tank volumes. During a test, the flow rates should not vary more than 10 
percent from one test chamber to another.
    (iv) Test chambers. Test chambers made of stainless steel should be 
welded, not soldered. Test chambers made of glass should be fused or 
bonded using clear silicone adhesive. As little adhesive as possible 
should be left exposed in the interior of the chamber.
    (v) Cleaning of test system. Test substance delivery systems and 
test chambers should be cleaned before each test. They should be washed 
with detergent and then rinsed in sequence with clean water, pesticide-
free acetone, clean water, and 5 percent nitric acid, followed by two or 
more changes of dilution water.
    (vi) Dilution water. (A) Clean surface or ground water reconstituted 
water, or dechlorinated tap water is acceptable as dilution water if the 
test fish will survive in it for the duration of the holding, 
acclimating, and testing periods without showing signs of stress, such 
as discoloration, hemorrhaging, disorientation or other unusual 
behavior. The quality of the dilution water should be constant and 
should meet the following specifications measured at least twice a year:

------------------------------------------------------------------------
                 Substance                             Maximum
------------------------------------------------------------------------
Particulate matter........................  20 mg/liter.
Total organic carbon or...................  2 mg/liter.
      chemical oxygen demand..............  5 mg/liter.
Un-ionized ammonia........................  1 [micro]g/liter.
Residual chlorine.........................  1 [micro]g/liter.
Total organochloring pesticides...........  50 [micro]g/liter.
Total organocholorine pesticides plus       50 [micro]g/liter.
 polychlorinated biphenyls (PCBs).
      or organic chlorine.................  25 [micro]g/liter.
------------------------------------------------------------------------

    (B) The concentration of dissolved oxygen in the dilution water 
should be between 90 and 100 percent saturation; 9.8 to 10.9 mg/l for 
tests with trout, and 8.0 to 8.9 mg/l for tests with bluegill or fathead 
minnow at sea level. If necessary, the dilution water can be aerated 
before the addition of the test substance. All reconstituted water 
should be aerated before use. Buffered soft water should be aerated 
before but not after the addition of buffers.
    (C) If disease organisms are present in the dilution water in 
sufficient numbers to cause infection, they should be killed or removed 
by suitable equipment.
    (D) Glass distilled or carbon filtered deionized water with a 
conductivity less than 1 micromho/cm is acceptable for use in making 
reconstituted water. If the reconstituted water is prepared from a 
ground or surface water source, conductivity, and total organic carbon 
(TOC) or chemical oxygen demand

[[Page 120]]

(COD) should be measured on each batch.
    (vii) Carriers. (A) Distilled water should be used in making stock 
solutions of the test substance. If the stock volume however is more 
than 10 percent of the test solution volume, dilution water should be 
used. If a carrier is absolutely necessary to dissolve the test 
substance, the volume used should not exceed the minimum volume 
necessary to dissolve or suspend the test substance in the test 
solution. If the test substance is a mixture, formulation, or commercial 
product, none of the ingredients is considered a carrier unless an extra 
amount is used to prepare the stock solution.
    (B) Triethylene glycol and dimethyl formamide are the prefered 
carriers, but acetone may also be used. The concentration of triethylene 
glycol in the test solution should not exceed 80 mg/1. The concentration 
of dimethyl formamide or acetone in the test solution should not exceed 
5.0 mg/1.
    (3) Test parameters--(i) Loading. The number of fish placed in a 
test chamber should not be so great as to affect the results of the 
test. The loading should not be so great that the test substance 
concentrations are decreased by more than 20 percent due to uptake by 
the fish. In static tests, loading should not exceed 0.5 grams of fish 
per liter of solution in the test chamber at any one time. In flow-
through tests loading should not exceed 0.5 grams of fish per liter of 
test solution passing through the chamber in 24 hours. These loading 
rates should be sufficient to maintain the dissolved oxygen 
concentration above the recommended levels and the ammonia concentration 
below 20 [micro]g/l.
    (ii) Dissolved oxygen concentration. (A) During static tests with 
rainbow trout the dissolved oxygen in each test chamber shall be greater 
than 5.5 mg/1. In tests with bluegill and fathead minnows, the DO shall 
be maintained above 4.5 mg/1.
    (B) During flow-through tests the dissolved oxygen concentration 
shall be maintained above 8.2 mg/1 in tests with trout and above 6.6 mg/
l in tests with bluegills or fathead minnows.
    (iii) Temperature. The test temperature shall be 22 [deg]C for 
bluegill and fathead minnow and 12 [deg]C for rainbow trout. Excursions 
from the test temperature shall be no greater than 2 [deg]C. The temperature shall be measured at least 
hourly in one test chamber.
    (iv) Light. A 16-hour light and 8-hour dark photoperiod should be 
maintained.
    (e) Reporting. The sponsor shall submit to the EPA all data 
developed by the test that are suggestive or predictive of toxicity. In 
addition to the reporting requirements prescribed in part 792--Good 
Laboratory Practice Standards of this chapter, the reported test data 
shall include the following:
    (1) The source of the dilution water, a description of any 
pretreatment, and the measured hardness, acidity, alkalinity, pH, 
conductivity, TOC or COD and particulate matter.
    (2) A description of the test chambers, the depth and volume of 
solution in the chamber, the specific way the test was begun (e.g., 
conditioning, test substance additions), and for flow-through tests, a 
description of the test substance delivery system.
    (3) Detailed information about the test fish, including the 
scientific name and method of verification, average weight (grams, wet 
weight), standard length, age, source, history, observed diseases, 
treatments, and mortalities, acclimation procedures, and food used.
    (4) The number of replicates used, the number of organisms per 
replicate, the loading rate, and the flow rate for flow-through tests.
    (5) The measured DO, pH and temperature and the lighting regime.
    (6) The solvent used, the test substance concentration in the stock 
solution, the highest solvent concentration in the test solution and a 
description of the solubility determinations in water and solvents if 
used.
    (7) The concentrations of the test substance at each test 
concentration just before the start of the test and at all subsequent 
sampling periods.
    (8) The number of dead and live tests organisms, the percentage of 
organisms that died, and the number that showed any abnormal effects in 
the control and in each test chamber at each observation period.
    (9) The 96-hour LC50, and when sufficient data have been 
generated, the 24-, 48-, 72-, and incipient LC50 values,

[[Page 121]]

their 95 percent confidence limits, and the methods used to calculate 
the LC50 values and their confidence limits.
    (10) When observed, the observed no effect concentration (the 
highest concentration tested at which there were no mortalities or 
abnormal behavioral or physiological effects).
    (11) The concentration-response curve at each observation period for 
which a LC50 was calculated.
    (12) Methods and data records of all chemical analyses of water 
quality parameters and test substance concentrations, including method 
validations and reagent blanks.

[50 FR 39321, Sept. 27, 1985, as amended at 52 FR 19062, May 20, 1987; 
54 FR 29715, July 14, 1989; 54 FR 33148, Aug. 11, 1989]



Sec. 797.1600  Fish early life stage toxicity test.

    (a) Purpose. This guideline is intended to be used for assessing the 
propensity of chemical substances to produce adverse effects to fish 
during the early stages of their growth and development. This guideline 
describes the conditions and procedures for the continuous exposure of 
several representative species to a chemical substance during egg, fry 
and early juvenile life stages. The Environmental Protection Agency 
(EPA) will use data from this test in assessing the potential hazard of 
the test substance to the aquatic environment.
    (b) Definitions. The definitions in section 3 of the Toxic 
Substances Control Act (TSCA) and the definitions in part 792--Good 
Laboratory Practice Standards, apply to this section. In addition, the 
following definitions are applicable to this specific test guideline:
    (1) ``Acclimation'' physiological or behavioral adaptation of 
organisms to one or more environmental conditions associated with the 
test method (e.g., temperature, hardness, pH).
    (2) ``Carrier'' solvent or other agent used to dissolve or improve 
the solubility of the test substance in dilution water.
    (3) ``Conditioning'' exposure of construction materials, test 
chambers, and testing apparatus to dilution water or to the test 
solution prior to the start of the test in order to minimize the 
sorption of test substance onto the test facilities or the leachig of 
substances from test facilities into the dilution water or the test 
solution.
    (4) ``Control'' an exposure of test organisms to dilution water only 
or dilution water containing the test solvent or carrier (no toxic agent 
is intentionally or inadvertently added).
    (5) ``Dilution water'' the water used to produce the flow-through 
conditions of the test to which the test substance is added and to which 
the test species is exposed.
    (6) ``Early life stage toxicity test'' a test to determine the 
minimum concentration of a substance which produces a statistically 
significant observable effect on hatching, survival, development and/or 
growth of a fish species continuously exposed during the period of their 
early development.
    (7) ``Embryo cup'' a small glass jar or similar container with a 
screened bottom in which the embryos of some species (i.e., minnow) are 
placed during the incubation period and which is normally oscillated to 
ensure a flow of water through the cup.
    (8) ``Flow through'' refers to the continuous or very frequent 
passage of fresh test solution through a test chamber with no recycling.
    (9) ``Hardness'' the total concentration of the calcium and 
magnesium ions in water expressed as calcium carbonate (mg 
CaCO3/liter).
    (10) ``Loading'' the ratio of biomass (grams of fish, wet weight) to 
the volume (liters) of test solution passing through the test chamber 
during a specific interval (normally a 24-hr. period).
    (11) ``No observed effect concentration (NOEC)'' the highest tested 
concentration in an acceptable early life stage test: (i) which did not 
cause the occurrence of any specified adverse effect (statistically 
different from the control at the 95 percent level); and (ii) below 
which no tested concentration caused such an occurrence.
    (12) ``Observed effect concentration (OEC)'' the lowest tested 
concentration in an acceptable early life stage test: (i) Which caused 
the occurrence of any specified adverse effect (statistically different 
from the control at the 95 percent level); and (ii) above which all

[[Page 122]]

tested concentrations caused such an occurrence.
    (13) ``Replicate'' two or more duplicate tests, samples, organisms, 
concentrations, or exposure chambers.
    (14) ``Stock solution'' the source of the test solution prepared by 
dissolving the test substance in dilution water or a carrier which is 
then added to dilution water at a specified, selected concentration by 
means of the test substance delivery system.
    (15) ``Test chamber'' the individual containers in which test 
organisms are maintained during exposure to test solution.
    (16) ``Test solution'' dilution water with a test substance 
dissolved or suspended in it.
    (17) ``Test substance'' the specific form of a chemical substance or 
mixture that is used to develop data.
    (c) Test Procedures--(1) Summary of test. (i) The early life stage 
toxicity test with fish involves exposure of newly fertilized embryos to 
various concentrations of a test substance. Exposure continues for 28 
days post hatch for the minnows and 60 days post hatch for the trout 
species. During this time various observations and measurements are made 
in a specific manner and schedule in order to determine the lowest 
effect and highest no-effect concentrations of the test substance.
    (ii) A minimum of five exposure (treatment) concentrations of a test 
substance and one control are required to conduct an early life stage 
toxicity test. The concentration of the test substance in each treatment 
is usually 50 percent of that in the next higher treatment level.
    (iii) For each exposure concentration of the test substance and for 
each control (i.e., regular control and carrier control is required) 
there shall be:
    (A) At least two replicate test chambers, each containing one or 
more embryo incubation trays or cups; and there shall be no water 
connections between the replicate test chambers;
    (B) At least 60 embryos divided equally in such a manner that test 
results show no significant bias from the distributions, between the 
embryo incubation trays or cups for each test concentration and control 
(i.e., 30 per embryo cup with 2 replicates);
    (C) All surviving larvae divided equally between the test chambers 
for each test concentration and control (e.g., 30 larvae per test 
chamber with 2 replicates).
    (iv) Duration. (A) For fathead minnow and sheepshead minnow a test 
begins when the newly fertilized minnow embryos (less than 48-hours old) 
are placed in the embryo cups and are exposed to the test solution 
concentrations. The test terminates following 28 days of post-hatch 
exposure, i.e., 28 days after the newly hatched fry are transferred from 
the embryo cups into the test chambers.
    (B) For brook trout and rainbow trout a test begins when newly 
fertilized trout embryos (less than 96-hours old) are placed in the 
embryo trays or cups and are exposed to the test solution 
concentrations. The test terminates following 60 days of post-hatch 
exposure (for an approximate total exposure period of 90 days).
    (C) For silverside a test begins with newly fertilized embryos (less 
than or equal to 48 hours old) and is terminated 28 days after hatching. 
The chorionic fibrils should be cut before randomly placing the embryos 
in the egg incubation cups.
    (2) [Reserved]
    (3) Range-finding test. (i) A range finding test is normally 
performed with the test substance to determine the test concentrations 
to be used in the early life stage toxicity test, especially when the 
toxicity is unknown. It is recommended that the test substance 
concentrations be selected based on information gained from a 4- to 10-
day flow-through toxicity test with juveniles of the selected test 
species.
    (ii) The highest concentration selected for the early life stage 
toxicity test should approximate the lowest concentration indicated in 
any previous testing to cause a significant reduction in survival. The 
range of concentrations selected is expected to include both observed 
effect and no-observed effect levels. The dilution factor between 
concentrations is normally 0.50, however, other dilution factors may be 
used as necessary.
    (4) Definitive test--(i) General. (A) A test shall not be initiated 
until after the test conditions have been met and

[[Page 123]]

the test substance delivery system has been observed functioning 
properly for 48-hours. This includes temperature stability, flow 
requirements of dilution water, lighting requirements, and the function 
of strainers and air traps included in the water-supply system, and 
other conditions as specified previously.
    (B) New holding and test facilities should be tested with sensitive 
organisms (i.e., juvenile test species or daphnids) before use to assure 
that the facilities or substances possibly leaching from the equipment 
will not adversely affect the test organisms during an actual test.
    (C) Embryos should be acclimated for as long as practical to the 
test temperature and dilution water prior to the initiation of the test.
    (D) When embryos are received from an outside culture source (i.e., 
rainbow and brook trout) at a temperature at variance with the 
recommended test temperature they shall be acclimated to the test 
temperature. When eggs are received, they should be immediately unpacked 
and the temperature of the surrounding water determined. Sudden 
temperature changes should be avoided. Acclimation to the appropriate 
test temperature should be accomplished within a period of 6 hours, and 
should incorporate the use of dilution water.
    (E) Embryos should be visually inspected prior to placement in the 
embryo cups or screen trays. All dead embryos shall be discarded. Dead 
embryos can be discerned by a change in coloration from that of living 
embryos (e.g., trout embryos turn white when dead). During visual 
inspection, empty shells, opaque embryos, and embryos with fungus or 
partial shells attached shall be removed and discarded. If less than 50 
percent of the eggs to be used appear to be healthy, all embryos in such 
a lot shall be discarded.
    (ii) Embryo incubation procedures. (A) Embryos can be distributed to 
the embryo cups or screen trays using a pipette with a large bore or a 
similar apparatus. Newly-hatched silverside fry are very sensitive to 
handling; the egg incubation cups should not be handled at all the first 
5 days after hatching begins. Just before hatching is expected to begin, 
the embryos should be transferred to clean incubation cups. Trout 
embryos can be distributed by using a small container which has been 
precalibrated to determine the approximate number of embryos it can 
hold; embryos are measured volumetrically in this manner, and are then 
poured onto the screen tray (or embryo cup). Trout embryos should be 
separated on the screen tray so that they are not in contact with each 
other. A final count will ensure the actual number on the screen tray. 
After random assignment, the screen trays or embryo cups are placed in 
the test chambers.
    (B) Each day until hatch the embryos are visually examined. Minnow 
embryos may be examined with the aid of a magnifying viewer. Trout 
embryos should not be touched. Trout embryos should be maintained in low 
intensity light or in darkness until 1-week post hatch, and are usually 
examined with the aid of a flashlight or under low intensity light. Dead 
embryos should be removed and discarded. Any embryos which are heavily 
infected with fungus shall be discarded and shall be subtracted from the 
initial number of embryos used as a basis for the calculations of 
percentage hatch.
    (C) When embryos begin to hatch they should not be handled.
    (iii) Initiation of fry exposure. (A) Forty-eight hours after the 
first hatch in each treatment level, or when hatching is completed, the 
live young fish shall be counted and transferred from each embryo cup 
into the appropriate test chamber. For silverside, all surviving fry are 
not counted until six days after hatching and are not transferred to 
embryo cups. All of the normal and abnormal fry shall be gently released 
into the test chamber by allowing the fry to swim out of each embryo 
cup; nets shall not be used. The trout embryos incubated on screen trays 
will hatch out in the test chambers, therefore handling of fish is not 
necessary.
    (B) If necessary, fry can be transferred from one replicate embryo 
cup to the other replicate within a test concentration to achieve equal 
numbers in each replicate chamber.
    (C) The number of live fry, live normal fry, live embryos, dead 
embryos and unaccounted for embryos for each

[[Page 124]]

cup shall be recorded when hatching is deemed complete. Those fry which 
are visibly (without the use of a dissecting scope or magnifying viewer) 
lethargic or grossly abnormal (either in swimming behavior or physical 
appearance) shall be counted. Late hatching embryos shall be left in the 
embryo cups to determine if they will eventually hatch or not. The range 
of time-to-hatch (to the nearest day) for each cup shall be recorded.
    (iv) Time to first feeding. (A) The first feeding for the fathead 
and sheepshead minnow fry shall begin shortly after transfer of the fry 
from the embryo cups to the test chambers. Silversides are fed the first 
day after hatch. Trout species initiate feeding at swim-up. The trout 
fry shall be fed trout starter mash three times a day ad libitum, with 
excess food siphoned off daily. The minnow fry shall be fed live newly-
hatched brine shrimp nauplii (Artemia salina) at least three times a 
day.
    (B) For the first seven days, feeding shall be done at minimum 
intervals of four hours (i.e., 8 am, 12 noon, and 4 pm); thereafter the 
fry shall be fed as indicated below.
    (v) Feeding. (A) The fathead and sheepshead minnow fry shall be fed 
newly hatched brine shrimp nauplii for the duration of the test at 
approximately 4-hour intervals three times a day during the week and 
twice on the weekend after the first week. Trout fry shall be fed at 
similar intervals and may receive live brine shrimp nauplii in addition 
to the trout starter food after the first week. Between days 1 and 8 
after first hatching, silverside fry are fed the rotifer, Brachionus 
plicatilis, three times daily at a concentration of 5,000 to 10,000 
organisms per egg cup (based on 15 fish/cup). From days 9 to 11, the fry 
shall be fed approximately 2,500 newly hatched brine shrimp (Artemia) 
nauplii and 5,000 to 10,000 rotifers twice daily. For the remainder of 
the test, the fish will be fed brine shrimp exclusively. The number of 
organisms used should be gradually increased to approximately 5,000 
nauplii by test day 28.
    (B) An identical amount of food should be provided to each chamber. 
Fish should be fed ad libitum for 30 minutes with excess food siphoned 
off the bottom once daily if necessary.
    (C) Fish should not be fed for the last 24 hours prior to 
termination of the test.
    (vi) Carriers. Water should be used in making up the test stock 
solutions. If carriers other than water are absolutely necessary, the 
amount used should be the minimum necessary to achieve solution of the 
test substance. Triethylene glycol and dimethyl formamide are preferred, 
but ethanol and acetone can be used if necessary. Carrier concentrations 
selected should be kept constant at all treatment levels.
    (vii) Controls. Every test requires a control that consists of the 
same dilution water, conditions, procedures, and test organisms from the 
same group used in the other test chambers, except that none of the test 
substance is added. If a carrier (solvent) is used, a separate carrier 
control is required in addition to the regular control. The carrier 
control shall be identical to the regular control except that the 
highest amount of carrier present in any treatment is added to this 
control. If the test substance is a mixture, formulation, or commercial 
product, none of the ingredients is considered a carrier unless an extra 
amount is used to prepare the stock solution.
    (viii) Randomization. The location of all test chambers within the 
test system shall be randomized. A representative sample of the test 
embryos should be impartially distributed by adding to each cup or 
screen tray no more than 20 percent of the number of embryos to be 
placed in each cup or screen tray and repeating the process until each 
cup or screen tray contains the specified number of embryos. 
Alternatively, the embryos can be assigned by random assignment of a 
small group (e.g., 1 to 5) of embryos to each embryo cup or screen tray, 
followed by random assignment of a second group of equal number to each 
cup or tray, which is continued until the appropriate number of embryos 
are contained in each embryo cup or screen tray. The method of 
randomization used shall be reported.
    (ix) Observations. During the embryo exposure period observations 
shall be made to check for mortality. During

[[Page 125]]

the exposure period of the fry, observations shall be made to check for 
mortality and to note the physical appearance and behavior of the young 
fish. The biological responses are used in combination with physical and 
chemical data in evaluating the overall lethal and sublethal effects of 
the test substance. Additional information on the specific methodology 
for the data obtained during the test procedure are discussed in the 
following sections.
    (x) Biological data. (A) Death of embryos shall be recorded daily.
    (B) When hatching commences, daily records of the number of embryos 
remaining in each embryo cup are required. This information is necessary 
to quantify the hatching success. A record of all deformed larvae shall 
be kept throughout the entire post-hatch exposure. Time to swim-up shall 
be recorded for the trout. Upon transfer of fry from the embryo cups to 
the test chambers, daily counts of the number of live fish should be 
made. At a minimum, live fish shall be counted on days 4, 11, 18, 25 and 
(weekly thereafter for the trout species) finally on termination of the 
test.
    (C) The criteria for death of young fish is usually immobility, 
especially absence of respiratory movement, and lack of reaction to 
gentle prodding. Deaths should be recorded daily and dead fish removed 
when discovered.
    (D) Daily and at termination of the test, the number of fish that 
appear (without the use of a magnifying viewer) to be abnormal in 
behavior (e.g., swimming erratic or uncoordinated, obviously lethargic, 
hyperventilating, or over excited, etc.) or in physical appearance 
(e.g., hemorrhaging, producing excessive mucous, or are discolored, 
deformed, etc.) shall be recorded and reported in detail.
    (E) All physical abnormalities (e.g., stunted bodies, scoliosis, 
etc.) shall be photographed and the deformed fish which die, or are 
sacrificed at the termination of the test, shall be preserved for 
possible future pathological examination.
    (F) At termination, all surviving fish shall be measured for growth. 
Standard length measurements should be made directly with a caliper, but 
may be measured photographically. Measurements shall be made to the 
nearest millimeter (0.1 mm is desirable). Weight measurements shall also 
be made for each fish alive at termination (wet, blotted dry, and to the 
nearest 0.01 g for the minnows and 0.1 g for the trout). If the fish 
exposed to the toxicant appear to be edematous compared to control fish, 
determination of dry, rather than wet, weight is recommended.
    (G) Special physiological, biochemical and histological 
investigations on embryos, fry, and juveniles may be deemed appropriate 
and shall be performed on a case by case basis.
    (5) Test results. (i) Data from toxicity tests are usually either 
continuous (e.g. length or weight measurements) or dichotomous (e.g. 
number hatching or surviving) in nature. Several methods are available 
and acceptable for statistical analysis of data derived from early life 
stage toxicity tests; however, the actual statistical methodology to 
analyze and interpret the test results shall be reported in detail.
    (ii) The significance level for all statistical testing shall be a 
minimum of P=0.05 (95 percent confidence level).
    (A) Example of statistical analysis. (1) Mortality data for the 
embryonic stage, fry stage and for both stages in replicate exposure 
chambers should first be analyzed using a two-way analysis of variance 
(ANOVA) with interaction model. This analysis will determine if 
replicates are significantly different from each other. If a significant 
difference between replicates or a significant interaction exists, cause 
for the difference should be determined. Modification should then be 
made in the test apparatus or in handling procedures for future toxicity 
tests. Further calculations should incorporate the separation of 
replicates. If no significant difference is observed, replicates may be 
pooled in further analyses.
    (2) After consideration of replicate responses, mortality data 
should then be subjected to one-way ANOVA. The purpose of this analysis 
is to determine if a significant difference exists in the percentage 
mortality between control fish and those exposed to the test material.

[[Page 126]]

    (3) If the one-way ANOVA results in a F ratio that is significant, 
it would be acceptable to perform t-tests on the control versus each 
concentration. A second technique is to identify treatment means that 
are significantly different; this method should involve the additional 
assumption that the true mean response decreases generally with 
increasing concentration. The researcher may also be interested in 
determining significant differences between concentrations.
    (4) Growth data should also be analyzed by one-way ANOVA with the 
inclusion of a covariate to account for possible differences in growth 
of surviving fry in embryo cup(s) that contain fewer individuals. This 
condition can occur in cases when the same amount of food is given to 
each test chamber regardless of the number of survivors.
    (B) Test data to be analyzed. Data to be statistically analyzed are:
    (1) Percentage of healthy, fertile embryos at 40-48 hours after 
initiation of the test. Percentage is based upon initial number used.
    (2) Percentage of embryos that produce live fry for release into 
test chambers. Percentage is based on number of embryos remaining after 
thinning.
    (3) Percentage of embryos that produce live, normal fry for release 
into test chambers. Percentage is based upon number of embryos remaining 
after thinning.
    (4) Percentage of fry survival at swim-up for trout. Percentage is 
based upon number of embryos remaining after thinning.
    (5) Percentage of embryos that produce live fish at end of test. 
Percentage is based upon number of embryos remaining after thinning.
    (6) Percentage of embryos that produce live, normal fish at end of 
test. Percentage is based upon number of embryos remaining after 
thinning.
    (7) Weights and lengths of individual fish alive at the end of the 
test.
    (C) It is important that fish length and weight measurements be 
associated with individual test chambers since the density of the fish 
and available food should be considered in the growth of the organism.
    (iii) Acceptability criteria. (A) An early life stage toxicity test 
is not acceptable unless at least one of the following criteria is 
significantly different (p=0.05) from control organisms when compared 
with treated organisms, and the responses are concentration-dependent: 
mortality of embryos, hatching success, mortality of fry (at swim-up for 
trout), total mortality throughout the test, and growth (i.e. weight). 
If no significant effects occur, but the concentrations tested were the 
highest possible due to solubility or other physio-chemical limitations, 
the data will be considered for acceptance.
    (B) In addition to obtaining significant effects on the exposed test 
species, a measure of acceptability in the response of control fish is 
also required.
    (C) A test is not acceptable if the average survival of the control 
fish at the end of the test is less than 80 percent or if survival in 
any one control chamber is less than 70 percent. For silversides, a test 
is not acceptable if the average overall survival of the control embryos 
and fish at the end of the test is less than 60 percent.
    (D) If a carrier is used, the criteria for effect (mortality of 
embryos and fry, growth, etc.) used in the comparison of control and 
exposed test organisms shall also be applied to the control and control 
with carrier chambers. For the test to be considered acceptable, no 
significant difference shall exist between these criteria.
    (E) A test is not acceptable if the relative standard deviation 
(RSD=100 times the standard deviation divided by the mean) of the 
weights of the fish that were alive at the end of the test in any 
control test chamber is greater than 40 percent.
    (6) Analytical measurements--(i) Analysis of water quality. 
Measurement of certain dilution water quality parameters shall be 
performed every 6 months, to determine the consistency of the dilution 
water quality. In addition, if data in 30-day increments are not 
available to show that freshwater dilution water is constant, 
measurements of hardness, alkalinity, pH, acidity, conductivity, TOC or 
COD and particulate matter should be conducted once a week in the 
highest test substance concentration. Measurement of

[[Page 127]]

calcium, magnesium, sodium, potassium, chloride, and sulfate is 
desirable.
    (ii) Dissolved oxygen measurement. The dissolved oxygen 
concentration shall be measured in each test chamber at the beginning of 
the test and at least once weekly thereafter (as long as live organisms 
are present) in two replicates of the control and the high, medium, and 
low test substance concentrations.
    (iii) Temperature measurement. Temperatures shall be recorded in all 
test chambers at the beginning of the test, once weekly thereafter and 
at least hourly in one test chamber. When possible, the hourly 
measurement shall be alternated between test chambers and between 
replicates.
    (iv) Test substance measurement. (A) Prior to the addition of the 
test substance to the dilution water, it is recommended that the test 
substance stock solution be analyzed to verify the concentration. After 
addition of the test substance, the concentration of test substance 
should be measured at the beginning of the test in each test 
concentration and control(s), and at least once a week thereafter. Equal 
aliquots of test solution may be removed from each replicate chamber and 
pooled for analysis. If a malfunction in the delivery system is 
discovered, water samples shall be taken from the affected test chambers 
immediately and analyzed.
    (B) The measured concentration of test substance in any chamber 
should be no more than 30 percent higher or lower than the concentration 
calculated from the composition of the stock solution and the 
calibration of the test substance delivery system. If the difference is 
more than 30 percent, the concentration of test substance in the 
solution flowing into the exposure chamber (influent) should be 
analyzed. These results will indicate whether the problem is in the 
stock solution, the test substance delivery system or in the test 
chamber. Measurement of degradation products of the test substance is 
recommended if a reduction of the test substance concentration occurs in 
the test chamber.
    (v) Sampling and analysis methodology. (A) Generally, total test 
substance measurements are sufficient; however, the chemical 
characteristics of the test substance may require both dissolved and 
suspended test substance measurements.
    (B) For measurement of the test substance, water samples shall be 
taken midway between the top, bottom, and sides of the test chamber and 
should not include any surface scum or material stirred up from the 
bottom or sides. Samples of test solutions shall be handled and stored 
appropriately to minimize loss of test substance by microbial 
degradation, photodegradation, chemical reaction, volatilization, or 
sorption.
    (C) Chemical and physical analyses shall be performed using 
standardized methods whenever possible. The analytical method used to 
measure the concentration of the test substance in the test solution 
shall be validated before the beginning of the test. At a minimum, a 
measure of the accuracy of the method should be obtained on each of two 
separate days by using the method of known additions, and using dilution 
water from a tank containing test organisms. Three samples should be 
analyzed at the next-to-lowest test substance concentration. It is also 
desirable to study the accuracy and precision of the analytical method 
for test guideline determination by use of reference (split) samples, or 
interlaboratory studies, and by comparison with alternative, reference, 
or corroborative methods of analysis.
    (D) An analytical method is not acceptable if likely degradation 
products of the test substance, such as hydrolysis and oxidation 
products, give positive or negative interferences, unless it is shown 
that such degradation products are not present in the test chambers 
during the test. In general, atomic absorption spectrophotometric 
methods for metals and gas chromatographic methods for organic compounds 
are preferable to colorimetric methods.
    (E) In addition to analyzing samples of test solution, at least one 
reagent blank also should be analyzed when a reagent is used in the 
analysis. Also, at least one sample for the method of known additions 
should be prepared by adding test substance at the concentration used in 
the toxicity test.

[[Page 128]]

    (d) Test conditions--(1) Test species. (i) One or more of the 
recommended test species will be specified in rules under part 799 of 
this chapter requiring testing of specific chemicals. The recommended 
test species are:
    (A) Fathead minnow (Pimephales promelas Rafinesque).
    (B) Sheepshead minnow (Cyprinodon variegatus).
    (C) Brook trout (Salvelinus fontinalis).
    (D) Rainbow trout (Salmo gairdneri).
    (E) Atlantic silverside (Menidia menidia).
    (F) Tidewater silverside (Menidia peninsulae).
    (ii) Embryos used to initiate the early life stage test shall be 
less than 48 hours old for the fathead and sheepshead minnows, 
silversides, and less than 96 hours old for the brook trout and rainbow 
trout. In addition, the following requirements shall be met:
    (A) All embryos used in the test shall be from the same source. 
Embryos shall be obtained from a stock cultured in-house when possible, 
and maintained under the same parameters as specified for the test 
conditions. When it is necessary to obtain embryos from an external 
source, caution should be exercised to ensure embryo viability and to 
minimize the possibility of fungal growth. A description of the brood 
stock history or embryo source shall be made available to EPA upon 
request.
    (B) Test species shall be cared for and handled properly in order to 
avoid unnecessary stress. To maintain test species in good condition and 
to maximize growth, crowding shall be prevented, and the dissolved 
oxygen level shall be maintained near saturation.
    (C) Embryos and fish shall be handled as little as possible. Embryos 
shall be counted and periodically inspected until hatching begins. When 
larvae begin to hatch, they shall not be handled. Transfer of minnow 
larvae from embryo cups to test chambers shall not involve the use of 
nets. No handling is necessary following introduction into the test 
chambers until termination of the test.
    (D) If fathead minnow embryos are obtained from in-house culture 
units, the embryos should be gently removed from the spawning substrate. 
The method for separating the fertilized eggs from the substrate is 
important and can affect the viability of the embryos; therefore the 
finger-rolling procedure is recommended.
    (E) Disease treatment. Chemical treatments to cure or prevent 
diseases should not be used before, and should not be used during a 
test. All prior treatments of brood stock should be reported in detail. 
Severely diseased organisms should be destroyed.
    (2) Test facilities--(i) Construction materials. Construction 
materials and equipment that contact stock solutions, test solutions, or 
dilution water into which test embryos or fish are placed should not 
contain any substances that can be leached or dissolved into aqueous 
solutions in quantities that can affect test results. Materials and 
equipment that contact stock or test solutions should be chosen to 
minimize sorption of test chemicals from dilution water. Glass, 
316 stainless steel, nylon screen and perfluorocarbon plastic 
(e.g., Teflon) are acceptable materials. Concrete or rigid 
(unplasticized) plastic may be used for holding and acclimation tanks, 
and for water supply systems, but they should be thoroughly conditioned 
before use. If cast iron pipe is used in freshwater supply systems, 
colloidal iron may leach into the dilution water and strainers should be 
used to remove rust particles. Natural rubber, copper, brass, galvanized 
metal, epoxy glues, and flexible tubing should not come in contact with 
dilution water, stock solutions, or test solutions.
    (ii) Test chambers (exposure chambers). (A) Stainless steel test 
chambers should be welded or glued with silicone adhesive, and not 
soldered. Glass should be fused or bonded using clear silicone adhesive. 
Epoxy glues are not recommended, but if used ample curing time should be 
allowed prior to use. As little adhesive as possible should be in 
contact with the water.
    (B) Many different sizes of test chambers have been used 
successfully. The size, shape and depth of the test chamber is 
acceptable if the specified flow rate and loading requirements can be 
achieved.
    (C) The actual arrangement of the test chambers can be important to 
the statistical analysis of the test data.

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Test chambers can be arranged totally on one level (tier) side by side, 
or on two levels with each level having one of the replicate test 
substance concentrations or controls. Regardless of the arrangement, it 
shall be reported in detail and considered in the data analysis.
    (iii) Embryo incubation apparatus. (A) Recommended embryo incubation 
apparatus include embryo cups for the minnow species and screen trays 
for the trout species, although embryo cups can be used for the trout 
species. Embryo cups are normally constructed from approximately 4-5 cm 
inside diameter, 7-8 cm high, glass jars with the end cut off or similar 
sized sections of polyethylene tubing. One end of the jar or tubing is 
covered with stainless steel or nylon screen (approximately 40 meshes 
per inch is recommended). Embryo cups for silversides are normally 
constructed by using silicone adhesive to glue a 10-cm high, 363-um 
nylon mesh tube inside a 9-cm I.D. glass Petri dish bottom. The embryo 
cups shall be appropriately labeled and then suspended in the test 
chamber in such a manner as to ensure that the test solution regularly 
flows through the cup and that the embryos are always submerged but are 
not agitated too vigorously. Cups may be oscillated by a rocker arm 
apparatus with a low rpm motor (e.g., 2 rpm) to maintain the required 
flow of test water. The vertical-travel distance of the rocker arm 
apparatus during oscillation is normally 2.5-4.0 cm. The water level in 
the test chambers may also be varied by means of a self-starting siphon 
in order to ensure exchange of water in the embryo cups.
    (B) The trout embryo incubation trays can be made from stainless 
steel screen (or other acceptable material such as plastic) of about 3-4 
mm mesh. The screen tray should be supported above the bottom of the 
test chamber by two folds of screen or other devices which function as 
legs or supports. The edges of the screen tray should be turned up to 
prevent bump spills and to prevent the embryos from rolling off in the 
event of excessive turbulence. Suspending or supporting the screen tray 
off the bottom ensures adequate water circulation around the embryos and 
avoids contact of embryos with possible bottom debris.
    (iv) Test substance delivery system. (A) The choice of a specific 
delivery system depends upon the specific properties and requirements of 
the test substance. The apparatus used should accurately and precisely 
deliver the appropriate amount of stock solution and dilution water to 
the test chambers. The system selected shall be calibrated before each 
test. Calibration includes determining the flow rate through each 
chamber, and the proportion of stock solution to dilution water 
delivered to each chamber. The general operation of the test substance 
delivery system shall be checked at least twice daily for normal 
operation throughout the test. A minimum of five test substance 
concentrations and one control shall be used for each test.
    (B) The proportional diluter and modified proportional diluter 
systems and metering pump systems have proven suitable and have received 
extensive use.
    (C) Mixing chambers shall be used between the diluter and the test 
chamber(s). This may be a small container or flow-splitting chamber to 
promote mixing of test substance stock solution and dilution water, and 
is positioned between the diluter and the test chambers for each 
concentration. If a proportional diluter is used, separate delivery 
tubes shall run from the flow-splitting chamber to each replicate test 
chamber. Daily checks on this latter system shall be made.
    (D) Silverside fry are injured easily and are susceptible to 
impingement on the mesh of the incubation cups. Consequently, water flow 
into and out of the cups when counting fry must be at a slow rate. This 
can be accomplished by using small diameter (e.g., 2 mm I.D.) capillary 
tubes to drain the test solution from spitter boxes into the replicate 
test chambers. The use of a self-starting siphon to gradually lower 
(i.e., less than or equal to 1 min.) the water level approximately 2 cm 
in the test chamber is recommended. A minimum water depth of 5 cm should 
be maintained in the cups. Although it may be satisfactory, a rocker-arm 
type apparatus has not yet been used with silversides.

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    (v) Other equipment required. (A) An apparatus for removing 
undesirable organisms, particulate matter and air bubbles.
    (B) An apparatus for aerating water.
    (C) A suitable magnifying viewer for examination of minnow embryos.
    (D) A suitable apparatus for the precise measurement of growth of 
the fish, including both length (e.g., with metric or ruler caliper or 
photographic equipment) and weight.
    (E) Facilities for providing a continuous supply of live brine 
shrimp nauplii (Artemia salina).
    (F) For silversides, facilities for providing a supply of rotifers 
(Brachionus plicatilis) for approximately 11 days.
    (G) Facilities (or access to facilities) for performing the required 
water chemistry analyses.
    (vi) Cleaning of equipment. (A) Test substance delivery systems and 
test chambers should be cleaned before use. Test chambers should be 
cleaned during the test as needed to maintain the dissolved oxygen 
concentration, and to prevent clogging of the embryo cup screens and 
narrow flow passages.
    (B) Debris can be removed with a rubber bulb and large pipette or by 
siphoning with a glass tube attached to a flexible hose. Debris should 
be run into a bucket light enough to observe that no live fish are 
accidentally discarded.
    (vii) Dilution water--(A) General. (1) A constant supply of 
acceptable dilution water should be available for use throughout the 
test. Dilution water shall be of a minimum quality such that the test 
species selected will survive in it for the duration of testing without 
showing signs of stress (e.g., loss of pigmentation, disorientation, 
poor response to external stimuli, excessive mucous secretion, lethargy, 
lack of feeding, or other unusual behavior). A better criterion for an 
acceptable dilution water for tests on early life stages should be such 
that the species selected for testing will survive, grow, and reproduce 
satisfactorily in it.
    (2) The concentration of dissolved oxygen in the dilution water 
(fresh or salt) shall be between 90 percent and 100 percent saturation. 
When necessary, dilution water should be aerated by means of airstones, 
surface aerators, or screen tubes before the introduction of the test 
substance.
    (3) Water that is contaminated with undesirable microoganisms (e.g., 
fish pathogens) shall not be used. If such contamination is suspected, 
the water should be passed through a properly maintained ultraviolet 
sterilizer equipped with an intensity meter before use. Efficacy of the 
sterilizer can be determined by using standard plate count methods.
    (B) Freshwater. (1) Natural water (clean surface or ground water) is 
preferred, however, dechlorinated tap water may be used as a last 
resort. Reconstituted freshwater is not recommended as a practical 
dilution water for the early life stage toxicity test because of the 
large volume of water required.
    (2) Particulate and dissolved substance concentrations should be 
measured at least twice a year and should meet the following 
specifications:

------------------------------------------------------------------------
                Substance                      Concentration maximum
------------------------------------------------------------------------
Particulate matter.......................  <20 mg/liter.
Total organic carbon (TOC)...............  <2 mg/liter.
Chemical oxygen demand (COD).............  <5 mg/liter.
Un-ionized ammonia.......................  <1 [micro]g/liter.
Residual chlorine........................  <1 [micro]g/liter.
Total organoposphorus pesticides.........  <50 ng/liter.
Total organochlorine pesticides plus       <50 ng/liter.
 polychlorinated biphenyls (PCBs).
Total organic chlorine...................  <25 ng/liter.
------------------------------------------------------------------------

    (3) During any one month, freshwater dilution water should not vary 
more than 10 percent from the respective monthly averages of hardness, 
alkalinity and specific conductance; the monthly pH range should be less 
than 0.4 pH units.
    (C) Saltwater. (1) Marine dilution water is considered to be of 
constant quality if the minimum salinity is greater than 15\0\/
00 and the weekly range of the salinity is less than 15\0\/
00. The monthly range of pH shall be less than 0.8 pH units. 
Saltwater shall be filtered to remove larval predators. A pore size of 
<=20 micrometers ([micro]m) is recommended. For silversides, the 
recommended salinity is 20 ppt and shall be maintained between 15 and 25 
ppt throughout testing.
    (2) Artificial sea salts may be added to natural seawater during 
periods of

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low salinity to maintain salinity above 15\0\/00.
    (3) Test parameters--(i) Dissolved oxygen concentration. It is 
recommended that the dissolved oxygen concentration be maintained 
between 90 and 100 percent saturation; but it shall be no less than 75 
percent saturation at all times for both minnow species and between 90 
and 100 percent saturation for the trout species in all test chambers. 
Dilution water in the head box may be aerated, but the test solution 
itself shall not be aerated.
    (ii) Loading and flow rate. (A) The loading in test chambers should 
not exceed 0.1 grams of fish per liter of test solution passing through 
the test chamber in 24 hours. The flow rate to each chamber should be a 
minimum of 6 tank volumes per 24 hours. During a test, the flow rates 
should not vary more than 10 percent from any one test chamber to any 
other.
    (B) A lower loading or higher flow rate or both shall be used if 
necessary to meet the following three criteria at all times during the 
test in each chamber containing live test organisms:
    (1) The concentration of dissolved oxygen shall not fall below 75 
percent saturation for the fathead and sheepshead minnows and 90 percent 
for the rainbow and brook trout;
    (2) The concentration of un-ionized ammonia should not exceed 1 
[micro]g/1; and
    (3) The concentration of toxicant should not be lowered (i.e., 
caused by uptake by the test organisms and/or materials on the sides and 
bottoms of the chambers) more than 20 percent of the mean measured 
concentration.
    (iii) Temperature. (A) The recommended test temperatures are:
    (1) Fathead minnow--25 [deg]C for all life stages.
    (2) Sheepshead minnow--30 [deg]C for all life stages.
    (3) Rainbow and brook trout--10 [deg]C for embryos. 12 [deg]C for 
fry and alevins.
    (4) Atlantic and tidewater silversides--25 [deg]C for all life 
stages.
    (B) Excursions from the test temperature shall be no greater than 
2.0[deg]C. It is recommended that the test system 
be equipped with an automatic alarm system to alert staff of 
instantaneous temperature changes in excess of 2 [deg]C. If the water is 
heated (i.e., for minnow species), precautions should be taken to ensure 
that supersaturation of dissolved gases is avoided. Temperatures shall 
be recorded in all test chambers at the beginning of the test and weekly 
thereafter. The temperature shall be recorded at least hourly in one 
test chamber throughout the test.
    (iv) Light. (A) Brook and rainbow trout embryos shall be maintained 
in darkness or very low light intensity through one week post-hatch, at 
which time a 14-hour light and 10-hour dark photoperiod shall be 
provided.
    (B) For fathead and sheepshead minnows, a 16-hour light and 8-hour 
dark (or 12:12) photoperiod shall be used throughout the test period.
    (C) For silversides, a 14-hour light and 10-hour dark photoperiod 
shall be used throughout the test period.
    (D) A 15-minute to 30-minute transition period between light and 
dark is optional.
    (E) Light intensities ranging from 30 to 100 lumens at the water 
surface shall be provided; the intensity selected should be duplicated 
as closely as possible for all test chambers.
    (e) Reporting. A report of the results of an early life stage 
toxicity test shall include the following:
    (1) Name of test, sponsor, investigator, laboratory, and dates of 
test duration.
    (2) Detailed description of the test substance including its source, 
lot number, composition (identity and concentration of major ingredients 
and major impurities), known physical and chemical properties, and any 
carriers (solvents) or other additives used.
    (3) The source of the dilution water, its chemical characteristics, 
and a description of any pretreatment.
    (4) Detailed information about the test organisms including 
scientific name and how verified and source history, observed diseases, 
treatments, acclimation procedure, and concentration of any contaminants 
and the method of measurement.
    (5) A description of the experimental design and the test chambers, 
the depth and volume of the solution in the chambers, the way the test 
was begun, the number of organisms per treatment, the number of 
replicates, the

[[Page 132]]

loading, the lighting, a description of the test substance delivery 
system, and the flow rate as volume additions per 24 hours.
    (6) Detailed information on feeding of fish during the toxicity 
test, including type of food used, its source, feeding frequency and 
results of analysis (i.e., concentrations) for contaminants.
    (7) Number of embryos hatched, number of healthy embryos, time to 
hatch, mortality of embryos and fry, measurements of growth (weight and 
length), incidence of pathological or histological effects and 
observations of other effects or clinical signs, number of healthy fish 
at end of test.
    (8) Number of organisms that died or showed an effect in the control 
and the results of analysis for concentration(s) of any contaminant in 
the control(s) should mortality occur.
    (9) Methods used for, and the results of (with standard deviation), 
all chemical analyses of water quality and test substance concentration, 
including validation studies and reagent blanks; the average and range 
of the test temperature(s).
    (10) Anything unusual about the test, any deviation from these 
procedures, and any other relevant information.
    (11) A description of any abnormal effects and the number of fish 
which were affected during each period between observations in each 
chamber, and the average concentration of test substance in each test 
chamber.
    (12) Reference to the raw data location.

[50 FR 39321, Sept. 27, 1985, as amended at 52 FR 19064, May 20, 1987]



Sec. 797.1930  Mysid shrimp acute toxicity test.

    (a) Purpose. This guideline is intended for use in developing data 
on the acute toxicity of chemical substances and mixtures 
(``chemicals'') subject to environmental effects test regulations under 
the Toxic Substances Control Act (TSCA) (Pub. L. 94-469, 90 Stat. 2003, 
15 U.S.C. 2601 et seq.). This guideline prescribes a test using mysid 
shrimp as test organisms to develop data on the acute toxicity of 
chemicals. The United States Environmental Protection Agency (EPA) will 
use data from these tests in assessing the hazard of a chemical to the 
aquatic environment.
    (b) Definitions. The definitions in section 3 of the Toxic 
Substances Control Act (TSCA) and in part 792--Good Laboratory Practice 
Standards of this chapter, apply to this test guideline. The following 
definitions also apply to this guideline.
    (1) ``Death'' means the lack of reaction of a test organism to 
gentle prodding.
    (2) ``Flow-through'' means a continuous or an intermittent passage 
of test solution or dilution water through a test chamber or a holding 
or acclimation tank, with no recycling.
    (3) ``LC50'' means that experimentally derived 
concentration of test substance that is calculated to kill 50 percent of 
a test population during continuous exposure over a specified period of 
time.
    (4) ``Loading'' means the ratio of test organisms biomass (grams, 
wet weight) to the volume (liters) of test solution in a test chamber.
    (5) ``Retention chamber'' means a structure within a flow-through 
test chamber which confines the test organisms, facilitating observation 
of test organisms and eliminating loss of organisms in outflow water.
    (6) ``Static system'' means a test chamber in which the test 
solution is not renewed during the period of the test.
    (c) Test procedures--(1) Summary of the test. In preparation for the 
test, test chambers are filled with appropriate volumes of dilution 
water. If a flow-through test is performed, the flow of dilution water 
through each chamber is adjusted to the rate desired. The test substance 
is introduced into each test chamber. In a flow-through test, the rate 
at which the test substance is added is adjusted to establish and 
maintain the desired concentration of test substance in each test 
chamber. The test is started by randomly introducing mysids acclimated 
in accordance with the test design into the test chambers. Mysids in the 
test chambers are observed periodically during the test, the dead mysids 
removed and the findings recorded. Dissolved oxygen

[[Page 133]]

concentration, pH, temperature, salinity, the concentration of test 
substance, and other water quality characteristics are measured at 
specified intervals in test chambers. Data collected during the test are 
used to develop concentration-response curves and LC50 values 
for the test substance.
    (2) [Reserved]
    (3) Range-finding test. (i) A range-finding test should be conducted 
to determine:
    (A) Which life stage (juvenile or young adult) is to be utilized in 
the definitive test.
    (B) The test solution concentrations for the definitive test.
    (ii) The mysids should be exposed to a series of widely spaced 
concentrations of test substance (e.g., 1, 10, 100 mg/l, etc.), usually 
under static conditions.
    (iii) This test should be conducted with both newly hatched juvenile 
(< 24 hours old) and young adult (5 to 6 days old) mysids. For each age 
class (juvenile or young adult), a minimum of 10 mysids should be 
exposed to each concentration of test substance for up to 96 hours. The 
exposure period may be shortened if data suitable for the purpose of the 
range-finding test can be obtained in less time. The age class which is 
most sensitive to the test substance in the range-finding test shall be 
utilized in the definitive test. When no apparent difference in 
sensitivity of the two life stages is found, juveniles shall be utilized 
in the definitive test. No replicates are required, and nominal 
concentrations of the chemical are acceptable.
    (4) Definitive test. (i) The purpose of the definitive test is to 
determine the concentration-response curves and the 48- and 96-hour 
LC50 values with the minimum amount of testing beyond the 
range-finding test.
    (ii) The definitive test shall be conducted on the mysid life stage 
(juveniles or young adults) which is most sensitive to the test 
substance being evaluated.
    (iii) A minimum of 20 mysids per concentration shall be exposed to 
five or more concentrations of the chemical chosen in a geometric series 
in which the ratio is between 1.5 and 2.0 (e.g., 2, 4, 8, 16, 32, and 64 
mg/l). An equal number of mysids shall be placed in two or more 
replicates. If solvents, solubilizing agents or emulsifiers have to be 
used, they shall be commonly used carriers and shall not possess a 
synergistic or antagonistic effect on the toxicity of the test 
substance. The concentration of solvent shall not exceed 0.1 ml/1. The 
concentration ranges shall be selected to determine the concentration-
response curves and LC50 values at 48 and 96 hours.
    (iv) Every test shall include controls consisting of the same 
dilution water, conditions, procedures, and mysids from the same 
population or culture container, except that none of the chemical is 
added.
    (v) The dissolved oxygen concentration temperature, salinity, and pH 
shall be measured at the beginning and end of the test in each chamber.
    (vi) The test duration is 96 hours. The test is unacceptable if more 
than 10 percent of the control organisms die or exhibit abnormal 
behavior during the 96 hour test period. Each test chamber should be 
checked for dead mysids at 24, 48, 72, and 96 hours after the beginning 
of the test. Concentration-response curves and 24-, 48-, 72- and 96-hour 
LC50 values should be determined along with their 95 percent 
confidence limits.
    (vii) In addition to death, any abnormal behavior or appearance 
shall also be reported.
    (viii) Test organisms shall be impartially distributed among test 
chambers in such a manner that test results show no significant bias 
from the distributions. In addition, test chambers within the testing 
area shall be positioned in a random manner or in a way in which 
appropriated statistical analyses can be used to determine the variation 
due to placement.
    (ix) The concentration of the test substance in the chambers should 
be measured as often as is feasible during the test. At a minimum, 
during static tests the concentration of test substance shall be 
measured at each concentration at the beginning and at the end of the 
test. During the flow-through test, the concentration of test substance 
should be measured at the beginning and end of the test and in at

[[Page 134]]

least one appropriate chamber whenever a malfunction is detected in any 
part of the test substance delivery system. Equal aliquots of test 
solution may be removed from each replicate chamber and pooled for 
analysis. Among replicate test chambers of a treatment concentration, 
the measured concentration of the test substance should not vary more 
than 20 percent.
    (5) [Reserved]
    (6) Analytical measurements--(i) Test chemical. Deionized water 
should be used in making stock solutions of the test substance. Standard 
analytical methods should be used whenever available in performing the 
analyses. The analytical method used to measure the amount of test 
substance in a sample shall be validated before beginning the test by 
appropriate laboratory practices. An analytical method is not acceptable 
if likely degradation products of the test substance, such as hydrolysis 
and oxidation products, give positive or negative interferences which 
cannot be systematically identified and corrected mathematically.
    (ii) Numerical. The number of dead mysids shall be counted during 
each definitive test. Appropriate statistical analyses should provide a 
goodness-of-fit determination for the concentration-response curves. A 
48- and 96-hour LC50 and corresponding 95 percent interval 
shall be calculated.
    (d) Test conditions--(1) Test species--(i) Selection. (A) The mysid 
shrimp, Mysidopsis bahia, is the organism specified for these tests. 
Either juvenile (<24 hours old) or young adult (5 to 6 days old) mysids 
are to be used to start the test.
    (B) Mysids to be used in chronic toxicity tests should originate 
from laboratory cultures in order to ensure the individuals are of 
similar age and experimental history. Mysids used for establishing 
laboratory cultures may be purchased commercially or collected from 
appropriate natural areas. Because of similarities with other mysids 
species, taxonomic verification should be obtained from the commercial 
supplier by experienced laboratory personnel or by an outside expert.
    (C) Mysids used in a particular test shall be of similar age and be 
of normal size and appearance for their age. Mysids shall not be used 
for a test if they exhibit abnormal behavior or if they have been used 
in a previous test, either in a treatment or in a control group.
    (ii) Acclimation. (A) Any change in the temperature and chemistry of 
the dilution water used for holding or culturing the test organisms to 
those of the test shall be gradual. Within a 24-hour period, changes in 
water temperature shall not exceed 1 [deg]C, while salinity changes 
shall not exceed 5 percent.
    (B) During acclimation mysids should be maintained in facilities 
with background colors and light intensities similar to those of the 
testing areas.
    (iii) Care and handling. Methods for the care and handling of mysids 
such as those described in paragraph (f)(1) of this section can be used 
during holding, culturing and testing periods.
    (iv) Feeding. Mysids should be fed during testing. Any food utilized 
should support survival, growth and reproduction of the mysids. A 
recommended food is live Artemia spp. (48-hour-old nauplii).
    (2) Facilities--(i) Apparatus. (A) Facilities which may be needed to 
perform this test include: (1) flow-through or recirculating tanks for 
holding and acclimating mysids; (2) a mechanism for controlling and 
maintaining the water temperature during the holding, acclimation and 
test periods; (3) apparatus for straining particulate matter, removing 
gas bubbles, or aerating the water, as necessary; and (4) an apparatus 
for providing a 14-hour light and 10-hour dark photoperiod with a 15 to 
30 minute transition period. In addition, for flow-through tests, flow-
through chambers and a test substance delivery system are required. 
Furthermore, it is recommended that mysids be held in retention chambers 
within test chambers to facilitate observations and eliminate loss of 
test organisms through outflow water. For static tests, suitable 
chambers for exposing test mysids to the test substance are required. 
Facilities should be well ventilated and free of fumes and disturbances 
that may affect the test organisms.

[[Page 135]]

    (B) Test chambers shall be loosely covered to reduce the loss of 
test solution or dilution water due to evaporation and to minimize the 
entry of dust or other particulates into the solutions.
    (ii) Cleaning. Test substance delivery systems and test chambers 
shall be cleaned before each test following standard laboratory 
practices.
    (iii) Construction materials. (A) Materials and equipment that 
contact test solutions should be chosen to minimize sorption of test 
chemicals from dilution water and should not contain substances that can 
be leached into aqueous solution in quantities that can affect test 
results.
    (B) For use in the flow-through test, retention chambers utilized 
for confinement of test organisms can be constructed with netting 
material of appropriate mesh size.
    (iv) Dilution water. (A) Natural or artificial seawater is 
acceptable as dilution water if mysids will survive and successfully 
reproduce in it for the duration of the holding, acclimating and testing 
periods without showing signs of stress, such as reduced growth and 
fecundity. Mysids shall be cultured and tested in dilution water from 
the same origin.
    (B) Natural seawater shall be filtered through a filter with a pore 
size of <20 microns prior to use in a test.
    (C) Artificial seawater can be prepared by adding commercially 
available formulations or by adding specific amounts of reagent-grade 
chemicals to deionized water. Deionized water with a conductivity less 
than 1 [micro]ohm/cm at 12 [deg]C is acceptable for making artificial 
seawater. When deionized water is prepared from a ground or surface 
water source, conductivity and total organic carbon (or chemical oxygen 
demand) shall be measured on each batch.
    (v) Test substance delivery system. In flow-through tests, 
proportional diluters, metering pumps, or other suitable systems should 
be used to deliver test substance to the test chambers. The system used 
shall be calibrated before each test. Calibration includes determining 
the flow rate through each chamber and the concentration of the test 
substance in each chamber. The general operation of the test substance 
delivery system should be checked twice daily during a test. The 24-hour 
flow through a test chamber shall be equal to at least 5 times the 
volume of the test chamber. During a test, the flow rates should not 
vary more than 10 percent among test chambers or across time.
    (3) Test parameters. Environmental parameters of the water contained 
in test chambers shall be maintained as specified below:
    (i) The test temperature shall be 25[deg]C. Excursions from the test 
temperature shall be not greater than 2[deg]C.
    (ii) Dissolved oxygen concentration between 60 and 105 percent 
saturation. Aeration, if needed to achieve this level, shall be done 
before the addition of the test substance. All treatment and control 
chambers shall be given the same aeration treatment.
    (iii) The number of mysids placed in a test solution shall not be so 
great as to affect results of the test. Loading shall not exceed 30 
mysids per liter for a static test. Loading requirements for the flow-
through test will vary depending on the flow rate of dilution water. The 
loading shall not cause the dissolved oxygen concentration to fall below 
the recommended levels.
    (iv) Photoperiod of 14 hours light and 10 hours darkness, with a 15 
to 30 minute transition period.
    (v) Salinity of 20 parts per thousand 3 
percent.
    (e) Reporting. The sponsor shall submit to the EPA all data 
developed during the test that are suggestive or predictive of acute 
toxicity and all concomitant toxicologic manifestations. In addition to 
the general reporting requirements prescribed in part 792--Good 
Laboratory Practice Standards of this chapter, the reporting of test 
data shall include the following:
    (1) The source of the dilution water, its chemical characteristics 
(e.g., salinity, pH, etc.) and a description of any pretreatment.
    (2) Detailed information about the test organisms, including the 
scientific name and method of verification, age, source, history, 
abnormal behavior, acclimation procedures and food used.
    (3) A description of the test chambers, the depth and volume of 
solution in the chamber, the way the test was

[[Page 136]]

begun (e.g., conditioning, test substance additions, etc.), the number 
of organisms per treatment, the number of replicates, the loading, the 
lighting, the test substance delivery system and the flow rate expressed 
as volume additions per 24 hours.
    (4) The measured concentration of test substance in test chambers at 
the times designated.
    (5) The number and percentage of organisms that died or showed any 
other adverse effects in the control and in each treatment at each 
observation period.
    (6) Concentration-response curves shall be fitted to mortality data 
collected at 24, 48, 72, and 96 hours. A statistical test of goodness-
of-fit shall be performed and the results reported.
    (7) The 96-hour LC50 and when sufficient data have been 
generated, the 24-, 48-, and 72-hour LC50's and the 
corresponding 95-percent confidence limits and the methods used to 
calculate the values. These calculations shall be made using the average 
measured concentration of the test substance.
    (8) Methods and data records of all chemical analyses of water 
quality and test substance concentrations, including method validations 
and reagent blanks.
    (9) The data records of the holding, acclimation and test 
temperature and salinity.
    (f) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) U.S. Environmental Protection Agency, ``Bioassay Procedures for 
the Ocean Disposal Permit Program,'' EPA Report No. 600-9-78-010 (Gulf 
Breeze, Florida, 1978).
    (2) [Reserved]

[50 FR 39321, Sept. 27, 1985, as amended at 52 FR 19068, May 20, 1987; 
52 FR 26150, July 13, 1987]



Sec. 797.1950  Mysid shrimp chronic toxicity test.

    (a) Purpose. This guideline is intended for use in developing data 
on the chronic toxicity of chemical substances and mixtures 
(``chemicals'') subject to environmental effects test regulations under 
the Toxic Substances Control Act (TSCA) (Pub. L. 94-469, 90 Stat. 2003, 
15 U.S.C. 2601 et seq.). This guideline prescribes tests using mysids as 
test organisms to develop data on the chronic toxicity of chemicals. The 
United States Environmental Protection Agency (EPA) will use data from 
these tests in assessing the hazard of a chemical to the aquatic 
environment.
    (b) Definitions. The definitions in section 3 of the Toxic 
Substances Control Act (TSCA) and in part 792--Good Laboratory Practice 
Standards of this chapter apply to this test guideline. The following 
definitions also apply to this guideline:
    (1) ``Chronic toxicity test'' means a method used to determine the 
concentration of a substance that produces an adverse effect from 
prolonged exposure of an organism to that substance. In this test, 
mortality, number of young per female and growth are used as measures of 
chronic toxicity.
    (2) ``Death'' means the lack of reaction of a test organism to 
gentle prodding.
    (3) ``Flow-through'' means a continuous or an intermittent passage 
of test solution or dilution water through a test chamber or a holding 
or acclimation tank, with no recycling.
    (4) ``G1 (Generation 1)'' means those mysids which are used to begin 
the test, also referred to as adults; G2 (Generation 2) are the young 
produced by G1.
    (5) ``LC50'' means that experimentally derived 
concentration of test substance that is calculated to kill 50 percent of 
a test population during continuous exposure over a specified period of 
time.
    (6) ``Loading'' means the ratio of test organism biomass (gram, wet 
weight) to the volume (liters) of test solution in a test chamber.
    (7) ``MATC'' (Maximum Acceptable Toxicant Concentration) means the 
maximum concentration at which a chemical can be present and not be 
toxic to the test organism.
    (8) ``Retention chamber'' means a structure within a flow-through 
test chamber which confines the test organisms, facilitating observation 
of test organisms and eliminating washout from test chambers.
    (c) Test procedures--(1) Summary of the test. (i) In preparation for 
the test, the flow of test solution through each

[[Page 137]]

chamber is adjusted to the rate desired. The test substance is 
introduced into each test chamber. The rate at which the test substance 
is added is adjusted to establish and maintain the desired concentration 
of test substance in each test chamber. The test is started by randomly 
introducing mysids acclimated in accordance with the test design into 
retention chambers within the test and the control chambers. Mysids in 
the test and control chambers are observed periodically during the test, 
the dead mysids removed and the findings reported.
    (ii) Dissolved oxygen concentration, pH, temperature, salinity, the 
concentration of test substance and other water quality characteristics 
are measured at specified intervals in selected test chambers.
    (iii) Data collected during the test are used to develop a MATC 
(Maximum Acceptable Toxicant Concentration) and quantify effects on 
specific chronic parameters.
    (2) [Reserved]
    (3) Range-finding test. (i) A range-finding test should be conducted 
to establish test solution concentrations for the definitive test.
    (ii) The mysids should be exposed to a series of widely spaced 
concentrations of the test substance (e.g., 1, 10, 100 mg/l), usually 
under static conditions.
    (iii) A minimum of 10 mysids should be exposed to each concentration 
of test substance for a period of time which allows estimation of 
appropriate chronic test concentrations. No replicates are required and 
nominal concentrations of the chemical are acceptable.
    (4) Definitive test. (i) The purpose of the definitive test is to 
determine concentration-response curves, LC50 values, and 
effects of a chemical on growth and reproduction during chronic 
exposure.
    (ii) A minimum of 40 mysids per concentration shall be exposed to 
four or more concentrations of the chemical chosen in a geometric series 
in which the ratio is between 1.5 and 2.0 (e.g., 2, 4, 8, 16, 32, and 64 
mg/1). An equal number of mysids shall be placed in two or more 
replicates. If solvents, solubilizing agents or emulsifiers have to be 
used, they shall be commonly used carriers and shall not possess a 
synergistic or antagonistic effect on the toxicity of the test 
substance. The concentration of solvent should not exceed 0.1 ml/1. The 
concentration ranges should be selected to determine the concentration 
response curves, LC50 values and MATC. Concentration of test 
substance in test solutions should be analyzed prior to use.
    (iii) Every test should include controls consisting of the same 
dilution water, conditions, procedures and mysids from the same 
population or culture container, except that none of the chemical is 
added.
    (iv) The dissolved oxygen concentration, temperature, salinity, and 
pH shall be measured weekly in each chamber.
    (v) The test duration is 28 days. The test is unacceptable if more 
than 20 percent of the control organisms die, appear stressed or are 
diseased during the test. The number of dead mysids in each chamber 
shall be recorded on days 7, 14, 21, and 28 of the test. At the time 
when sexual characteristics are discernible in the mysids (approximately 
10 to 12 days in controls; possible delays may occur in mysids exposed 
to test substances), the number of males and females (identified by 
ventral brood pouch) in each chamber shall be recorded. Body length (as 
measured by total midline body length, from the anterior tip of the 
carapace to the posterior margin of the uropod) shall be recorded for 
males and females at the time when sex can be determined simultaneously 
for all mysids in control and treatment groups. This time cannot be 
specified because of possible delays in sexual maturation of mysids 
exposed to test substances. A second observation of male and female body 
lengths shall be conducted on day 28 of the test. To reduce stress on 
the mysids, body lengths can be recorded by photography through a 
stereomicroscope with appropriate scaling information. As offspring are 
produced by the G1 mysids (approximately 13 to 16 days in controls), the 
young shall be counted and separated into retention chambers at the same 
test substance concentration as the chambers where they originated. If

[[Page 138]]

available prior to termination of the test, observations on the 
mortality, number of males and females and male and female body length 
shall be recorded for the G2 mysids. Concentration-response curves, 
LC50 values and associated 95 percent confidence limits for 
the number of dead mysids (G1) shall be determined for days 7, 14, 21, 
and 28. An MATC shall be determined for the most sensitive test criteria 
measured (cumulative mortality of adult mysids, number of young per 
female, and body lengths of adult males and females).
    (vi) In addition to death, any abnormal behavior or appearance shall 
also be reported.
    (vii) Test organisms shall be impartially distributed among test 
chambers in such a manner that test results show no significant bias 
from the distributions. In addition, test chambers within the testing 
area shall be positioned in a random manner or in a way in which 
appropriate statistical analyses can be used to determined the variation 
due to placement.
    (viii) The concentration of the test substance in the chambers 
should be measured as often as is feasible during the test. The 
concentration of test substance shall be measured:
    (A) At each test concentration at the beginning of the test and on 
days 7, 14, 21, and 28; and
    (B) In at least one appropriate chamber whenever a malfunction is 
detected in any part of the test substance delivery system.

Equal aliquots of test solutions may be removed from each test chamber 
and pooled for analysis. Among replicate test chambers of a treatment 
concentration, the measured concentration of the test substance should 
not vary more than 20 percent.
    (5) [Reserved]
    (6) Analytical measurements--(i) Test chemical. Deionized water 
should be used in making stock solutions of the test substance. Standard 
analytical methods should be employed whenever available in performing 
the analyses. The analytical method used to measure the amount of test 
substance in a sample shall be validated before beginning the test by 
appropriate laboratory practices. An analytical method is not acceptable 
if likely degradation products of the test substance, such as hydrolysis 
and oxidation products, give positive or negative interferences which 
cannot be systematically identified and corrected mathematically.
    (ii) Numerical. (A) The number of dead mysids, cumulative young per 
female, and body lengths of male and female mysids shall be recorded 
during each definitive test. Appropriate statistical analyses shall 
provide a goodness-of-fit determination for the day 7, 14, 21 and 28 
adult (Gl) death concentration-response curves.
    (B) A 7-, 14-, 21- and 28-day LC50, based on adult (Gl) 
death, and corresponding 95 percent confidence intervals shall be 
calculated. Appropriate statistical tests (e.g., analysis of variance, 
mean separation test) should be used to test for significant chemical 
effects on chronic test criteria (cumulative mortality of adults, 
cumulative number of young per female and body lengths of adult male and 
females) on designated days. An MATC shall be calculated using these 
chronic tests criteria.
    (d) Test conditions--(1) Test species--(i) Selection. (A) The mysid 
shrimp, Mysidopsis bahia, is the organism specified for these tests. 
Juvenile mysids, <=24 hours old, are to be used to start the test.
    (B) Mysids to be used in chronic toxicity tests should originate 
from laboratory cultures in order to ensure the individuals are of 
similar age and experimental history. Mysids used for establishing 
laboratory cultures may be purchased commercially or collected from 
appropriate natural areas. Because of similarities with other mysid 
species, taxonomic verification should be obtained from the commercial 
supplier, by experienced laboratory personnel, or by an outside expert.
    (C) Mysids used in a particular test shall be of similar age and be 
of normal size and appearance for their age.
    (D) Mysids shall not be used for a test if they exhibit abnormal 
behavior, or if they have been used in a previous test, either in a 
treatment or in a control group.
    (ii) Acclimation. (A) Any change in the temperature and chemistry of 
the water used for holding or culturing the

[[Page 139]]

test organisms to those of the test should be gradual. Within a 24-hour 
period, changes in water temperature should not exceed 1 [deg]C, while 
salinity changes should not exceed 5 percent.
    (B) During acclimation mysids should be maintained in facilities 
with background colors and light intensities similar to those of the 
testing areas.
    (iii) Care and handling. Methods for the care and handling of mysids 
such as those described in paragraph (f)(1) of this section can be used 
during holding, culturing and testing periods.
    (iv) Feeding. Mysids should be fed during testing. Any food utilized 
should support survival, growth and reproduction of the mysids. A 
recommended food is live Artemia spp. nauplii (approximately 48 hours 
old).
    (2) Facilities--(i) Apparatus. (A) Facilities which may be needed to 
perform this test include: (1) flow-through or recirculating tanks for 
holding and acclimating mysids; (2) a mechanism for controlling and 
maintaining the water temperature during the holding, acclimation and 
test periods; (3) apparatus for straining particulate matter, removing 
gas bubbles, or aerating the water, as necessary; and (4) an apparatus 
for providing a 14-hour light and 10-hour dark photoperiod with a 15- to 
30-minute transition period. In addition, flow-through chambers and a 
test substance delivery system are required. It is recommended that 
mysids be held in retention chambers within test chambers to facilitate 
observations and eliminate loss through outflow water.
    (B) Facilities should be well ventilated and free of fumes and 
disturbances that may affect test organisms.
    (C) Test chambers shall be loosely covered to reduce the loss of 
test solution or dilution water due to evaporation and to minimize the 
entry of dust or other particulates into the solutions.
    (ii) Cleaning. Test substance delivery systems and test chambers 
shall be cleaned before each use following standard laboratory 
practices.
    (iii) Construction materials. (A) Materials and equipment that 
contact test solutions should be chosen to minimize sorption of test 
chemicals from the dilution water and should not contain substances that 
can be leached into aqueous solution in quantities that can affect the 
test results.
    (B) Retention chambers utilized for confinement of test organisms 
can be constructed with netting material of appropriate mesh size.
    (iv) Dilution water. (A) Natural or artificial seawater is 
acceptable as dilution water if mysids will survive and successfully 
reproduce in it for the duration of the holding, acclimating and testing 
periods without showing signs of stress, such as reduced growth and 
fecundity. Mysids shall be cultured and tested in dilution water from 
the same origin.
    (B) Natural seawater shall be filtered through a filter with a pore 
size of  20 microns prior to use in a test.
    (C) Artificial seawater can be prepared by adding commercially 
available formulations or by adding specific amounts of reagent-grade 
chemicals to deionized or glass-distilled water. Deionized water with a 
conductivity less than 1 [micro]ohm/cm at 12 [deg]C is acceptable as the 
diluent for making artificial seawater. When deionized water is prepared 
from a ground or surface water source, conductivity and total organic 
carbon (or chemical oxygen demand) shall be measured on each batch.
    (v) Test substance delivery system. Proportional diluters, metering 
pumps, or other suitable systems should be used to deliver test 
substance to the test chambers. The system used shall be calibrated 
before each test. Calibration includes determining the flow rate and the 
concentration of the test substance in each chamber. The general 
operation of the test substance delivery system should be checked twice 
daily during a test. The 24-hour flow rate through a chamber shall be 
equal to at least 5 times the volume of the chamber. The flow rates 
should not vary more than 10 percent among chambers or across time.
    (3) Test parameters. Environmental parameters of the water contained 
in test chambers shall be maintained as specified below:
    (i) The test temperature shall be 25 [deg]C. Excursions from the 
test temperature shall be no greater than 2 
[deg]C.
    (ii) Dissolved oxygen concentration between 60 and 105 percent 
saturation.

[[Page 140]]

Aeration, if needed to achieve this level, shall be done before the 
addition of the test substance. All treatment and control chambers shall 
be given the same aeration treatment.
    (iii) The number of mysids placed in a test solution shall not be so 
great as to affect results of the test. Loading requirements for the 
test will vary depending on the flow rate of dilution water. The loading 
shall not cause the dissolved oxygen concentration to fall below the 
recommended levels.
    (iv) Photoperiod of 14 hours light and 10 hours darkness, with a 15-
30 minute transition period.
    (v) Salinity of 20 parts per thousand 3 
percent.
    (e) Reporting. The sponsor shall submit to the EPA all data 
developed by the test that are suggestive or predictive of chronic 
toxicity and all concomitant toxicologic manifestations. In addition to 
the general reporting requirements prescribed in part 792--Good 
Laboratory Practice Standards of this chapter, the reporting of test 
data shall include the following:
    (1) The source of the dilution water, its chemical characteristics 
(e.g., salinity, pH, etc.) and a description of any pretreatment.
    (2) Detailed information about the test organisms, including the 
scientific name and method of verification, average length, age, source, 
history, observed diseases, treatments, acclimation procedures and food 
used.
    (3) A description of the test chambers, the depth and volume of 
solution in the chamber, the way the test was begun (e.g., conditioning, 
test substance additions, etc.), the number of organisms per treatment, 
the number of replicates, the loading, the lighting, the test substance 
delivery system, and the flow rate expressed as volume additions per 24 
hours.
    (4) The measured concentration of test substance in test chambers at 
the times designated.
    (5) The first time (day) that sexual characteristics can be observed 
in controls and in each test substance concentration.
    (6) The length of time for the appearance of the first brood for 
each concentration.
    (7) The means (average of replicates) and respective 95 percent 
confidence intervals for:
    (i) Body length of males and females at the first observation day 
(depending on time of sexual maturation) and on day 28.
    (ii) Cumulative number of young produced per female on day 28.
    (iii) Cumulative number of dead adults on day 7, 14, 21 and 28.
    (iv) If available prior to test termination (day 28), effects on G2 
mysids (number of males and females, body length of males and females 
and cumulative mortality).
    (8) The MATC is calculated as the geometric mean between the lowest 
measured test substance concentration that had a significant (P<0.05) 
effect and the highest measured test substance concentration that had no 
significant (P<0.05) effect in the chronic test. The most sensitive of 
the test criteria for adult (Gl) mysids (cumulative number of dead 
mysids, body lengths of males and females or the number of young per 
female) is used to calculate the MATC. The criterion selected for MATC 
computation is the one which exhibits an effect (a statistically 
significant difference between treatment and control groups; P<0.05) at 
the lowest test substance concentration for the shortest period of 
exposure. Appropriate statistical tests (analysis of variance, mean 
separation test) should be used to test for significant chemical 
effects. The statistical tests employed and the results of these tests 
shall be reported.
    (9) Concentration-response curves shall be fitted to the cumulative 
number of adult dead for days 7, 14, 21, and 28. A statistical test of 
goodness-of-fit shall be performed and the results reported.
    (10) An LC50 value based on the number of dead adults 
with corresponding 95 percent confidence intervals for days 7, 14, 21 
and 28. These calculations shall be made using the average measured 
concentration of the test substance.
    (11) Methods and data records of all chemical analyses of water 
quality and test substance concentrations, including method validations 
and reagent blanks.

[[Page 141]]

    (12) The data records of the holding, acclimation and test 
temperature and salinity.
    (f) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) U.S. Environmental Protection Agency, ``Bioassay Procedures for 
the Ocean Disposal Permit Program,'' EPA Report No. 600/9-78-010 (Gulf 
Breeze, Florida, 1978).
    (2) [Reserved]

[50 FR 39321, Sept. 27, 1985, as amended at 52 FR 19069, May 20, 1987]



PART 798_HEALTH EFFECTS TESTING GUIDELINES--Table of Contents




Subparts A-B [Reserved]

                      Subpart C_Subchronic Exposure

Sec.
798.2250 Dermal toxicity.
798.2450 Inhalation toxicity.
798.2650 Oral toxicity.

                       Subpart D_Chronic Exposure

798.3260 Chronic toxicity.
798.3300 Oncogenicity.
798.3320 Combined chronic toxicity/oncogenicity.

                Subpart E_Specific Organ/Tissue Toxicity

798.4100 Dermal sensitization.
798.4350 Inhalation developmental toxicity study.
798.4700 Reproduction and fertility effects.
798.4900 Developmental toxicity study.

                       Subpart F_Genetic Toxicity

798.5195 Mouse biochemical specific locus test.
798.5200 Mouse visible specific locus test.
798.5265 The salmonella typhimurium reverse mutation assay.
798.5275 Sex-linked recessive lethal test in drosophila melanogaster.
798.5300 Detection of gene mutations in somatic cells in culture.
798.5375 In vitro mammalian cytogenetics.
798.5385 In vivo mammalian bone marrow cytogenetics tests: Chromosomal 
          analysis.
798.5395 In vivo mammalian bone marrow cytogenetics tests: Micronucleus 
          assay.
798.5450 Rodent dominant lethal assay.
798.5460 Rodent heritable translocation assays.
798.5500 Differential growth inhibition of repair proficient and repair 
          deficient bacteria: ``Bacterial DNA damage or repair tests.''
798.5955 Heritable translocation test in drosophila melanogaster.

                         Subpart G_Neurotoxicity

798.6050 Functional observational battery.
798.6200 Motor activity.
798.6400 Neuropathology.
798.6500 Schedule-controlled operant behavior.
798.6560 Subchronic delayed neurotoxicity of organophosphorus 
          substances.

    Authority: 15 U.S.C. 2603.

    Source: 50 FR 39397, Sept. 27, 1985, unless otherwise noted.

Subparts A-B [Reserved]



                      Subpart C_Subchronic Exposure



Sec. 798.2250  Dermal toxicity.

    (a) Purpose. In the assessment and evaluation of the toxic 
characteristics of a chemical, the determination of subchronic dermal 
toxicity may be carried out after initial information on toxicity has 
been obtained by acute testing. The subchronic dermal study has been 
designed to permit the determination of the no-observed-effect level and 
toxic effects associated with continuous or repeated exposure to a test 
substance for a period of 90 days. The test is not capable of 
determining those effects that have a long latency period for 
development (e.g., carcinogenicity and life shortening). It provides 
information on health hazards likely to arise from repeated exposure by 
the dermal route over a limited period of time. It will provide 
information on target organs, the possibilities of accumulation, and can 
be of use in selecting dose levels for chronic studies and for 
establishing safety criteria for human exposure.
    (b) Definitions. (1) Subchronic dermal toxicity is the adverse 
effects occurring as a result of the repeated daily exposure of 
experimental animals to a chemical by dermal application for part 
(approximately 10 percent) of a life span.
    (2) Dose in a dermal test is the amount of test substance applied to 
the skin (applied daily in subchronic tests).

[[Page 142]]

Dose is expressed as weight of the substance (g, mg) per unit weight of 
test animal (e.g., mg/kg).
    (3) No-effect level/No-toxic-effect level/No-adverse-effect level/
No-observed-effect level is the maximum dose used in a test which 
produces no observed adverse effects. A no-observed-effect level is 
expressed in terms of the weight of a test substance given daily per 
unit weight of test animal (mg/kg).
    (4) Cumulative toxicity is the adverse effects of repeated doses 
occurring as a result of prolonged action on, or increased concentration 
of the administered test substance or its metabolites in susceptible 
tissues.
    (c) Principle of the test method. The test substance is applied 
daily to the skin in graduated doses to several groups of experimental 
animals, one dose level per unit group, for a period of 90 days. During 
the period of application the animals are observed daily to detect signs 
of toxicity. Animals which die during the test are necropsied, and at 
the conclusion of the test the surviving animals are sacrificed and 
necropsied and appropriate histopathological examinations carried out.
    (d) Limit test. If a test at one dose level of at least 1,000 mg/kg 
body weight (expected human exposure may indicate the need for a higher 
dose level), using the procedures described for this study, produces no 
observable toxic effects and if toxicity would not be expected based 
upon data of structurally related compounds, then a full study using 
three dose levels might not be necessary.
    (e) Test procedures--(1) Animal selection--(i) Species and strain. A 
mammalian species shall be used for testing. The rat, rabbit, or guinea 
pig may be used, although the albino rabbit is preferred. The albino 
rabbit is preferred because of its size, skin permeability, and 
extensive data base. Commonly used laboratory strains shall be employed. 
If another mammalian species is used, the tester shall provide 
justification/reasoning for its selection.
    (ii) Age. Young adult animals shall be used. The following weight 
ranges at the start of the test are suggested in order to provide 
animals of a size which facilitates the conduct of the test: rats, 200 
to 300 g; rabbits, 2.0 to 3.0 kg; guinea pigs, 350 to 450 g.
    (iii) Sex. (A) Equal numbers of animals of each sex with healthy 
skin shall be used at each dose level.
    (B) The females shall be nulliparous and nonpregnant.
    (iv) Numbers. (A) At least 20 animals (10 females and 10 males) 
shall be used at each dose level.
    (B) If interim sacrifices are planned, the number shall be increased 
by the number of animals scheduled to be sacrificed before completion of 
the study.
    (2) Control groups. A concurrent control group is required. This 
group shall be an untreated or sham-treated control group or, if a 
vehicle is used in administering the test substance, a vehicle control 
group. If the toxic properties of the vehicle are not known or cannot be 
made available, both untreated and vehicle control groups are required.
    (3) Satellite group. A satellite group of 20 animals (10 animals per 
sex) may be treated with the high dose level for 90 days and observed 
for reversibility, persistence, or delayed occurrence, of toxic effects 
for a posttreatment period of appropriate length, normally not less than 
28 days.
    (4) Dose level and dose selection. (i) In subchronic toxicity tests, 
it is desirable to have a dose-response relationship as well as a no-
observed-toxic-effect level. Therefore, at least 3 dose levels with a 
control and, where appropriate, a vehicle control (corresponding to the 
concentration of vehicle at the highest exposure level) shall be used. 
Doses should be spaced appropriately to produce test groups with a range 
of toxic effects. The data shall be sufficient to produce a dose-
response curve.
    (ii) The highest dose level should result in toxic effects but not 
produce severe skin irritation or an incidence of fatalities which would 
prevent a meaningful evaluation.
    (iii) The lowest dose level should not produce any evidence of 
toxicity. Where there is a usable estimation of human exposure, the 
lowest dose level should exceed this.
    (iv) Ideally, the intermediate dose level(s) should produce minimal 
observable toxic effects. If more than one

[[Page 143]]

intermediate dose is used, the dose levels should be spaced to produce a 
gradation of toxic effects.
    (v) In the low and intermediate groups and in the controls the 
incidence of fatalities should be low, to permit a meaningful evaluation 
of the results.
    (5) Exposure conditions. The animals are treated with test 
substance, ideally for at least 6 hours per day on a 7-day per week 
basis, for a period of 90 days. However, based primarily on practical 
considerations, application on a 5-day per week basis is considered to 
be acceptable.
    (6) Observation period. (i) Duration of observation shall be at 
least 90 days.
    (ii) Animals in the satellite group scheduled for followup 
observations should be kept for at least 28 days further without 
treatment to detect recovery from, or persistence of, toxic effects.
    (7) Preparation of animal skin. (i) Shortly before testing, fur 
shall be clipped from the dorsal area of the trunk of the test animals. 
Shaving may be employed, but it should be carried out approximately 24 
hours before the test. Repeat clipping or shaving is usually needed at 
approximately weekly intervals. When clipping or shaving the fur, care 
should be taken to avoid abrading the skin, which could alter its 
permeability.
    (ii) Not less than 10 percent of the body surface area should be 
clear for the application of the test substance. The weight of the 
animal should be taken into account when deciding on the area to be 
cleared and on the dimensions of any covering used.
    (iii) When testing solids, which may be pulverized if appropriate, 
the test substance should be moistened sufficiently with water or, where 
necessary, a suitable vehicle to ensure good contact with the skin. When 
a vehicle is used, the influence of the vehicle on toxicity of and 
penetration of the skin by the test substance should be taken into 
account.
    (8) Application of the test substance. (i) The test substance shall 
be applied uniformly over an area which is approximately 10 percent of 
the total body surface area. With highly toxic substances, the surface 
area covered may be less, but as much of the area shall be covered with 
as thin and uniform a film as possible.
    (ii) During the exposure period, the test substance shall be held in 
contact with the skin with a porous gauze dressing and nonirritating 
tape. The test site shall be further covered in a suitable manner to 
retain the gauze dressing and test substance and ensure that the animals 
cannot ingest the test substance. Restrainers may be used to prevent the 
ingestion of the test substance, but complete immobilization is not a 
recommended method.
    (9) Observation of animals. (i) Each animal shall be observed daily, 
and if necessary handled to appraise its physical condition.
    (ii) Additional observations shall be made daily with appropriate 
actions taken to minimize loss of animals to the study (e.g., necropsy 
or refrigeration of those animals found dead and isolation or sacrifice 
of weak or moribund animals).
    (iii) Signs of toxicity shall be recorded as they are observed, 
including the time of onset, the degree, and duration.
    (iv) Cage-side observations shall include, but not be limited to, 
changes in skin and fur, eyes and mucous membranes, respiratory, 
circulatory, autonomic and central nervous systems, somatomotor activity 
and behavior pattern.
    (v) Animals shall be weighed weekly. Feed consumption shall also be 
determined weekly if abnormal body weight changes are observed.
    (vi) At the end of the study period, all survivors in the 
nonsatellite treatment groups shall be sacrificed. Moribund animals 
shall be removed and sacrificed when noticed.
    (10) Clinical examinations. (i) The following examinations shall be 
made on all animals of each sex in each group:
    (A) Certain hematology determinations shall be carried out at least 
two times during the test period on all groups of animals including 
concurrent controls: After 30 days of test and just prior to terminal 
sacrifice at the end of the test period. Hematology determinations which 
are appropriate to all studies: Hematocrit, hemoglobin concentration, 
erythrocyte count, total

[[Page 144]]

and differential leukocyte count, and a measure of clotting potential 
such as clotting time, prothrombin time, thromboplastin time, or 
platelet count.
    (B) Certain clinical biochemistry determinations on blood should be 
carried out at least two times during the test period on all groups of 
animals including concurrent controls: After 30 days of test and just 
prior to terminal sacrifice at the end of the test period. Clinical 
biochemistry test areas which are considered appropriate to all studies: 
Electrolyte balance, carbohydrate metabolism, and liver and kidney 
function. The selection of specific tests will be influenced by 
observations on the mode of action of the substance. Suggested 
determinations: Calcium, phosphorus, chloride, sodium, potassium, 
fasting glucose (with period of fasting appropriate to the species), 
serum glutamic pyruvic transaminase (now known as serum alanine 
aminotransferase), serum glutamic oxaloacetic transaminase (now known as 
serum aspartate aminotransferase), ornithine decarboxylase, gamma 
glutamyl transpeptidase, urea nitrogen, albumen blood creatinine, total 
bilirubin, and total serum protein measurements. Other determinations 
which may be necessary for an adequate toxicological evaluation include: 
Analyses of lipids, hormones, acid/base balance, methemoglobin, and 
cholinesterase activity. Additional clinical biochemistry may be 
employed, where necessary, to extend the investigation of observed 
effects.
    (ii) The following examinations shall be made on high dose and 
control groups. If changes in the eyes are detected all animals should 
be examined.
    (A) Ophthalmological examination, using an ophthalmoscope or 
equivalent suitable equipment, shall be made prior to exposure to the 
test substance and at the termination of the study.
    (B) Urinalysis is not recommended on a routine basis, but only when 
there is an indication based on expected or observed toxicity.
    (11) Gross necropsy. (i) All animals shall be subjected to a full 
gross necropsy which includes examination of the external surface of the 
body, all orifices, and the cranial, thoracic, and abdominal cavities 
and their contents.
    (ii) The liver, kidneys, adrenals, brain, and gonads shall be 
weighed wet, as soon as possible after dissection, to avoid drying. In 
addition, for the rodent, the brain; for the non-rodent, the thyroid 
with parathyroids also shall be weighed wet.
    (iii) The following organs and tissues, or representative samples 
thereof, shall be preserved in a suitable medium for possible future 
histopathological examination: All gross lesions; lungs--which should be 
removed intact, weighed, and treated with a suitable fixative to ensure 
that lung structure is maintained (perfusion with the fixative is 
considered to be an effective procedure); nasopharyngeal tissues; 
brain--including sections of medulla/pons, cerebellar cortex, and 
cerebral cortex; pituitary; thyroid/parathyroid; thymus; trachea; heart; 
sternum with bone marrow; salivary glands; liver; spleen; kidneys; 
adrenals; pancreas; gonads; uterus; accessory genital organs 
(epididymis, prostate, and, if present, seminal vesicles); aorta; 
(skin); gall bladder (if present); esophagus; stomach; duodenum; 
jejunum; ileum; cecum; colon; rectum; urinary bladder; representative 
lymph node; (mammary gland); (thigh musculature); peripheral nerve; 
(eyes); (femur--including articular surface); (spinal cord at three 
levels--cervical, midthoracic, and lumbar); and (zymbal and exorbital 
lachrymal glands).
    (12) Histopathology. The following histopathology shall be 
performed:
    (i) Full histopathology on normal and treated skin and on organs and 
tissues, listed above, of all animals in the control and high dose 
groups.
    (ii) All gross lesions in all animals.
    (iii) Target organs in all animals.
    (iv) The tissues listed in parenthesis in paragraph (e)(11)(iii) of 
this section, if indicated by signs of toxicity or expected target organ 
involvement.
    (v) Lungs of animals (rodents) in the low and intermediate dose 
groups shall be subjected to histopathological examination for evidence 
of infection, since this provides a convenient assessment of the state 
of health of the animals.
    (vi) When a satellite group is used, histopathology shall be 
performed on

[[Page 145]]

tissues and organs identified as showing effects in the treated groups.
    (f) Data and reporting--(1) Treatment of results. (i) Data shall be 
summarized in tabular form, showing for each test group the number of 
animals at the start of the test, the number of animals showing lesions, 
the types of lesions, and the percentage of animals displaying each type 
of lesion.
    (ii) All observed results, quantitative and incidental, should be 
evaluated by an appropriate statistical method. Any generally accepted 
statistical method may be used; the statistical methods should be 
selected during the design of the study.
    (2) Evaluation of results. The findings of a subchronic dermal 
toxicity study should be evaluated in conjunction with the findings of 
preceding studies and considered in terms of the observed toxic effects 
and the necropsy and histopathological findings. The evaluation should 
include the relationship between the dose of the test substance and the 
presence or absence, the incidence and severity, of abnormalities, 
including behavioral and clinical abnormalities, gross lesions, 
identified target organs, body weight changes, effect on mortality and 
any other general or specific toxic effects. A properly conducted 
subchronic test should provide a satisfactory estimation of a no-effect 
level.
    (3) Test report. In addition to the reporting requirements as 
specified in the EPA Good Laboratory Practice Standards under 40 CFR 
part 792, subpart J, the following specific information shall be 
reported.
    (i) Group animal data. Tabulation of toxic response data by species, 
strain, sex and exposure level for:
    (A) Number of animals dying.
    (B) Number of animals showing signs of toxicity.
    (C) Number of animals exposed.
    (ii) Individual animal data. (A) Date of death during the study or 
whether animals survived to termination.
    (B) Date of observation of each abnormal sign and its subsequent 
course.
    (C) Body weight data.
    (D) Feed consumption data when collected.
    (E) Hematological tests employed and all results.
    (F) Clinical biochemistry tests employed and all results.
    (G) Necropsy findings.
    (H) Detailed description of all histopathological findings.
    (I) Statistical treatment of results where appropriate.
    (g) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) Draize, J.H. ``Dermal toxicity,'' Appraisal of Chemicals in 
Food, Drugs and Cosmetics. The Association of Food and Drug Officials of 
the United States (1959, 3rd printing 1975). pp. 46-59.
    (2) Fitzhugh, O.G. ``Subacute toxicity,'' Appraisal of the Safety of 
Chemicals in Foods, Drugs and Cosmetics. The Association of Food and 
Drug Officials of the United States (1959, 3rd printing 1975). pp. 26-
35.
    (3) National Academy of Sciences. ``Principles and Procedures for 
Evaluating the Toxicity of Household Substances,'' a report prepared by 
the Committee for the Revision of NAS Publication 1138, under the 
auspices of the Committee on Toxicology, National Research Council, 
National Academy of Sciences, Washington, DC (1977).
    (4) World Health Organization. ``Part I. Environmental Health 
Criteria 6,''Principles and Methods for Evaluating the Toxicity of 
Chemicals. (Geneva: World Health Organization, 1978).

[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19072, May 20, 1987; 
53 FR 49149, Dec. 6, 1988; 54 FR 21064, May 16, 1989]



Sec. 798.2450  Inhalation toxicity.

    (a) Purpose. In the assessment and evaluation of the toxic 
characteristics of a gas, volatile substance, or aerosol/particulate, 
determination of subchronic inhalation toxicity may be carried out after 
initial information on toxicity has been obtained by acute testing. The 
subchronic inhalation study has been designed to permit the 
determination of the no-observed-effect level and toxic effects 
associated with continuous or repeated exposure to a test substance for 
a period of 90 days. The test is not capable of determining those 
effects that have a long latency

[[Page 146]]

period for development (e.g., carcinogenicity and life shortening). It 
provides information on health hazards likely to arise from repeated 
exposures by the inhalation route over a limited period of time. It will 
provide information on target organs, the possibilities of accumulation, 
and can be of use in selecting dose levels for chronic studies and for 
establishing safety criteria for human exposure. Hazards of inhaled 
substances are influenced by the inherent toxicity and by physical 
factors such as volatility and particle size.
    (b) Definitions. (1) Subchronic inhalation toxicity is the adverse 
effects occurring as a result of the repeated daily exposure of 
experimental animals to a chemical by inhalation for part (approximately 
10 percent) of a life span.
    (2) Aerodynamic diameter applies to the size of particles of 
aerosols. It is the diameter of a sphere of unit density which behaves 
aerodynamically as the particle of the test substance. It is used to 
compare particles of different size and densities and to predict where 
in the respiratory tract such particles may be deposited. This term is 
used in contrast to measured or geometric diameter which is 
representative of actual diameters which in themselves cannot be related 
to deposition within the respiratory tract.
    (3) The geometric mean diameter or the median diameter is the 
calculated aerodynamic diameter which divides the particles of an 
aerosol in half based on the weight of the particles. Fifty percent of 
the particles by weight will be larger than the median diameter and 50 
percent of the particles will be smaller than the median diameter. The 
median diameter describes the particle size distribution of any aerosol 
based on the weight and size of the particles.
    (4) Inhalable diameter refers to that aerodynamic diameter of a 
particle which is considered to be inhalable for the organism. It is 
used to refer to particles which are capable of being inhaled and may be 
deposited anywhere within the respiratory tract from the trachea to the 
alveoli. For man, inhalable diameter is considered as 15 micrometers or 
less.
    (5) Dose refers to an exposure level. Exposure is expressed as 
weight or volume of test substance per volume of air (mg/l), or as parts 
per million (ppm).
    (6) No-effect level/No-toxic-effect level/No-adverse-effect level/
No-observed-effect level is the maximum dose used in a test which 
produces no observed adverse effects. A no-observed-effect level is 
expressed in terms of weight or volume of test substance given daily per 
unit volume of air (mg/l or ppm).
    (7) Cumulative toxicity is the adverse effects of repeated doses 
occuring as a result of prolonged action on, or increased concentration 
of the administered test substance or its metabolites in susceptible 
tissues.
    (c) Principle of the test method. Several groups of experimental 
animals are exposed daily for a defined period to the test substance in 
graduated concentrations, one concentration being used per group, for a 
period of 90 days. During the period of administration, the animals are 
observed daily to detect signs of toxicity. Animals which die during the 
test are necropsied and at the conclusion of the test, surviving animals 
are sacrificed and necropsied and appropriate histopathological 
examinations carried out.
    (d) Test procedures--(1) Animal selection--(i) Species and strain. A 
mammalian species shall be used for testing. A variety of rodent species 
may be used, although the rat is the preferred species. Commonly used 
laboratory strains shall be employed. If another mammalian species is 
used, the tester shall provide justification/ reasoning for its 
selection.
    (ii) Age. Young adult animals shall be used. At the commencement of 
the study the weight variation of animals shall not exceed 20 percent of the mean weight for each sex.
    (iii) Sex. (A) Equal numbers of animals of each sex shall be used at 
each dose level.
    (B) Females shall be nulliparous and nonpregnant.
    (iv) Numbers. (A) At least 20 rodents (10 females and 10 males) 
shall be used for each test group. If another mammalian species is 
selected (e.g. dog, rabbit, or non-human primate), at least 8 animals (4 
males and 4 females) shall be used.

[[Page 147]]

    (B) If interim sacrifices are planned, the number of animals shall 
be increased by the number of animals scheduled to be sacrificed before 
the completion of the study.
    (2) Control groups. A concurrent control group is required. This 
group shall be an untreated or sham-treated control group. Except for 
treatment with the test substance, animals in the control group shall be 
handled in a manner identical to the test group animals. Where a vehicle 
is used to help generate an appropriate concentration of the substance 
in the atmosphere, a vehicle control group shall be used. If the toxic 
properties of the vehicle are not known or cannot be made available, 
both untreated and vehicle control groups are required.
    (3) Satellite group. A satellite group of 20 animals (10 animals per 
sex) may be treated with the high concentration level for 90 days and 
observed for reversibility, persistence, or delayed occurrence of toxic 
effects for a post-treatment period of appropriate length, normally not 
less than 28 days.
    (4) Dose levels and dose selection. (i) In subchronic toxicity 
tests, it is desirable to have a concentration-response relationship as 
well as a no-observed-toxic-effect level. Therefore, at least 3 
concentration levels with a control and, where appropriate, a vehicle 
control (corresponding to the concentration of vehicle at the highest 
exposure level) shall be used. Concentrations should be spaced 
appropriately to produce test groups with a range of toxic effects. The 
data should be sufficient to produce a concentration-response curve.
    (ii) The highest concentration should result in toxic effects but 
not produce an incidence of fatalities which would prevent a meaningful 
evaluation.
    (iii) The lowest concentration should not produce any evidence of 
toxicity. Where there is a usable estimation of human exposure the 
lowest concentration should exceed this.
    (iv) Ideally, the intermediate concentration level(s) should produce 
minimal observable toxic effects. If more than one intermediate 
concentration level is used, the concentrations should be spaced to 
produce a gradation of toxic effects.
    (v) In the low and intermediate groups and in the controls the 
incidence of fatalities should be low, to permit a meaningful evaluation 
of the results.
    (vi) In the case of potentially explosive test substances, care 
should be taken to avoid generating explosive concentrations.
    (5) Exposure conditions. The animals should be exposed to the test 
substance, ideally for 6 hours per day on a 7-day per week basis, for a 
period of 90 days. However, based primarily on practical considerations, 
exposure on a 5-day-per-week basis for 6 hours per day is the minimum 
acceptable exposure period.
    (6) Observation period. (i) Duration of observation shall be for at 
least 90 days.
    (ii) Animals in a satellite group scheduled for followup 
observations should be kept for at least 28 days further without 
treatment to detect recovery from, or persistence of, toxic effects.
    (7) Inhalation exposure. (i) The animals shall be tested in 
inhalation equipment designed to sustain a minimum dynamic air flow of 
12 to 15 air changes per hour and ensure an adequate oxygen content of 
19 percent and an evenly distributed exposure atmosphere. Where a 
chamber is used, its design should minimize crowding of the test animals 
and maximize their exposure to the test substance. This is best 
accomplished by individual caging. To ensure stability of a chamber 
atmosphere, the total ``volume'' of the test animals shall not exceed 5 
percent of the volume of the test chamber. Oronasal or head-only 
exposure may be used if it is desirable to avoid concurrent exposure by 
the dermal or oral routes.
    (ii) A dynamic inhalation system with a suitable flow control system 
shall be used. The rate of air flow shall be adjusted to ensure that 
conditions throughout the exposure chamber are essentially the same. 
Maintenance of slight negative pressure inside the chamber will prevent 
leakage of the test substance into surrounding areas.
    (iii) The temperature at which the test is performed should be 
maintained

[[Page 148]]

at 22 [deg]C (2[deg]). Ideally, the relative 
humidity should be maintained between 40 to 60 percent, but in certain 
instances (e.g., tests of aerosols, use of water vehicle) this may not 
be practicable.
    (8) Physical measurements. Measurements or monitoring shall be made 
of the following:
    (i) The rate of air flow shall be monitored continuously and 
recorded at least every 30 minutes.
    (ii) The actual concentrations of the test substance shall be 
measured in the breathing zone. During the exposure period the actual 
concentrations of the test substance shall be held as constant as 
practicable, monitored continuously or intermittently depending on the 
method of analysis, and recorded at least at the beginning, at an 
intermediate time, and at the end of the exposure period.
    (iii) During the development of the generating system, particle size 
analysis shall be performed to establish the stability of aerosol 
concentrations with respect to particle size. During exposure, analysis 
shall be conducted as often as necessary to determine the consistency of 
particle size distribution.
    (iv) Temperature and humidity shall be monitored continuously but 
shall be recorded at least every 30 minutes.
    (9) Feed and water during exposure period. Feed shall be withheld 
during exposure. Water may also be withheld during exposure.
    (10) Observation of animals. (i) Each animal shall be observed daily 
and, if necessary, handled to appraise its physical condition.
    (ii) Additional observations should be made daily with appropriate 
actions taken to minimize loss of animals to the study (e.g., necropsy 
or refrigeration of those animals found dead and isolation or sacrifice 
of weak or moribund animals).
    (iii) Signs of toxicity shall be recorded as they are observed 
including the time of onset, the degree, and duration.
    (iv) Cage-side observations should include, but not be limited to, 
changes in the skin and fur, eyes and mucous membranes, respiratory, 
circulatory, autonomic and central nervous systems, somatomotor activity 
and behavior pattern.
    (v) Animals shall be weighed weekly. Feed consumption shall also be 
determined weekly if abnormal body weight changes are observed.
    (vi) At the end of the study period all survivors in the 
nonsatellite treatment groups shall be sacrificed. Moribund animals 
shall be removed and sacrificed when noticed.
    (11) Clinical examinations. (i) The following examinations shall be 
made on all animals of each sex in each group:
    (A) Certain hematology determinations shall be carried out at least 
two times during the test period on all groups of animals including 
concurrent controls: After 30 days of test and just prior to terminal 
sacrifice at the end of the test period. Hematology determinations which 
are appropriate to all studies: Hematocrit, hemoglobin concentration, 
erythrocyte count, total and differential leukocyte count, and a measure 
of clotting potential such as clotting time, prothrombin time, 
thromboplastin time, or platelet count.
    (B) Certain clinical biochemistry determinations on blood should be 
carried out at least two times during the test period on all groups of 
animals including concurrent controls: After 30 days of test and just 
prior to terminal sacrifice at the end of the test period. Clinical 
biochemistry test areas which are considered appropriate to all studies: 
Electrolyte balance, carbohydrate metabolism, and liver and kidney 
function. The selection of specific tests will be influenced by 
observations on the mode of action of the substance. Suggested 
determinations: calcium, phosphorus, chloride, sodium, potassium, 
fasting glucose (with period of fasting appropriate to the species), 
serum glutamic-pyruvic transaminase, (now known as serum alanine 
aminotransferase), serum glutamic-oxaloacetic transaminase (now known as 
serum aspartate aminotransferase), ornithine decarboxylase, gamma 
glutamyl transpeptidase, urea nitrogen, albumen, blood creatinine, total 
bilirubin, and total serum protein measurements. Other determinations 
which may be necessary for an adequate toxicological evaluation include: 
Analyses of lipids, hormones, acid/base

[[Page 149]]

balance, methemoglobin, and cholinesterase activity. Additional clinical 
biochemistry may be employed, where necessary, to extend the 
investigation of observed effects.
    (ii) The following examinations shall be made on high dose and 
control groups. If changes in the eyes are detected, all animals shall 
be examined:
    (A) Ophthalmological examination, using an ophthalmoscope or 
equivalent suitable equipment, shall be made prior to exposure to the 
test substance and at the termination of the study.
    (B) Urinalysis is not recommended on a routine basis, but only when 
there is an indication based on expected and/or observed toxicity.
    (12) Gross pathology. (i) All animals shall be subjected to a full 
gross necropsy which includes examination of the external surface of the 
body, all orifices and the cranial, thoracic, and abdominal cavities and 
their contents.
    (ii) At least the liver, kidneys, adrenals, brain, and gonads shall 
be weighed wet, as soon as possible after dissection to avoid drying. In 
addition, for the rodent, the brain; for the non-rodent, the thyroid 
with parathyroids also shall be weighed wet.
    (iii) The following organs and tissues, or representative samples 
thereof, shall be preserved in a suitable medium for possible future 
histopathological examination: All gross lesions; lungs--which should be 
removed intact, weighed, and treated with a suitable fixative to ensure 
that lung structure is maintained (perfusion with the fixative is 
considered to be an effective procedure); nasopharyngeal tissues; 
brain--including sections of medulla/pons cerebellar cortex and cerebral 
cortex; pituitary; thyroid/parathyroid; thymus; trachea; heart; sternum 
with bone marrow; salivary glands; liver; spleen; kidneys; adrenals; 
pancreas; gonads; uterus; accessory genital organs (epididymis, 
prostate, and, if present, seminal vesicles); aorta; (skin); gall 
bladder (if present); esophagus; stomach; duodenum; jejunum; ileum; 
cecum; colon; rectum; urinary bladder; representative lymph node; 
(mammary gland); (thigh musculature); peripheral nerve; (eyes); (femur--
including articular surface); (spinal cord at three levels--cervical, 
midthoracic, and lumbar); and (zymbal and exorbital lachrymal glands).
    (13) Histopathology. The following histopathology shall be 
performed:
    (i) Full histopathology on the respiratory tract and other organs 
and tissues, listed above, of all animals in the control and high dose 
groups.
    (ii) All gross lesions in all animals.
    (iii) Target organs in all animals.
    (iv) The tissues mentioned in brackets (listed above) if indicated 
by signs of toxicity or target organ involvement.
    (v) Lungs of animals (rodents) in the low and intermediate dose 
groups shall also be subjected to histopathological examination, 
primarily for evidence of infection since this provides a convenient 
assessment of the state of health of the animals.
    (vi) When a satellite group is used, histopathology shall be 
performed on tissues and organs identified as showing effects in the 
treated groups.
    (e) Data and reporting--(1) Treatment of results. (i) Data shall be 
summarized in tabular form, showing for each test group the number of 
animals at the start of the test, the number of animals showing lesions, 
the types of lesions, and the percentage of animals displaying each type 
of lesion.
    (ii) All observed results, quantitative and incidental, should be 
evaluated by an appropriate statistical method. Any generally accepted 
statistical method may be used; the statistical methods should be 
selected during the design of the study.
    (2) Evaluation of results. The findings of the subchronic inhalation 
toxicity study should be evaluated in conjunction with the findings of 
preceding studies and considered in terms of the observed toxic effects 
and the necropsy and histopathological findings. The evaluation will 
include the relationship between the concentration of the test substance 
and duration of exposure, and the presence or absence, the incidence and 
severity, of abnormalities, including behavioral and clinical 
abnormalities, gross lesions, identified target organs, body weight 
changes, effects on mortality and any other general or specific toxic 
effects. A properly

[[Page 150]]

conducted subchronic test should provide a satisfactory estimation of a 
no-effect level.
    (3) Test report. In addition to the reporting requirements as 
specified under EPA Good Laboratory Practice Standards, 40 CFR part 792, 
subpart J, the following specific information shall be reported:
    (i) Test conditions. (A) Description of exposure apparatus, 
including design, type, dimensions, source of air, system for generating 
particulates and aerosols, method of conditioning air, treatment of 
exhaust air, and the method of housing animals in a test chamber.
    (B) The equipment for measuring temperature, humidity, and 
particulate aerosol concentrations and size shall be described.
    (ii) Exposure data. These shall be tabulated and presented with mean 
values and measure of variability (e.g., standard deviation) and shall 
include:
    (A) Airflow rates through the inhalation equipment.
    (B) Temperature and humidity of air.
    (C) Nominal concentration (total amount of test substance fed into 
the inhalation equipment divided by volume of air).
    (D) Actual concentration in test breathing zone.
    (E) Particle size distribution (e.g., median aerodynamic diameter of 
particles with standard deviation from the mean).
    (iii) Group animal data. Tabulation of toxic response data by 
species, strain, sex, and exposure level for:
    (A) Number of animals dying.
    (B) Number of animals showing signs of toxicity.
    (C) Number of animals exposed.
    (iv) Individual animal data. (A) Date of death during the study or 
whether animals survived to termination.
    (B) Date of observation of each abnormal sign and its subsequent 
course.
    (C) Body weight data.
    (D) Feed consumption data when collected.
    (E) Hematological tests employed and all results.
    (F) Clinical biochemistry tests employed and all results.
    (G) Necropsy findings.
    (H) Detailed description of all histopathological findings.
    (I) Statistical treatment of results where appropriate.
    (f) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) Cage, J.C. ``Experimental Inhalation Toxicology,'' Methods in 
Toxicology. Ed. G.E. Paget. (Philadelphia: F.A. Davis Co. 1970, pp. 258-
277.
    (2) Casarett, L.J., Doull, J. ``Chapter 9.'' Toxicology: The Basic 
Science of Poisons (New York: Macmillan Publishing Co. Inc. 1975).
    (3) MacFarland, H.N. ``Respiratory Toxicology,'' Essays in 
Toxicology. Ed. W.J. Hayes. Vol. 7 (New York: Academic Press, 1976) pp. 
121-154.
    (4) National Academy of Sciences. ``Principles and Procedures for 
Evaluating the Toxicity of Household Substances,'' a report prepared by 
the Committee for the Revision of NAS Publication 1138, under the 
auspices of the Committee on Toxicology, National Research Council, 
National Academy of Sciences, Washington, DC (1977).
    (5) World Health Organization. ``Part I. Environmental Health 
Criteria 6,'' Principles and Methods for Evaluating the Toxicity of 
Chemicals. (Geneva: World Health Organization, 1978).

[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19073, May 20, 1987; 
52 FR 26150, July 13, 1987; 53 FR 49150, Dec. 6, 1988; 54 FR 21064, May 
16, 1989]



Sec. 798.2650  Oral toxicity.

    (a) Purpose. In the assessment and evaluation of the toxic 
characteristics of a chemical, the determination of subchronic oral 
toxicity may be carried out after initial information on toxicity has 
been obtained by acute testing. The subchronic oral study has been 
designed to permit the determination of the no-observed-effect level and 
toxic effects associated with continuous or repeated exposure to a test 
substance for a period of 90 days. The test is not capable of 
determining those effects that have a long latency period for 
development (e.g., carcinogenicity and life shortening). It provides 
information on health hazards likely to arise from repeated exposure

[[Page 151]]

by the oral route over a limited period of time. It will provide 
information on target organs, the possibilities of accumulation, and can 
be of use in selecting dose levels for chronic studies and for 
establishing safety criteria for human exposure.
    (b) Definitions. (1) Subchronic oral toxicity is the adverse effects 
occurring as a result of the repeated daily exposure of experimental 
animals to a chemical by the oral route for a part (approximately 10 
percent) of a life span.
    (2) Dose is the amount of test substance administered. Dose is 
expressed as weight of test substance (g, mg) per unit weight of test 
animal (e.g., mg/kg), or as weight of test substance per unit weight of 
food or drinking water.
    (3) No-effect level/No-toxic-effect level/No-adverse-effect level/
No-observed-effect level is the maximum dose used in a test which 
produces no observed adverse effects. A no-observed-effect level is 
expressed in terms of the weight of a substance given daily per unit 
weight of test animal (mg/kg). When administered to animals in food or 
drinking water the no-observed-effect level is expressed as mg/kg of 
food or mg/ml of water.
    (4) Cumulative toxicity is the adverse effects of repeated doses 
occurring as a result of prolonged action on, or increased concentration 
of, the administered test substance or its metabolites in susceptible 
tissue.
    (c) Principle of the test method. The test substance is administered 
orally in graduated daily doses to several groups of experimental 
animals, one dose level per group, for a period of 90 days. During the 
period of administration the animals are observed daily to detect signs 
of toxicity. Animals which die during the period of administration are 
necropsied. At the conclusion of the test all animals are necropsied and 
histo-pathological examinations carried out.
    (d) Limit test. If a test at one dose level of at least 1,000 mg/kg 
body weight (expected human exposure may indicate the need for a higher 
dose level), using the procedures described for this study, produces no 
observable toxic effects and if toxicity would not be expected based 
upon data of structurally related compounds, then a full study using 
three dose levels might not be necessary.
    (e) Test procedures--(1) Animal selection--(i) Species and strain. A 
mammalian species shall be used for testing. A variety of rodent species 
may be used, although the rat is the preferred species. Commonly used 
laboratory strains shall be employed. The commonly used nonrodent 
species is the dog, preferably of a defined breed; the beagle is 
frequently used. If other mammalian species are used, the tester shall 
provide justification/reasoning for his or her selection.
    (ii) Age--(A) General. Young adult animals shall be employed. At the 
commencement of the study the weight variation of animals used shall not 
exceed 20 percent of the mean weight for each sex.
    (B) Rodents. Dosing shall begin as soon as possible after weaning, 
ideally before the rats are 6, and in any case, not more than 8 weeks 
old.
    (C) Non-rodent. In the case of the dog, dosing shall commence after 
acclimatization, preferably at 4 to 6 months and not later than 9 months 
of age.
    (iii) Sex. (A) Equal numbers of animals of each sex shall be used at 
each dose level.
    (B) The females shall be nulliparous and nonpregnant.
    (iv) Numbers--(A) Rodents. At least 20 animals (10 females and 10 
males) shall be used at each dose level.
    (B) Non-rodents. At least eight animals (four females and four 
males) shall be used at each dose level.
    (C) If interim sacrifices are planned, the number shall be increased 
by the number of animals scheduled to be sacrificed before the 
completion of the study.
    (2) Control groups. A concurrent control group is required. This 
group shall be an untreated or sham-treated control group or, if a 
vehicle is used in administering the test substance, a vehicle control 
group. If the toxic properties of the vehicle are not known or cannot be 
made available, both untreated and vehicle control groups are required.
    (3) Satellite group. (Rodent) A satellite group of 20 animals (10 
animals per sex) may be treated with the high dose level

[[Page 152]]

for 90 days and observed for reversibility, persistence, or delayed 
occurrence of toxic effects for a post-treatment period of appropriate 
length, normally not less than 28 days.
    (4) Dose levels and dose selection. (i) In subchronic toxicity 
tests, it is desirable to have a dose response relationship as well as a 
no-observed-toxic-effect level. Therefore, at least 3 dose levels with a 
control and, where appropriate, a vehicle control (corresponding to the 
concentration of vehicle at the highest exposure level) shall be used. 
Doses should be spaced appropriately to produce test groups with a range 
of toxic effects. The data should be sufficient to produce a dose-
response curve.
    (ii) The highest dose level in rodents should result in toxic 
effects but not produce an incidence of fatalities which would prevent a 
meaningful evaluation; for non-rodents there should be no fatalities.
    (iii) The lowest dose level should not produce any evidence of 
toxicity. Where there is a usable estimation of human exposure the 
lowest dose level should exceed this.
    (iv) Ideally, the intermediate dose level(s) should produce minimal 
observable toxic effects. If more than one intermediate dose is used, 
the dose levels should be spaced to produce a gradation of toxic 
effects.
    (v) For rodents, the incidence of fatalities in low and intermediate 
dose groups and in the controls should be low, to permit a meaningful 
evaluation of the results; for non-rodents, there should be no 
fatalities.
    (5) Exposure conditions. The animals are dosed with the test 
substance ideally on a 7-day per week basis over a period of 90 days. 
However, based primarily on practical considerations, dosing in gavage 
or capsule studies on a 5-day per week basis is considered to be 
acceptable.
    (6) Observation period. (i) Duration of observation shall be for at 
least 90 days.
    (ii) Animals in the satellite group scheduled for followup 
observations should be kept for at least 28 days further without 
treatment to detect recovery from, or persistence of, toxic effects.
    (7) Administration of the test substance. (i) The test substance may 
be administered in the diet or in capsules. In addition, for rodents it 
may also be administered by gavage or in the drinking water.
    (ii) All animals shall be dosed by the same method during the entire 
experimental period.
    (iii) Where necessary, the test substance is dissolved or suspended 
in a suitable vehicle. If a vehicle or diluent is needed, ideally it 
should not elicit important toxic effects itself nor substantially alter 
the chemical or toxicological properties of the test substance. It is 
recommended that wherever possible the usage of an aqueous solution be 
considered first, followed by consideration of a solution of oil and 
then by possible solution in other vehicles.
    (iv) For substances of low toxicity, it is important to ensure that 
when administered in the diet the quantities of the test substance 
involved do not interfere with normal nutrition. When the test substance 
is administered in the diet either a constant dietary concentration 
(ppm) or a constant dose level in terms of the animals' body weight 
shall be used; the alternative used shall be specified.
    (v) For a substance administered by gavage or capsule, the dose 
shall be given at approximately the same time each day, and adjusted at 
intervals (weekly or bi-weekly) to maintain a constant dose level in 
terms of animal body weight.
    (8) Observation of animals. (i) Each animal shall be observed daily 
and, if necessary, handled to appraise its physical condition.
    (ii) Additional observations shall be made daily with appropriate 
actions taken to minimize loss of animals to the study (e.g., necropsy 
or refrigeration of those animals found dead and isolation or sacrifice 
of weak or moribund animals).
    (iii) Signs of toxicity shall be recorded as they are observed 
including the time of onset, degree and duration.
    (iv) Cage-side observations shall include, but not be limited to, 
changes in skin and fur, eyes and mucous membranes, respiratory, 
circulatory, autonomic and central nervous systems,

[[Page 153]]

somatomotor activity and behavior pattern.
    (v) Measurements shall be made weekly of feed consumption or water 
consumption when the test substance is administered in the feed or 
drinking water, respectively.
    (vi) Animals shall be weighed weekly.
    (vii) At the end of the 90-day period all survivors in the 
nonsatellite treatment groups shall be sacrificed. Moribund animals 
shall be removed and sacrificed when noticed.
    (9) Clinical examinations. (i) The following examinations shall be 
made on all animals of each sex in each group for rodents and all 
animals when non-rodents are used as test animals.
    (A) Certain hematology determinations shall be carried out at least 
two times during the test period on all groups of animals including 
concurrent controls: After 30 days of test and just prior to terminal 
sacrifice at the end of the test period. Hematology determinations which 
are appropriate to all studies: Hematocrit, hemoglobin concentration, 
erythrocyte count, total and differential leukocyte count, and a measure 
of clotting potential such as clotting time, prothrombin time, 
thromboplastin time, or platelet count.
    (B) Certain clinical biochemistry determinations on blood should be 
carried out at least two times during the test period on all groups of 
animals including concurrent controls: After 30 days of test and just 
prior to terminal sacrifice at the end of the test period. Clinical 
biochemistry test areas which are considered appropriate to all studies: 
Electrolyte balance, carbohydrate metabolism, and liver and kidney 
function. The selection of specific tests will be influenced by 
observations on the mode of action of the substance. Suggested 
determinations: Calcium, phosphorus, chloride, sodium, potassium, 
fasting glucose (with period of fasting appropriate to the species), 
serum glutamic-pyruvic transaminase (now known as serum alanine 
aminotransferase), serum glutamic oxaloacetic transaminase (now known as 
serum aspartate aminotransferase), ornithine decarboxylase, gamma 
glutamyl transpeptidase, urea nitrogen, albumen, blood creatinine, total 
bilirubin, and total serum protein measurements. Other determinations 
which may be necessary for an adequate toxicological evaluation include: 
Analyses of lipids, hormones, acid/base balance, methemoglobin, and 
cholinesterase activity. Additional clinical biochemistry may be 
employed, where necessary, to extend the investigation of observed 
effects.
    (ii) The following examinations shall be made on high dose and 
control groups. If changes in the eyes are detected, all animals should 
be examined.
    (A) Ophthalmological examination, using an ophthalmoscope or 
equivalent suitable equipment, shall be made prior to the administration 
of the test substance and at the termination of the study.
    (B) Urinalysis is not recommended on a routine basis, but only when 
there is an indication based on expected and or observed toxicity.
    (10) Gross necropsy. (i) All animals shall be subjected to a full 
gross necropsy which includes examination of the external surface of the 
body, all orifices, and the cranial, thoracic and abdominal cavities and 
their contents.
    (ii) At least the liver, kidneys, adrenals, and gonads shall be 
weighed wet, as soon as possible after dissection to avoid drying. In 
addition, for the rodent, the brain; for the non-rodent, the thyroid 
with parathyroids also shall be weighed wet.
    (iii) The following organs and tissues, or representative samples 
thereof, shall be preserved in a suitable medium for possible future 
histopathological examination: All gross lesions; lungs--which should be 
removed intact, weighed, and treated with a suitable fixative to ensure 
that lung structure is maintained (perfusion with the fixative is 
considered to be an effective procedure); nasopharyngeal tissues; 
brain--including sections of medulla/pons, cerebellar cortex, and 
cerebral cortex; pituitary; thyroid/parathyroid; thymus; trachea; heart; 
sternum with bone marrow; salivary glands; liver; spleen; kidneys; 
adrenals; pancreas; gonads; uterus; accessory genital organs 
(epididymis, prostate, and, if present, seminal vesicles); aorta; 
(skin); gall bladder (if present); esophagus; stomach; duodenum; 
jejunum; ileum; cecum; colon;

[[Page 154]]

rectum; urinary bladder; representative lymph node; (mammary gland); 
(thigh musculature); peripheral nerve; (eyes); (femur--including 
articular surface); (spinal cord at three levels--cervical, midthoracic, 
and lumbar); and (zymbal and exorbital lachrymal glands); and (rodent-
zymbal glands).
    (11) Histopathology. The following histopathology shall be 
performed:
    (i) Full histopathology on the organs and tissues, listed above, of 
all rodents in the control and high dose groups, all non-rodents, and 
all rodents that died or were killed during the study.
    (ii) All gross lesions in all animals.
    (iii) Target organs in all animals.
    (iv) The tissues mentioned in brackets (listed above) if indicated 
by signs of toxicity of target organ involvement.
    (v) Lungs, liver and kidneys of all animals. Special attention to 
examination of the lungs of rodents shall be made for evidence of 
infection since this provides a convenient assessment of the state of 
health of the animals.
    (vi) When a satellite group is used (rodents), histopathology shall 
be performed on tissues and organs identified as showing effects in the 
treated groups.
    (f) Data and reporting--(1) Treatment of results. (i) Data shall be 
summarized in tabular form, showing for each test group the number of 
animals at the start of the test, the number of animals showing lesions, 
the types of lesions and the percentage of animals displaying each type 
of lesion.
    (ii) All observed results, quantitative and incidental, should be 
evaluated by an appropriate statistical method. Any generally accepted 
statistical methods may be used; the statistical methods should be 
selected during the design of the study.
    (2) Evaluation of the study results. (i) The findings of a 
subchronic oral toxicity study should be evaluated in conjunction with 
the findings of preceding studies and considered in terms of the toxic 
effects and the necropsy and histopathological findings. The evaluation 
will include the relationship between the dose of the test substance and 
the presence or absence, the incidence and severity, of abnormalities, 
including behavioral and clinical abnormalities, gross lesions, 
identified target organs, body weight changes, effects on mortality and 
any other general or specific toxic effects. A properly conducted 
subchronic test should provide a satisfactory estimation of a no-effect 
level.
    (ii) In any study which demonstrates an absence of toxic effects, 
further investigation to establish absorption and bioavailability of the 
test substance should be considered.
    (3) Test report. In addition to the reporting requirements as 
specified under EPA Good Laboratory Practice Standards, 40 CFR part 792, 
subpart J, the following specific information shall be reported:
    (i) Group animal data. Tabulation of toxic response data by species, 
strain, sex and exposure level for:
    (A) Number of animals dying.
    (B) Number of animals showing signs of toxicity.
    (C) Number of animals exposed.
    (ii) Individual animal data. (A) Date of death during the study or 
whether animals survived to termination.
    (B) Date of observation of each abnormal sign and its subsequent 
course.
    (C) Body weight data.
    (D) Feed consumption data when collected.
    (E) Hematological tests employed and all results.
    (F) Clinical biochemistry tests employed and all results.
    (G) Necropsy findings.
    (H) Detailed description of all histopathological findings.
    (I) Statistical treatment of results where appropriate.
    (g) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) Boyd, E.M. ``Chapter 14--Pilot Studies, 15--Uniposal Clinical 
Parameters, 16--Uniposal Autopsy Parameters.'' Predictive Toxicometrics. 
(Baltimore: Williams and Wilkins, 1972).
    (2) Fitzhugh, O.G. ``Subacute Toxicity,'' Appraisal of the Safety of 
Chemicals in Foods, Drugs and Cosmetics. The Association of Food and 
Drug Officials of the United States (1959, 3rd Printing 1975) pp. 26-35.
    (3) Food Safety Council. ``Subchronic Toxicity Studies,'' Proposed 
System for

[[Page 155]]

Food Safety Assessment. (Columbia: Food Safety Council, 1978) pp. 83-96.
    (4) National Academy of Sciences. ``Principles and Procedures for 
Evaluating the Toxicity of Household Substances,'' a report prepared by 
the Committee for the Revision of NAS Publication 1138, under the 
auspices of the Committee on Toxicology, National Research Council, 
National Academy of Sciences, Washington, DC (1977).
    (5) World Health Organization. ``Part I. Environmental Health 
Criteria 6,'' Principles and Methods for Evaluating the Toxicity of 
Chemicals. (Geneva: World Health Organization, 1978).

[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19074, May 20, 1987; 
53 FR 49150, Dec. 6, 1988; 54 FR 21064, May 16, 1989]



                       Subpart D_Chronic Exposure



Sec. 798.3260  Chronic toxicity.

    (a) Purpose. The objective of a chronic toxicity study is to 
determine the effects of a substance in a mammalian species following 
prolonged and repeated exposure. Under the conditions of the chronic 
toxicity test, effects which require a long latency period or which are 
cumulative should become manifest. The application of this guideline 
should generate data on which to identify the majority of chronic 
effects and shall serve to define long term dose-response relationships. 
The design and conduct of chronic toxicity tests should allow for the 
detection of general toxic effects, including neurological, 
physiological, biochemical, and hematological effects and exposure-
related morphological (pathology) effects.
    (b) Test procedures--(1) Animal selection--(i) Species and strain. 
Testing should be performed with two mammalian species, one a rodent and 
another a non-rodent. The rat is the preferred rodent species and the 
dog is the preferred non-rodent species. Commonly used laboratory 
strains should be employed. If other mammalian species are used, the 
tester should provide justification/reasoning for their selection.
    (ii) Age. (A) Dosing of rats should begin as soon as possible after 
weaning, ideally before the rats are 6, but in no case more than 8 weeks 
old.
    (B) Dosing of dogs should begin between 4 and 6 months of age and in 
no case later than 9 months of age.
    (C) At commencement of the study the weight variation of animals 
used should not exceed 20 percent of the mean 
weight for each sex.
    (iii) Sex. (A) Equal numbers of animals of each sex should be used 
at each dose level.
    (B) The females should be nulliparous and non-pregnant.
    (iv) Numbers. (A) For rodents, at least 40 animals (20 females and 
20 males) and for non-rodents (dogs) at least 8 animals (4 females and 4 
males) should be used at each dose level.
    (B) If interim sacrifices are planned, the number should be 
increased by the number of animals scheduled to be sacrificed during the 
course of the study.
    (C) The number of animals at the termination of the study must be 
adequate for a meaningful and valid statistical evaluation of chronic 
effects.
    (2) Control groups. (i) A concurrent control group is suggested. 
This group should be an untreated or sham treated control group or, if a 
vehicle is used in administering the test substance, a vehicle control 
group. If the toxic properties of the vehicle are not known or cannot be 
made available, both untreated and vehicle control groups are strongly 
suggested.
    (ii) In special circumstances such as in inhalation studies 
involving aerosols or the use of an emulsifier of uncharacterized 
biological activity in oral studies, a concurrent negative control group 
should be utilized. The negative control group should be treated in the 
same manner as all other test animals except that this control group 
should not be exposed to either the test substance or any vehicle.
    (3) Dose levels and dose selections. (i) In chronic toxicity tests, 
it is necessary to have a dose-response relationship as well as a no-
observed-toxic-effect level. Therefore, at least three dose levels 
should be used in addition to the concurrent control group. Dose levels 
should be spaced to produce a gradation of effects.
    (ii) The high dose level in rodents should elicit some signs of 
toxicity without causing excessive lethality; for non-rodents, there 
should be signs of

[[Page 156]]

toxicity but there should be no fatalities.
    (iii) The lowest dose level should not produce any evidence of 
toxicity. Where there is a usable estimation of human exposure the 
lowest dose level should exceed this even though this dose level may 
result in some signs of toxicity.
    (iv) Ideally, the intermediate dose level(s) should produce minimal 
observable toxic effects. If more than one intermediate dose is used, 
the dose level should be spaced to produce a gradation of toxic effects.
    (v) For rodents, the incidence of fatalities in low and intermediate 
dose groups and in the controls should be low to permit a meaningful 
evaluation of the results. For non-rodents, there should be no 
fatalities.
    (4) Exposure conditions. The animals are dosed with the test 
substance ideally on a 7-day per week basis over a period of at least 12 
months. However, based primarily on practical considerations, dosing on 
a 5-day per week basis is considered to be acceptable.
    (5) Observation period. Duration of observation should be for at 
least 12 months, and may be concurrent with or subsequent to dosing. If 
there is a post-exposure observation period, an interim sacrifice should 
be performed on no fewer than half of the animals of each sex at each 
dose level immediately upon termination of exposure.
    (6) Administration of the test substance. The three main routes of 
administration are oral, dermal, and inhalation. The choice of the route 
of administration depends upon the physical and chemical characteristics 
of the test substance and the form typifying exposure in humans.
    (i) Oral studies. (A) The animals should receive the test substance 
in their diet, dissolved in drinking water, or given by gavage or 
capsule for a period of at least 12 months.
    (B) If the test substance is administered in the drinking water, or 
mixed in the diet, exposure is continuous.
    (C) For a diet mixture, the highest concentration should not exceed 
5 percent.
    (ii) Dermal studies. (A) The animals are treated by topical 
application with the test substance, ideally for at least 6 hours per 
day.
    (B) Fur should be clipped from the dorsal area of the trunk of the 
test animals. Care must be taken to avoid abrading the skin which could 
alter its permeability.
    (C) The test substance should be applied uniformly over a shaved 
area which is approximately 10 percent of the total body surface area. 
With highly toxic substances, the surface area covered may be less, but 
as much of the area should be covered with as thin and uniform a film as 
possible.
    (D) During the exposure period, the test substance may be held if 
necessary, in contact with the skin with a porous gauze dressing and 
non-irritating tape. The test site should be further covered in a 
suitable manner to retain the gauze dressing and test substance and 
ensure that the animals cannot ingest the test substance.
    (iii) Inhalation studies. (A) The animals should be tested with 
inhalation equipment designed to sustain a dynamic air flow of 12 to 15 
air changes per hour, ensure an adequate oxygen content of 19 percent 
and an evenly distributed exposure atmosphere. Where a chamber is used, 
its design should minimize crowding of the test animals and maximize 
their exposure to the test substance. This is best accomplished by 
individual caging. As a general rule to ensure stability of a chamber 
atmosphere, the total ``volume'' of the test animals should not exceed 5 
percent of the volume of the test chamber. Alternatively, oro-nasal, 
head-only or whole body individual chamber exposure may be used.
    (B) The temperature at which the test is performed should be 
maintained at 22 [deg]C (2[deg]). Ideally, the 
relative humidity should be maintained between 40 to 60 percent, but in 
certain instances (e.g., tests of aerosols, use of water vehicle) this 
may not be practicable.
    (C) Feed and water should be withheld during each daily 6 hour 
exposure period.
    (D) A dynamic inhalation system with a suitable analytical 
concentration control system should be used. The rate of air flow should 
be adjusted to ensure that conditions throughout

[[Page 157]]

the equipment are essentially the same. Maintenance of slight negative 
pressure inside the chamber will prevent leakage of the test substance 
into the surrounding areas.
    (7) Observation of animals. (i) Each animal should be handled and 
its physical condition appraised at least once each day.
    (ii) Additional observations should be made daily with appropriate 
actions taken to minimize loss of animals to the study (e.g., necropsy 
or refrigeration of those animals found dead and isolation or sacrific 
of weak or moribund animals).
    (iii) Clinical signs of toxicity including suspected tumors and 
mortality should be recorded as they are observed, including the time of 
onset, the degree and duration.
    (iv) Cage-side observations should include, but not be limited to, 
changes in skin and fur, eyes and mucous membranes, respiratory, 
circulatory, autonomic and central nervous systems, somatomotor activity 
and behavior pattern.
    (v) Body weights should be recorded individually for all animals 
once a week during the first 13 weeks of the test period and at least 
once every 4 weeks thereafter unless signs of clinical toxicity suggest 
more frequent weighings to facilitate monitoring of health status.
    (vi) When the test substance is administered in the feed or drinking 
water, measurements of feed or water consumption, respectively, should 
be determined weekly during the first 13 weeks of the study and then at 
approximately monthly intervals unless health status or body weight 
changes dictate otherwise.
    (vii) At the end of the study period all survivors should be 
sacrificed. Moribund animals should be removed and sacrificed when 
noticed.
    (8) Physical measurements. For inhalation studies, measurements or 
monitoring should be made of the following:
    (i) The rate of air flow should be monitored continuously, but 
should be recorded at intervals of at least once every 30 minutes.
    (ii) During each exposure period the actual concentrations of the 
test substance should be held as constant as practicable, monitored 
continuously and measured at least three times during the test period: 
at the beginning, at an intermediate time and at the end of the period.
    (iii) During the development of the generating system, particle size 
analysis should be performed to establish the stability of aerosol 
concentrations. During exposure, analysis should be conducted as often 
as necessary to determine the consistency of particle size distribution 
and homogeneity of the exposure stream.
    (iv) Temperature and humidity should be monitored continuously, but 
should be recorded at intervals of at least once every 30 minutes.
    (9) Clinical examinations. The following examinations should be made 
on at least 10 rats of each sex per dose and on all non-rodents.
    (i) Certain hematology determinations (e.g., hemoglobin content, 
packed cell volume, total red blood cells, total white blood cells, 
platelets, or other measures of clotting potential) should be performed 
at termination and should be performed at 3 months, 6 months and at 
approximately 6 month intervals thereafter (for studies extending beyond 
12 months) on blood samples collected from all non-rodents and from 10 
rats per sex of all groups. These collections should be from the same 
animals at each interval. If clinical observations suggest a 
deterioration in health of the animals during the study, a differential 
blood count of the affected animals should be performed. A differential 
blood count should be performed on samples from those animals in the 
highest dosage group and the controls. Differential blood counts should 
be performed for the next lower group(s) if there is a major discrepancy 
between the highest group and the controls. If hematological effects 
were noted in the subchronic test, hematological testing should be 
performed at 3, 6, 12, 18, and 24 months for a two year study and at 3, 
6, and 12 months for a 1-year study.
    (ii) Certain clinical biochemistry determinations on blood should be 
carried out at least three times during the test period: just prior to 
initiation of dosing (base line data), near the middle and at the end of 
the test period. Blood

[[Page 158]]

samples should be drawn for clinical chemistry measurements from all 
non-rodents and at least ten rodents per sex of all groups; if possible, 
from the same rodents at each time interval. Test areas which are 
considered appropriate to all studies: electrolyte balance, carbohydrate 
metabolism and liver and kidney function. The selection of specific 
tests will be influenced by observations on the mode of action of the 
substance and signs of clinical toxicity. Suggested chemical 
determinations: calcium, phosphorus, chloride, sodium, potassium, 
fasting glucose (with period of fasting appropriate to the species), 
serum glutamic-pyruvic transaminase (now known as serum alanine 
aminotransferase), serum glutamic oxaloacetic transaminase (now known as 
serum aspartate aminotransferase), ornithine decarboxylase, gamma 
glutamyl transpeptidase, blood urea nitrogen, albumen, blood creatinine, 
creatinine phosphokinase, total cholesterol, total bilirubin and total 
serum protein measurements. Other determinations which may be necessary 
for an adequate toxicological evaluation include analyses of lipids, 
hormones, acid/base balance, methemoglobin and cholinesterase activity. 
Additional clinical biochemistry may be employed where necessary to 
extend the investigation of observed effects.
    (iii) Urine samples from rodents at the same intervals as the 
hematological examinations under paragraph (b)(9)(i) of this section 
should be collected for analysis. The following determinations should be 
made from either individual animals or on a pooled sample/sex/group for 
rodents: appearance (volume and specific gravity), protein, glucose, 
ketones, bilirubin, occult blood (semi-quantitatively); and microscopy 
of sediment (semi-quantitatively).
    (iv) Ophthalmological examination, using an ophthalmoscope or 
equivalent suitable equipment, should be made prior to the 
administration of the test substance and at the termination of the 
study. If changes in eyes are detected all animals should be examined.
    (10) Gross necropsy. (i) A complete gross examination should be 
performed on all animals, including those which died during the 
experiment or were killed in moribund conditions.
    (ii) The liver, kidneys, adrenals, brain and gonads should be 
weighed wet, as soon as possible after dissection to avoid drying. For 
these organs, at least 10 rodents per sex per group and all non-rodents 
should be weighed.
    (iii) The following organs and tissues, or representative samples 
thereof, should be preserved in a suitable medium for possible future 
histopathological examination: All gross lesions and tumors; brain--
including sections of medulla/pons, cerebellar cortex, and cerebral 
cortex; pituitary; thyroid/parathyroid; thymus; lungs; trachea; heart; 
sternum and/or femur with bone marrow; salivary glands; liver; spleen; 
kidneys; adrenals; esophagus; stomach; duodenum; jejunum; ileum; cecum; 
colon; rectum; urinary bladder; representative lymph nodes; pancreas; 
gonads; uterus; accessory genital organs (epididymis, prostate, and, if 
present, seminal vesicles; female mammary gland; aorta; gall bladder (if 
present); skin; musculature; peripheral nerve; spinal cord at three 
levels--cervical, midthoracic, and lumbar; and eyes. In inhalation 
studies, the entire respiratory tract, including nose, pharynx, larynx, 
and paranasal sinuses should be examined and preserved. In dermal 
studies, skin from sites of skin painting should be examined and 
preserved.
    (iv) Inflation of lungs and urinary bladder with a fixative is the 
optimal method for preservation of these tissues. The proper inflation 
and fixation of the lungs in inhalation studies is considered essential 
for appropriate and valid histopathological examination.
    (v) If other clinical examinations are carried out, the information 
obtained from these procedures should be available before microscopic 
examination, since they may provide significant guidance to the 
pathologist.
    (11) Histopathology. (i) The following histopathology should be 
performed:
    (A) Full histopathology on the organs and tissues, listed above, of 
all non-rodents, of all rodents in the control and high dose groups and 
of all rodents that died or were killed during the study.

[[Page 159]]

    (B) All gross lesions in all animals.
    (C) Target organs in all animals.
    (D) Lungs, liver and kidneys of all animals. Special attention to 
examination of the lungs of rodents should be made for evidence of 
infection since this provides an assessment of the state of health of 
the animals.
    (ii) If excessive early deaths or other problems occur in the high 
dose group compromising the significance of the data, the next dose 
level should be examined for complete histopathology.
    (iii) In case the results of an experiment give evidence of 
substantial alteration of the animals' normal longevity or the induction 
of effects that might affect a toxic response, the next lower dose level 
should be examined fully, as described under paragraph (b)(11)(i) of 
this section.
    (iv) An attempt should be made to correlate gross observations with 
microscopic findings.
    (c) Data and reporting--(1) Treatment of results. (i) Data should be 
summarized in tabular form, showing for each test group the number of 
animals at the start of the test, the number of animals showing lesions, 
the types of lesions and the percentage of animals displaying each type 
of lesion.
    (ii) All observed results, quantitative and incidental, should be 
evaluated by an appropriate statistical method. Any generally accepted 
statistical methods may be used; the statistical methods should be 
selected during the design of the study.
    (2) Evaluation of study results. (i) The findings of a chronic 
toxicity study should be evaluated in conjunction with the findings of 
preceding studies and considered in terms of the toxic effects, the 
necropsy and histopathological findings. The evaluation will include the 
relationship between the dose of the test substance and the presence, 
incidence and severity of abnormalities (including behavioral and 
clinical abnormalities), gross lesions, identified target organs, body 
weight changes, effects on mortality and any other general or specific 
toxic effects.
    (ii) In any study which demonstrates an absence of toxic effects, 
further investigation to establish absorption and bioavailability of the 
test substance should be considered.
    (3) Test report. (i) In addition to the reporting requirements as 
specified under 40 CFR part 792 subpart J, the following specific 
information should be reported:
    (A) Group animal data. Tabulation of toxic response data by species, 
strain, sex and exposure level for:
    (1) Number of animals dying.
    (2) Number of animals showing signs of toxicity.
    (3) Number of animals exposed.
    (B) Individual animal data. (1) Time of death during the study or 
whether animals survived to termination.
    (2) Time of observation of each abnormal sign and its subsequent 
course.
    (3) Body weight data.
    (4) Feed and water consumption data, when collected.
    (5) Results of ophthalmological examination, when performed.
    (6) Hematological tests employed and all results.
    (7) Clinical biochemistry tests employed and all results.
    (8) Necropsy findings.
    (9) Detailed description of all histopathological findings.
    (10) Statistical treatment of results, where appropriate.
    (ii) In addition, for inhalation studies the following should be 
reported:
    (A) Test conditions. (1) Description of exposure apparatus including 
design, type, dimensions, source of air, system for generating 
particulates and aerosols, method of conditioning air, treatment of 
exhaust air and the method of housing the animals in a test chamber.
    (2) The equipment for measuring temperature, humidity, and 
particulate aerosol concentrations and size should be described.
    (B) Exposure data. These should be tabulated and presented with mean 
values and a measure of variability (e.g., standard deviation) and 
should include:
    (1) Airflow rates through the inhalation equipment.
    (2) Temperature and humidity of air.
    (3) Nominal concentration (total amount of test substance fed into 
the inhalation equipment divided by volume of air).

[[Page 160]]

    (4) Actual concentration in test breathing zone.
    (5) Particle size distribution (e.g., median aerodynamic diameter of 
particles with standard deviation from the mean).
    (d) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) Benitz, K.F. ``Measurement of Chronic Toxicity,'' Methods of 
Toxicology. Ed. G.E. Paget. (Oxford: Blackwell Scientific Publications, 
1970) pp. 82-131.
    (2) D'Aguanno, W. ``Drug Safety Evaluation--Pre-Clinical 
Considerations,'' Industrial Pharmacology: Neuroleptics. Vol. I, Ed. S. 
Fielding and H. Lal. (Mt. Kisco: Futura Publishing Co. 1974) pp. 317-
332.
    (3) Fitzhugh, O.G. Third Printing: 1975. ``Chronic Oral Toxicity,'' 
Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. The 
Association of Food and Drug Officials of the United States (1959, 3rd 
Printing 1975) pp. 36-45.
    (4) Goldenthal, E.I., D'Aguanno, W. ``Evaluation of Drugs,'' 
Appraisal of the Safety of Chemicals in Foods, Drugs, and Cosmetics. The 
Association of Food and Drug Officials of the United States (1959, 3rd 
Printing 1975) pp. 60-67.
    (5) National Academy of Sciences. ``Principles and Procedures for 
Evaluating the Toxicity of Household Substances,'' a report prepared by 
the Committee for the Revision of NAS Publication 1138, under the 
auspices of the Committee on Toxicology, National Research Council, 
National Academy of Sciences, Washington, DC (1977).
    (6) National Center for Toxicological Research. ``Appendix B,'' 
Report of Chronic Studies Task Force Committee, April 13-21, 1972. 
(Rockville: National Center for Toxicological Research, 1972).
    (7) Page, N.P. ``Chronic Toxicity and Carcinogenicity Guidelines,'' 
Journal of Environmental Pathology and Toxicology, 1:161-182 (1977).
    (8) Schwartz, E. ``Toxicology of Neuroleptic Agents,'' Industrial 
Pharmacology: Neuroleptics Ed. S. Fielding and H. Lal. (Mt. Kisco, 
Futura Publishing Co., 1974) pp. 203-221.
    (9) United States Pharmaceutical Manufacturers Association. 
Guidelines for the Assessment of Drug and Medical Device Safety in 
Animals. (1977).
    (10) World Health Organization. ``Guidelines for Evaluation of Drugs 
for Use in Man,'' WHO Technical Report Series No. 563. (Geneva: World 
Health Organization, 1975).
    (11) World Health Organization. ``Part I. Environmental Health 
Criteria 6,'' Principles and Methods for Evaluating the Toxicity of 
Chemicals. (Geneva: World Health Organization, 1978).
    (12) World Health Organization. ``Principles for Pre-Clinical 
Testing of Drug Safety,'' WHO Technical Report Series No. 341. (Geneva: 
World Health Organization, 1966).

[50 FR 39397, Sept. 27, 1985, as amended at 54 FR 21064, May 16, 1989]



Sec. 798.3300  Oncogenicity.

    (a) Purpose. The objective of a long-term oncogenicity study is to 
observe test animals for a major portion of their life span for the 
development of neoplastic lesions during or after exposure to various 
doses of a test substance by an appropriate route of administration.
    (b) Test procedures--(1) Animal selection--(i) Species and strain. A 
compound of unknown activity shall be tested on two mammalian species. 
Rats and mice are the species of choice because of their relatively 
short life spans, the limited cost of their maintenance, their 
widespread use in pharmacological and toxicological studies, their 
susceptibility to tumor induction, and the availability of inbred or 
sufficiently characterized strains. Commonly used laboratory strains 
shall be employed. If other species are used, the tester shall provide 
justification/reasoning for their selection.
    (ii) Age. (A) Dosing of rodents shall begin as soon as possible 
after weaning, ideally before the animals are 6 weeks old, but in no 
case more than 8 weeks old.
    (B) At commencement of the study, the weight variation of animals 
used shall not exceed 20 percent of the mean 
weight for each sex.
    (C) Studies using prenatal or neonatal animals may be recommended 
under special conditions.

[[Page 161]]

    (iii) Sex. (A) Animals of each sex shall be used at each dose level.
    (B) The females shall be nulliparous and non-pregnant.
    (iv) Numbers. (A) For rodents, at least 100 animals (50 females and 
50 males) shall be used at each dose level and concurrent control.
    (B) If interim sacrifices are planned the number shall be increased 
by the number of animals scheduled to be sacrificed during the course of 
the study.
    (C) The number of animals at the termination of the study should be 
adequate for a meaningful and valid statistical evaluation of long term 
exposure. For a valid interpretation of negative results, it is 
essential that survival in all groups does not fall below 50 percent at 
the time of termination.
    (2) Control groups. (i) A concurrent control group is required. This 
group shall be an untreated or sham treated control group or, if a 
vehicle is used in administering the test substance, a vehicle control 
group. If the toxic properties of the vehicle are not known or cannot be 
made available, both untreated and vehicle control groups are required.
    (ii) In special circumstances such as in inhalation studies 
involving aerosols or the use of an emulsifier of uncharacterized 
biological activity in oral studies, a concurrent negative control group 
shall be utilized. The negative control group shall be treated in the 
same manner as all other test animals except that this control group 
shall not be exposed to either the test substance or any vehicle.
    (iii) The use of historical control data (i.e., the incidence of 
tumors and other suspect lesions normally occurring under the same 
laboratory conditions and in the same strain of animals employed in the 
test) is desirable for assessing the significance of changes observed in 
exposed animals.
    (3) Dose levels and dose selection. (i) For risk assessment 
purposes, at least 3 dose levels shall be used, in addition to the 
concurrent control group. Dose levels should be spaced to produce a 
gradation of chronic effects.
    (ii) The high dose level should elicit signs of minimal toxicity 
without substantially altering the normal life span.
    (iii) The lowest dose should not interfere with normal growth, 
development and longevity of the animal; and it should not otherwise 
cause any indication of toxicity. In general, this should not be lower 
than ten percent of the high dose.
    (iv) The intermediate dose(s) should be established in a mid-range 
between the high and low doses, depending upon the toxicokinetic 
properties of the chemical, if known.
    (v) The selection of these dose levels should be based on existing 
data, preferably on the results of subchronic studies.
    (4) Exposure conditions. The animals are dosed with the test 
substance ideally on a 7 day per week basis over a period of at least 24 
months for rats, and 18 months for mice. However, based primarily on 
practical considerations, dosing on a 5 day per week basis is considered 
to be acceptable.
    (5) Observations period. It is necessary that the duration of an 
oncogenicity test comprise the majority of the normal life span of the 
strain of animals to be used. This time period shall not be less than 24 
months for rats and 18 months for mice, and ordinarily not longer than 
30 months for rats and 24 months for mice. For longer time periods, and 
where any other species are used, consultation with the Agency in regard 
to the duration of the test is advised.
    (6) Administration of the test substance. The three main routes of 
administration are oral, dermal, and inhalation. The choice of the route 
of administration depends upon the physical and chemical characteristics 
of the test substance and the form typifying exposure in humans.
    (i) Oral studies. (A) The animals shall receive the test substance 
in their diet, dissolved in drinking water at levels that do not exceed 
the maximum solubility of the test chemical under testing condition.
    (B) If the test substance is administered in the drinking water, or 
mixed in the diet, exposure shall be continuous.
    (C) For a diet mixture, the highest concentration should not exceed 
5 percent.

[[Page 162]]

    (ii) Dermal studies. (A) The animals are treated by topical 
application with the test substance, ideally for at least 6 hours per 
day.
    (B) Fur should be clipped from the dorsal area of the trunk of the 
test animals. Care should be taken to avoid abrading the skin which 
could alter its permeability.
    (C) The test substance shall be applied uniformly over a shaved area 
which is approximately 10 percent of the total body surface area. With 
highly toxic substances, the surface area covered may be less, but as 
much of the area shall be covered with as thin and uniform a film as 
possible.
    (D) During the exposure period, the test substance may be held, if 
necessary, in contact with the skin with a porous gauze dressing and 
non-irritating tape. The test site should be further covered in a 
suitable manner to retain the gauze dressing and test substance and 
ensure that the animals cannot ingest the test substance.
    (iii) Inhalation studies. (A) The animals shall be tested with 
inhalation equipment designed to sustain a minimum dynamic air flow of 
12 to 15 air changes per hour, ensure an adequate oxygen content of 19 
percent and an evenly distributed exposure atmosphere. Where a chamber 
is used, its design should minimize crowding of the test animals and 
maximize their exposure to the test substance. This is best accomplished 
by individual caging. To ensure stability of a chamber atmosphere, the 
total ``volume'' of the test animals shall not exceed 5 percent of the 
volume of the test chamber. Alternatively, oro-nasal, head-only, or 
whole-body individual chamber exposure may be used.
    (B) The temperature at which the test is performed should be 
maintained at 22 [deg]C (2[deg]). Ideally, the 
relative humidity should be maintained between 40 to 60 percent, but in 
certain instances (e.g. tests of aerosols, use of water vehicle) this 
may not be practicable.
    (C) Feed and water shall be withheld during each daily 6-hour 
exposure period.
    (D) A dynamic inhalation system with a suitable flow control system 
shall be used. The rate of air flow shall be adjusted to ensure that 
conditions throughout the equipment are essentially the same. 
Maintenance of slight negative pressure inside the chamber will prevent 
leakage of the test substance into the surrounding areas.
    (7) Observations of animals. (i) Each animal shall be observed daily 
and if necessary should be handled to appraise its physical condition.
    (ii) Additional observations shall be made daily with appropriate 
actions taken to minimize loss of animals to the study (e.g., necropsy 
or refrigeration of those animals found dead and isolation or sacrifice 
of weak or moribund animals).
    (iii) Clinical signs and mortality shall be recorded for all 
animals. Special attention should be paid to tumor development. The day 
of onset, location, dimensions, appearance and progression of each 
grossly visible or palpable tumor shall be recorded.
    (iv) Body weights shall be recorded individually for all animals 
once a week during the first 13 weeks of the test period and at least 
once every 4 weeks thereafter unless signs of clinical toxicity suggest 
more frequent weighings to facilitate monitoring of health status.
    (v) When the test substance is administered in the feed or drinking 
water, measurements of feed or water consumption, respectively, shall be 
determined weekly during the first 13 weeks of the study and then at 
approximately monthly intervals unless health status or body weight 
changes dictate otherwise.
    (vi) At the end of the study period all survivors are sacrificed. 
Moribund animals shall be removed and sacrificed when noticed.
    (8) Physical measurements. For inhalation studies, measurements or 
monitoring should be made of the following:
    (i) The rate of air flow shall be monitored continuously and 
recorded at intervals of at least once every 30 minutes.
    (ii) During each exposure period the actual concentrations of the 
test substance shall be held as constant as practicable, monitored 
continuously and recorded at least three times during the test period: 
at the beginning, at

[[Page 163]]

an intermediate time and at the end of the period.
    (iii) During the development of the generating system, particle size 
analysis shall be performed to establish the stability of aerosol 
concentrations with respect to particle size. During exposure, analyses 
shall be conducted as often as necessary to determine the consistency of 
particle size, distribution, and homogeneity of the exposure stream.
    (iv) Temperature and humidity shall be monitored continuously, but 
shoud be recorded at intervals of at least once every 30 minutes.
    (9) Clinical examinations. At 12 months, 18 months, and at 
sacrifice, a blood smear shall be obtained from all animals. A 
differential blood count shall be performed on blood smears from those 
animals in the highest dosage group and the controls. If these data, or 
data from the pathological examination indicate a need, then the 12- and 
18-month blood smears from other dose levels shall also be examined. 
Differential blood counts shall be performed for the next lower group(s) 
if there is a major discrepancy between the highest group and the 
controls. If clinical observations suggest a deterioration in health of 
the animals during the study, a differential blood count of the affected 
animals shall be performed.
    (10) Gross necropsy. (i) A complete gross examination shall be 
performed on all animals, including those which died during the 
experiment or were killed in moribund conditions.
    (ii) The following organs and tissues or representative samples 
thereof, shall be preserved in a suitable medium for possible future 
histopathological examination: All gross lesions and tumors of all 
animals shall be preserved; brain--including sections of medulla/pons, 
cerebellar cortex and cerebral cortex; pituitary; thyroid/parathyroid; 
thymus; lungs; trachea; heart; spinal cord at three levels--cervical, 
midthoracic and lumbar; sternum and/or femur with bone marrow; salivary 
glands; liver; spleen; kidneys; adrenals; esophagus; stomach; duodenum; 
jejunum; ileum; cecum; colon; rectum; urinary bladder; representative 
lymph nodes; pancreas; gonads; uterus; accessory genital organs 
(epididymis, prostate, and, if present, seminal vesicles); mammary 
gland; skin; musculature; peripheral nerve; and eyes. In inhalation 
studies, the entire respiratory tract shall be preserved, including 
nasal cavity, pharynx, larynx and paranasal sinuses. In dermal studies, 
skin from sites of skin painting shall be examined and preserved.
    (iii) Inflation of lungs and urinary bladder with a fixative is the 
optimal method for preservation of these tissues. The proper inflation 
and fixation of the lungs in inhalation studies is required for 
appropriate and valid histopathological examination.
    (iv) If other clinical examinations are carried out, the information 
obtained from these procedures shall be available before microscopic 
examination, since they may provide significant guidance to the 
pathologist.
    (11) Histopathology. (i) The following histopathology shall be 
performed:
    (A) Full histopathology on organs and tissues listed above of all 
animals in the control and high dose groups and all animals that died or 
were killed during the study.
    (B) All gross lesions in all animals.
    (C) Target organs in all animals.
    (ii) If a significant difference is observed in hyperplastic, pre-
neoplastic or neoplastic lesions between the highest dose and control 
groups, microscopic examination shall be made on that particular organ 
or tissue of all animals in the study.
    (iii) If excessive early deaths or other problems occur in the high 
dose group, compromising the significance of the data, the next lower 
dose level shall be examined for complete histopathology.
    (iv) In case the results of an experiment give evidence of 
substantial alteration of the animals' normal longevity or the induction 
of effects that might affect a neoplastic response, the next lower dose 
level shall be examined fully as described in this section.
    (v) An attempt shall be made to correlate gross observations with 
microscopic findings.
    (c) Data and reporting--(1) Treatment of results. (i) Data shall be 
summarized in tabular form, showing for each test group the number of 
animals at the

[[Page 164]]

start of the test, the number of animals showing lesions, the types of 
lesions and the percentage of animals displaying each type of lesion.
    (ii) All observed results, quantitative and incidental, shall be 
evaluated by an appropriate statistical method. Any generally accepted 
statistical method may be used; the statistical methods shall be 
selected during the design of the study.
    (2) Evaluation of study results. (i) The findings of an oncogenic 
toxicity study shall be evaluated in conjunction with the findings of 
preceding studies and considered in terms of the toxic effects, the 
necropsy and histopathological findings. The evaluation shall include 
the relationship between the dose of the test substance and the 
presence, incidence and severity of abnormalities (including behavioral 
and clinical abnormalities), gross lesions, identified target organs, 
body weight changes, effects on mortality and any other general or 
specific toxic effects.
    (ii) In any study which demonstrates an absence of toxic effects, 
further investigation to establish absorption and bioavailability of the 
test substance should be considered.
    (iii) In order for a negative test to be acceptable, it shall meet 
the following criteria: no more than 10 percent of any group is lost due 
to autolysis, cannibalism, or management problems; and survival in each 
group should be no less than 50 percent at 18 months for mice and 
hamsters and at 24 months for rats.
    (3) Test report. (i) In addition to the reporting requirements as 
specified under 40 CFR part 792, subpart J the following specific 
information shall be reported:
    (A) Group animal data. Tabulation of toxic response data by species, 
strain, sex and exposure level for:
    (1) Number of animals dying.
    (2) Number of animals showing signs of toxicity.
    (3) Number of animals exposed.
    (B) Individual animal data. (1) Time of death during the study or 
whether animals survived to termination.
    (2) Time of observation of each abnormal sign and its subsequent 
course.
    (3) Body weight data.
    (4) Feed and water consumption data, when collected.
    (5) Results of ophthalmological examination, when performed.
    (6) Hematological tests employed and all results.
    (7) Clinical biochemistry tests employed and all results.
    (8) Necropsy findings.
    (9) Detailed description of all histopathological findings.
    (10) Statistical treatment of results, where appropriate.
    (11) Historical control data, if taken into account.
    (ii) In addition, for inhalation studies the following shall be 
reported:
    (A) Test conditions. (1) Description of exposure apparatus including 
design, type, dimensions, source of air, system for generating 
particulates and aerosols, method of conditioning air, treatment of 
exhaust air and the method of housing the animals in a test chamber.
    (2) The equipment for measuring temperature, humidity, and 
particulate aerosol concentrations and size shall be described.
    (B) Exposure data. These shall be tabulated and presented with mean 
values and a measure of variability (e.g., standard deviation) and shall 
include:
    (1) Airflow rates through the inhalation equipment.
    (2) Temperature and humidity of air.
    (3) Nominal concentration (total amount of test substance fed into 
the inhalation equipment divided by volume of air).
    (4) Actual concentration in test breathing zone.
    (5) Particle size distribution (e.g., median aerodynamic diameter of 
particles with standard deviation from the mean).
    (d) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) Department of Health and Welfare. The Testing of Chemicals for 
Carcinogenicity, Mutagenicity, Teratogenicity. Minister of Health and 
Welfare. (Canada: Department of Health and Welfare, 1975).
    (2) Food and Drug Administration Advisory Committee on Protocols for

[[Page 165]]

Safety Evaluation: Panel on Carcinogenesis. ``Report on Cancer Testing 
in the Safety of Food Additives and Pesticides,'' Toxicology and Applied 
Pharmacology. 20:419-438 (1971).
    (3) International Union Against Cancer. ``Carcinogenicity Testing,'' 
IUCC Technical Report Series. Vol. 2., Ed. I. Berenblum. (Geneva: 
International Union Against Cancer, 1969).
    (4) Leong, B.K.J., Laskin, S. ``Number and Species of Experimental 
Animals for Inhalation Carcinogenicity Studies'' Paper presented at 
Conference on Target Organ Toxicity, September 1975, Cincinnati, Ohio.
    (5) National Academy of Sciences. ``Principles and Procedures for 
Evaluating the Toxicity of Household Substances.'' A report prepared by 
the Committee for the Revision of NAS Publication 1138, under the 
auspices of the Committee on Toxicology, National Research Council, 
National Academy of Sciences, Washington, DC (1977).
    (6) National Cancer Institute. Report of the Subtask Group on 
Carcinogen Testing to the Interagency Collaborative Group on 
Environmental Carcinogenesis. (Bethesda: United States National Cancer 
Institute, 1976).
    (7) National Center for Toxicological Research. ``Appendix B,'' 
Report of Chronic Studies Task Force Committee. April 13-21 (Rockville: 
National Center for Toxicological Research, 1972).
    (8) Page, N.P. ``Chronic Toxicity and Carcinogenicity Guidelines,'' 
Journal of Environmental Pathology and Toxicology. 1:161-182 (1977).
    (9) Page, N.P. ``Concepts of a Bioassay Program in Environmental 
Carcinogenesis,'' Advances in Modern Toxicology Vol. 3, Ed. Kraybill and 
Mehlman. (Washington, DC: Hemisphere Publishing Corporation, 1977) pp. 
87-171.
    (10) Sontag, J.M., Page N.P., Saffiotti, U. Guidelines for 
Carcinogen Bioassay in Small Rodents. NCI-CS-TR-1. (Bethesda: United 
States Cancer Institute, Division of Cancer Control and Prevention, 
Carcinogenesis Bioassay Program, 1976).
    (11) United States Pharmaceutical Manufacturers Association. 
Guidelines for the Assessment of Drug and Medical Device Safety in 
Animals. (1977).
    (12) World Health Organization. ``Principles for the Testing and 
Evaluation of Drugs for Carcinogenicity,'' WHO Technical Report Series 
No. 426. (Geneva: World Health Organization, 1969).
    (13) World Health Organization. ``Part I. Environmental Health 
Criteria 6,'' Principles and Methods for Evaluating the Toxicity of 
Chemicals. (Geneva: World Health Organization, 1978).

[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19075, May 20, 1987; 
54 FR 21064, May 16, 1989]



Sec. 798.3320  Combined chronic toxicity/oncogenicity.

    (a) Purpose. The objective of a combined chronic toxicity/
oncogenicity study is to determine the effects of a substance in a 
mammalian species following prolonged and repeated exposure. The 
application of this guideline should generate data which identify the 
majority of chronic and oncogenic effects and determine dose-response 
relationships. The design and conduct should allow for the detection of 
neoplastic effects and a determination of oncogenic potential as well as 
general toxicity, including neurological, physiological, biochemical, 
and hematological effects and exposure-related morphological (pathology) 
effects.
    (b) Test procedures--(1) Animal selection--(i) Species and strain. 
Preliminary studies providing data on acute, subchronic, and metabolic 
responses should have been carried out to permit an appropriate choice 
of animals (species and strain). As discussed in other guidelines, the 
mouse and rat have been most widely used for assessment of oncogenic 
potential, while the rat and dog have been most often studied for 
chronic toxicity. The rat is the species of choice for combined chronic 
toxicity and oncogenicity studies. The provisions of this guideline are 
designed primarily for use with the rat as the test species. If other 
species are used, the tester should provide justification/reasoning for 
their selection. The strain selected should be susceptible to the 
oncogenic or toxic effect of the class of substances being tested, if 
known, and provided it does not have a spontaneous background too high 
for

[[Page 166]]

meaningful assessment. Commonly used laboratory strains should be 
employed.
    (ii) Age. (A) Dosing of rats should begin as soon as possible after 
weaning, ideally before the rats are 6 weeks old, but in no case more 
than 8 weeks old.
    (B) At commencement of the study, the weight variation of animals 
used should not exceed 20 percent of the mean 
weight for each sex.
    (C) Studies using prenatal or neonatal animals may be recommended 
under special conditions.
    (iii) Sex. (A) Equal numbers of animals of each sex should be used 
at each dose level.
    (B) The females should be nulliparous and nonpregnant.
    (iv) Numbers. (A) At least 100 rodents (50 females and 50 males) 
should be used at each dose level and concurrent control for those 
groups not intended for early sacrifice. At least 40 rodents (20 females 
and 20 males) should be used for satellite dose group(s) and the 
satellite control group. The purpose of the satellite group is to allow 
for the evaluation of pathology other than neoplasia.
    (B) If interim sacrifices are planned, the number of animals should 
be increased by the number of animals scheduled to be sacrificed during 
the course of the study.
    (C) The number of animals at the termination of each phase of the 
study should be adequate for a meaningful and valid statistical 
evaluation of long term exposure. For a valid interpretation of negative 
results, it is essential that survival in all groups not fall below 50 
percent at the time of termination.
    (2) Control groups. (i) A concurrent control group (50 females and 
50 males) and a satellite control group (20 females and 20 males) are 
recommended. These groups should be untreated or sham treated control 
groups or, if a vehicle is used in administering the test substance, 
vehicle control groups. If the toxic properties of the vehicle are not 
known or cannot be made available, both untreated and vehicle control 
groups are recommended. Animals in the satellite control group should be 
sacrificed at the same time the satellite test group is terminated.
    (ii) In special circumstances such as inhalation studies involving 
aerosols or the use of an emulsifier of uncharacterized biological 
activity in oral studies, a concurrent negative control group should be 
utilized. The negative control group should be treated in the same 
manner as all other test animals, except that this control group should 
not be exposed to the test substance or any vehicle.
    (iii) The use of historical control data (i.e., the incidence of 
tumors and other suspect lesions normally occuring under the same 
laboratory conditions and in the same strain of animals employed in the 
test) is desirable for assessing the significance of changes observed in 
exposed animals.
    (3) Dose levels and dose selection. (i) For risk assessment 
purposes, at least three dose levels should be used, in addition to the 
concurrent control group. Dose levels should be spaced to produce a 
gradation of effects.
    (ii) The highest dose level in rodents should elicit signs of 
toxicity without substantially altering the normal life span due to 
effects other than tumors.
    (iii) The lowest dose level should produce no evidence of toxicity. 
Where there is a usable estimation of human exposure, the lowest dose 
level should exceed this even though this dose level may result in some 
signs of toxicity.
    (iv) Ideally, the intermediate dose level(s) should produce minimal 
observable toxic effects. If more than one intermediate dose is used the 
dose levels should be spaced to produce a gradation of toxic effects.
    (v) For rodents, the incidence of fatalities in low and intermediate 
dose groups and in the controls should be low to permit a meaningful 
evaluation of the results.
    (vi) For chronic toxicological assessment, a high dose treated 
satellite and a concurrent control satellite group should be included in 
the study design. The highest dose for satellite animals should be 
chosen so as to produce frank toxicity, but not excessive lethality, in 
order to elucidate a chronic toxicological profile of the test 
substance. If more than one dose level is selected for satellite dose 
groups, the doses should be spaced to produce a gradation of toxic 
effects.

[[Page 167]]

    (4) Exposure conditions. The animals are dosed with the test 
substance ideally on a 7-day per week basis over a period of at least 24 
months for rats, and 18 months for mice and hamsters, except for the 
animals in the satellite groups which should be dosed for 12 months.
    (5) Observation period. It is necessary that the duration of the 
oncogenicity test comprise the majority of the normal life span of the 
animals to be used. It has been suggested that the duration of the study 
should be for the entire lifetime of all animals. However, a few animals 
may greatly exceed the average lifetime and the duration of the study 
may be unnecessarily extended and complicate the conduct and evaluation 
of the study. Rather, a finite period covering the majority of the 
expected life span of the strain is preferred since the probability is 
high that, for the great majority of chemicals, induced tumors will 
occur within such an observation period. The following guidelines are 
recommended:
    (i) Generally, the termination of the study should be at 18 months 
for mice and hamsters and 24 months for rats; however, for certain 
strains of animals with greater longevity and/or low spontaneous tumor 
rate, termination should be at 24 months for mice and hamsters and at 30 
months for rats. For longer time periods, and where any other species 
are used, consultation with the Agency in regard to duration of the test 
is advised.
    (ii) However, termination of the study is acceptable when the number 
of survivors of the lower doses or of the control group reaches 25 
percent. In the case where only the high dose group dies prematurely for 
obvious reasons of toxicity, this should not trigger termination of the 
study.
    (iii) The satellite groups and the concurrent satellite control 
group should be retained in the study for at least 12 months. These 
groups should be scheduled for sacrifice for an estimation of test-
substance-related pathology uncomplicated by geriatric changes.
    (6) Administration of the test substance. The three main routes of 
administration are oral, dermal, and inhalation. The choice of the route 
of administration depends upon the physical and chemical characteristics 
of the test substance and the form typifying exposure in humans.
    (i) Oral studies. (A) The animals should receive the test substance 
in their diet, dissolved in drinking water, or given by gavage or 
capsule for a period of at least 24 months for rats and 18 months for 
mice and hamsters.
    (B) If the test substance is administered in the drinking water, or 
mixed in the diet, exposure is continuous.
    (C) For a diet mixture, the highest concentration should not exceed 
5 percent.
    (ii) Dermal studies. (A) The animals are treated by topical 
application with the test substance, ideally for at least 6 hours per 
day.
    (B) Fur should be clipped from the dorsal area of the trunk of the 
test animals. Care should be taken to avoid abrading the skin which 
could alter its permeability.
    (C) The test substance should be applied uniformly over a shaved 
area which is approximately 10 percent of the total body surface area. 
With highly toxic substances, the surface area covered may be less, but 
as much of the area as possible should be covered with as thin and 
uniform a film as possible.
    (D) During the exposure period, the test substance may be held, if 
necessary, in contact with the skin with a porous gauze dressing and 
nonirritating tape. The test site should be further covered in a 
suitable manner to retain the gauze dressing and test substance and 
ensure that the animals cannot ingest the test substance.
    (iii) Inhalation studies. (A) The animals should be tested with 
inhalation equipment designed to sustain a dynamic air flow of 12 to 15 
air changes per hour, to ensure an adequate oxygen content of 19 percent 
and an evenly distributed exposure atmosphere. Where a chamber is used, 
its design should minimize crowding of the test animals and maximize 
their exposure to the test substance. This is best accomplished by 
individual caging. As a general rule, to ensure stability of a chamber 
atmosphere, the total ``volume'' of the test animals should not exceed 5 
percent of the volume of the test chamber. Alternatively, oro-nasal, 
head only, or whole

[[Page 168]]

body individual chamber exposure may be used.
    (B) The temperature at which the test is performed should be 
maintained at 22 [deg]C (2[deg]). Ideally, the 
relative humidity should be maintained between 40 to 60 percent, but in 
certain instances (e.g., tests of aerosols, use of water vehicle) this 
may not be practicable.
    (C) Feed and water should be withheld during each daily 6-hour 
exposure period.
    (D) A dynamic inhalation system with a suitable analytical 
concentration control system should be used. The rate of air flow should 
be adjusted to ensure that conditions throughout the equipment are 
essentially the same. Maintenance of slight negative pressure inside the 
chamber will prevent leakage of the test substance into the surrounding 
areas.
    (7) Observation of animals. (i) Each animal should be handled and 
its physical condition appraised at least once each day.
    (ii) Additional observations should be made daily with appropriate 
actions taken to minimize loss of animals to the study (e.g., necropsy 
or refrigeration of those animals found dead and isolation or sacrifice 
of weak or moribund animals).
    (iii) Clinical signs and mortality should be recorded for all 
animals. Special attention should be paid to tumor development. The time 
of onset, location, dimensions, appearance and progression of each 
grossly visible or palpable tumor should be recorded.
    (iv) Body weights should be recorded individually for all animals 
once a week during the first 13 weeks of the test period and at least 
once every 4 weeks thereafter, unless signs of clinical toxicity suggest 
more frequent weighings to facilitate monitoring of health status.
    (v) When the test substance is administered in the feed or drinking 
water, measurements of feed or water consumption, respectively, should 
be determined weekly during the first 13 weeks of the study and then at 
approximately monthly intervals unless health status or body weight 
changes dictate otherwise.
    (vi) At the end of the study period, all survivors are sacrificed. 
Moribund animals should be removed and sacrificed when noticed.
    (8) Physical measurements. For inhalation studies, measurements or 
monitoring should be made of the following:
    (i) The rate of airflow should be monitored continuously, but should 
be recorded at intervals of at least once every 30 minutes.
    (ii) During each exposure period the actual concentrations of the 
test substance should be held as constant as practicable, monitored 
continuously and recorded at least three times during the test period: 
At the beginning, at an intermediate time and at the end of the period.
    (iii) During the development of the generating system, particle size 
analysis should be performed to establish the stability of aerosol 
concentrations. During exposure, analyses should be conducted as often 
as necessary to determine the consistency of particle size distribution 
and homogeneity of the exposure stream.
    (iv) Temperature and humidity should be monitored continuously, but 
should be recorded at intervals of at least once every 30 minutes.
    (9) Clinical examinations. (i) The following examinations should be 
made on at least 20 rodents of each sex per dose level:
    (A) Certain hematology determinations (e.g., hemoglobin content, 
packed cell volume, total red blood cells, total white blood cells, 
platelets, or other measures of clotting potential) should be performed 
at termination and should be performed at 3 months, 6 months and at 
approximately 6-month intervals thereafter (for those groups on test for 
longer than 12 months) on blood samples collected from 20 rodents per 
sex of all groups. These collections should be from the same animals at 
each interval. If clinical observations suggest a deterioration in 
health of the animals during the study, a differential blood count of 
the affected animals should be performed. A differential blood count 
should be performed on samples from animals in the highest dosage group 
and the controls. Differential blood counts should be performed for the 
next lower group(s) if

[[Page 169]]

there is a major discrepancy between the highest group and the controls. 
If hematological effects were noted in the subchronic test, 
hematological testing should be performed at 3, 6, 12, 18 and 24 months 
for a year study.
    (B) Certain clinical biochemistry determinations on blood should be 
carried out at least three times during the test period: Just prior to 
initiation of dosing (baseline data), near the middle and at the end of 
the test period. Blood samples should be drawn for clinical measurements 
from at least ten rodents per sex of all groups; if possible, from the 
same rodents at each time interval. Test areas which are considered 
appropriate to all studies: electrolyte balance, carbohydrate metabolism 
and liver and kidney function. The selection of specific tests will be 
influenced by observations on the mode of action of the substance and 
signs of clinical toxicity. Suggested chemical determinations: Calcium, 
phosphorus, chloride, sodium, potassium, fasting glucose (with period of 
fasting appropriate to the species), serum glutamic-pyruvic transaminase 
(now known as serum alanine aminotransferase), serum glutamic 
oxaloacetic transaminase (now known as serum aspartate 
aminotransferase), ornithine decarboxylase, gamma glutamyl 
transpeptidase, blood urea nitrogen, albumen, creatinine phosphokinase, 
total cholesterol, total bilirubin and total serum protein measurements. 
Other determinations which may be necessary for an adequate 
toxicological evaluation include analyses of lipids, hormones, acid/base 
balance, methemoglobin and cholinesterase activity. Additional clinical 
biochemistry may be employed where necessary to extend the investigation 
of observed effects.
    (ii) The following should be performed on at least 10 rodents of 
each sex per dose level:
    (A) Urine samples from the same rodents at the same intervals as 
hematological examination above, should be collected for analysis. The 
following determinations should be made from either individual animals 
or on a pooled sample/sex/group for rodents: appearance (volume and 
specific gravity), protein, glucose, ketones, bilirubin, occult blood 
(semi-quantitatively) and microscopy of sediment (semi-quantitatively).
    (B) Ophthalmological examination, using an ophthalmoscope or 
equivalent suitable equipment, should be made prior to the 
administration of the test substance and at the termination of the 
study. If changes in the eyes are detected, all animals should be 
examined.
    (10) Gross necropsy. (i) A complete gross examination should be 
performed on all animals, including those which died during the 
experiment or were killed in moribund conditions.
    (ii) The liver, kidneys, adrenals, brain and gonads should be 
weighed wet, as soon as possible after dissection to avoid drying. For 
these organs, at least 10 rodents per sex per group should be weighed.
    (iii) The following organs and tissues, or representative samples 
thereof, should be preserved in a suitable medium for possible future 
histopathological examination: All gross lesions and tumors; brain-
including sections of medulla/pons, cerebellar cortex, and cerebral 
cortex; pituitary; thyroid/parathyroid; thymus; lungs; trachea; heart; 
sternum and/or femur with bone marrow; salivary glands; liver; spleen; 
kidneys; adrenals; esophagus; stomach; duodenum; jejunum; ileum; cecum; 
colon; rectum; urinary bladder; representative lymph nodes; pancreas; 
gonads; uterus; accessory genital organs (epididymis, prostate, and, if 
present, seminal vesicles); female mammary gland; aorta; gall bladder 
(if present); skin; musculature; peripheral nerve; spinal cord at three 
levels--cervical, midthoracic, and lumbar; and eyes. In inhalation 
studies, the entire respiratory tract, including nose, pharynx, larynx 
and paranasal sinuses should be examined and preserved. In dermal 
studies, skin from sites of skin painting should be examined and 
preserved.
    (iv) Inflation of lungs and urinary bladder with a fixative is the 
optimal method for preservation of these tissues. The proper inflation 
and fixation of the lungs in inhalation studies is considered essential 
for appropriate and valid histopathological examination.

[[Page 170]]

    (v) If other clinical examinations are carried out, the information 
obtained from these procedures should be available before microscopic 
examination, since they may provide significant guidance to the 
pathologist.
    (11) Histopathology. (i) The following histopathology should be 
performed:
    (A) Full histopathology on the organs and tissues, listed above, of 
all non-rodents, of all rodents in the control and high dose groups and 
of all rodents that died or were killed during the study.
    (B) All gross lesions in all animals.
    (C) Target organs in all animals.
    (D) Lungs, liver and kidneys of all animals. Special attention to 
examination of the lungs of rodents should be made for evidence of 
infection since this provides an assessment of the state of health of 
the animals.
    (ii) If excessive early deaths or other problems occur in the high 
dose group compromising the significance of the data, the next dose 
level should be examined for complete histopathology.
    (iii) In case the results of the experiment give evidence of 
substantial alteration of the animals' normal longevity or the induction 
of effects that might affect a toxic response, the next lower dose level 
should be examined as described above.
    (iv) An attempt should be made to correlate gross observations with 
microscopic findings.
    (c) Data and reporting--(1) Treatment of results. (i) Data should be 
summarized in tabular form, showing for each test group the number of 
animals at the start of the test, the number of animals showing lesions, 
the types of lesions and the percentage of animals displaying each type 
of lesion.
    (ii) All observed results, quantitative and incidental, should be 
evaluated by an appropriate statistical method. Any generally accepted 
statistical methods may be used; the statistical methods should be 
selected during the design of the study.
    (2) Evaluation of study results. (i) The findings of a combined 
chronic toxicity/oncogenicity study should be evaluated in conjunction 
with the findings of preceding studies and considered in terms of the 
toxic effects, the necropsy and histopathological findings. The 
evaluation will include the relationship between the dose of the test 
substance and the presence, incidence and severity of abnormalities 
(including behavioral and clinical abnormalities), gross lesions, 
identified target organs, body weight changes, effects on mortality and 
any other general or specific toxic effects.
    (ii) In any study which demonstrates an absence of toxic effects, 
further investigation to establish absorption and bioavailablity of the 
test substance should be considered.
    (iii) In order for a negative test to be acceptable, it should meet 
the following criteria: No more than 10 percent of any group is lost due 
to autolysis, cannibalism, or management problems; and survival in each 
group is no less than 50 percent at 18 months for mice and hamsters and 
at 24 months for rats.
    (3) Test report. (i) In addition to the reporting requirements as 
specified under 40 CFR part 792, subpart J the following specific 
information should be reported:
    (A) Group animal data. Tabulation of toxic response data by species, 
strain, sex and exposure level for:
    (1) Number of animals dying.
    (2) Number of animals showing signs of toxicity.
    (3) Number of animals exposed.
    (B) Individual animal data. (1) Time of death during the study or 
whether animals survived to termination.
    (2) Time of observation of each abnormal sign and its subsequent 
course.
    (3) Body weight data.
    (4) Feed and water consumption data, when collected.
    (5) Results of ophthalmological examination, when performed.
    (6) Hematological tests employed and all results.
    (7) Clinical biochemistry tests employed and all results.
    (8) Necropsy findings.
    (9) Detailed description of all histopathological findings.
    (10) Statistical treatment of results where appropriate.
    (11) Historical control data, if taken into account.
    (ii) In addition, for inhalation studies the following should be 
reported:

[[Page 171]]

    (A) Test conditions. (1) Description of exposure apparatus including 
design, type, dimensions, source of air, system for generating 
particulates and aerosols, method of conditioning air, treatment of 
exhaust air and the method of housing the animals in a test chamber.
    (2) The equipment for measuring temperature, humidity, and 
particulate aerosol concentrations and size should be described.
    (B) Exposure data. These should be tabulated and presented with mean 
values and a measure of variability (e.g. standard deviation) and should 
include:
    (1) Airflow rates through the inhalation equipment.
    (2) Temperature and humidity of air.
    (3) Nominal concentration (total amount of test substance fed into 
the inhalation equipment divided by volume of air).
    (4) Actual concentration in test breathing zone.
    (5) Particle size distribution (e.g. median aerodynamic diameter of 
particles with standard deviation from the mean).
    (d) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) Benitz, K.F. ``Measurement of Chronic Toxicity,'' Methods of 
Toxicology. Ed. G.E. Paget. (Oxford: Blackwell Scientific Publications, 
1970) pp. 82-131.
    (2) D'Aguanno, W. ``Drug Safety Evaluation--Pre-Clinical 
Considerations,'' ``Industrial Pharmacology: Neuroleptics. Vol. I Ed. S. 
Fielding and H. Lal. (Mt. Kisco, New York: Futura Publishing Co., 1974) 
pp. 317-332.
    (3) Department of Health and Welfare. The Testing of Chemicals for 
Carcinogenicity, Mutagenicity, Teratogenicity. Minister of Health and 
Welfare. (Canada: Department of Health and Welfare, 1975).
    (4) Fitzhugh, O.G. ``Chronic Oral Toxicity,'' Appraisal of the 
Safety of Chemicals in Foods, Drugs and Cosmetics. The Association of 
Food and Drug Officials of the United States (1959, 3rd Printing 1975). 
pp. 36-45.
    (5) Food and Drug Administration Advisory Committee on Protocols for 
Safety Evaluation: Panel on Carcinogenesis. ``Report on Cancer Testing 
in the Safety of Food Additives and Pesticides,'' Toxicology and Applied 
Pharmacology. 20:419-438 (1971).
    (6) Goldenthal, E.I., and D'Aguanno, W. ``Evaluation of Drugs,'' 
Appraisal of the Safety of Chemicals in Foods, Drugs, and Cosmetics. The 
Association of Food and Drug Officials of the United States (1959, 3rd 
printing 1975) pp.60-67.
    (7) International Union Against Cancer. ``Carcinogenicity Testing,'' 
IUCC Technical Report Series Vol. 2, Ed. I. Berenblum. (Geneva: 
International Union Against Cancer, 1969).
    (8) Leong, B.K.J., and Laskin, S. ``Number and Species of 
Experimental Animals for Inhalation Carcinogenicity Studies,'' Paper 
presented at Conference on Target Organ Toxicity. September, 1975, 
Cincinnati, Ohio.
    (9) National Academy of Sciences. ``Principles and Procedures for 
Evaluating the Toxicity of Household Substances,'' A report prepared by 
the Committee for the Revision of NAS Publication 1138, under the 
auspices of the Committee on Toxicology, National Research Council, 
National Academy of Sciences, Washington, DC (1977).
    (10) National Cancer Institute. Report of the Subtask Group on 
Carcinogen Testing to the Interagency Collaborative Group on 
Environmental Carcinogenesis. (Bethesda: United States National Cancer 
Institute, 1976).
    (11) National Center for Toxicological. Report of Chronic Studies 
Task Force Research Committee. ``Appendix B, (Rockville: National Center 
for Toxicological Research, 1972)).
    (12) Page, N.P. ``Chronic Toxicity and Carcinogenicity Guidelines,'' 
Journal Environmental Pathology and Toxicology. 1:161-182 (1977).
    (13) Page, N.P. ``Concepts of a Bioassay Program in Environmental 
Carcinogenesis,'' Advances in Modern Toxicology Volume 3, Ed. Kraybill 
and Mehlman. (Washington, D.C.: Hemisphere Publishing Corp., 1977) pp. 
87-171.
    (14) Schwartz, E. 1974. ``Toxicology of Neuroleptic Agents,'' 
Industrial Pharmacology: Neuroleptics. Ed. S. Fielding and H. Lal. (Mt. 
Kisco, New York: Futura Publishing Co, 1974) pp. 203-221.

[[Page 172]]

    (15) Sontag, J.M., Page, N.P., and Saffiotti, U. Guidelines for 
Carcinogen Bioassay in Small Rodents. NCI-CS-TR-1 (Bethesda: United 
States Cancer Institute, Division of Cancer Control and Prevention, 
Carcinogenesis Bioassay Program, 1976).
    (16) United States Pharmaceutical Manufacturers Association. 
Guidelines for the Assessment of Drug and Medical Device Safety in 
Animals. (1977).
    (17) World Health Organization. ``Principles for the Testing and 
Evaluation of Drugs for Carcinogenicity,'' WHO Technical Report Series 
No. 426. (Geneva: World Health Organization, 1969).
    (18) World Health Organization. ``Guidelines for Evaluation of Drugs 
for Use in Man,'' WHO Technical Report Series No. 563. (Geneva: World 
Health Organization, 1975).
    (19) World Health Organization. ``Part I. Environmental Health 
Criteria 6,'' Principles and Methods for Evaluating the Toxicity of 
Chemicals. (Geneva: World Health Organization, 1978).
    (20) World Health Organization. ``Principles for Pre-Clinical 
Testing of Drug Safety,'' WHO Technical Report Series No. 341. (Geneva: 
World Health Organization, 1966).

[50 FR 39397, Sept. 27, 1985, as amended at 54 FR 21064, May 16, 1989]



                Subpart E_Specific Organ/Tissue Toxicity



Sec. 798.4100  Dermal sensitization.

    (a) Purpose. In the assessment and evaluation of the toxic 
characteristics of a substance, determination of its potential to 
provoke skin sensitization reactions is important. Information derived 
from tests for skin sensitization serves to identify the possible hazard 
to a population repeatedly exposed to a test substance. While the 
desirability of skin sensitization testing is recognized, there are some 
real differences of opinion about the best method to use. The test 
selected should be a reliable screening procedure which should not fail 
to identify substances with significant allergenic potential, while at 
the same time avoiding false negative results.
    (b) Definitions. (1) Skin sensitization (allergic contact 
dermatitis) is an immunologically mediated cutaneous reaction to a 
substance. In the human, the responses may be characterized by pruritis, 
erythema, edema, papules, vesicles, bullae, or a combination of these. 
In other species the reactions may differ and only erythema and edema 
may be seen.
    (2) Induction period is a period of at least 1 week following a 
sensitization exposure during which a hypersensitive state is developed.
    (3) Induction exposure is an experimental exposure of a subject to a 
test substance with the intention of inducing a hypersensitive state.
    (4) Challenge exposure is an experimental exposure of a previously 
treated subject to a test substance following an induction period, to 
determine whether the subject will react in a hypersensitive manner.
    (c) Principle of the test method. Following initial exposure(s) to a 
test substance, the animals are subsequently subjected, after a period 
of not less than 1 week, to a challenge exposure with the test substance 
to establish whether a hypersensitive state has been induced. 
Sensitization is determined by examining the reaction to the challenge 
exposure and comparing this reaction to that of the initial induction 
exposure.
    (d) Test procedures. (1) Any of the following seven test methods is 
considered to be acceptable. It is realized, however, that the methods 
differ in their probability and degree of reaction to sensitizing 
substances.
    (i) Freund's complete adjuvant test.
    (ii) Guinea-pig maximization test.
    (iii) Split adjuvant technique.
    (iv) Buehler test.
    (v) Open epicutaneous test.
    (vi) Mauer optimization test.
    (vii) Footpad technique in guinea pig.
    (2) Removal of hair is by clipping, shaving, or possibly by 
depilation, depending on the test method used.
    (3) Animal selection--(i) Species and strain. The young adult guinea 
pig is the preferred species. Commonly used laboratory strains should be 
employed. If other species are used, the tester should provide 
justification/reasoning for their selection.

[[Page 173]]

    (ii) Number and sex. (A) The number and sex of animals used will 
depend on the method employed.
    (B) The females should be nulliparous and nonpregnant.
    (4) Control animals. (i) Periodic use of a positive control 
substance with an acceptable level of reliability for the test system 
selected is recommended;
    (ii) Animals may act as their own controls or groups of induced 
animals can be compared to groups which have received only a challenge 
exposure.
    (5) Dose levels. The dose level will depend upon the method 
selected.
    (6) Observation of animals. (i) Skin reactions should be graded and 
recorded after the challenge exposures at the time specified by the 
methodology selected. This is usually at 24, 48, and 72, hours. 
Additional notations should be made as necessary to fully describe 
unusual responses;
    (ii) Regardless of method selected, initial and terminal body 
weights should be recorded.
    (7) Procedures. The procedures to be used are those described by the 
methodology chosen.
    (e) Data and reporting. (1) Data should be summarized in tabular 
form, showing for each individual animal the skin reaction, results of 
the induction exposure(s) and the challenge exposure(s) at times 
indicated by the method chosen. As a minimum, the erythema and edema 
should be graded and any unusual finding should be recorded.
    (2) Evaluation of the results. The evaluation of results will 
provide information on the proportion of each group that became 
sensitized and the extent (slight, moderate, severe) of the 
sensitization reaction in each individual animal.
    (3) Test report. In addition to the reporting requirements as 
specified under 40 CFR part 792, subpart J, the following specific 
information should be reported:
    (i) A description of the method used and the commonly accepted name.
    (ii) Information on the positive control study, including positive 
control used, method used, and time conducted.
    (iii) The number and sex of the test animals.
    (iv) Species and strain.
    (v) Individual weights of the animals at the start of the test and 
at the conclusion of the test.
    (vi) A brief description of the grading system.
    (vii) Each reading made on each individual animal.
    (f) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) Buehler, E.V. ``Delayed Contact Hypersensitivity in the Guinea 
Pig,'' Archives Dermatology. 91:171 (1965).
    (2) Draize, J.H. ``Dermal Toxicity,'' Food Drug Cosmetic Law 
Journal. 10:722-732 (1955).
    (3) Klecak, G. ``Identification of Contact Allergens: Predictive 
Tests in Animals,'' Advances in Modern Toxicology: Dermatology and 
Pharmacology. Ed. F.N. Marzulli and H.I. Maibach. (Washington, D.C.: 
Hemisphere Publishing Corp., 1977) 4:305-339).
    (4) Klecak, G., Geleick, H., Grey, J.R. ``Screening of Fragrance 
Materials for Allergenicity in the Guinea Pig.-1. Comparison of Four 
Testing Methods,'' Journal of the Society of Cosmetic Chemists. 28:53-64 
(1977).
    (5) Magnusson, B., Kligman, A.M. ``The Identification of Contact 
Allergens by Animal Assay,'' The Guinea Pig Maximization Test. The 
Journal of Investigative Dermatology. 52:268-276 (1973).
    (6) Maguire, H.C. ``The Bioassay of Contact Allergens in the Guinea 
Pig'' Journal of the Society of Cosmetic Chemists. 24:151-162 (1973).
    (7) Maurer, T., Thomann, P., Weirich, E.G., Hess, R. ``The 
Optimization Test in the Guinea Pig. A Method for the Predictive 
Evaluation of the Contact Allergenicity of Chemicals,'' Agents and 
Actions. (Basel: Birkhauser Verlag, 1975) Vol. 5/2.
    (8) Maurer, T., Thomann, P., Weirich, E.G., Hess, R. ``The 
Optimization Test in the Guinea Pig: A Method for the Predictive 
Evaluation of the Contact Allergenicity of Chemicals,'' International 
Congress Series Excerpta Medica No. 376, (1975) Vol. 203.



Sec. 798.4350  Inhalation developmental toxicity study.

    (a) Purpose. In the assessment and evaluation of the toxic 
characteristics

[[Page 174]]

of an inhalable material such as a gas, volatile substance, or aerosol/
particulate, determination of the potential developmental toxicity is 
important. The inhalation developmental toxicity study is designed to 
provide information on the potential hazard to the unborn which may 
arise from exposure of the mother during pregnancy.
    (b) Definitions. (1) Developmental toxicity is the property of a 
chemical that causes in utero death, structural or functional 
abnormalities or growth retardation during the period of development.
    (2) ``Aerodynamic diameter'' applies to the behavioral size of 
particles of aerosols. It is the diameter of a sphere of unit density 
which behaves aerodynamically like the particles of the test substance. 
It is used to compare particles of different sizes, shapes, and 
densities and to predict where in the respiratory tract such particles 
may be deposited. This term is used in contrast to ``optical,'' 
``measured'' or ``geometric'' diameters which are representation of 
actual diameters which in themselves cannot be related to deposition 
within the respiratory tract.
    (3) ``Geometric mean diameter'' or ``median diameter'' is the 
calculated aerodynamic diameter which divides the particles of an 
aerosol in half based on the weight of the particles. Fifty percent of 
the particles by weight will be larger than the median diameter and 50 
percent of the particles will be smaller than the median diameter. The 
median diameter and its geometeric standard deviation are used to 
statistically describe the particle size distribution of any aerosol 
based on the weight and size of the particles.
    (4) ``Inhalable diameter'' refers to that aerodynamic diameter of a 
particle which is considered to be inhalable for the organism. It is 
used to refer to particles which are capable of being inhaled and may be 
deposited anywhere within the respiratory tract from the trachea to the 
deep lung (the alveoli). For man, the inhalable diameter is considered 
here as 15 micrometers or less.
    (5) ``Concentration'' refers to an exposure level. Exposure is 
expressed as weight or volume of test substance per volume of air (mg/
1), or as parts per million (ppm).
    (6) ``No-observed-effect level'' is the maximum concentration in a 
test which produces no observed adverse effects. A no-observed-effect 
level is expressed in terms of weight or volume of test substance given 
daily per unit volume of air.
    (c) Principle of the test method. The test substance is administered 
in graduated concentrations, for at least that part of the pregnancy 
covering the major period of organogenesis, to several groups of 
pregnant experimental animals, one exposure level being used per group. 
Shortly before the expected date of delivery, the pregnant females are 
sacrificed, the uteri removed, and the contents examined for embryonic 
or fetal deaths, and live fetuses.
    (d) Limit test. If a test at an exposure of 5 mg/1 (actual 
concentration of respirable substances) or, where this is not possible 
due to physical or chemical properties of the test substance, the 
maximum attainable concentration, produces no observable developmental 
toxicity, then a full study using three exposure levels might not be 
necessary.
    (e) Test procedures--(1) Animal selection--(i) Species and strain. 
Testing shall be performed in at least two mamalian species. Commonly 
used species include the rat, mouse, rabbit, and hamster. If other 
mamalian species are used, the tester shall provide justification/
reasoning for their selection. Commonly used laboratory strains shall be 
employed. The strain shall not have low fecundity and shall preferably 
be characterized for its sensitivity to developmental toxins.
    (ii) Age. Young adult animals (nulliparous females) shall be used.
    (iii) Sex. Pregnant female animals shall be used at each exposure 
level.
    (iv) Number of animals. At least 20 pregnant rats, mice, or hamsters 
or 12 pregnant rabbits are required at each exposure level. The 
objective is to ensure that sufficient pups are produced to permit 
meaningful evaluation of the potential developmental toxicity of the 
test substance.
    (2) Control group. A concurrent control group shall be used. This 
group shall be exposed to clean, filtered air

[[Page 175]]

under conditions identical to those used for the group exposed to the 
substance of interest. In addition, a vehicle-exposed group may be 
necessary when the substance under study requires a vehicle for 
delivery. It is recommended that during preliminary range finding 
studies, air vs. vehicle exposure be compared. If there is no 
substantial difference, air exposure itself would be an appropriate 
control. If vehicle and air exposure yield different results, both 
vehicle and air exposed control groups are recommended.
    (3) Concentration levels and concentration selection. (i) At least 
three concentration levels with a control and, where appropriate, a 
vehicle control, shall be used.
    (ii) The vehicle shall neither be developmentally toxic nor have 
effects on reproduction.
    (iii) To select the appropriate concentration levels, a pilot or 
trial study may be advisable. Since pregnant animals have an increased 
minute ventilation as compared to non-pregnant animals, it is 
recommended that the trial study be conducted in pregnant animals. 
Similarly, since presumably the minute ventilation will vary with 
progression of pregnancy, the animals should be exposed during the same 
period of gestation as in the main study. In the trial study, the 
concentration producing embryonic or fetal lethalities or maternal 
toxicity should be determined.
    (iv) Unless limited by the physical/chemical nature or biological 
properties of the substance, the highest concentration level shall 
induce some overt maternal toxicity such as reduced body weight or body 
weight gain, but not more than 10 percent maternal deaths.
    (v) The lowest concentration level should not produce any grossly 
observable evidence of either maternal or developmental toxicity.
    (vi) Ideally, the intermediate concentration level(s) shall produce 
minimal observable toxic effects. If more than one intermediate 
concentration is used, the concentration levels shall be spaced to 
produce a gradation of toxic effects.
    (4) Exposure duration. The duration of exposure shall be at least 
six hours daily allowing appropriate additional time for chamber 
equilibrium.
    (5) Observation period. Day 0 in the test is the day on which a 
vaginal plug and/or sperm are observed. The exposure period shall cover 
the period of major organogenesis. This may be taken as days 6 to 15 for 
rat and mouse, 6 to 14 for hamster, or 6 to 18 for rabbit.
    (6) Inhalation exposure. (i)(A) The animals shall be tested in 
inhalation equipment designed to sustain a minimum dynamic air flow of 
12 to 15 air changes per hour and ensure an adequate oxygen content of 
19 percent and an evenly distributed exposure atmosphere. Where a 
chamber is used, its design should minimize crowding of the test animals 
and maximize their exposure to the test substance. This is best 
accomplished by individual caging. To ensure stability of a chamber 
atmosphere, the total ``volume'' of the test animals shall not exceed 5 
percent of the volume of the test chamber.
    (B) Pregnant animals shall not be subjected to beyond the minimum 
amount of stress. Since whole-body exposure appears to be the least 
stressful mode of exposure, it is the method preferred. In general oro-
nasal or head-only exposure, which is sometimes used to avoid concurrent 
exposure by the dermal or oral routes, is not recommended because of the 
associated stress accompanying the restraining of the animals. However, 
there may be specific instances where it may be more appropriate than 
whole-body exposure. The tester shall provide justification/reasoning 
for its selection.
    (ii) A dynamic inhalation system with a suitable flow control system 
shall be used. The rate of air flow shall be adjusted to ensure that 
conditions throughout the exposure chamber are essentially the same. 
Test material distribution should be established before animals are 
committed to dosing. Maintenance of slight negative pressure inside the 
chamber will prevent leakage of the test substance into the surrounding 
areas.
    (iii) The temperature at which the test is performed should be 
maintained at 22 [deg]C (2[deg]) for rodents or 20 
[deg]C (3[deg]) for rabbits. Ideally, the relative 
humidity should be maintained between 40 to 60 percent, but in certain 
instances

[[Page 176]]

(e.g., tests of aerosols, use of water vehicle) this may not be 
practicable.
    (7) Physical measurements. Measurements or monitoring should be made 
of the following:
    (i) The rate of airflow shall be monitored continuously but shall be 
recorded at least every 30 minutes.
    (ii) The actual concentration of the test substance shall be 
measured in the breathing zone. During the exposure period the actual 
concentrations of the test substance shall be held as constant as 
practicable, monitored continously or intermittently depending on the 
method of analysis and measured at least at the beginning, at an 
intermediate time and at the end of the exposure period.
    (iii) During the development of the generating system, particle size 
analysis shall be performed to establish the stability of aerosol 
concentrations with respect to particle size. During exposure, analysis 
shall be conducted as often as necessary to determine the consistency of 
particle size distribution.
    (iv) Temperature and humidity shall be monitored continuously and be 
recorded at least every 30 minutes.
    (8) Food and water during exposure period. Food should be withheld 
during exposure. Water may or may not be withheld. If it is not withheld 
it should not come in direct contact with the test atmospheres.
    (9) Observation of animals. (i) A gross examination shall be made at 
least once each day.
    (ii) Additional observations should be made daily with appropriate 
actions taken to minimize loss of animals to the study (e.g., necropsy 
or refrigeration of animals found dead and isolation or sacrifice of 
weak or moribund animals).
    (iii) Signs of toxicity shall be recorded as they are observed, 
including the time of onset, the degree and duration.
    (iv) Cage-side observations shall include, but not be limited to: 
Changes in skin and fur, eye and mucous membranes, as well as 
respiratory, autonomic and central nervous systems, somatomotor activity 
and behavioral pattern. Particular attention should be directed to 
observation of tremors, convulsions, salivation, diarrhea, lethargy, 
sleep, and coma.
    (v) Measurements should be made weekly of food consumption for all 
animals in the study.
    (vi) Animals shall be weighed at least weekly.
    (vii) Females showing signs of abortion or premature delivery shall 
be sacrificed and subjected to a thorough macroscopic examination.
    (10) Gross necropsy. (i) At the time of sacrifice or death during 
the study, the dam shall be examined macroscopically for any structural 
abnormalities or pathological changes which may have influenced the 
pregnancy.
    (ii) Immediately after sacrifice or death, the uterus shall be 
removed, weighed, and the contents examined for embryonic or fetal 
deaths and the number of viable fetuses. Gravid uterine weights should 
not be obtained from dead animals if autolysis or where decomposition 
has occurred. The degree of resorption shall be described in order to 
help estimate the relative time of death.
    (iii) The number of corpora lutea shall be determined for all 
species except mice.
    (iv) The sex of the fetuses shall be determined and they shall be 
weighed individually, the weights recorded, and the mean fetal weight 
derived.
    (v) Following removal, each fetus shall be examined externally.
    (vi) For rats, mice and hamsters, one-third to one-half of each 
litter shall be prepared and examined for skeletal anomalies, and the 
remaining part of each litter shall be prepared and examined for soft 
tissue anomalies using appropriate methods.
    (vii) For rabbits, each fetus shall be examined by careful 
dissection for visceral anomalies and then examined for skeletal 
anomalies.
    (f) Data and reporting--(1) Treatment of results. Data shall be 
summarized in tabular form, showing for each test group: the number of 
animals at the start of the test, the number of pregnant animals, the 
number and percentages of live fetuses and the number of fetuses with 
any soft tissue or skeletal abnormalities.
    (2) Evaluation of results. The findings of a developmental toxicity 
study shall

[[Page 177]]

be evaluated in terms of the observed effects and the exposure levels 
producing effects. It is necessary to consider the historical 
developmental toxicity data on the species/strain tested. A properly 
conducted developmental toxicity study should provide a satisfactory 
estimation of a no-effect level.
    (3) Test report. In addition to the reporting requirements as 
specified under 40 CFR part 792, subpart J, the following specific 
information shall be reported:
    (i) Test conditions. (A) Description of exposure apparatus including 
design, type, dimensions, source of air, system for generating 
particulates and aerosols, methods of conditioning air, and the method 
of housing the animals in a test chamber when this apparatus is used.
    (B) The equipment for measuring temperature, humidity, and 
particulate aerosol concentrations and size shall be described.
    (ii) Exposure data. These shall be tabulated and presented with mean 
values and a measure of variability (e.g., standard deviation) and shall 
include:
    (A) Airflow rates through the inhalation equipment.
    (B) Temperature of air.
    (C) Nominal concentration--total amount of test substance fed into 
the inhalation equipment divided by volume of air (no standard 
deviation).
    (D) Measured total concentrations (particulate and/or gaseous 
phases) in test breathing zone.
    (E) Particle size distribution (e.g., median aerodynamic diameter of 
particles with geometric standard deviation) including estimates of the 
percents of inhalable and non-inhalable portions for the test animals.
    (iii) Animal data. (A) Toxic response data by concentration.
    (B) Species and strain.
    (C) Date of death during the study or whether animals survived to 
termination.
    (D) Date of onset and duration of each abnormal sign and its 
subsequent course.
    (E) Feed, body weight and uterine weight data.
    (F) Pregnancy and litter data.
    (G) Fetal data (live/dead, sex, soft tissue and sketetal defects, 
resorptions).
    (g) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) Department of Health and Welfare. The Testing of Chemicals for 
Carcinogenicity, Mutagenicity and Teratogenicity. Minister of Health and 
Welfare (Canada: Department of Health and Welfare, 1975).
    (2) National Academy of Sciences. ``Principles and Procedures for 
Evaluating the Toxicity of Household Substances.'' A report prepared by 
the Committee for the Revision of NAS Publication 1138, under the 
auspices of the Committee on Toxicology, National Research Council, 
National Academy of Sciences, Washington, DC (1977).
    (3) World Health Organization. Principles for the Testing of Drugs 
for Teratogenicity. WHO Technical Report Series No. 364. (Geneva: World 
Health Organization, 1967).

[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19076, May 20, 1987; 
52 FR 26150, July 13, 1987; 54 FR 21064, May 16, 1989]



Sec. 798.4700  Reproduction and fertility effects.

    (a) Purpose. This guideline for two-generation reproduction testing 
is designed to provide general information concerning the effects of a 
test substance on gonadal function, conception, parturition, and the 
growth and development of the offspring. The study may also provide 
information about the effects of the test substance on neonatal 
morbidity, mortality, and preliminary data on teratogenesis and serve as 
a guide for subsequent tests.
    (b) Principle of the test method. The test substance is administered 
to parental (P) animals prior to their mating, during the resultant 
pregnancies, and through the weaning of their F1 offspring. 
The substance is then administered to selected F1 offspring 
during their growth into adulthood, mating, and production of an 
F2 generation, up until the F2 generation is 
weaned.
    (c) Test procedures--(1) Animal selection--(i) Species and strain. 
The rat is the preferred species. If another mammalian species is used, 
the tester shall provide justification/reasoning for its

[[Page 178]]

selection. Strains with low fecundity shall not be used.
    (ii) Age. Parental (P) animals shall be about 5 to 8 weeks old at 
the start of dosing.
    (iii) Sex. (A) For an adequate assessment of fertility, both males 
and females shall be studied.
    (B) The females shall be nulliparous and non-pregnant.
    (iv) Number of animals. Each test and control group shall contain at 
least 20 males and a sufficient number of females to yield at least 20 
pregnant females at or near term.
    (2) Control groups. (i) A concurrent control group shall be used. 
This group shall be an untreated or sham treated control group or if a 
vehicle is used in administering the test substance, a vehicle control 
group.
    (ii) If a vehicle is used in administering the test substance, the 
control group shall receive the vehicle in the highest volume used.
    (iii) If a vehicle or other additive is used to facilitate dosing, 
it shall not interfere significantly with absorption of the test 
substance or produce toxic effects.
    (3) Dose levels and dose selection. (i) At least three dose levels 
and a concurrent control shall be used.
    (ii) The highest dose level should induce toxicity but not high 
levels of mortality in the parental (P) animals.
    (iii) The lowest dose level should not produce any grossly 
observable evidence of toxicity.
    (iv) Ideally the intermediate dose level(s) should produce minimal 
observable toxic effects. If more than one intermediate dose is used, 
dose levels should be spaced to produce a gradation of toxic effects.
    (4) Exposure conditions. The animals should be dosed with the test 
substance, ideally, on a 7 days per week basis.
    (i) Dosing, mating, delivery, and sacrifice schedule.
    (A) Daily dosing of the parental (P) males and females shall begin 
when they are 5 to 8 weeks old. For both sexes, dosing shall be 
continued for at least 10 weeks before the mating period.
    (B) Dosing of P males shall continue through the 3 week mating 
period. At the end of the mating period, P males may be sacrificed and 
examined, or may be retained for possible production of a second litter. 
If these animals are retained for a second litter, dosing shall be 
continued. Dosing of the F1 males saved for mating shall 
continue from the time they are weaned through the period they are mated 
with the F1 females (11 weeks). F1 males may be 
sacrificed after the F1 mating period.
    (C) Daily dosing of the P females shall continue through the three 
week mating period, pregnancy, and to the weaning of the F1 
offspring. Dosing of the F1 females saved for mating shall 
continue from the time they are weaned, through the period they are 
mated with the F1 males (11 weeks from the time of weaning) 
pregnancy, and to the weaning of the F2 offspring.
    (ii) All animals are sacrificed as scheduled.
    (A) All P males should be sacrificed at the end of the 3-week mating 
period, or may be retained for possible production of a second litter. 
If these animals are retained for a second litter, dosing shall be 
continued.
    (B) F1 males selected for mating should be sacrificed at 
the end of the three week period of the F1 mating.
    (C) F1 males and females not selected for mating should 
be sacrified when weaned.
    (D) The P females should be sacrificed upon weaning of their 
F1 offspring.
    (E) F1 dams and their F2 offspring are 
sacrificed when the offspring are weaned.
    (5) Administration of the test substance--(i) Oral studies. (A) It 
is recommended that the test substance be administered in the diet or 
drinking water.
    (B) If administered by gavage or capsule, the dosage administered to 
each animal prior to mating shall be based on the individual animal's 
body weight and adjusted weekly. During pregnancy the dosage shall be 
based on the body weight at day 0 and 6 of pregnancy.
    (ii) If another route of administration is used, the tester should 
provide justification and reasoning for its selection.
    (6) Mating procedure--(i) Parental. (A) For each mating, each female 
shall be

[[Page 179]]

placed with a single male from the same dose level until pregnancy 
occurs or 1 week has elapsed. If mating has not occurred after 1 week, 
the female shall be placed with a different male. Paired matings should 
be clearly identified.
    (B) Those pairs that fail to mate should be evaluated to determine 
the cause of the apparent infertility. This may involve such procedures 
as additional opportunities to mate with proven fertile males or 
females, histological examination of the reproductive organs, and 
examination of the estrus or spermatogenic cycles.
    (C) Each day, the females shall be examined for presence of sperm or 
vaginal plugs. Day 0 of pregnancy is defined as the day vaginal plugs or 
sperm are found.
    (ii) F1 cross. (A) For mating the F1 offspring, one male 
and one female are randomly selected at weaning from each litter for 
cross mating with another pup of the same dose level but different 
litter, to produce the F2 generation.
    (B) F1 males and females not selected for mating are 
sacrificed upon weaning.
    (iii) Special housing. After evidence of copulation, pregnant 
animals shall be caged separately in delivery or maternity cages. 
Pregnant animals shall be provided with nesting materials when 
parturition is near.
    (iv) Standardization of litter sizes. (A) On day 4 after birth, the 
size of each litter should be adjusted by eliminating extra pups by 
random selection to yield, as nearly as possible, 4 males and 4 females 
per litter.
    (B) Whenever the number of male or female pups prevents having 4 of 
each sex per litter, partial adjustment (for example, 5 males and 3 
females) is permitted. Adjustments are not appropriate for litters of 
less than 8 pups.
    (C) Elimination of runts only is not appropriate.
    (D) Adjustments of the F2 litters is conducted in the 
same manner.
    (7) Observation of animals. (i) A gross examination shall be made at 
least once each day. Pertinent behavioral changes, signs of difficult or 
prolonged parturition, and all signs of toxicity, including mortality, 
shall be recorded. These observations shall be reported for each 
individual animal. Food consumption for all animals shall be monitored 
weekly except during the mating period.
    (ii) The duration of gestation shall be calculated from day 0 of 
pregnancy.
    (iii) Each litter should be examined as soon as possible after 
delivery for the number of pups, stillbirths, live births, sex, and the 
presence of gross anomalies. Live pups should be counted and litters 
weighed at birth or soon thereafter, and on days 4, 7, 14, and 21 after 
parturition.
    (iv) Physical or behavioral abnormalities observed in the dams of 
offspring shall be recorded.
    (v) P males and females shall be weighed on the first day of dosing 
and weekly thereafter. F1 litters shall be weighed at birth, 
or soon thereafter, and on days 4, 7, 14, and 21. In all cases, litter 
weights shall be calculated from the weights of the individual pups.
    (8) Gross necropsy. (i) A complete gross examination shall be 
performed on all adult animals, including those which died during the 
experiment or were killed in moribund conditions.
    (ii) Special attention shall be directed to the organs of the 
reproductive system.
    (iii) The following organs and tissues, or representative samples 
thereof, shall be preserved in a suitable medium for possible future 
histopathological examination: Vagina; uterus; ovaries; testes; 
epididymides; seminal vesicles; prostate, pituitary gland; and, target 
organ(s) when previously identified of all P and F1 animals 
selected for mating.
    (9) Histopathology. Except if carried out in other studies of 
comparable duration and dose levels the following histopathology shall 
be performed:
    (i) Full histopathology on the organs listed above for all high 
dose, and control P1 and F1 animals selected for 
mating.
    (ii) Organs demonstrating pathology in these animals shall then be 
examined in animals from the other dose groups.
    (iii) Microscopic examination shall be made of all tissues showing 
gross pathological changes.
    (d) Data and reporting--(1) Treatment of results. Data shall be 
summarized in

[[Page 180]]

tabular form, showing for each test group the number of animals at the 
start of the test, the number of animals pregnant, the types of change 
and the percentage of animals displaying each type of change.
    (2) Evaluation of study results. (i) An evaluation of test results, 
including the statistical analysis, based on the clinical findings, the 
gross necropsy findings, and the microscopic results shall be made and 
supplied. This should include an evaluation of the relationship, or lack 
thereof, between the animals' exposure to the test substance and the 
incidence and severity of all abnormalities.
    (ii) In any study which demonstrates an absence of toxic effects, 
further investigation to establish absorption and bioavailability of the 
test substance should be considered.
    (3) Test report. In addition to the reporting requirements as 
specified under 40 CFR part 792, subpart J the following specific 
information shall be reported:
    (i) Toxic response data by sex and dose, including fertility, 
gestation, viability and lactation indices, and length of gestation.
    (ii) Species and strain.
    (iii) Date of death during the study or whether animals survived to 
termination.
    (iv) Toxic or other effects on reproduction, offspring, or postnatal 
growth.
    (v) Date of observation of each abnormal sign and its subsequent 
course.
    (vi) Body weight data for P, F1, and F2 
animals.
    (vii) Necropsy findings.
    (viii) Detailed description of all histopathological findings.
    (ix) Statistical treatment of results where appropriate.
    (e) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) Clermont, Y., Perry, B. ``Quantitative Study of the Cell 
Population of the Seminiferous Tubules in Immature Rats,'' American 
Journal of Anatomy. 100:241-267 (1957).
    (2) Goldenthal, E.I. Guidelines for Reproduction Studies for Safety 
Evaluation of Drugs for Human Use. Drug Review Branch, Division of 
Toxicological Evaluation, Bureau of Science, Food and Drug 
Administration, Washington, DC (1966).
    (3) Hasegawa, T., Hayashi, M., Ebling, F.J.G., Henderson, I.W. 
Fertility and Sterility. (New York: American Elsevier Publishing Co., 
Inc., 1973).
    (4) Oakberg, E.F. ``Duration of Spermatogenesis in the Mouse and 
Timing of Stages of the Cycle of the Seminiferous Epithelium,'' American 
Journal of Anatomy. 9:507-516 (1956).
    (5) Roosen-Runge, E.C. ``The Process of Spermatogenesis in 
Mammals,'' Biological Review. 37:343-377 (1962).

[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19077, May 20, 1987]



Sec. 798.4900  Developmental toxicity study.

    (a) Purpose. In the assessment and evaluation of the toxic 
characteristics of a chemical, determination of the potential 
developmental toxicity is important. The developmental toxicity study is 
designed to provide information on the potential hazard to the unborn 
which may arise from exposure of the mother during pregnancy.
    (b) Definitions. (1) Developmental toxicity is the property of a 
chemical that causes in utero death, structural or functional 
abnormalities or growth retardation during the period of development.
    (2) Dose is the amount of test substance administered. Dose is 
expressed as weight of test substance (g, mg) per unit weight of a test 
animal (e.g., mg/kg).
    (3) No-observed-effect level is the maximum concentration in a test 
which produces no observed adverse effects. A no-observed-effect level 
is expressed in terms of weight of test substance given daily per unit 
weight of test animal (mg/kg)
    (c) Principle of the test method. The test substance is administered 
in graduated doses for at least part of the pregnancy covering the major 
period of organogenesis, to several groups of pregnant experimental 
animals, one dose level being used per group. Shortly before the 
expected date of delivery, the pregnant females are sacrificed, the 
uteri removed, and the contents examined for embryonic or fetal deaths, 
and live fetuses.

[[Page 181]]

    (d) Limit test. If a test at an exposure of at least 1000 mg/kg body 
weight, using the procedures described for this study, produces no 
observable developmental toxicity, then a full study using three dose 
levels might not be necessary.
    (e) Test procedures--(1) Animal selection--(i) Species and strain. 
Testing shall be performed in at least 2 mammalian species. Commonly 
used species include the rat, mouse, rabbit, and hamster. If other 
mammalian species are used, the tester shall provide justification/
reasoning for their selection. Commonly used laboratory strains shall be 
employed. The strain shall not have low fecundity and shall preferably 
be characterized for its sensitivity to developmental toxins.
    (ii) Age. Young adult animals (nulliparous females) shall be used.
    (iii) Sex. Pregnant female animals shall be used at each dose level.
    (iv) Number of animals. At least 20 pregnant rats, mice or hamsters 
or 12 pregnant rabbits are required at each dose level. The objective is 
to ensure that sufficient pups are produced to permit meaningful 
evaluation of the potential developmental toxicity of the test 
substance.
    (2) Control group. A concurrent control group shall be used. This 
group shall be an untreated or sham treated control group, or, if a 
vehicle is used in administering the test substance, a vehicle control 
group. Except for treatment with the test substance, animals in the 
control group(s) shall be handled in an identical manner to test group 
animals.
    (3) Dose levels and dose selection. (i) At least 3 dose levels with 
a control and, where appropriate, a vehicle control, shall be used.
    (ii) The vehicle shall neither be developmentally toxic nor have 
effects on reproduction.
    (iii) To select the appropriate dose levels, a pilot or trial study 
may be advisable. It is not always necessary to carry out a trial study 
in pregnant animals. Comparison of the results from a trial study in 
non-pregnant, and the main study in pregnant animals will demonstrate if 
the test substance is more toxic in pregnant animals. If a trial study 
is carried out in pregnant animals, the dose producing embryonic or 
fetal lethalities or maternal toxicity shall be determined.
    (iv) Unless limited by the physical/chemical nature or biological 
properties of the substance, the highest dose level shall induce some 
overt maternal toxicity such as reduced body weight or body weight gain, 
but not more than 10 percent maternal deaths.
    (v) The lowest dose level should not produce any grossly observable 
evidence of either maternal or developmental toxicity.
    (vi) Ideally, the intermediate dose level(s) should produce minimal 
observable toxic effects. If more than one intermediate concentration is 
used, the concentration levels should be spaced to produce a gradation 
of toxic effects.
    (4) Observation period. Day 0 in the test is the day on which a 
vaginal plug and/or sperm are observed. The dose period shall cover the 
period of major organogenesis. This may be taken as days 6 to 15 for rat 
and mouse, 6 to 14 for hamster, or 6 to 18 for rabbit.
    (5) Administration of test substance. The test substance or vehicle 
is usually administered orally, by oral intubation unless the chemical 
or physical characteristics of the test substance or pattern of human 
exposure suggest a more appropriate route of administration. The test 
substance shall be administered approximately the same time each day.
    (6) Exposure conditions. The female test animals are treated with 
the test substance daily throughout the appropriate treatment period. 
When given by gavage, the dose may be based on the weight of the females 
at the start of substance administration, or, alternatively, in view of 
the rapid weight gain which takes place during pregnancy, the animals 
may be weighed periodically and the dosage based on the most recent 
weight determination.
    (7) Observation of animals. (i) A gross examination shall be made at 
least once each day.
    (ii) Additional observations shall be made daily with appropriate 
actions taken to minimize loss of animals to the study (e.g., necropsy 
or refrigeration of those animals found dead and isolation or sacrifice 
of weak or moribund animals).

[[Page 182]]

    (iii) Signs of toxicity shall be recorded as they are observed, 
including the time of onset, the degree and duration.
    (iv) Cage-side observations shall include, but not be limited to: 
changes in skin and fur, eye and mucous membranes, as well as 
respiratory, autonomic and central nervous systems, somatomotor activity 
and behavioral pattern.
    (v) Measurements should be made weekly of food consumption for all 
animals in the study.
    (vi) Animals shall be weighed at least weekly.
    (vii) Females showing signs of abortion or premature delivery shall 
be sacrificed and subjected to a thorough macroscopic examination.
    (8) Gross necropsy. (i) At the time of sacrifice or death during the 
study, the dam shall be examined macroscopically for any structural 
abnormalities or pathological changes which may have influenced the 
pregnancy.
    (ii) Immediately after sacrifice or as soon as possible after death, 
the uterus shall be removed and the contents examined for embryonic or 
fetal deaths and the number of viable fetuses. The degree of resorption 
shall be described in order to help estimate the relative time of death 
of the conceptus. The weight of the gravid uterus should be recorded for 
dams that are sacrificed. Gravid uterine weights should not be obtained 
from dead animals if autolysis or decomposition has occurred.
    (iii) The number of corpora lutea shall be determined for all 
species except mice.
    (iv) The sex of the fetuses shall be determined and they shall be 
weighed individually, the weights recorded, and the mean fetal weight 
derived.
    (v) Following removal, each fetus shall be examined externally.
    (vi) For rats, mice and hamsters, one-third to one-half of each 
litter shall be prepared and examined for skeletal anomalies, and the 
remaining part of each litter shall be prepared and examined for soft 
tissue anomalies using appropriate methods.
    (vii) For rabbits, each fetus shall be examined by careful 
dissection for visceral anomalies and then examined for skeletal 
anomalies.
    (f) Data and reporting--(1) Treatment of results. Data shall be 
summarized in tablular form, showing for each test group: the number of 
animals at the start of the test, the number of pregnant animals, the 
number and percentages of live fetuses and the number of fetuses with 
any soft tissue or skeletal abnormalities.
    (2) Evaluation of results. The findings of a developmental toxicity 
study shall be evaluated in terms of the observed effects and the 
exposure levels producing effects. It is necessary to consider the 
historical developmental toxicity data on the species/strain tested. A 
properly conducted developmental toxicity study should provide a 
satisfactory estimation of a no-effect level.
    (3) Test report. In addition to the reporting requirements as 
specified under 40 CFR part 792, subpart J the following specific 
information shall be reported:
    (i) Toxic response data by concentration.
    (ii) Species and strain.
    (iii) Date of death during the study or whether animals survived to 
termination.
    (iv) Date of onset and duration of each abnormal sign and its 
subsequent course.
    (v) Food, body weight and uterine weight data.
    (vi) Pregnancy and litter data.
    (vii) Fetal data (live/dead, sex, soft tissue and skeletal defects, 
resorptions).
    (g) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) Department of Health and Welfare. The Testing of Chemicals for 
Carcinogenicity, mutagenicity and Teratogenicity. Minister of Health and 
Welfare (Canada: Department of Health and Welfare, 1975).
    (2) National Academy of Sciences. ``Principles and Procedures for 
Evaluating the Toxicity of Household Substances.'' A report prepared by 
the Committee for the Revision of NAS Publication 1138, under the 
auspices of the Committee on Toxicology, National Research Council, 
National

[[Page 183]]

Academy of Sciences, Washington, DC (1977).
    (3) World Health Organization. Principles for the Testing of Drugs 
for Teratogenicity. WHO Technical Report Series No. 364. (Geneva: World 
Health Organization, (1967).

[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19077, May 20, 1987]



                       Subpart F_Genetic Toxicity



Sec. 798.5195  Mouse biochemical specific locus test.

    (a) Purpose. The mouse biochemical specific locus test (MBSL) may be 
used to detect and quantitate mutations originating in the germ line of 
a mammalian species.
    (b) Definitions. (1) A biochemical specific locus mutation is a 
genetic change resulting from a DNA lesion causing alterations in 
proteins that can be detected by electrophoretic methods.
    (2) The germ line is comprised of the cells in the gonads of higher 
eukaryotes, which are the carriers of the genetic information for the 
species.
    (c) Reference substances. Not applicable.
    (d) Test method--(1) Principle. The principle of the MBSL is that 
heritable damage to the genome can be detected by electrophoretic 
analysis of proteins in the tissues of the progeny of mice treated with 
germ cell mutagens.
    (2) Description. For technical reasons, males rather than females 
are generally treated with the test chemical. Treated males are then 
mated to untreated females to produce F1 progeny. Both blood and kidney 
samples are taken from progeny for electrophoretic analysis. Up to 33 
loci can be examined by starch-gel electrophoresis and broad-range 
isoelectric focussing. Mutants are identified by variations from the 
normal electrophoretic pattern. Presumed mutants are bred to confirm the 
genetic nature of the change.
    (3) Animal selection--(i) Species and strain. Mice shall be used as 
the test species. Although the biochemical specific locus test could be 
performed in a number of in bred strains, in the most frequently used 
cross, C57BL/6 females are mated to DBA/2 males to produce (C57BL/6xDBA/
2) F1 progeny for screening.
    (ii) Age. Healthy, sexually-mature (at least 8 weeks old) animals 
shall be used for treatment and breeding.
    (iii) Number. A decision on the minimum number of treated animals 
should take into account possible effects of the test chemical on the 
fertility of the treated animals. Other considerations should include:
    (A) The production of concurrent spontaneous controls.
    (B) The use of positive controls.
    (C) The power of the test.
    (4) Control groups--(i) Concurrent controls. An appropriate number 
of concurrent control loci shall be analyzed in each experiment. These 
should be partly derived from matings of untreated animals (from 5 to 20 
percent ofthe treated matings), although some data on control loci can 
be taken from the study of the alleles transmitted from the untreated 
parent in the experimental cross. However, any laboratory which has had 
no prior experience with the test shall produce a spontaneous control 
sample of about 5,000 progeny animals and a positive control (using 100 
mg/kg ethylnitrosourea) sample of at least 1,200 offspring.
    (ii) Historical controls. Long-term, accumulated spontaneous control 
data (currently, 1 mutation in 1,200,000 control loci screened) are 
available for comparative purposes.
    (5) Test chemicals--(i) Vehicle. When possible, test chemicals shall 
be dissolved or suspended in distilled water or buffered isotonic 
saline. Water-insoluble chemicals shall be dissolved or suspended in 
appropriate vehicles. The vehicle used shall neither interfere with the 
test chemical nor produce major toxic effects. Fresh preparations of the 
test chemical should be employed.
    (ii) Dose levels. Usually, only one dose need be tested. This should 
be the maximum tolerated dose (MTD), the highest dose tolerated without 
toxic effects. Any temporary sterility induced due to elimination of 
spermatogonia at this dose must be of only moderate duration, as 
determined by are turn of males to fertility within 80 days after

[[Page 184]]

treatment. For evaluation of dose-response, it is recommended that at 
least two dose levels be tested.
    (iii) Route of administration. Acceptable routes of administration 
include, but are not limited to, gavage, inhalation, and mixture with 
food or water, and intraperitoneal or intravenous injections.
    (e) Test performance--(1) Treatment and mating. Male DBA/2 mice 
shall be treated with the test chemical and mated to virgin C57BL/6 
females immediately after cessation of treatment. Each treated male 
shall be mated to new virgin C57BL/6 females each week. Each pairing 
will continue for a week until the next week's mating is to begin. This 
mating schedule permits sampling of all post-spermatogonial stages of 
germ-cell development during the first 7 weeks after exposure. 
Spermatogonial stem cells are studied thereafter. Repeated mating cycles 
should be conducted until sufficient offspring have been obtained to 
meet the power criterion of the assay for spermatogonial stem cells.
    (2) Examination of offspring--(i) Birth and weaning. Offspring shall 
be examined at birth and at weaning for externally detectable changes in 
morphology and behavior; these could be due to dominant mutations. Such 
characteristics may include, but are not limited to, variations in coat 
color, appearance of eyes, size (in which case weighing of variant 
animals and littermates should be carried out), fur texture, etc. Gross 
changes in external form and behavior shall also be sought. Scrutiny of 
such visible characteristics of all animals shall be made during all 
subsequent manipulations of the animals.
    (ii) Tissue sampling. Blood (about 0.1 mL) and one kidney shall be 
removed from progeny mice under anesthesia. Both tissues are then 
prepared for analysis by electrophoresis.
    (iii) Electrophoresis. The gene products of 6 loci shall be analyzed 
in the blood sample by broad-range isoelectric focussing and of 27 loci 
in the kidney sample by starch-gel electrophoresis and enzyme-specific 
staining. Details on these procedures are included in paragraphs (g)(1) 
through (g)(3) of this section.
    (iv) Mutant identification. Presumptive electrophoretic mutants 
shall be identified by variation from the normal electrophoretic banding 
patterns. Reruns of all variant samples shall be performed to confirm 
the presence of altered banding patterns. Samples from parents of 
progeny exhibiting banding pattern variations shall be assayed to 
determine whether the variant was induced by the experimental treatment 
or was pre-existing. All treatment-induced variants are bred to 
determine the genetic nature of the change.
    (f) Data and reports--(1) Treatment of results. Data shall be 
presented in tabular form and shall permit independent analysis of cell 
stage-specific effects, and dose-dependent phenomena. The data shall be 
recorded and analyzed in such a way that clusters of identical mutations 
are clearly identified. The individual mutants detected shall be 
thoroughly described. In addition, concurrent positive control data (if 
employed) and spontaneous control data shall also be tabulated. These 
concurrent controls shall be added to, as well as compared with, the 
historical control data.
    (2) Statistical evaluation. Data shall be evaluated by appropriate 
statistical methods.
    (3) Interpretation of results. (i) There are several criteria for 
determining a positive response, one of which is a statistically 
significant dose-related increase in the frequency of electrophoretic 
mutations. Another criterion may be based upon detection of a 
reproducible and statistically significant positive response for at 
least one of these test points.
    (ii) A test chemical which does not produce a statistically 
significant increase in the frequency of electrophoretic mutations over 
the spontaneous frequency, or a statistically significant and 
reproducible positive response for at least one of the test points, is 
considered nonmutagenic in this system, provided that the sample size is 
sufficient to exclude a biologically significant increase in mutation 
frequency.
    (iii) Both biological and statistical significance should be 
considered together in the evaluation.

[[Page 185]]

    (4) Test evaluation. (i) Positive results in the MBSL indicate that, 
under the test conditions, the test chemical induces heritable gene 
mutations in a mammalian species.
    (ii) Negative results indicate that, under the test conditions, the 
test chemical does not induce heritable genemutations in a mammalian 
species.
    (5) Test report. In addition to the reporting requirements as 
specified under 40 CFR part 792, subpart J, and paragraph (h) of this 
section, the following specific information shall be reported:
    (i) Strain, age and weight of animals used; numbers of animals of 
each sex in experimental and control groups.
    (ii) Test chemical vehicle, doses used, rationale for dose 
selection, and toxicity data, if available.
    (iii) Route and duration of exposure.
    (iv) Mating schedule.
    (v) Number of loci screened for both treated and spontaneous data.
    (vi) Criteria for scoring mutants.
    (vii) Number of mutants found/locus.
    (viii) Loci at which mutations were found.
    (ix) Use of concurrent negative and positive controls.
    (x) Dose-response relationship, if applicable.
    (g) References. For additional background information on this test 
guideline, the following references should be consulted:
    (1) Personal communication from Susan E. Lewis, Ph.D. to Dr. Michael 
Cimino, U.S. EPA, OPPT, October 5, 1989.
    (2) Johnson, F.M., G.T. Roberts, R.K. Sharma, F.Chasalow, R. 
Zweidinger, A. Morgan, R.W. Hendren, and S.E.Lewis. ``The detection of 
mutants in mice by electrophoresis: Results of a model induction 
experiment with procarbazine.'' Genetics 97:113-124 (1981).
    (3) Johnson, F.M. and S.E. Lewis. ``Mutation rate determinations 
based on electrophoretic analysis of laboratory mice.'' Mutation 
Research 82:125-135 (1981a).
    (4) Johnson, F.M. and S.E. Lewis. ``Electrophoretically detected 
germinal mutations induced by ethylnitrosourea in the mouse.'' 
Proceedings of the National Academy of Sciences 78:3138-93141 (1981b).
    (5) Lewis, S.E., C. Felton, L.B. Barnett, W. Generoso, N. Cacheiro, 
and M.D. Shelby. ``Dominant visible and electrophoretically expressed 
mutations induced in male mice exposed to ethylene oxide by 
inhalation.'' Environmental Mutagenesis 8:867-872 (1986).
    (h) Additional requirements. Testing facilities conducting the mouse 
biochemical specific locus test in accordance with this section shall, 
in addition to adhering to the provisions of Sec. Sec. 792.190 and 
792.195 of this chapter, obtain, adequately identify, and retain for at 
least 10 years, acceptable 35-mm photographs (and their negatives) of 
the stained isoelectric-focussing columns and the stained starch-gels 
obtained following analyses of blood and kidney preparations, 
respectively, from mutant mice, their siblings, and their parents.

[55 FR 12641, Apr. 5, 1990]



Sec. 798.5200  Mouse visible specific locus test.

    (a) Purpose. The mouse visible specific locus test (MSLT) may be 
used to detect and quantitate mutations in the germ line of a mammalian 
species.
    (b) Definitions. (1) A visible specific locus mutation is a genetic 
change that alters factors responsible for coat color and other visible 
characteristics of certain mouse strains.
    (2) The germ line is the cells in the gonads of higher eukaryotes 
which are the carriers of the genetic information for the species.
    (c) Reference substances. Not applicable.
    (d) Test method--(1) Principle. (i) The principle of the MSLT is to 
cross individuals who differ with respect to the genes present at 
certain specific loci, so that a genetic alteration involving the 
standard gene at any one of these loci will produce an offspring 
detectably different from the standard heterozygote. The genetic change 
may be detectable by various means, depending on the loci chosen to be 
marked.
    (ii) Three variations of the method currently exist for detecting 
newly arising point mutations in mouse germ cells:

[[Page 186]]

    (A) The visible specific locus test using either 5 or 7 loci.
    (B) The biochemical specific locus test using up to 20 enzymes.
    (C) The test for mutations at histocompatibility loci.
    (iii) Of the three tests, the visible specific locus test has been 
most widely used in assessing genetic hazard due to environmental 
agents. It is the method described in this guideline.
    (2) Description. For technical reasons, males rather than females 
are generally treated with the test agent. Treated males are then mated 
to females which are genetically homozygous for certain specific visible 
marker loci. Offspring are examined in the next generation for evidence 
that a new mutation has arisen.
    (3) Animal selection--(i) Species and strain. Mice shall be used as 
the test species. Male mice shall be either 
(C3Hx101)F1 or (101xC3H)F1 
hybrids. Females shall be T stock virgins.
    (ii) Age. Healthy sexually mature animals shall be used.
    (iii) Number. A decision on the minimum number of treated animals 
should take into account the spontaneous variation of the biological 
characterization being evaluated. Other considerations should include:
    (A) The use of either historical or concurrent controls.
    (B) The power of the test.
    (C) The minimal rate of induction required.
    (D) The use of positive controls.
    (E) The level of significance desired.
    (iv) Assignment to groups. Animals shall be randomized and assigned 
to treatment and control groups.
    (4) Control groups--(i) Concurrent controls. The use of positive or 
spontaneous controls is left to the discretion of the investigator. 
However, any laboratory which has had no prior experience with the test 
shall, at its first attempt, produce a negative control sample of 20,000 
and a positive control, using 100 mg/kg 1-ethyl-nitrosourea, in a sample 
of 5,000 offspring.
    (ii) Historical controls. Long term, accumulated spontaneous control 
data of 43/801,406 are available for comparative purposes.
    (5) Test chemicals--(i) Vehicle. When possible, test chemicals 
should be dissolved or suspended in distilled water or isotonic saline 
buffered appropriately, if needed, for stability. Water-insoluble 
chemicals shall be dissolved or suspended in appropriate vehicles. The 
vehicle used shall neither interfere with the test compound nor produce 
major toxic effects. Fresh preparations of the test chemical should be 
employed.
    (ii) Dose levels. Usually, only one dose level need be tested. This 
should be the highest dose tolerated without toxic effects, provided 
that any temporary sterility induced due to elimination of spermatagonia 
is of only moderate duration, as determined by a return of males to 
fertility within 80 days after treatment. For evaluation of dose-
response, it is recommended that at least two dose levels be tested.
    (iii) Route of administration. Acceptable routes of administration 
include gavage, inhalation, admixture with food or water, and IP or IV 
injections.
    (e) Test performance--(1) Treatment and mating. Hybrid F1 
(C3 Hx101 or 101xC3 H) male mice shall be treated 
with the test substance and immediately mated to virgin T stock females. 
Each treated male shall be mated to a fresh group of 2 to 4 virgin 
females each week for 7 weeks, after which he shall be returned to the 
first group of females and rotated through the seven sets of females 
repeatedly. This mating schedule generally permits sampling of all 
postspermatagonial stages of germ cell development during the first 7 
weeks and rapid accumulation of data for exposed spermatagonial stem 
cells thereafter. Repeated mating cycles should be conducted until the 
entire spermatogonial cycle has been evaluated and enough offspring have 
been obtained to meet the power criterion of the assay.
    (2) Examination of offspring. (i) Offspring may be examined at (or 
soon after) birth but must be examined at about 3 weeks of age at which 
time the numbers of mutant and nonmutant offspring in each litter shall 
be recorded.
    (ii) Nonmutant progeny should be discarded. Mutant progeny shall be 
subjected to genetic tests for verification.

[[Page 187]]

    (f) Data and report--(1) Treatment of results. Data shall be 
presented in tabular form and shall permit independent analysis of cell 
stage specific effects and dose dependent phenomena. The data shall be 
recorded and analyzed in such a way that clusters of identical mutations 
are clearly identified. The individual mutants detected shall be 
thoroughly described. In addition, concurrent positive and negative 
control data, if they are available, shall be tabulated so that it is 
possible to differentiate between concurrent (when available) and long-
term accumulated mutation frequencies.
    (2) Statistical evaluation. Data shall be evaluated by appropriate 
statistical methods.
    (3) Interpretation of results. (i) There are several criteria for 
determining a positive result, one of which is a statistically 
significant dose-related increase in the number of specific locus 
mutations. Another criterion may be based upon detection of a 
reproducible and statistically significant positive response for at 
least one of the test points.
    (ii) A test substance which does not produce either a statistically 
significant dose-related increase in the number of specific locus 
mutations or a statistically significant and reproducible positive 
response at any one of the test points is considered nonmutagenic in 
this system.
    (iii) Both biological and statistical significance should be 
considered together in the evaluation.
    (4) Test evaluation. (i) Positive results in the MSLT indicate that 
under the test conditions the test substance induces heritable gene 
mutations in the test species.
    (ii) Negative results indicate that under the test conditions the 
test substance does not induce heritable gene mutations in the test 
species.
    (5) Test report. In addition to the reporting requirements as 
specified under 40 CFR part 792, subpart J, and paragraph (h) of this 
section, the following specific information shall be reported:
    (i) Strain, age and weight of animals used, number of animals of 
each sex in experimental and control groups.
    (ii) Test chemical vehicle, doses used and rationale for dose 
selection, toxicity data.
    (iii) Route and duration of exposure.
    (iv) Mating schedule.
    (v) Time of examination for mutant progeny.
    (vi) Criteria for scoring mutants.
    (vii) Use of concurrent or negative controls.
    (viii) Dose response relationship, if applicable.
    (g) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) Russell, L.B., Shelby, P.B., von Halle, E., Sheridan, W., 
Valcovic, L. The mouse specific locus test with agents other than 
radiations: interpretation of data and recommendations for future work: 
A report of the U.S. EPA's Gene-Tox Program,'' Mutation Research, 
86:329-354 (1981).
    (2) [Reserved]
    (h) Additional requirements. Testing facilities conducting the mouse 
visible specific locus test in accordance with this section shall, in 
addition to adhering to the provisions of Sec. Sec. 792.190 and 792.195 
of this chapter, obtain, and retain for at least 10 years, acceptable 
35-mm color photographs (and their negatives) demonstrating the visible 
mutations observed in mutant animals and the lack of such mutations in 
their siblings and parents.

[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19078, May 20, 1987; 
55 FR 12643, Apr. 5, 1990]



Sec. 798.5265  The salmonella typhimurium reverse mutation assay.

    (a) Purpose. The Salmonella typhimurium histidine (his) reversion 
system is a microbial assay which measures his-[rarr] 
his= reversion induced by chemicals which cause base changes 
or frameshift mutations in the genome of this organism.
    (b) Definitions. (1) A reverse mutation assay in Salmonella 
typhimurium detects mutation in a gene of a histidine requiring strain 
to produce a histidine independent strain of this organism.
    (2) Base pair mutagens are agents which cause a base change in the 
DNA. In a reversion assay, this change may

[[Page 188]]

occur at the site of the original mutation or at a second site in the 
chromosome.
    (3) Frameshift mutagens are agents which cause the addition or 
deletion of single or multiple base pairs in the DNA molecule.
    (c) Reference substances. These may include, but need not be limited 
to, sodium azide, 2-nitrofluorene, 9-aminoacridine, 2-aminoanthracene, 
congo red, benzopurpurin 4B, trypan blue or direct blue 1.
    (d) Test method--(1) Principle. Bacteria are exposed to test 
chemical with and without a metabolic activation system and plated onto 
minimal medium. After a suitable period of incubation, revertant 
colonies are counted and compared to the number of spontaneous 
revertants in an untreated and/or vehicle control culture.
    (2) Description. Several methods for performing the test have been 
described. Among those used are:
    (i) The direct plate incorporation method.
    (ii) The preincubation method.
    (iii) The azo-reduction method.


The procedures described here are for the direct plate incorporation 
method and the azo-reduction method.
    (3) Strain selection--(i) Designation. At the present time four 
strains, TA 1535, TA 1537, TA 98 and TA 100 should be used. The use of 
other strains in addition to these four is left to the discretion of the 
investigator.
    (ii) Preparation and storage. Recognized methods of stock culture 
preparation and storage should be used. The requirement of histidine for 
growth should be demonstrated for each strain. Other phenotypic 
characteristics should be checked using such methods as crystal violet 
sensitivity and resistance to ampicillin. Spontaneous reversion 
frequency should be in the range expected either as reported in the 
literature or as established in the laboratory by historical control 
values.
    (iii) Bacterial growth. Fresh cultures of bacteria should be grown 
up to the late exponential or early stationary phase of growth 
(approximately 10\8\-10\9\ cells per ml).
    (4) Metabolic activation. Bacteria should be exposed to the test 
substance both in the presence and absence of an appropriate metabolic 
activation system. For the direct plate incorporation method, the most 
commonly used system is a cofactor supplemented postmitochondrial 
fraction prepared from the livers of rodents treated with enzyme 
inducing agents such as Aroclor 1254. For the azo-reduction method, a 
cofactor supplemented postmitochondrial fraction prepared from the 
livers of untreated hamsters is preferred. For this method, the cofactor 
supplement should contain flavin mononucleotide, exogenous glucose 6-
phosphate dehydrogenase, NADH and excess of glucose-6-phosphate.
    (5) Control groups--(i) Concurrent controls. Concurrent positive and 
negative (untreated and/or vehicle) controls shall be included in each 
experiment. Positive controls shall ensure both strain responsiveness 
and efficacy of the metabolic activation system.
    (ii) Strain specific positive controls. Strain specific positive 
controls shall be included in the assay. Examples of strain specific 
positive controls are as follows:
    (A) Strain TA 1535, TA 100, sodium azide.
    (B) TA 98, 2-nitrofluorene.
    (C) TA 1537, 9-aminoacridine.
    (iii) Positive controls to ensure the efficacy of the activation 
system. The positive control reference substance for tests including a 
metabolic activation system should be selected on the basis of the type 
of activation system used in the test. 2-Aminoanthracene is an example 
of a positive control compound in plate-incorporation tests using 
postmitochondrial fractions from the livers of rodents treated with 
enzyme inducing agents such as Aroclor-1254. Congo red is an example of 
a positive control compound in the azo-reduction method. Other positive 
control reference substances may be used.
    (iv) Class-specific positive controls. The azo-reduction method 
should include positive controls from the same class of compounds as the 
test agent wherever possible.
    (6) Test chemicals--(i) Vehicle. Test chemicals and positive control 
reference substances should be dissolved or suspended in an appropriate 
vehicle and then further diluted in vehicle for use in the assay.

[[Page 189]]

    (ii) Exposure concentrations. (A) The test should initially be 
performed over a broad range of concentrations. Among the criteria to be 
taken into consideration for determining the upper limits of test 
chemical concentration are cytotoxicity and solubility. Cytotoxicity of 
the test chemical may be altered in the presence of metabolic activation 
systems. Toxicity may be evidenced by a reduction in the number of 
spontaneous revertants, a clearing of the background lawn or by the 
degree of survival of treated cultures. Relatively insoluble compounds 
should be tested up to the limits of solubility. For freely soluble 
nontoxic chemicals, the upper test chemical concentration should be 
determined on a case by case basis.
    (B) Generally, a maximum of 5 mg/plate for pure substances is 
considered acceptable. At least 5 different amounts of test substance 
shall be tested with adequate intervals between test points.
    (C) When appropriate, a single positive response shall be confirmed 
by testing over a narrow range of concentrations.
    (e) Test performance--(1) Direct plate incorporation method. For 
this test without metabolic activation, test chemica1 and 0.1 m1 of a 
fresh bacterial culture should be added to 2.0 ml of overlay agar. For 
tests with metabolic activation, 0.5 ml of activation mixture containing 
an adequate amount of postmitochondrial fraction should be added to the 
agar overlay after the addition of test chemical and bacteria. Contents 
of each tube shall be mixed and poured over the surface of a selective 
agar plate. Overlay agar shall be allowed to solidify before incubation. 
At the end of the incubation period, revertant colonies per plate shall 
be counted.
    (2) Azo-reduction method. (i) For this test with metabolic 
activation, 0.5 ml of S-9 mix containing 150 ul of S-9 and 0.1 ml of 
bacterial culture should be added to a test tube kept on ice. One-tenth 
milliliter of chemical should be added, and the tubes should be 
incubated with shaking at 30 [deg]C for 30 min. At the end of the 
incubation period, 2.0 ml of agar should be added to each tube, the 
contents mixed and poured over the surface of a selective agar plate. 
Overlay agar shall be allowed to solidify before incubation. At the end 
of the incubation period, revertant colonies per plate shall be counted.
    (ii) For tests without metabolic activation, 0.5 ml of buffer should 
be used in place of the 0.5 ml of S-9 mix. All other procedures shall be 
the same as those used for the test with metabolic activation.
    (3) Other methods. Other methods may also be appropriate.
    (4) Media. An appropriate selective medium with an adequate overlay 
agar shall be used.
    (5) Incubation conditions. All plates within a given experiment 
shall be incubated for the same time period. This incubation period 
shall be for 48-72 hours at 37 [deg]C.
    (6) Number of cultures. All plating should be done at least in 
triplicate.
    (f) Data and report--(1) Treatment of results. Data shall be 
presented as number of revertant colonies per plate for each replicate 
and dose. The numbers of revertant colonies on both negative (untreated 
and/or vehicle) and positive control plates shall also be presented. 
Individual plate counts, the mean number of revertant colonies per plate 
and standard deviation shall be presented for test chemical and positive 
and negative (untreated and/or vehicle) controls.
    (2) Statistical evaluation. Data should be evaluated by appropriate 
statistical methods.
    (3) Interpretation of results. (i) There are several criteria for 
determining a positive result, one of which is a statistically 
significant dose-related increase in the number of revertants. Another 
criterion may be based upon detection of a reproducible and 
statistically significant positive response for at least one of the test 
substance concentrations.
    (ii) A test substance which does not produce either a statistically 
significant dose-related increase in the number of revertants or a 
statistically significant and reproducible positive response at any one 
of the test points is considered nonmutagenic in this system.

[[Page 190]]

    (iii) Both biological and statistical significance should be 
considered together in the evaluation.
    (4) Test evaluation. (i) Positive results from the S. typhimurium 
reverse mutation assay indicate that, under the test conditions, the 
test substance induces point mutations by base changes or frameshifts in 
the genome of this organism.
    (ii) Negative results indicate that under the test conditions the 
test substance is not mutagenic in S. typhimurium.
    (5) Test report. In addition to the reporting recommendations as 
specified under 40 CFR part 792, subpart J, the following specific 
information shall be reported:
    (i) Bacterial strain used.
    (ii) Metabolic activation system used (source, amount and cofactor); 
details of preparations of S-9 mix.
    (iii) Dose levels and rationale for selection of dose.
    (iv) Positive and negative controls.
    (v) Individual plate counts, mean number of revertant colonies per 
plate, standard deviation.
    (vi) Dose-response relationship, if applicable.
    (g) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) Ames, B.N., McCann, J., Yamasaki, E. ``Methods for detecting 
carcinogens and mutagens with the Salmonella/ mammalian-microsome 
mutagenicity test,'' Mutation Research 31:347-364 (1975).
    (2) de Serres, F.J., Shelby, M.D. ``The Salmonella mutagenicity 
assay: recommendations,'' Science 203:563-565 (1979).
    (3) Prival, M.J., Mitchell, V.D. ``Analysis of a method for testing 
azo dyes for mutagenic activity in Salmonella typhimurium in the 
presence of flavin mononucleotide and hamster liver S-9,'' Mutation 
Research 97:103-116 (1982).
    (4) Vogel, H.J., Bonner, D.M. ``Acetylornithinase of E. coli: 
partial purification and some properties,'' Journal of Biological 
Chemistry. 218:97-106 (1956).

[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19078, May 20, 1987]



Sec. 798.5275  Sex-linked recessive lethal test in drosophila melanogaster.

    (a) Purpose. The sex-linked recessive lethal (SLRL) test using 
Drosophila melanogaster detects the occurrence of mutations, both point 
mutations and small deletions, in the germ line of the insect. This test 
is a forward mutation assay capable of screening for mutations at about 
800 loci on the X-chromosome. This represents about 80 percent of all X-
chromosome loci. The X-chromosome represents approximately one-fifth of 
the entire haploid genome.
    (b) Definitions. (1) Lethal mutation is a change in the genome 
which, when expressed, causes death to the carrier.
    (2) Recessive mutation is a change in the genome which is expressed 
in the homozygous or hemizygous condition.
    (3) Sex-Linked genes are present on the sex (X or Y) chromosomes. 
Sex-linked genes in the context of this guideline refer only to those 
located on the X-chromosome.
    (c) Reference substances. These may include, but need not be limited 
to, ethyl methanesulfonate or N-nitroso-dimethylamine.
    (d) Test method--(1) Principle. Mutations in the X-chromosome of D. 
melanogaster are phenotypically expressed in males carrying the mutant 
gene. When the mutation is lethal in the hemizygous condition, its 
presence is inferred from the absence of one class of male offspring out 
of the two that are normally produced by a heterozygous female. The SLRL 
test takes advantage of these facts by means of specially marked and 
arranged chromosomes.
    (2) Description. Wild-type males are treated and mated to 
appropriate females. Female offspring are mated individually to their 
brothers, and in the next generation the progeny from each separate dose 
are scored for phenotypically wild-type males. Absence of these males 
indicates that a sex-linked recessive lethal mutation has occurred in a 
germ cell of the P1 male.
    (3) Drosophila stocks. Males of a well-defined wild type stock and 
females of the Muller-5 stock may be used. Other appropriately marked 
female stocks

[[Page 191]]

with multiple inverted X-chromosomes may also be used.
    (4) Control groups--(i) Concurrent controls. Concurrent positive and 
negative (vehicle) controls shall be included in each experiment.
    (ii) Positive controls. Examples of positive controls include ethyl 
methanesulfonate and N-nitroso-dimethylamine.
    (iii) Other positive controls. Other positive control reference 
substances may be used.
    (iv) Negative controls. Negative (vehicle) controls shall be 
included. The size of the negative (vehicle) control group shall be 
determined by the availability of appropriate laboratory historical 
control data.
    (5) Test chemicals--(i) Vehicle. Test chemicals should be dissolved 
in water. Compounds which are insoluble in water may be dissolved or 
suspended in appropriate vehicles (e.g., a mixture of ethanol and Tween-
60 or 80) and then diluted in water or saline prior to administration. 
Dimethylsulfoxide should br avoided as a vehicle.
    (ii) Dose levels. For the initial assessment of mutagenicity, it is 
sufficient to test a single dose of the test substance for screening 
purposes. This dose should be the maximum tolerated dose, or that which 
produces some indication of toxicity, or shall be the highest dose 
attainable. For dose-response purposes, at least three additional dose 
levels should be used.
    (iii) Route of administration. Exposure may be oral, by injection or 
by exposure to gases or vapors. Feeding of the test compound may be done 
in sugar solution. When necessary, substances may be dissolved in 0.7 
percent NaCl solution and injected into the thorax or abdomen.
    (e) Test performance--(1) Treatment and mating. Wild-type males (3 
to 5 days old) shall be treated with the test substance and mated 
individually to an appropriate number of virgin females from the Muller-
5 stock or females from another appropriately marked (with multiply-
inverted X-chromosomes) stock. The females shall be replaced with fresh 
virgins every 2 to 3 days to cover the entire germ cell cycle. The 
offspring of these females are scored for lethal effects corresponding 
to the effects on mature sperm, mid or late stage spermatids, early 
spermatids, spermatocytes and spermatogonia at the time of treatment.
    (2) F11 matings. Heterozygous F1 females from 
the above crosses shall be allowed to mate individually (i.e., one 
female per vial) with their brothers. In the F2 generation, 
each culture shall be scored for the absence of wild-type males. If a 
culture appears to have arisen from an F1 female carrying a 
lethal in the parental X-chromosome (i.e., no males with the treated 
chromosome are observed), daughters of that female with the same 
genotype shall be tested to ascertain if the lethality is repeated in 
the next generation.
    (3) Number of matings. (i) The test should be designed with a 
predetermined sensitivity and power. The number of flies in each group 
should reflect these defined parameters. The spontaneous mutant 
frequency observed in the appropriate control group will strongly 
influence the number of treated chromosomes that must be analysed to 
detect substances which show mutation rates close to those of the 
controls.
    (ii) Test results should be confirmed in a separate experiment.
    (f) Data and report--(1) Treatment of results. Data shall be 
tabulated to show the number of chromosomes tested, the number of 
nonfertile males and the number of lethal chromosomes at each exposure 
concentration and for each mating period for each male treated. Numbers 
of clusters of different size per male shall be reported.
    (2) Statistical evaluation. Data shall be evaluated by appropriate 
statistical techniques.
    (3) Interpretation of results. (i) There are several criteria for 
determining a positive result, one of which is a statistically 
significant dose-related increase in the number of sex-lined recessive 
lethals. Another criterion may be based upon detection of a reproducible 
and statistically significant positive response for at least one of the 
test points.

[[Page 192]]

    (ii) A test substance which does not produce either a statistically 
significant dose-related increase in the number of sex-linked recessive 
lethals or a statistically significant and reproducible positive 
response at any one of the test points is considered non-mutagenic in 
this system.
    (iii) Both biological and statistical significance should be 
considered together in the evaluation.
    (4) Test evaluation. (i) Positive results in the SLRL test in D. 
melanogaster indicate that under the test conditions the test agent 
causes mutations in germ cells of this insect.
    (ii) Negative results indicate that under the test conditions the 
test substance is not mutagenic in D. melanogaster.
    (5) Test report. In addition to the reporting recommendations as 
specified under 40 CFR part 792, subpart J the following specific 
information shall be reported.
    (i) Drosophila stock used in the assay, age of insects, number of 
males treated, number of sterile males, number of F2 cultures 
established, number of F2 cultures without progeny.
    (ii) Test chemical vehicle, treatment and sampling schedule, 
exposure levels, toxicity data, negative (vehicle) and positive 
controls, if appropriate.
    (iii) Criteria for scoring lethals.
    (iv) Number of chromosomes tested, number of chromosomes scored, 
number of chromosomes carrying a lethal mutation.
    (v) Historical control data, if available.
    (vi) Dose-response relationship, if applicable.
    (g) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) Sobels, F.H., Vogel, E. ``The capacity of Drosophila for 
detecting relevant genetic damage,'' Mutation Research 41:95-106 (1976).
    (2) Wurgler F.E., Sobels F.H., Vogel E. ``Drosophila as assay system 
for detecting genetic changes,'' Handbook of mutagenicity test 
procedures. Eds. Kilbey, B.J., Legator, M., Nichols, W., Ramel, C., 
(Amsterdam: Elsevier/North Holland Biomedical Press, 1977) pp. 335-373.

[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19079, May 20, 1987]



Sec. 798.5300  Detection of gene mutations in somatic cells in culture.

    (a) Purpose. Mammalian cell culture systems may be used to detect 
mutations induced by chemical substances. Widely used cell lines include 
L5178Y mouse lymphoma cells and the CHO and V-79 lines of Chinese 
hamster cells. In these cell lines the most commonly used systems 
measure mutation at the thymidine kinase (TK), hypoxanthine-guanine-
phosphoribosyl transferase (HPRT) and Na=/K= 
ATPase loci. The TK and HPRT mutational systems detect base pair 
mutations, frameshift mutations, and small deletions; the 
Na=/K= ATPase system detects base pair mutations 
only.
    (b) Definitions. (1) A forward mutation assay detects a gene 
mutation from the parental type to the mutant form which gives rise to a 
change in an enzymatic or functional protein.
    (2) Base pair mutagens are agents which cause a base change in the 
DNA.
    (3) Frameshift mutagens are agents which cause the addition or 
deletion of single or multiple base pairs in the DNA molecule.
    (4) Phenotypic expression time is a period during which unaltered 
gene products are depleted from newly mutated cells.
    (c) Reference substances. These may include, but need not be limited 
to, ethyl methanesulfonate, N-nitroso-dimethylamine, 2-
acetylaminofluorene, 7,12-dimethylbenzanthracene or hycanthone.
    (d) Test method--(1) Principle. Cells are exposed to test substance, 
both with and without metabolic activation, for a suitable period of 
time and subcultured to determine cytotoxicity and to allow phenotypic 
expression prior to mutant selection. Cells deficient in thymidine 
kinase (TK) due to the forward mutation TK=[rarr] 
TK- are resistant to the cytotoxic effects of pyrimidine 
analogues such as bromodeoxyuridine (BrdU), fluorodeoxyuridine (FdU) or

[[Page 193]]

trifluorothymidine (TFT). The deficiency of the ``salvage'' enzyme 
thymidine kinase means that these antimetabolites are not incorporated 
into cellular nucleotides and the nucleotides needed for cellular 
metabolism are obtained solely from de novo synthesis. However, in the 
presence of thymidine kinase, BrdU, FdU or TFT are incorporated into the 
nucleotides, resulting in inhibition of cellular metabolism and 
cytotoxicity. Thus mutant cells are able to proliferate in the presence 
of BrdU, FdU or TFT whereas normal cells, which contain thymidine 
kinase, are not. Similarly cells deficient in HPRT are selected by 
resistance to 8-azaguanine (AG) or 6-thioguanine (TG) and cells with 
altered Na=/K= ATPase are selected by resistance 
to ouabain.
    (2) Description. Cells in suspension or monolayer culture are 
exposed to the test substance, both with and without metabolic 
activation, for a defined period of time. Cytotoxicity is determined by 
measuring the colony forming ability or growth rate of the cultures 
after the treatment period. The treated cultures are maintained in 
growth medium for a sufficient period of time--characteristic of each 
selected locus--to allow near-optimal phenotypic expression of induced 
mutations. Mutant frequency is determined by seeding known numbers of 
cells in medium containing the selective agent to detect mutant cells, 
and in medium without selective agent to determine the cloning 
efficiency. After a suitable incubation time, cell colonies are counted. 
The number of mutant colonies in selective medium is adjusted by the 
number of colonies in nonselective medium to derive the mutant 
frequency.
    (3) Cells--(i) Type of cells used in the assay. A variety of cell 
lines are available for use in this assay including subclones of L5178Y, 
CHO cells or V-79 cells. Cell types used in this assay should have a 
demonstrated sensitivity to chemical mutagens, a high cloning efficiency 
and a low spontaneous mutation frequency. Cells should be checked for 
Mycoplasma contamination and may be periodically checked for karyotype 
stability.
    (ii) Cell growth and maintenance. Appropriate culture media and 
incubation conditions (culture vessels, CO2 concentrations, 
temperature and humidity) shall be used.
    (4) Metabolic activation. Cells shall be exposed to test substance 
both in the presence and absence of an appropriate metabolic activation 
system.
    (5) Control groups. Positive and negative (untreated and/or vehicle) 
controls shall be included in each experiment. When metabolic activation 
is used, the positive control substance shall be known to require such 
activation.
    (6) Test chemicals--(i) Vehicle. Test substances may be prepared in 
culture media or dissolved or suspended in appropriate vehicles prior to 
treatment of the cells. The final concentration of the vehicle shall not 
interfere with cell viability or growth rate. Treatment vessels should 
be chosen to ensure that there is no visible interaction, such as 
etching, between the solvent, the test chemical, and the vessel.
    (ii) Exposure concentrations. (A) The test should be designed to 
have a predetermined sensitivity and power. The number of cells, 
cultures, and concentrations of test substance used should reflect these 
defined parameters. The number of cells per culture is based on the 
expected background mutant frequency; a general guide is to use a number 
which is 10 times the inverse of this frequency.
    (B) Several concentrations (usually at least 4) of the test 
substance shall be used. Generally, these shall yield a concentration-
related toxic effect. The highest concentration shall produce a low 
level of survival (approximately 10 percent), and the survival in the 
lowest concentration shall approximate the negative control. 
Cytotoxicity shall be determined after treatment with the test substance 
both in the presence and in the absence of an exogenous metabolic 
activation system. Relatively insoluble substances should be tested up 
to their limit of solubility under culture conditions. For freely-
soluble nontoxic substances the highest concentration used should be 
determined on a case-by-case basis.
    (e) Test performance. (1) Cells shall be exposed to the test 
substance both with

[[Page 194]]

and without exogenous metabolic activation. Exposure shall be for a 
suitable period of time, in most cases 1 to 5 hours is effective; 
exposure time may be extended over one or more cell cycles.
    (2) At the end of the exposure period, cells shall be washed and 
cultured to determine viability and to allow for expression of the 
mutant phenotype.
    (3) At the end of the expression period, which shall be sufficient 
to allow near optimal phenotypic expression of induced mutants, cells 
should be grown in medium with and without selective agent(s) for 
determination of number of mutants and cloning efficiency, respectively.
    (4) Results shall be confirmed in an independent experiment. When 
appropriate, a single positive response should be confirmed by testing 
over a narrow range of concentrations.
    (f) Data and report--(1) Treatment of results. Data shall be 
presented in tabular form. Individual colony counts for the treated and 
control groups shall be presented for both mutation induction and 
survival. Survival and cloning efficiencies shall be given as a 
percentage of the controls. Mutant frequency shall be expressed as 
number of mutants per number of surviving cells.
    (2) Statistical evaluation. Data should be evaluated by appropriate 
statistical methods.
    (3) Interpretation of results. (i) There are several criteria for 
determining a positive result, one of which is a statistically 
significant concentration-related increase in the mutant frequency. 
Another criterion may be based upon detection of a reproducible and 
statistically significant positive response for at least one of the test 
substance concentrations.
    (ii) A test substance which does not produce either a statistically 
significant concentration-related increase in the mutant frequency or a 
statistically significant and reproducible positive response at any one 
of the test points is considered nonmutagenic in this system.
    (iii) Both biological and statistical significance should be 
considered together in the evaluation.
    (4) Test evaluation. (i) Positive results for an in vitro mammalian 
cell gene mutation test indicate that, under the test conditions, a 
substance induces gene mutations in the cultured mammalian cells used.
    (ii) Negative results indicate that, under the test conditions, the 
test substance does not induce gene mutations in the cultured mammalian 
cells used.
    (5) Test report. In addition to the reporting recommendations as 
specified under 40 CFR part 792, subpart J the following specific 
information shall be reported:
    (i) Cell type used, number of cell cultures, methods used for 
maintenance of cell cultures.
    (ii) Rationale for selection of concentrations and number of 
cultures.
    (iii) Test conditions: composition of media, CO2 
concentration, concentration of test substance, vehicle, incubation 
temperature, incubation time, duration of treatment, cell density during 
treatment, type of metabolic activation system, positive and negative 
controls, length of expression period (including number of cells seeded 
and subculture and feeding schedules, if appropriate), selective 
agent(s).
    (iv) Methods used to enumerate numbers of viable and mutant cells.
    (v) Dose-response relationship, where possible.
    (g) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) Amacher, D.E., Paillet, S.C., Ray, V. ``Point mutations at the 
thymidine kinase locus in L5178Y mouse lymphoma cells. I. Application to 
genetic toxicology testing,'' Mutation Research, 64:391-406 (1979).
    (2) Amacher, D.E., Paillet, S.C., Turner, G.N., Ray, V.A. Salsburg, 
V.A. ``Point mutations at the thymidine kinase locus in L5178Y mouse 
lymphoma cells. II. Test validation and interpretation,'' Mutation 
Research, 72:447-474 (1980).
    (3) Bradley, M.O., Bhuyan B., Francis, M.C., Langenback, R., 
Peterson, A., Huberman, E. ``Mutagenesis by chemical agents in V-79 
Chinese hamster cells: a review and analysis of the literature: a report 
of the Gene-Tox Program,'' Mutation Research, 87:81-142 (1981).

[[Page 195]]

    (4) Clive, D., Johnson, K.O., Spector, J.F.S., Batson, A.G., Brown, 
M.M. ``Validation and characterization of the L5178Y TK=/
- mouse lymphoma mutagen assay system,'' Mutation Research, 
59:61-108 (1979).
    (5) Clive, D., Spector, J.F.S. ``Laboratory procedures for assessing 
specific locus mutations at the TK locus in cultured L5178Y mouse 
lymphoma cells,'' Mutation Research, 31:17-29 (1975).
    (6) Hsie, A.W., Casciano, D.A., Couch, D.B., Krahn, D.F., O'Neill, 
J.P., Whitfield, B.L. ``The use of Chinese hamster ovary cells to 
quantify specific locus mutation and to determine mutagenicity of 
chemicals: a report of the U.S. EPA's Gene-Tox Program,'' Mutation 
Research, 86:193-214 (1981).

[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19079, May 20, 1987]



Sec. 798.5375  In vitro mammalian cytogenetics.

    (a) Purpose. The in vitro cytogenetics test is a mutagenicity test 
system for the detection of chromosomal aberrations in cultured 
mammalian cells. Chromosomal aberrations may be either structural or 
numerical. However, because cytogenetic assays are usually designed to 
analyse cells at their first post-treatment mitosis and numerical 
aberrations require at least one cell division to be visualized, this 
type of aberration is generally not observed in a routine cytogenetics 
assay. Structural aberrations may be of two types, chromosome or 
chromatid.
    (b) Definitions. (1) Chromosome-type aberrations are changes which 
result from damage expressed in both sister chromatids at the same time.
    (2) Chromatid-type aberrations are damage expressed as breakage of 
single chromatids or breakage and/or reunion between chromatids.
    (c) Reference substances. Not applicable.
    (d) Test method--(1) Principle. In vitro cytogenetics assays may 
employ cultures of established cell lines, cell strains or primary cell 
cultures. Cell cultures are exposed to the test substance both with and 
without metabolic activation. Following exposure of cell cultures to 
test substances, they are treated with a spindle inhibitor (e.g., 
colchicine or Colcemid) to arrest cells in a metaphase-like 
stage of mitosis (c-metaphase). Cells are then harvested and chromosome 
preparations made. Preparations are stained and metaphase cells are 
analyzed for chromosomal aberrations.
    (2) Description. Cell cultures are exposed to test compounds and 
harvested at various intervals after treatment. Prior to harvesting, 
cells are treated with a spindle inhibitor (e.g., colchicine or 
Colcemid) to accumulate cells in c-metaphase. Chromosome 
preparations from cells are made, stained and scored for chromosomal 
aberrations.
    (3) Cells--(i) Type of cells used in the assay. There are a variety 
of cell lines or primary cell cultures, including human cells, which may 
be used in the assay. Established cell lines and strains should be 
checked for Mycoplasma contamination and may be periodically checked for 
karyotype stability.
    (ii) Cell growth and maintenance. Appropriate culture media, and 
incubation conditions (culture vessels CO2 concentrations, 
temperature and humidity) shall be used.
    (4) Metabolic activation. Cells shall be exposed to test substance 
both in the presence and absence of an appropriate metabolic activation 
system.
    (5) Control groups. Positive and negative (untreated and/or vehicle) 
controls both with and without metabolic activation shall be included in 
each experiment. When metabolic activation is used, the positive control 
substance shall be known to require such activation.
    (6) Test chemicals--(i) Vehicle. Test substances may be prepared in 
culture media or dissolved or suspended in appropriate vehicles prior to 
treatment of the cells. Final concentration of the vehicle shall not 
interfere with cell viability or growth rate. Treatment vessels should 
be chosen to ensure that there is no visible interaction, such as 
etching, between the solvent, the test chemical, and the vessel.
    (ii) Exposure concentrations. Multiple concentrations of the test 
substance over a range adequate to define the response should be tested. 
Generally the highest test substance concentrations

[[Page 196]]

tested with and without metabolic activation should show evidence of 
cytotoxicity or reduced mitotic activity. Relatively insoluble 
substances should be tested up to the limit of solubility. For freely 
soluble nontoxic chemicals, the upper test chemical concentration should 
be determined on a case by case basis.
    (e) Test performance--(1) Established cell lines and strains. Prior 
to use in the assay, cells should be generated from stock cultures, 
seeded in culture vessels at the appropriate density and incubated at 37 
[deg]C.
    (2) Human lymphocyte cultures. Heparinized or acid-citrate-dextrose 
whole blood should be added to culture medium containing a mitogen, 
e.g., phytohemagglutinin (PHA) and incubated at 37 [deg]C. White cells 
sedimented by gravity (buffy coat) or lymphocytes which have been 
purified on a density gradient may also be utilized.
    (3) Treatment with test substance. For established cell lines and 
strains, cells in the exponential phase of growth shall be treated with 
test substances in the presence and absence of an exogenous metabolic 
activation system. Mitogen-stimulated human lymphocyte cultures may be 
treated with the test substance in a similar manner.
    (4) Number of cultures. At least two independent cultures shall be 
used for each experimental point.
    (5) Culture harvest time. (i) For established cell lines and 
strains, multiple harvest times are recommended. However, for screening 
purposes, a single harvest time may be appropriate. If the test chemical 
changes the cell cycle length, the fixation intervals should be changed 
accordingly. If a single harvest time is selected, supporting data for 
the harvest time should be presented in such a study.
    (ii) For human lymphocyte cultures, the substance to be tested may 
be added to the cultures at various times after mitogen stimulation so 
that there is a single harvest time after the initiation of the cell 
culture. Alternatively, a single treatment may be followed by multiple 
harvest times. Harvest time should be extended for those chemicals which 
induce an apparent cell cycle delay. Because the population of human 
lymphocytes is only partially synchronized, a single treatment, at, or 
close to, the time when metaphase stages first appear in the culture 
will include cells in all phases of the division cycle. Therefore, a 
single harvest at the time of second mitosis may be carried out for 
screening purposes.
    (iii) Cell cultures shall be treated with a spindle inhibitor, 
(e.g., colchicine or Colcemid [reg]), 1 or 2 hours prior to 
harvesting. Each culture shall be harvested and processed separately for 
the preparation of chromosomes.
    (6) Chromosome preparation. Chromosome preparation involves 
hypotonic treatment of the cells, fixation and staining.
    (7) Analysis. Slides shall be coded before analysis. In human 
lymphocytes, only cells containing 46 centromeres shall be analyzed. In 
established cell lines and strains, only metaphases containing 2 centromeres of the modal number shall be analyzed. 
Uniform criteria for scoring aberrations shall be used.
    (8) Confirmatory tests. When appropriate, a single positive response 
shall be confirmed by testing over a narrow range of concentrations.
    (f) Data and report--(1) Treatment of results. Data shall be 
presented in a tabular form. Different types of structural chromosomal 
aberrations shall be listed with their numbers and frequencies for 
experimental and control groups. Data should be evaluated by appropriate 
statistical methods. Gaps or achromatic lesions are recorded separately 
and not included in the total aberration frequency.
    (2) Statistical evaluation. Data should be evaluated by appropriate 
statistical methods.
    (3) Interpretation of results. (i) There are several criteria for 
determining a positive result, one of which is a statistically 
significant dose-related increase in the number of structural 
chromosomal aberrations. Another criterion may be based upon detection 
of a reproducible and statistically significant positive response for at 
least one of the test substance concentrations.

[[Page 197]]

    (ii) A test substance which does not produce either a statistically 
significant dose-related increase in the number of structural 
chromosomal aberrations or a statistically significant and reproducible 
positive response at any one of the test points is considered 
nonmutagenic in this system.
    (iii) Both biological and statistical significance should be 
considered together in the evaluation.
    (4) Test evaluation. (i) Positive results in the in vitro 
cytogenetics assay indicate that under the test conditions the test 
substance induces chromosomal aberrations in cultured mammalian somatic 
cells.
    (ii) Negative results indicate that under the test conditions the 
test substance does not induce chromosomal aberrations in cultured 
mammalian somatic cells.
    (5) Test report. In addition to the reporting recommendations as 
specified under 40 CFR part 792, subpart J the following specific 
information shall be reported:
    (i) Cells used, density and passage number at time of treatment, 
number of cell cultures.
    (ii) Methods used for maintenance of cell cultures including medium, 
temperature and CO2 concentration.
    (iii) Test chemical vehicle, concentration and rationale for the 
selection of the concentrations used in the assay, duration of 
treatment.
    (iv) Details of both the protocol used to prepare the metabolic 
activation system and of its use in the assay.
    (v) Identity of spindle inhibitor, its concentration and duration of 
treatment.
    (vi) Date of cell harvest.
    (vii) Positive and negative controls.
    (viii) Methods used for preparation of slides for microscopic 
examination.
    (ix) Number of metaphases analysed.
    (x) Mitotic index where applicable.
    (xi) Criteria for scoring aberrations.
    (xii) Type and number of aberrations, given separately for each 
treated and control culture, total number of aberrations per group; 
frequency distribution of number of chromosomes in established cell 
lines and strains.
    (xiii) Dose-response relationship, if applicable.
    (g) References. For additional background information on this test 
guideline the following references should be consulted.
    (1) Ames, B.N., McCann, J., Yamasaki, E. ``Methods for detecting 
carcinogens and mutagens with the Salmonella/ mammalian-microsome 
mutagenicity test,'' Mutation Research, 31:347-364 (1975).
    (2) Evans, H.J. ``Cytological methods for detecting chemical 
mutagens,'' Chemical mutagens, principles and methods for their 
detection, Vol. 4, Ed. A. Hollaender (New York, London: Plenum Press, 
1976) pp. 1-29.
    (3) Howard, P.N., Bloom, A.D., Krooth, R.S. ``Chromosomal 
aberrations induced by N-methyl-N'-nitro-N-nitrosoguanidine in mammalian 
cells,'' In Vitro 7:359-365 (1972).
    (4) Ishidate, M. Jr., Odashima, S. ``Chromosome tests with 134 
compounds on Chinese hamster cells in vitro: A screening for chemical 
carcinogens,'' Mutation Research, 48:337-354 (1975).
    (5) Preston, R.J., Au, W., Bender, M.A., Brewen, J.G., Carrano, 
A.V., Heddle, J.A., McFee, A.F., Wolff, S., Wassom, J.S., ``Mammalian in 
vivo and in vitro cytogenetic assays: A report of the Gene-tox 
Program,'' Mutation Research, 87:143-188 (1981).

[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19079, May 20, 1987]



Sec. 798.5385  In vivo mammalian bone marrow cytogenetics tests: Chromosomal 

analysis.

    (a) Purpose. The in vivo bone marrow cytogenetic test is a 
mutagenicity test for the detection of structural chromosomal 
aberrations. Chromosomal aberrations are generally evaluated in first 
post-treatment mitoses. With the majority of chemical mutagens, induced 
aberrations are of the chromatid type but chromosome type aberrations 
also occur.
    (b) Definitions. (1) Chromosome-type aberrations are changes which 
result from damage expressed in both sister chromatids at the same time.
    (2) Chromatid-type aberrations are damage expressed as breakage of 
single chromatids or breakage and/or reunion between chromatids.

[[Page 198]]

    (c) Reference substances. Not applicable.
    (d) Test method--(1) Principle. Animals are exposed to test 
chemicals by appropriate routes and are sacrificed at sequential 
intervals. Chromosome preparations are made from bone marrow cells. The 
stained preparations are examined and metaphase cells are scored for 
chromosomal aberrations.
    (2) Description. The method employs bone marrow of laboratory 
rodents which have been exposed to test chemicals. Prior to sacrifice, 
animals are further treated with a spindle inhibitor, (e.g., colchicine 
or Colcemid [reg]) to arrest the cells in c-metaphase. 
Chromosome preparations from the cells are stained and scored for 
chromosomal aberrations.
    (3) Animal selection--(i) Species and strain. Any appropriate 
mammalian species may be used. Examples of commonly used rodent species 
are rats, mice, and hamsters.
    (ii) Age. Healthy young adult animals shall be used.
    (iii) Number and sex. At least five female and five male animals per 
experimental and control group shall be used. Thus, 10 animals would be 
sacrificed per time per group treated with the test compound if several 
test times after treatment are included in the experimental schedule. 
The use of a single sex or smaller number of animals should be 
justified.
    (iv) Assignment to groups. Animals shall be randomized and assigned 
to treatment and control groups.
    (4) Control groups--(1) Concurrent controls. (i) Concurrent positive 
and negative (vehicle) controls shall be included in the assay.
    (ii) Positive controls. A single dose positive control showing a 
significant response at any one time point is adequate. A compound known 
to produce chromosomal aberrations in vivo shall be employed as the 
positive control.
    (5) Test chemicals--(i) Vehicle. When possible, test chemicals shall 
be dissolved in isotonic saline or distilled water. Water insoluble 
chemicals may be dissolved or suspended in appropriate vehicles. The 
vehicles used shall neither interfere with the test chemical nor produce 
toxic effects. Fresh preparations of the test compound should be 
employed.
    (ii) Dose levels. For an initial assessment, one dose of the test 
substance may be used, the dose being the maximum tolerated dose (to a 
maximum of 5,000 mg/kg) or that producing some indication of 
cytotoxicity (e.g., partial inhibition of mitosis) or shall be the 
highest dose attainable (to a maximum of 5,000 mg/kg). Additional dose 
levels may be used. For determination of dose-response, at least three 
dose levels should be used.
    (iii) Route of administration. The usual routes are oral or by 
intraperitoneal injection. Other routes may be appropriate.
    (iv) Treatment schedule. In general, test substances should be 
administered once only. However, based on toxicological information a 
repeated treatment schedule may be employed.
    (e) Test performance--(1) Generally the test may be performed in two 
assays. (i) Animals should be treated with the test substance once at 
the selected dose(s). Samples should be taken at three times after 
treatment. For rodents, the central sampling interval is 24 hours. Since 
cell cycle kinetics can be influenced by the test substance, one earlier 
and one later sampling interval adequately spaced within the range of 6 
to 48 hours shall be applied. Where the additional dose levels are 
tested in a subsequent experiment, samples shall be taken at the 
predetermined most sensitive interval or, if this is not established, at 
the central sampling time. If the most sensitive interval is known and 
documented with data, only this one time point shall be sampled.
    (ii) If a repeated treatment schedule is used at the selected 
dose(s), samples shall be taken 6 and 24 hours after the last treatment; 
other sampling times may be used if justified. Where the additional dose 
levels are tested in a subsequent experiment, samples shall be taken at 
the predetermined most sensitive interval or, if this is not 
established, at 6 hours after the last treatment.
    (2) Administration of spindle inhibitor. Prior to sacrifice, animals 
shall be injected IP with an appropriate dose of a spindle inhibitor 
(e.g., colchicine or

[[Page 199]]

Colcemid [reg]) to arrest cells in c-metaphase.
    (3) Preparation of slides. Immediately after sacrifice, the bone 
marrow shall be obtained, exposed to hypotonic solution, and fixed. The 
cells shall then be spread on slides and stained. Chromosome 
preparations shall be made following standard procedures.
    (4) Analysis. The number of cells to be analyzed per animal should 
be based upon the number of animals used, the negative control 
frequency, the predetermined sensitivity, and the power chosen for the 
test. Slides shall be coded before microscopic analysis.
    (f) Data and report--(1) Treatment of results. Data should be 
presented in tabular form for both cells and animals. Different types of 
structural chromosomal aberrations should be listed with their numbers 
and a mean frequency per cell for each animal in all treated and control 
groups. Gaps (achromatic lesions) should be recorded separately and not 
included in the total abberration frequency. Differences among animals 
within each group should be considered before making comparisons between 
treated and control groups.
    (2) Statistical evaluation. Data should be evaluated by appropriate 
statistical methods.
    (3) Interpretation of results. (i) There are several criteria for 
determining a positive result, one of which is a statistically 
significant dose-related increase in the number of structual chromosomal 
aberrations or abnormal metaphase figures. Another criterion may be 
based upon detection of a reproducible and statistically significant 
positive response for a least one of the test points.
    (ii) A test substance which does not produce either a statistically 
significant dose-related increase in the number of chromosomal 
aberrations or abnormal metaphase figures or a statistically significant 
and reproducible positive response at any one of the test points is 
considered nonmutagenic in this system.
    (iii) Both biological and statistical significance should be 
considered together in the evaluation.
    (4) Test evaluation. (i) Positive results in the in vivo bone marrow 
cytogenetics assay indicate that under the test conditions the test 
substance induces chromosomal aberrations in the bone marrow of the test 
species.
    (ii) Negative results indicate that under the test conditions, the 
test substance does not induce chromosomal aberrations in the bone 
marrow of the test species.
    (5) Test report. In addition to the reporting recommendations as 
specified under 40 CFR part 792, subpart J the following specific 
information shall be reported:
    (i) Species, strain, age, weight, number and sex of animals in each 
treatment and control group.
    (ii) Test chemical vehicle, dose levels used, rationale for dose 
selection.
    (iii) Route of administration, treatment and sampling schedules, 
toxicity data, negative and positive controls.
    (iv) Identity of spindle-inhibitor, its concentration and duration 
of treatment.
    (v) Details of the protocol used for chromosome preparation, number 
of cells scored per animal, type and number of aberrations given 
separately for each treated and control animal.
    (vi) Mitotic index, where applicable.
    (vii) Criteria for scoring aberrations.
    (viii) Number and frequency of aberrant cells per animal in each 
treatment and control groups.
    (ix) Total number of aberrations per group.
    (x) Number of cells with aberrations per group.
    (xi) Dose-response relationship, if applicable.
    (g) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) Adler, I.D., Ramarao, G., Epstein, S.S. ``In vivo cytogenetic 
effects of trimethyl-phosphate and of TEPA on bone marrow cells of male 
rats,'' Mutation Research, 13:263-273 (1971).
    (2) Evans, H.J. ``Cytological methods for detecting chemical 
mutagens,'' Chemical Mutagens: Principles and Methods for Their 
Detection, Vol. 4. Ed. A. Hollaender (New York and London: Plenum Press, 
1976) pp. 1-29.
    (3) Kilian, J.D., Moreland, F.E. Benge, M.C., Legator, M.S., 
Whorton, E.B. Jr. ``A collaborative study to

[[Page 200]]

measure intralaboratory variation with the in vivo bone morrow metaphase 
procedure,'' Handbook of mutagenicity test procedures. Eds. Kilby, B.J., 
Legator, M. Nichols, C., Ramel, D., (Amsterdam: Elsevier/North Holland 
Biomedical Press, 1977) 243-260.
    (4) Preston, J.R., Au, W., Bender, M.A., Brewen, J.G., Carrano, A.V. 
Heddle, J.A., McFee, A.F., Wolff, S., Wassom, J. ``Mammalian in vivo and 
vitro cytogenetics assays: Report of the Gene-Tox Program,'' Mutation 
Research, 87:143-188 (1981).

[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19080, May 20, 1987]



Sec. 798.5395  In vivo mammalian bone marrow cytogenetics tests: Micronucleus 

assay.

    (a) Purpose. The micronucleus test is a mammalian in vivo test which 
detects damage of the chromosomes or mitotic apparatus by chemicals. 
Polychromatic erythrocytes in the bone marrow of rodents are used in 
this assay. When the erythroblast develops into an erythrocyte the main 
nucleus is extruded and may leave a micronucleus in the cytoplasm. The 
visualization of micronuclei is facilitated in these cells because they 
lack a nucleus. Micronuclei form under normal conditions. The assay is 
based on an increase in the frequency of micronucleated polychromatic 
erythrocytes in bone marrow of treated animals.
    (b) Definition. Micronuclei are small particles consisting of 
acentric fragments of chromosomes or entire chromosomes, which lag 
behind at anaphase of cell division. After telophase, these fragments 
may not be included in the nuclei of daughter cells and form single or 
multiple micronuclei in the cytoplasm.
    (c) Reference substances. Not applicable.
    (d) Test method--(1) Principle. (i) Animals are exposed to test 
substance by an appropriate route. They are sacrificed, the bone marrow 
extracted and smear preparations made and stained. Polychromatic 
erythrocytes are scored for micronuclei under the microscope.
    (ii) Micronuclei may also be detected in other test systems:
    (A) Tissue culture.
    (B) Plants.
    (C) Blood smears.
    (D) Fetal tissues.
    (E) Meiotic cells.
    (F) Hepatic cells.
    (iii) The present guideline is based on the mammalian bone marrow 
assay.
    (2) Description. The method employs bone marrow of laboratory 
mammals which are exposed to test substances.
    (3) Animal selection--(i) Species and strain. Mice are recommended. 
However, any appropriate mammalian species may be used.
    (ii) Age. Young adult animals shall be used.
    (iii) Number and sex. At least five female and five male animals per 
experimental and control group shall be used. Thus, 10 animals would be 
sacrificed per time per group if several test times after treatment were 
included in the experimental schedule. The use of a single sex or a 
smaller number of animals should be justified.
    (iv) Assignment to groups. Animals shall be randomized and assigned 
to treatment and control groups.
    (4) Control groups--(i) Concurrent controls. Concurrent positive and 
negative (vehicle) controls shall be included in each assay.
    (ii) Positive controls. A compound known to produce micronuclei in 
vivo shall be employed as the positive control.
    (5) Test chemicals--(i) Vehicle. When appropriate for the route of 
administration, solid and liquid test substances should be dissolved or 
suspended in distilled water or isotonic saline. Water insoluble 
chemicals may be dissolved or suspended in appropriate vehicles. The 
vehicle used shall neither interfere with the test compound nor produce 
toxic effects. Fresh preparations of the test compound should be 
employed.
    (ii) Dose levels. For an initial assessment, one dose of the test 
substance may be used, the dose being the maximum tolerated dose (to a 
maximum of 5,000 mg/kg) or that producing some indication of 
cytotoxicity, e.g., a change in the ratio of polychromatic to 
normochromatic erythrocytes. Additional dose levels may be used. For 
determination of dose response, at least three dose levels shall be 
used.

[[Page 201]]

    (iii) Route of administration. The usual routes of administration 
are IP or oral. Other routes may be appropriate.
    (iv) Treatment schedule. Test substances should generally be 
administered only once. However, based upon toxicological information a 
repeated treatment schedule may be employed.
    (e) Test performance--(1) Treatment and sampling times. (i) Animals 
shall be treated with the test substance once at the highest tolerated 
dose. Sampling times should coincide with the maximum responses of the 
assay which varies with the test substance. Therefore, using the highest 
dose, bone marrow samples should be taken at least three times, starting 
not earlier than 12 hours after treatment, with appropriate intervals 
following the first sample but not extending beyond 72 hours. When other 
doses are used sampling shall be at the maximum sensitive period, or, if 
that is not known, approximately 24 hours after treatment. Other 
appropriate sampling times may be used in addition. If the most 
sensitive interval is known and documented with data, only this one time 
point need be sampled.
    (ii) If a repeated treatment schedule is used, samples shall be 
taken at least three times, starting not earlier than 12 hours after the 
last treatment and at appropriate intervals following the first sample, 
but not extending beyond 72 hours.
    (iii) Bone marrow shall be obtained immediately after sacrifice. 
Cells shall be prepared, put on slides, spread as a smear and stained.
    (2) Analysis. Slides shall be coded before microscopic analysis. At 
least 1,000 polychromatic erythrocytes per animal shall be scored for 
the incidence of micronuclei. The ratio of polychromatic to 
normochromatic erythrocytes should be determined for each animal by 
counting a total of 200 erythrocytes. To ensure consistency with OECD 
and other guidelines, 1,000 polychromatic erythrocytes are recommended. 
Additional information may be obtained by scoring normochromatic 
erythrocytes for micronuclei.
    (f) Data and report--(1) Treatment of results. Criteria for scoring 
micronuclei shall be given. Individual data shall be presented in a 
tabular form including positive and negative (vehicle) controls and 
experimental groups. The number of polychromatic erythrocytes scored, 
the number of micronucleated polychromatic erythrocytes, the percentage 
of micronucleated cells, the number of micronucleated normochromatic 
erythrocytes, and, if applicable, the percentage of micronucleated 
erythrocytes and the ratio of normochromatic to polychromatic 
erythrocytes shall be listed separately for each experimental and 
control animal. Absolute numbers shall be included if percentages are 
reported.
    (2) Statistical evaluation. Data should be evaluated by appropriate 
statistical methods.
    (3) Interpretation of results. (i) There are several criteria for 
determining a positive response, one of which is a statistically 
significant dose-related increase in the number of micronucleated 
polychromatic erythrocytes. Another criterion may be based upon 
detection of a reproducible and statistically significant positive 
response for at least one of the test substance concentrations.
    (ii) A test substance which does not produce either a statistically 
significant dose-related increase in the number of micronucleated 
polychromatic erythrocytes or a statistically significant and 
reproducible positive response at any one of the test points is 
considered nonmutagenic in this system.
    (iii) Both biological and statistical significance should be 
considered together in the evaluation.
    (4) Test evaluation. (i) The results of the micronucleus test 
provide information on the ability of a chemical to induce micronuclei 
in polychromatic erythrocytes of the test species under the conditions 
of the test. This damage may have been the result of chromosomal damage 
or damage to the mitotic apparatus.
    (ii) Negative results indicate that under the test conditions the 
test substance does not produce micronuclei in the bone marrow of the 
test species.
    (5) Test report. In addition to the reporting recommendations as 
specified under 40 CFR part 792, subpart J, the following specific 
information shall be reported:

[[Page 202]]

    (i) Species, strain, age, weight, number and sex of animals in each 
treatment and control group.
    (ii) Test chemical vehicle, dose levels used, rationale for dose 
selection.
    (iii) Rationale for and description of treatment and sampling 
schedules, toxicity data, negative and positive controls.
    (iv) Details of the protocol used for slide preparation.
    (v) Criteria for identifying micronucleated erythrocytes.
    (vi) Dose-response relationship, if applicable.
    (g) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) Cihak, R. ``Evaluation of benzidine by the micronucleus test,'' 
Mutation Research, 67: 383-384 (1979).
    (2) Cole, R.J., Taylor, N., Cole, J., Arlett, C.F. ``Short-term 
tests for transplacentally active carcinogens. 1. Micronucleus formation 
in fetal and maternal mouse erythroblasts,'' Mutation Research, 80: 141-
157 (1981).
    (3) Kliesch, U., Danford, N., Adler, I.D. ``Micronucleus test and 
bone-marrow chromosome analysis. A comparison of 2 methods in vivo for 
evaluating chemically induced chromosomal alterations,'' Mutation 
Research, 80: 321-332 (1981).
    (4) Matter, B., Schmid, W. ``Trenimon-induced chromosomal damage in 
bone-marrow cells of six mammalian species, evaluated by the 
micronucleus test,'' Mutation Research, 12: 417-425 (1971).
    (5) Schmid, W. ``The micronucleus test,'' Mutation Research, 31:9-15 
(1975).
    (6) Schmid, W. ``The micronucleus test for cytogenetic analysis,'' 
Chemical Mutagens, Principles and Methods for their Detection. Vol. 4 
Hollaender A, (Ed. A ed. (New York and London: Plenum Press, (1976) pp. 
31-53.
    (7) Heddle, J.A., Hite, M., Kurkhart, B., Mavournin, K., MacGregor, 
J.T., Newell, G.W., Salamone, M.F. ``The induction of micronuclei as a 
measure of genotoxicity. A report of the U.S. Environmental Protection 
Agency Gene-Tox Program,'' Mutation Research, 123: 61-118 (1983).

[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19080, May 20, 1987; 
52 FR 26150, July 13, 1987; 52 FR 34654, Sept. 14, 1987]



Sec. 798.5450  Rodent dominant lethal assay.

    (a) Purpose. Dominant lethal (DL) effects cause embryonic or fetal 
death. Induction of a dominant lethal event after exposure to a chemical 
substance indicates that the substance has affected germinal tissue of 
the test species. Dominant lethals are generally accepted to be the 
result of chromosomal damage (structural and numerical anomalies) but 
gene mutations and toxic effects cannot be excluded.
    (b) Definition. A dominant lethal mutation is one occurring in a 
germ cell which does not cause dysfunction of the gamete, but which is 
lethal to the fertilized egg or developing embryo.
    (c) Reference substances. These may include, but need not be limited 
to, triethylenemelamine, cyclophosphamide or ethyl methanesulfonate.
    (d) Test method--(1) Principle. Generally, male animals are exposed 
to the test substance and mated to untreated virgin females. The various 
germ cell stages can be tested separately by the use of sequential 
mating intervals. The females are sacrificed after an appropriate period 
of time and the contents of the uteri are examined to determine the 
numbers of implants and live and dead embryos. The calculation of the 
dominant lethal effect is based on comparison of the live implants per 
female in the treated group to the live implants per female in the 
control group. The increase of dead implants per female in the treated 
group over the dead implants per female in the control group reflects 
the post-implantation loss. The post-implantation loss is calculated by 
determining the ratio of dead to total implants from the treated group 
compared to the ratio of dead to total implants from the control group. 
Pre-implantation loss can be estimated on the basis of corpora lutea 
counts or by comparing the total implants per female in treated and 
control groups.

[[Page 203]]

    (2) Description. (i) Several treatment schedules are available. The 
most widely used requires single administration of the test substance. 
Other treatment schedules, such as treatment on five consecutive days, 
may be used if justified by the investigator.
    (ii) Individual males are mated sequentially to virgin females at 
appropriate intervals. The number of matings following treatment is 
governed by the treatment schedule and should ensure that germ cell 
maturation is adequately covered. Females are sacrificed in the second 
half of pregnancy and the uterine contents examined to determine the 
total number of implants and the number of live and dead embryos.
    (3) Animal selection--(i) Species. Rats or mice are generally used 
as the test species. Strains with low background dominant lethality, 
high pregnancy frequency and high implant numbers are recommended.
    (ii) Age. Healthy, sexually mature animals shall be used.
    (iii) Number. An adequate number of animals shall be used taking 
into account the spontaneous variation of the biological characteristics 
being evaluated. The number chosen should be based on the predetermined 
sensitivity of detection and power of significance. For example, in a 
typical experiment, the number of males in each group shall be 
sufficient to provide between 30 and 50 pregnant females per mating 
interval.
    (iv) Assignment to groups. Animals shall be randomized and assigned 
to treatment and control groups.
    (4) Control groups--(i) Concurrent controls. Generally concurrent 
positive and negative (vehicle) controls shall be included in each 
experiment. When acceptable positive control results are available from 
experiments conducted recently (within the last 12 months) in the same 
laboratory these results can be used instead of a concurrent positive 
control.
    (ii) Positive controls. Positive control substances shall be used at 
a dose which demonstrates the test sensitivity.
    (5) Test chemicals--(i) Vehicle. When possible, test substances 
shall be dissolved or suspended in isotonic saline or distilled water. 
Water-insoluble chemicals may be dissolved or suspended in appropriate 
vehicles. The vehicle used shall neither interfere with the test 
chemical nor produce toxic effects. Fresh preparations of the test 
chemical should be employed.
    (ii) Dose levels. Normally, three dose levels shall be used. The 
highest dose shall produce signs of toxicity (e.g., slightly reduced 
fertility and slightly reduced body weight). However, in an initial 
assessment of dominant lethality a single high dose may be sufficient. 
Nontoxic substances shall be tested at 5g/kg or, if this is not 
practicable, then as the highest dose attainable.
    (iii) Route of administration. The usual routes of administration 
are oral or by IP injection. Other routes may be appropriate.
    (e) Test performance. (1) Individual males are mated sequentially at 
appropriate predetermined intervals to one or two virgin females. 
Females should be left with the males for at least the duration of one 
estrus cycle or alternatively until mating has occurred as determined by 
the presence of sperm in the vagina or by the presence of a vaginal 
plug.
    (2) The number of matings following treatment should be governed by 
the treatment schedule and should ensure that germ cell maturation is 
adequately covered.
    (3) Females should be sacrificed in the second half of pregnancy and 
uterine contents examined to determine the number of implants and live 
and dead embryos. The ovaries may be examined to determine the number of 
corpora lutea.
    (f) Data and report--(1) Treatment of results. Data shall be 
tabulated to show the number of males, the number of pregnant females, 
and the number of nonpregnant females. Results of each mating, including 
the identity of each male and female, shall be reported individually. 
For each female, the dose level and week of mating and the frequencies 
of live implants and of dead implants shall be enumerated. If the data 
are recorded as early and late deaths, the tables shall make that clear. 
If preimplantation loss is estimated, it shall be reported.

[[Page 204]]

Preimplantation loss can be calculated as the difference between the 
number of corpora lutea and the number of implants or as a reduction in 
the average number of implants per female in comparison with control 
matings.
    (2) Statistical evaluation. Data shall be evaluated by appropriate 
statistical methods. Differences among animals within the control and 
treatment groups shall be considered before making comparisons between 
treated and control groups.
    (3) Interpretation of results. (i) There are several criteria for 
determining a positive result, one of which is a statistically 
significant dose-related increase in the number of dominant lethals. 
Another criterion may be based upon detection of a reproducible and 
statistically significant positive response for at least one of the test 
points.
    (ii) A test substance which does not produce either a statistically 
significant dose-related increase in the number of dominant lethals or a 
statistically significant and reproducible positive response at any one 
of the test points is considered nonmutagenic in this system.
    (iii) Both biological and statistical significance should be 
considered together in the evaluation.
    (4) Test evaluation. (i) A positive DL assay suggests that under the 
test conditions the test substance may be genotoxic in the germ cells of 
the treated sex of the test species.
    (ii) A negative result suggests that under the conditions of the 
test the test substance may not be genotoxic in the germ cells of the 
treated sex of the test species.
    (5) Test report. In addition to the reporting recommendations as 
specified under 40 CFR part 792, subpart J the following specific 
information shall be reported:
    (i) Species, strain, age and weights of animals used, number of 
animals of each sex in experimental and control groups.
    (ii) Test substance, vehicle used, dose levels and rationale for 
dosage selection, negative (vehicle) and positive controls, experimental 
observations, including signs of toxicity.
    (iii) Route and duration of exposure.
    (iv) Mating schedule.
    (v) Methods used to determine that mating has occurred (where 
applicable).
    (vi) Criteria for scoring dominant lethals including the number of 
early and late embryonic deaths.
    (vii) Dose-response relationship, if applicable.
    (g) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) Brewen, J.G., Payne, H.S., Jones, K.P., Preston, R.J. ``Studies 
on chemically induced dominant lethality. I. The cytogenetic basis of 
MMS-induced dominant lethality in post-meiotic germ cells'' Mutation 
Research, 33:239-250 (1975).
    (2) Ehling, U.H., Machemer, L., Buselmaier, E., Dycka, D., Frohberg, 
H., Kratochvilova, J., Lang, R., Lorke, D., Muller, D., Pheh, J., 
Rohrborn, G., Roll, R., Schulze-Schencking, M., Wiemann, H. ``Standard 
protocol for the dominant lethal test on male mice. Set up by the Work 
Group ``Dominant lethal mutations of the ad hoc Committee 
Chemogenetics,'' Archives of Toxicology, 39:173-185 (1978).

[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19081, May 20, 1987]



Sec. 798.5460  Rodent heritable translocation assays.

    (a) Purpose. This test detects transmitted chromosomal damage which 
manifests as balanced reciprocal translocations in progeny descended 
from parental males treated with chemical mutagens.
    (b) Definitions. (1) A heritable translocation is one in which 
distal segments of nonhomologous chromosomes are involved in a 
reciprocal exchange.
    (2) Diakinesis and metaphase I are stages of meiotic prophase scored 
cytologically for the presence of multivalent chromosome association 
characteristic of translocation carriers.
    (c) Reference substances. Not applicable.
    (d) Test method--(1) Principle. When a balanced reciprocal 
translocation is induced in a parental male germ cell, the resulting 
progeny is translocation heterozygote.

[[Page 205]]

    (i) Basis for fertility screening. Male translocation heterozygotes 
may be completely sterile. This class consists of two types of 
translocations:
    (A) Translocations between non-homologous chromosomes in which at 
least one of the breaks occurs close to one end of a chromosome.
    (B) Those that carry multiple translocations. The majority of male 
translocation heterozygotes are semisterile--they carry one or (rarely) 
two translocations. The degree of semisterility is dependent upon the 
proportions of balanced and unbalanced (duplication-deficiency) gametes 
produced in the ejaculate as a function of meiotic segregation. Balanced 
and unbalanced sperm are equally capable of fertilizing an egg. Balanced 
sperm lead to viable progeny. Unbalanced sperm result in early embryonic 
lethality.
    (ii) Basis for cytological screening. The great majority of male 
translocation heterozygotes can be identified cytologically through 
analysis of diakinesis metaphase I spermatocytes. Translocation 
heterozygotes are characterized by the presence of multivalent 
chromosome association such as a ring or chain of four chromosomes held 
together by chiasmata in paired homologous regions. Some translocation 
carriers can be identified by the presence of extra long and/or extra 
short chromosomes in spermatogonial and somatic cell metaphase 
preparations.
    (2) Description. Essentially, two methods have been used to screen 
for translocation heterozygosity; one method uses a mating sequence to 
identify sterile and semisterile males followed by cytological 
examination of suspect male individuals; the other method deletes the 
mating sequence altogether and all F1 male progeny are 
examined cytologically for presence of translocation. In the former 
approach, the mating sequence serves as a screen which eliminates most 
fully fertile animals for cytological confirmation as translocation 
heterozygotes.
    (3) Animal selection--(i) Species. The mouse is the species 
generally used, and is recommended.
    (ii) Age. Healthy sexually mature animals shall be used.
    (iii) Number. (A) The number of male animals necessary is determined 
by the following factors:
    (1) The use of either historical or concurrent controls.
    (2) The power of the test.
    (3) The minimal rate of induction required.
    (4) Whether positive controls are used.
    (5) The level of significance desired.
    (B) [Reserved]
    (iv) Assignment to groups. Animals shall be randomized and assigned 
to treatment and control groups.
    (4) Control groups--(i) Concurrent controls. No concurrent positive 
or negative (vehicle) controls are recommended as routine parts of the 
heritable translocation assay. However, investigators not experienced in 
performing translocation testing shall include a substance known to 
produce translocations in the assay as a positive control reference 
chemical.
    (ii) Historical controls. At the present time, historical control 
data must be used in tests for significance. When statistically reliable 
historical controls are not available, negative (vehicle) controls shall 
be used.
    (5) Test chemicals--(i) Vehicle. When appropriate for the route of 
administration, solid and liquid test substances should be dissolved or 
suspended in distilled water or isotonic saline. Water-insoluble 
chemicals may be dissolved or suspended in appropriate vehicles. The 
vehicle used shall neither interfere with the test chemical nor produce 
toxic effects. Fresh preparations of the test chemical should be 
employed.
    (ii) Dose levels. At least two dose levels shall be used. The 
highest dose level shall result in toxic effects (which shall not 
produce an incidence of fatalities which would prevent a meaningful 
evaluation) or shall be the highest dose attainable or 5g/kg body 
weight.
    (iii) Route of administration. Acceptable routes of administration 
include oral, inhalation, admixture with food or water, and IP or IV 
injection.
    (e) Test performance--(1) Treatment and mating. The animals shall be 
dosed with the test substances 7 days per week over a period of 35 days. 
After treatment, each male shall be caged

[[Page 206]]

with 2 untreated females for a period of 1 week. At the end of 1 week, 
females shall be separated from males and caged individually. When 
females give birth, the day of birth, litter size, and sex of progeny 
shall be recorded. All male progeny should be weaned, and all female 
progeny should be discarded.
    (2) Testing for translocation heterozygosity. When males are 
sexually mature, testing for translocation heterozygosity shall begin. 
One of two methods shall be used; the first method involves mating, 
determining those F1 progeny which are sterile or semisterile 
and subsequent cytological analysis of suspect progeny; the other method 
does not involve mating and determining sterility or semisterility; all 
progeny are examined cytologically.
    (i) Determination of sterility or semisterility--(A) Conventional 
method. Females are mated, usually three females for each male, and each 
female is killed at midpregnancy. Living and dead implantations are 
counted. Criteria for determining normal and semisterile males are 
usually established for each new strain because the number of dead 
implantations varies considerably among strains.
    (B) Sequential method. Males to be tested are caged individually 
with females and the majority of the presumably normal males are 
identified on the basis of a predetermined size of 1 or 2 litters. 
Breeding pens are examined daily on weekdays beginning 18 days after 
pairing. Young are discarded immediately after they are scored. Males 
that sire a litter whose size is the same as or greater than the minimum 
set for a translocation-free condition are discarded with their litter. 
If the litter size is smaller than the predetermined number, a second 
litter is produced with the same rule applying. Males that cannot be 
classified as normal after production of a second litter are tested 
further by the conventional method or by cytological confirmation of 
translocation.
    (ii) Cytological analysis. For cytological analysis of suspected 
semisteriles, the air-drying technique is used. Observation of at least 
2 diakinesis-metaphase 1 cells with mutivalent association constitutes 
the required evidence for the presence of a translocation. Sterile males 
are examined by one of two methods, those with testes of normal size and 
sperm in the epididymis are examined by the same techniques used for 
semisteriles. Animals with small testes are examined by squash 
preparations or, alternatively, by examination of mitotic metaphase 
preparations. If squash preparations do not yield diakinesis-metaphase 1 
cells, analysis of spermatogonia or bone marrow for the presence of 
unusually long or short chromosomes should be performed.
    (f) Data and report--(1) Treatment of results. (i) Data shall be 
presented in tabular form and shall include the number of animals at 
risk, the germ cell stage treated, the number of partial steriles and 
semisteriles (if the fertility test is used), the number of 
cytogenetically confirmed translocation heterozygotes (if the fertility 
test is used, report the number of confirmed steriles and confirmed 
partial steriles), the translocation rate, and either the standard error 
of the rate or the upper 95 percent confidence limit on the rate.
    (ii) These data shall be presented for both treated and control 
groups. Historical or concurrent controls shall be specified, as well as 
the randomization procedure used for concurrent controls.
    (2) Statistical evaluation. Data shall be evaluated by appropriate 
statistical methods.
    (3) Interpretation of results. (i) There are several criteria for 
determining a positive result, one of which is a statistically 
significant dose-related increase in the number of heritable 
translocations. Another criterion may be based upon detection of a 
reproducible and statistically significant positive response for at 
least one of the test points.
    (ii) A test substance which does not produce either a statistically 
significant dose-related increase in the number of heritable 
translocations or a statistically significant and reproducible positive 
response at any one of the test points is considered nonmutagenic in 
this system.
    (iii) Both biological and statistical significance should be 
considered together in the evaluation.

[[Page 207]]

    (4) Test evaluation. (i) Positive results in the heritable 
translocation assay indicate that under the test conditions the test 
substance causes heritable chromosomal damage in the test species.
    (ii) Negative results indicate that under the test conditions the 
test substance does not cause heritable chromosomal damage in the test 
species.
    (5) Test report. In addition to the reporting recommendations as 
specified under 40 CFR part 792, subpart J, the following specific 
information shall be reported:
    (i) Species, strain, age, weight and number of animals of each sex 
in each group.
    (ii) Test chemical vehicle, route and schedule of administration, 
toxicity data.
    (iii) Dosing regimen, doses tested and rationale for dosage 
selection.
    (iv) Mating schedule, number of females mated to each male.
    (v) The use of historical or concurrent controls.
    (vi) Screening procedure including the decision criteria used and 
the method by which they were determined.
    (vii) Dose-response relationship, if applicable.
    (g) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) Generoso, W.M., Bishop, J.B., Goslee, D.G., Newell, G.W., Sheu, 
G-J, von Halle, E. ``Heritable translocation test in mice,'' Mutation 
Research, 76:191-215 (1980).
    (2) [Reserved]

[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19081, May 20, 1987]



Sec. 798.5500  Differential growth inhibition of repair proficient and repair 

deficient bacteria: ``Bacterial DNA damage or repair tests.''

    (a) Purpose. Bacterial DNA damage or repair tests measure DNA damage 
which is expressed as differential cell killing or growth inhibition of 
repair deficient bacteria in a set of repair proficient and deficient 
strains. These tests do not measure mutagenic events per se. They are 
used as an indication of the interaction of a chemical with genetic 
material implying the potential for genotoxicity.
    (b) Definition. Test for differential growth inhibition of repair 
proficient and repair deficient bacteria measure differences in 
chemically induced cell killing between wild-type strains with full 
repair capacity and mutant strains deficient in one or more of the 
enzymes which govern repair of damaged DNA.
    (c) Reference substances. These may include, but need not be limited 
to, chloramphenicol or methyl methanesulfonate.
    (d) Test method--(1) Principle. The tests detect agents that 
interact with cellular DNA to produce growth inhibition or killing. This 
interaction is recognized by specific cellular repair systems. The 
assays are based upon the use of paired bacterial strains that differ by 
the presence of absence of specific DNA repair genes. The response is 
expressed in the preferential inhibition of growth or the preferential 
killing of the DNA repair deficient strain since it is incapable of 
removing certain chemical lesions from its DNA.
    (2) Description. Several methods for performing the test have been 
described. Those described here are:
    (i) Tests performed on solid medium (diffusion tests).
    (ii) Tests performed in liquid culture (suspension tests).
    (3) Strain selection--(i) Designation. At the present time, 
Escherichia coli polA (W3110/p3478) or Bacillus subtilis rec (H17/M45) 
pairs are recommended. Other pairs may be utilized when appropriate.
    (ii) Preparation and storage. Stock culture preparation and storage, 
growth requirements, method of strain identification and demonstration 
of appropriate phenotypic requirements should be performed using good 
microbiological techniques and should be documented.
    (4) Bacterial growth. Good microbiological techniques should be used 
to grow fresh cultures of bacteria. The phase of growth and cell density 
should be documented and should be adequate for the experimental design.
    (5) Metabolic activation. Bacteria should be exposed to the test 
substance both in the presence and absence of an appropriate metabolic 
activation system. The most commonly used system

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is a cofactor supplemented postmitochondrial fraction prepared from the 
livers of rodents treated with enzyme inducing agents. The use of other 
species, tissues or techniques may also be appropriate.
    (6) Control groups--(i) Concurrent controls. Concurrent positive, 
negative, and vehicle controls should be included in each assay.
    (ii) Negative controls. The negative control should show 
nonpreferential growth inhibition (i.e., should affect both strains 
equally). Chloramphenicol is an example of a negative control.
    (iii) Genotype specific controls. Examples of genotype specific 
positive controls are methyl methanesulfonate for polA strains and 
mitomycin C for rec strains.
    (iv) Positive controls to ensure the efficacy of the activation 
system. The positive control reference substance for tests including a 
metabolic activation system should be selected on the basis of the type 
of activation system used in the test.
    (v) Other positive controls. Other positive control reference 
substances may be used.
    (7) Test chemicals--(i) Vehicle. Test chemicals and positive and 
negative control reference substances should be dissolved in an 
appropriate vehicle and then further diluted in vehicle for use in the 
assay.
    (ii) Exposure concentrations. The test should initially be performed 
over a broad range of concentrations. Among the criteria to be taken 
into consideration for determining the upper limits of test chemical 
concentration are cytotoxicity and solubility. Cytotoxicity of the test 
chemical may be altered in the presence of metabolic activation systems. 
For freely soluble nontoxic chemicals, the upper test chemical 
concentration should be determined on a case by case basis. Because 
results are expressed as diameters of zones of growth inhibition in the 
diffusion test, it is most important that the amounts of chemical on the 
disc (or in the wells) are exact replicates. When appropriate, a 
positive response should be confirmed by testing over a narrow range of 
concentrations.
    (e) Test performance--(1) Diffusion assay--(i) Disc diffusion 
assays. Disc diffusion assays. may be performed in two ways:
    (A) A single strain of bacteria may be added to an agar overlay or 
spread on the surface of the agar and the test chemical placed on a 
filter disc on the surface of the agar or;
    (B) DNA repair proficient and DNA repair deficient bacteria may be 
streaked in a line on the surface of the agar of the same plate and a 
disc saturated with test chemical placed on the surface of the agar in 
contact with the streaks.
    (ii) Well diffusion assays. In well diffusion assays, bacteria may 
be either added to the agar overlay or spread onto the surface of the 
agar. A solution of the test chemical is then placed into a well in the 
agar.
    (2) Suspension assays. (i) A bacterial suspension may be exposed to 
the test chemical and the number of surviving bacteria determined (as 
colony-forming units) either as a function of time of treatment or as a 
function of the concentration of test agent.
    (ii) Nonturbid suspensions of bacteria may be exposed to serial 
dilutions of the test agent and a minimal inhibitory concentration for 
each strain determined, as evidenced by the presence or absence of 
visible growth after a period of incubation.
    (iii) Paired bacterial suspensions (usually with some initial 
turbidity) may be treated with a single dose of the chemical. Positive 
results are indicated by a differential inhibition in the rate of 
increase of turbidity of the paired cultures.
    (3) Number of cultures. When using a plate diffusion procedure, at 
least two independent plates should be used at each dilution. In liquid 
suspension assays, at least two independent specimens for determination 
of the number of viable cells should be plated.
    (4) Incubation conditions. All plates in a given test should be 
incubated for the same time period. This incubation period should be for 
18 to 24 hrs at 37 [deg] C.
    (f) Data and report--(1) Treatment of results--(i) Diffusion assays. 
Results should be expressed in diameters of zones of growth inhibition 
in millimeters or as areas derived therefrom as

[[Page 209]]

mm\2\. Dose-response data, if available, should be presented using the 
same units.
    (ii) Liquid suspension assays. (A) Survival data can be presented as 
dose responses, preferably as percentage of survivors or fractional 
survival of each strain or as a relative survival (ratio) of the two 
strains.
    (B) Results can also be expressed as the concentrations required to 
effect a predetermined survival rate (e.g., D37, the dose 
permitting 37 percent survival). These data are derived from the 
survival curve. The concentration should be expressed as weight per 
volume, as moles, or as molarity.
    (C) Similarly, results can be expressed as minimal inhibitory 
concentration or as minimal lethal dose. The former is determined by the 
absence of visible growth in liquid medium and the latter is determined 
by plating dilutions onto semisolid media.
    (iii) In all tests, concentrations must be given as the final 
concentrations during the treatment. Raw data, prior to transformation, 
should be provided. These should include actual quantities measured, 
e.g., neat numbers. For measurement of diffusion, the diameters of the 
discs and/or well should be indicated and the measurements should 
indicate whether the diameter of the discs and/or well was subtracted. 
Moreover, mention should be made as to whether the test chemical gave a 
sharp, diffuse, or double-zone of growth inhibition. If it is the 
latter, the investigator should indicate whether the inner or the outer 
zone was measured.
    (iv) Viability data should be given as the actual plate counts with 
an indication of the dilution used and the volume plated or as derived 
titers (cells per ml). Transformed data alone in the absence of 
experimental data are not acceptable (i.e, ratios, differences, survival 
fraction).
    (2) Statistical evaluation. Data should be evaluated by appropriate 
statistical methods.
    (3) Interpretation of results. (i) There are several criteria for 
determining a positive result, one of which is a statistically 
significant dose-related preferential inhibition or killing of the 
repair deficient strain. Another criterion may be based upon detection 
of a reproducible and statistically significant positive response for at 
least one of the test points.
    (ii) A test substance which does not produce either a statistically 
significant dose-related preferential inhibition or killing of the 
repair deficient strain or a statistically significant and reproducible 
positive response at any one of the test points is considered not to 
interact with the genetic material of the organisms used in assay.
    (iii) Both biological and statistical significance should be 
considered together in the evaluation.
    (4) Test evaluation. DNA damage tests in bacteria do not measure DNA 
repair per se nor do they measure mutations. They measure DNA damage 
which is expressed as cell killing or growth inhibition. A positive 
result in a DNA damage test in the absence of a positive result in 
another system is difficult to evaluate in the absence of a better data 
base.
    (5) Test report. In addition to the reporting recommendations as 
specified under 40 CFR part 792, subpart J the following specific 
information should be reported:
    (i) Bacterial strains used.
    (ii) Phase of bacterial cell growth at time of use in the assay.
    (iii) Media composition.
    (iv) Details of both the protocol used to prepare the metabolic 
activation system and its use in the assay.
    (v) Treatment protocol, including doses used and rationale for dose 
selection, positive and negative controls.
    (vi) Method used for determination of degree of cell kill.
    (vii) Dose-response relationship, if applicable.
    (g) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) Ames, B.N., McCann, J., Yamasaki, E. ``Methods for detecting 
carcinogens and mutagens with the Salmonella/mammalian-microsome 
mutagenicity test,'' Mutation Research, 31:347-364 (1975).
    (2) Kada, T., Sadie, Y., Tutikawa, K. ``In vitro and host-mediated 
``rec-

[[Page 210]]

assay'' procedures for screening chemical mutagens; and phloxine, a 
mutagenic red dye detected,'' Mutation Research, 16:165-174 (1972).
    (3) Leifer, Z., Kada, T., Mandel, M., Zeiger, E., Stafford, R., 
Rosenkranz, H.S. ``An evaluation of bacterial DNA repair tests for 
predicting genotoxicity and carcinogenicity: A report of the U.S. EPA's 
Gene-Tox Program,'' Mutation Research, 87:211-297 (1981).
    (4) Slater, E.E., Anderson, M.D., Rosenkranz, H.S. ``Rapid detection 
of mutagens and carcinogens.'' Cancer Research, 31:970-973 (1971).



Sec. 798.5955  Heritable translocation test in drosophila melanogaster.

    (a) Purpose. The heritable translocation test in Drosophila measures 
the induction of chromosomal translocations in germ cells of insects. 
Stocks carrying genetic markers on two or more chromosomes are used to 
follow the assortment of chromosomes in meiosis. The F1 male 
progeny of treated parents are individually mated to females and the 
F2 progeny phenotypes are scored. The observed spectrum of 
phenotypes is used to determine the presence or absence of a 
translocation. This is usually indicated by a lack of independent 
assortment of genes on different chromosomes.
    (b) Definitions--(1) Chromosome mutations are chromosomal changes 
resulting from breakage and reunion of chromosomes. Chromosomal 
mutations are also produced through nondisjunction of chromosomes during 
cell division.
    (2) Reciprocal translocations are chromosomal translocations 
resulting from reciprocal exchanges between two or more chromosomes.
    (3) Heritable translocations are reciprocal translocations 
transmitted from parent to the succeeding progeny.
    (c) Reference substances. These may include, but need not be limited 
to, ethyl methanesulfonate or N-dimethyl-nitrosamine.
    (d) Test method--(1) Principle. The method is based on the principle 
that balanced reciprocal chromosomal translocations can be induced by 
chemicals in the germ cells of treated flies and that these 
translocations are detected in the F2 progeny using genetic 
markers (mutations). Different mutations may be used as genetic markers 
and two or more of the four chromosomes may be genetically marked for 
inclusion in this test.
    (2) Description. Wild-type males are treated with chemical and bred 
with females of known genetic markers. The F1 males are 
collected and individually bred with virgin females of the female 
parental stock. The resulting F2 progeny are scored. Putative 
translocation carriers are confirmed with an F3 cross.
    (i) Illustrative example. The following example serves to illustrate 
the method. Males carrying genes for red eye color on chromosomes II and 
III are bred with females of white eye color carrying alleles for brown 
(bw) on the second chromosome and scarlet (st) and pink (pp) on the 
third chromosome. The F1 male progeny are bred with virgin 
females of the female parental stock and the resulting F2 
progeny are examined for eye color phenotypes. If there is no 
translocation in the F1 male, then the resulting 
F2 progeny will have four eye color phenotypes: red, white, 
orange, and brown. If the F1 male carries a translocation 
between chromosomes II and III, only red and white eye phenotypes are 
obtained in the F2 generation. This happens because the 
F1 translocation heterozygote produces two balanced (carrying 
either the parental or the translocated configuration of markers) and 
two unbalanced gametes. The unbalanced gametes (carrying one normal and 
one translocated chromosome) are unable to develop into normal 
individuals in the F2 generation.
    (ii) [Reserved]
    (3) Drosophila stocks. Wild-type males and females of the genotype 
bw:st:pp (white eyes) may be used in the heritable translocation test. 
Other appropriately marked Drosophila stocks may also be used.
    (4) Control groups. (i) Concurrent positive and negative (vehicle) 
controls should be included in each experiment.
    (ii) Negative (vehicle) controls should be included. The size of the 
negative (vehicle) control group should be determined by the 
availability of appropriate laboratory historical control data.

[[Page 211]]

    (iii) If the historical control data are of sufficient numbers, 
concurrent controls may not be necessary.
    (5) Test chemicals--(i) Vehicle. Test chemicals should be dissolved 
in water. Compounds which are insoluble in water may be dissolved or 
suspended in appropriate vehicles (e.g., a mixture of ethanol and Tween-
60 or 80), and then diluted in water or saline prior to administration. 
Dimethylsulfoxide should be avoided as a vehicle.
    (ii) Dose levels. For the initial assessment of mutagenicity, it may 
be sufficient to test a single dose of the test substance. This dose 
should be the maximum tolerated dose or that which produces some 
indication of toxicity. If the test is being used to verify mutagenic 
activity, at least two additional exposure levels should be used.
    (iii) Route of administration. Exposure may be oral, by injection or 
by exposure to gases or vapours. Feeding of the test compound may be 
done in sugar solution. When necessary, substances may be dissolved in 
0.7 percent NaCl solution and injected into the thorax or abdomen.
    (e) Test performance--(1) P1 mating. (i) In the primary screen of a 
chemical, it is enough to sample one germ cell stage, either mature 
sperm or spermatids (for indirect acting mutagens). Other stages may be 
sampled if needed, i.e., when mature germ cells give a positive result 
and data from earlier germ cells are needed for the purpose of risk 
assessment. Thus, the treated males may be mated only once for a period 
of 3 days to sample sperm or transferred every 2 to 3 days to cover the 
entire germ cell cycle.
    (ii) Mass matings may be performed because the control rate for 
translocations in the available literature is very low (near 0) and 
clustered events are extremely rare. Mated females may be aged for 2 
weeks in order to recover an enhanced incidence of translocation due to 
the storage effect. The females are then allowed to lay eggs and 
F1 males are collected for test mating.
    (2) F1 mating. F1 males should be bred with 
virgin females of the parental female stock. Since each F1 
male represents one treated gamete of the male parent, the F1 
males have to be mated individually to virgin females. Each 
F1 male should be mated to three females to ensure sufficient 
progeny.
    (3) Scoring the F2 generation. F2 cultures 
(each representing 1 F1 male tested) should be scored for the 
presence or absence of phenotype variations (linkage of markers) from 
the expected types. The test should be designed with a predetermined 
sensitivity and power. The number of flies in each group should reflect 
these defined parameters. The spontaneous mutant frequency observed in 
the appropriate control group will strongly influence the number of 
treated chromosomes that must be analyzed to detect substances which 
show mutation rates close to those of the controls. A positive test 
should be confirmed by F3 mating trials.
    (4) Number of replicate experiments. Replicate experiments are 
usually performed for each dose of the compound tested. If a chemical is 
a potent inducer of translocations, one experiment may be sufficient. 
Otherwise two or three replicate experiments should be done.
    (f) Data and report--(1) Treatment of results. Data should be 
tabulated to show the number of translocations and the number of fertile 
F1 males at each exposure for each germ cell stage sampled.
    (2) Statistical evaluation. Data should be evaluated by appropriate 
statistical methods.
    (3) Interpretation of results. (i) There are several criteria for 
determining a positive result, one of which is a statistically 
significant dose-related increase in the number of heritable 
translocations. Another criterion may be based upon detection of a 
reproducible and statistically significant positive response for at 
least one of the test points.
    (ii) A test substance which does not produce either a statistically 
significant dose-related increase in the number of heritable 
translocations or a statistically significant and reproducible positive 
response at any one of the test points is considered nonmutagenic in 
this system.
    (iii) Both biological and statistical significance should be 
considered together in the evaluation.

[[Page 212]]

    (4) Test evaluation. (i) Positive results in the heritable 
translocation test in Drosophila indicate that under the test conditions 
the test substance causes chromosome damage in germ cells of this 
insect.
    (ii) Negative results indicate that under the test conditions the 
test substance does not cause chromosomal damage in D. melanogaster.
    (5) Test report. In addition to the reporting recommendations as 
specified under 40 CFR part 792, subpart J, the following specific 
information should be reported:
    (i) Drosophila stock used in the assay, age of insects, number of 
males treated, number of F2 cultures established, number of 
replicate experiments.
    (ii) Test chemical vehicle, treatment and mating schedule, exposure 
levels, toxicity data, dose and route of exposure.
    (iii) Positive and negative (vehicle) controls.
    (iv) Historical control data, if available.
    (v) Number of chromosomes scored.
    (vi) Criteria for scoring mutant chromosomes.
    (vii) Dose-response relationship, if applicable.
    (g) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) Wurgler, F.E., Sobels, F.H., Vogel, E. ``Drosophila as assay 
system for detecting genetic changes,'' Handbook of mutagenicity test 
procedures. Eds. Kilby, B.J., Legator, M., Nichols, W., Ramel, C. 
(Amsterdam: Elsevier/North Holland Biomedical Press, 1979) pp. 335-374.
    (2) [Reserved]



                         Subpart G_Neurotoxicity



Sec. 798.6050  Functional observational battery.

    (a) Purpose. In the assessment and evaluation of the potential human 
health effects of substances, it may be necessary to test for neurotoxic 
effects. Substances that have been observed to cause neurotoxic signs 
(e.g., convulsions, tremors, ataxia) in other toxicity tests, as well as 
those having a structural similarity to known neurotoxicants, should be 
evaluated for neurotoxicity. The functional observational battery is a 
noninvasive procedure designed to detect gross functional deficits in 
young adults resulting from exposure to chemicals and to better quantify 
neurotoxic effects detected in other studies. This battery of tests is 
not intended to provide a detailed evaluation of neurotoxicity. It is 
designed to be used in conjunction with neuropathologic evaluation and/
or general toxicity testing. Additional functional tests may be 
necessary to assess completely the neurotoxic potential of a chemical.
    (b) Definitions. (1) Neurotoxicity is any adverse effect on the 
structure or function of the central and/or peripheral nervous system 
related to exposure to a chemical substance.
    (2) A toxic effect is an adverse change in the structure or function 
of an experimental animal as a result of exposure to a chemical 
substance.
    (c) Principle of the test method. The material is administered by an 
appropriate route to laboratory rodents. The animals are observed under 
carefully standardized conditions with sufficient frequency to ensure 
the detection of behavioral and/or neurologic abnormalities, if present. 
Various functions that could be affected by neurotoxicants are assessed 
during each observation period.
    (d) Test procedures--(1) Animal selection--(i) Species and strain. 
The laboratory rat or mouse is recommended. Although information will 
generally be lacking, whenever possible the choice of species should 
take into consideration such factors as the comparative metabolism of 
the chemical and species sensitivity to the toxic effects of the test 
substance, as evidenced by the results of other studies. The potential 
for combined studies should also be considered. Standard strains should 
be used.
    (ii) Age. Young adult animals (at least 42 days old for the rat or 
mouse) shall be used.
    (iii) Sex. (A) Equal numbers of animals of each sex are required for 
each dose level.
    (B) The females shall be nulliparous and nonpregnant.
    (2) Number of animals. At least eight animals of each sex should be 
used at

[[Page 213]]

each dose level and should be designated for behavioral testing. If 
interim sacrifices are planned, the number should be increased by the 
number of animals scheduled to be sacrificed before the end of the 
study. Animals shall be randomly assigned to treatment and control 
groups.
    (3) Control groups. (i) A concurrent (``sham'' exposure or vehicle) 
control group is required. Subjects shall be treated in the same way as 
for an exposure group except that administration of the test substance 
is omitted.
    (ii) Concurrent or historic data from the laboratory performing the 
testing shall provide evidence of the ability of the procedures used to 
detect major neurotoxic endpoints such as limb weakness or paralysis 
(e.g., acrylamide), CNS stimulation (e.g., [beta], [beta]'-
iminodiproprionitrile) autonomatic signs (e.g., physostigmine).
    (iii) A satellite group may be treated with the high dose level for 
the duration of exposure and observed for reversibility, persistence, or 
delayed occurrence of toxic effects for a post-treatment period of 
appropriate duration, normally not less than 28 days.
    (4) Dose levels and dose selection. At least 3 doses, equally spaced 
on a log scale (e.g., \1/2\ log units) over a range of at least 1 log 
unit shall be used in addition to a zero dose or vehicle administration. 
The data should be sufficient to produce a dose-effect curve.
    (i) The highest dose shall produce (A) clear behavioral effects or 
(B) life-threatening toxicity.
    (ii) The data from the lower doses must show either (A) graded dose-
dependent effects at 2 dose levels or (B) no effects at 2 dose levels, 
respectively.
    (5) Duration and frequency of exposure. The duration and frequency 
of exposure will be specified in the test rule.
    (6) Route of exposure. The test substance shall be administered by 
the route specified in the test rule. This route will usually be the one 
most closely approximating the expected route of human exposure. The 
exposure potocol shall conform to that outlined in the appropriate acute 
or subchronic toxicity study guideline under subpart B or subpart C of 
this part.
    (7) Combined protocol. Subjects used for other toxicity studies may 
be used if none of the requirements of either study are violated by the 
combination.
    (8) Study conduct. (i) All animals in a given study should be 
observed carefully by trained technicians who are blind with respect to 
the animals' treatments. Standard procedures to minimize observer 
variability shall be followed. Where possible, it is advisable that the 
same observer be used to evaluate the animals in a given study. If this 
is not possible, some demonstration of inter-observer reliability is 
required. All animals should be observed prior to initiation of 
exposure. Subsequent observations should be made with sufficent 
frequency to ensure the detection of behavioral and/or neurologic 
abnormalities, if present. At minimum, observations at 1 hour, 6 hours, 
24 hours, 7 days, and 14 days and monthly thereafter are recommended. In 
a subchronic study, subsequent to the first exposure all observations 
should be made before the daily exposure. The animals should be removed 
from the home cage to a standard arena for observation. Effort should be 
made to ensure that variations in the test conditions are minimal and 
are not systematically related to treatment. Among the variables that 
can affect behavior are sound level, temperature, humidity, lighting, 
odors, time of day, and environmental distractions. Explicit, 
operationally defined scales for each function should be used. The 
development of objective quantitative measures of the observational 
endpoints specified is encouraged.
    (ii) The following is a minimal list of observations that shall be 
noted:
    (A) Any unusual responses with respect to body position, activity 
level, coordination of movement, and gait.
    (B) Any unusual or bizarre behavior including, but not limited to, 
headflicking, head searching, compulsive biting or licking, self-
mutilation, circling, and walking backwards.
    (C) The presence of:
    (1) Convulsions.
    (2) Tremors.
    (3) Increased levels of lacrimation and/or red-colored tears.
    (4) Increased levels of salivation.
    (5) Piloerection.
    (6) Pupillary dilation or constriction.

[[Page 214]]

    (7) Unusual respiration (shallow, labored, dyspneic, gasping, and 
retching) and/or mouth breathing.
    (8) Diarrhea.
    (9) Excessive or diminished urination.
    (10) Vocalization.
    (D) Forelimb/hindlimb grip strength. The procedure described by 
Meyer et al. (1979), under paragraph (f)(9) of this section is 
recommended.
    (E) Sensory function. A simple assessment of sensory function 
(vision, audition, pain perception) shall be made. Marshall et al. 
(1971) under paragraph (f)(8) of this section have described a 
neurologic exam for this purpose; these procedures are also discussed by 
Deuel (1977), under paragraph (f)(4) of this section. Irwin (1968) under 
paragraph (f)(7) of this section described a number of reflex tests 
intended to detect gross sensory deficits, including the visual placing 
response, Preyer reflex, and tail pinch. Many procedures have been 
developed for assessing pain perception (e.g., Ankier, 1974 under 
paragraph (f)(1) of this section; D'Amour and Smith 1941 under paragraph 
(f)(3) of this section; Evans 1971 under paragraph (f)(6) of this 
section).
    (e) Data reporting and evaluation. In addition to the reporting 
requirements specified under 40 CFR part 792 subpart J the final test 
report must include the following information.
    (1) Description of system and test methods. (i) A detailed 
description of the procedures used to standardize observation, including 
the arena and operational definitions for scoring observations.
    (ii) Positive control data from the laboratory performing the test 
that demonstrate the sensitivity of the procedures being used. Historic 
data may be used if all aspects of the experimental protocol are the 
same, including personnel.
    (2) Results. The following information must be arranged by test 
group dose level.
    (i) In tabular form, data for each animal must be provided showing:
    (A) Its identification number.
    (B) Its body weight and score on each sign at each observation time, 
the time and cause of death (if appropriate).
    (ii) Summary data for each group must include:
    (A) The number of animals at the start of the test.
    (B) The number of animals showing each observation score at each 
observation time.
    (C) The percentage of animals showing each abnormal sign at each 
observation time.
    (D) The mean and standard deviation for each continuous endpoint at 
each observation time.
    (3) Evaluation of data. The findings of a functional observational 
battery should be evaluated in the context of preceding and/or 
concurrent toxicity studies and any correlative histopathological 
findings. The evaluation shall include the relationship between the 
doses of the test substance and the presence or absence, incidence and 
severity, of any neurotoxic effects. The evaluation should include 
appropriate statistical analyses. Choice of analyses should consider 
tests appropriate to the experimental design and needed adjustments for 
multiple comparisons.
    (f) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) Ankier, S.I. ``New hot plate tests to quantify antinociceptic 
and narcotic antagonist activities,'' European Journal of Pharmacology, 
27: 1-4 (1974).
    (2) Coughenour, L.L., McLean, J.R. and Parker, R.B. ``A new device 
for the rapid measurement of impaired motor function in mice,'' 
Pharmacology, Biochemistry and Behavior, 6: 351-353 (1977).
    (3) D'Amour, F.E., Smith, D.L. ``A method for determining loss of 
pain sensation,'' Journal of Pharmacology and Experimental Therapeutics, 
72: 74-79 (1941).
    (4) Deuel, R.K. ``Determining sensory deficits in animals,'' Methods 
in Psychobiology Ed. Myers R.D. (New York: Academic Press, 1977) pp. 99-
125.
    (5) Edwards, P.M., Parker, V.H. ``A simple, sensitive and objective 
method for early assessment of acrylamide neuropathy in rats,'' 
Toxicology and Applied Pharmacology, 40: 589-591 (1977).
    (6) Evans, W.O. ``A new technique for the investigation of some 
analgesic

[[Page 215]]

drugs on reflexive behavior in the rat,'' Psychopharmacologia, 2: 318-
325 (1961).
    (7) Irwin, S. ``Comprehensive observational assessment: Ia. A 
systematic quantitative procedure for assessing the behavioral and 
physiologic state of the mouse,'' Psychopharmacologia, 13: 222-257 
(1968).
    (8) Marshall, J.F., Turner, B.H., Teitlbaum, P. ``Sensory neglect 
produced by lateral hypothalamic damage,'' Science, 174: 523-525 (1971).
    (9) Meyer, O.A., Tilson, H.A., Byrd, W.C., Riley, M.T. ``A method 
for the routine assessment of fore- and hindlimb grip strength of rats 
and mice,'' Neurobehavioral Toxicology, 1: 233-236 (1979).

[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19082, May 20, 1987]



Sec. 798.6200  Motor activity.

    (a) Purpose--(1) General. In the assessment and evaluation of the 
toxic characteristics of a substance, determination of the effects of 
administration of the substance on motor activity is useful when 
neurotoxicity is suspected.
    (2) Acute Motor Activity Test. The purpose of the acute motor 
activity test is to examine changes in motor activity occurring over a 
range of acute exposure levels. These changes may then be evaluated in 
the context of changes occurring in other organ systems. This test is an 
initial step in determining the potential of a substance to produce 
acute neurotoxicity and may be used to screen members of a class of 
substances for known neurotoxicity, and/or to establish a dosage regimen 
prior to the initiation of subchronic neurotoxicity testing.
    (3) Subchronic Motor Activity Test. The purpose of the subchronic 
motor activity test is to determine whether the repeated administration 
of a suspected neurotoxicant results in changes in motor activity. These 
changes may be evaluated in the context of changes occurring in other 
organ systems. This test is an initial step in determining the potential 
of a substance to produce subchronic neurotoxicity.
    (b) Definitions. (1) Neurotoxicity is the adverse effect on the 
structure or function of the central and/or peripheral nervous system 
related to exposure to a chemical substance.
    (2) Motor activity is any movement of the experimental animal.
    (3) A toxic effect is an adverse change in the structure or function 
of an experimental animal as a result of exposure to a chemical 
substance.
    (c) Principle of the test method. The test substance is administered 
to several groups of experimental animals, one dose being used per 
group. Measurements of motor activity are made. The exposure levels at 
which significant changes in motor activity are produced are compared to 
those levels which produce toxic effects not originating in the central 
and/or peripheral nervous system.
    (d) Test procedures--(1) Animal selection--(i) Species and strain. 
Testing shall be performed in a laboratory rat or mouse. The choice of 
species should take into consideration such factors as the comparative 
metabolism of the chemical and species sensitivity to the toxic effects 
of the test substance, as evidenced by the results of other studies, the 
potential for combined studies, and the availability of other toxicity 
data for the species.
    (ii) Age. Young adult animals (at least 42 days old for rat or 
mouse) should be used.
    (iii) Sex. (A) Equal numbers of animals of each sex are required for 
each dose level for the motor activity test.
    (B) The females shall be nulliparous and nonpregnant.
    (2) Number of animals. Animals shall be randomly assigned to test 
and control groups. Each test or control group must be designed to 
contain a sufficient number of animals at the completion of the study to 
detect a 40 percent change in activity of the test groups relative to 
the control group with 90 percent power at the 5 percent level. For most 
designs, calculations can be made according to Dixon and Massey (1957) 
under paragraph (f)(1) of this section, Neter and Wasserman (1974) under 
paragraph (f)(5) of this section, Sokal and Rohlf (1969) under paragraph 
(f)(9) of this section, or Jensen (1972) under paragraph (f)(3) of this 
section.
    (3) Control groups. (i) A concurrent control group is required. This 
group must be an untreated group, or, if a vehicle is used in 
administering the test substance, a vehicle control group. If

[[Page 216]]

the toxic properties of the vehicle are not known or cannot be made 
available, both untreated and vehicle control group are required.
    (ii) Positive control data are required to demonstrate the 
sensitivity and reliability of the activity measuring device and testing 
procedure. These data should demonstrate the ability to detect increases 
or decreases in activity and to generate a dose-effect curve or its 
equivalent using three values of the dose or equivalent independent 
variable. A single administration of the dose (or equivalent) is 
sufficient. It is recommended that chemical exposure be used to collect 
positive control data. Positive control data shall be collected at the 
time of the test study unless the laboratory can demonstrate the 
adequacy of historical data for this purpose.
    (iii) A satellite group may be treated with the high dose level for 
90 days and observed for reversibility, persistence or delayed 
occurrence of toxic effects for a post-treatment period of appropriate 
length, normally not less than 28 days.
    (4) Dose levels and dose selection. At least 3 doses, equally spaced 
on a log scale (e.g., \1/2\ log units) over a range of at least 1 log 
unit shall be used in addition to a zero dose or vehicle administration. 
The data should be sufficient to produce a dose-effect curve.
    (i) The highest dose shall produce (A) clear effects on motor 
activity or (B) life-threatening toxicity.
    (ii) The data from the lower doses must show either (A) graded dose-
dependent effects at 2 dose levels or (B) no effects at 2 dose levels, 
respectively.
    (5) Duration of testing. The duration of exposure will be specified 
in the test rule.
    (6) Route of administration. The test substance shall be 
administered by the method specified in the test rule. This will usually 
be the route most closely approximating the route of human exposure. The 
exposure protocol shall conform to that outlined in the appropriate 
acute or subchronic toxicity study guideline.
    (7) Combined protocol. The tests described herein may be combined 
with any other toxicity study, as long as none of the requirements of 
either are violated by the combination.
    (8) Study conduct--(i) General. Motor activity must be monitored by 
an automated activity recording apparatus. The device used must be 
capable of detecting both increases and decreases in activity, i.e. 
baseline activity as measured by the device must not be so low as to 
preclude decreases nor so high as to preclude increases. Each device 
shall be tested by standard procedure to ensure, to the extent possible, 
reliability of operation across devices and across days for any one 
device. In addition, treatment groups must be balanced across devices. 
Each animal shall be tested individually. The test session shall be long 
enough for motor activity to approach asymptotic levels by the last 20 
percent of the session for most treatments and animals. All sessions 
should have the same duration. Treatment groups shall be counter-
balanced across test times. Effort should be made to ensure that 
variations in the test conditions are minimal and are not systematically 
related to treatment. Among the variables which can affect motor 
activity are sound level, size and shape of the test cage, temperature, 
relative humidity, lighting conditions, odors, use of home cage or novel 
test cage and environmental distractions. Tests shall be executed by an 
appropriately trained individual.
    (ii) Acute. Testing shall be timed to include the time of peak 
signs.
    (iii) Subchronic. All animals shall be tested prior to initiation of 
exposure and at 30 2, 60 2 
and 90 2 days during the exposure period. Testing 
shall occur prior to the daily exposure. Animals shall be weighed on 
each test day and at least once weekly during the exposure period.
    (e) Data reporting and evaluation. In addition to the reporting 
requirements specified under 40 CFR part 792, subpart J the final test 
report must include the following information:
    (1) Description of system and test methods. (i) Positive control 
data from the laboratory performing the test which demonstrate the 
sensitivity of the procedure being used.
    (ii) Procedures for calibrating and assuring the equivalence of 
devices and balancing treatment groups.

[[Page 217]]

    (2) Results. The following information must be arranged by test 
group (dose level).
    (i) In tabular form, data must be provided showing for each animal:
    (A) Its identification number.
    (B) Body weight, total session activity counts, and intrasession 
subtotals for each date measured.
    (ii) Group summary data should also be reported.
    (3) Evaluation of data. An evaluation of the test results (including 
statistical analysis comparing total activity counts at the end of 
exposure of treatment vs control animals must be made and supplied. This 
submission must include dose-effect curves for motor activity expressed 
as activity counts.
    (f) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) Dixon, W.J., Massey, E.J. Introduction to Statistical Analysis 
2nd Ed. (New York: McGraw-Hill, 1957).
    (2) Finger, F.W. ``Measuring behavioral activity,'' Methods in 
Psychobiology Vol. 2. Ed. R.D. Myers (New York: Academic, 1972) pp. 1-
19.
    (3) Jensen, D.R. ``Some simultaneous multivariate procedures using 
Hotelling's T\2\ Statistics,'' Biometrics, 28:39-53 (1972).
    (4) Kinnard, E.J. and Watzman, N. ``Techniques utilized in the 
evaluation of psychotropic drugs on animals activity,'' Journal of 
Pharmaceutical Sciences, 55:995-1012 (1966).
    (5) Neter, J. and Wasserman, W. Applied Linear Statistical Models. 
Homewood, Richard D. Irwin, Inc., 1974.
    (6) Reiter, L.E. ``Use of activity measures in behavioral 
toxicology,'' Environmental Health Perspectives, 26:9-20 (1978).
    (7) Reiter, L.W. and MacPhail, R.C. ``Motor Activity: A survey of 
methods with potential use in toxicity testing,'' Neurobehavioral 
Toxicology, 1: Suppl. 1, 53-66 (1979).
    (8) Robbins, T.W. ``A critique of the methods available for the 
measurement of spontaneous motor activity,'' Handbook of 
Psychopharmacology. Vol. 7. Eds. Iversen, L.L., Iversen, D.S., Snyder, 
S.H. (New York: Plenum, 1977) pp. 37-82.
    (9) Sokal, R.P. and Rohlf, E.J. Biometry. (San Francisco: W.H. 
Freeman and Co., 1969).

[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19082, May 20, 1987]



Sec. 798.6400  Neuropathology.

    (a) Purpose. The techniques in this guideline are designed to 
develop data on morphologic changes in the nervous system for chemical 
substances and mixtures subject to such testing under the Toxic 
Substances Control Act. The data will detect and characterize 
morphologic changes, if and when they occur, and determine a no-effect 
level for such changes. Neuropathological evaluation should be 
complemented by other neurotoxicity studies, e.g. behavioral and 
neurophysiological studies. Neuropathological evaluation may be done 
following acute, subchronic or chronic exposure.
    (b) Definition. Neurotoxicity or a neurotoxic effect is an adverse 
change in the structure or function of the nervous system following 
exposure to a chemical agent.
    (c) Principle of the test method. The test substance is administered 
to several groups of experimental animals, one dose being used per 
group. The animals are sacrificed and tissues in the nervous system are 
examined grossly and prepared for microscopic examination. Starting with 
the highest dosage level, tissues are examined under the light 
microscope for morphologic changes, until a no effect level is 
determined. In cases where light microscopy has revealed neuropathology, 
the no effect level may be confirmed by electron microscopy.
    (d) Test procedure--(1) Animal selection--(i) Species and strain. 
Testing shall be performed in the species being used in other tests for 
neurotoxicity. This will generally be the laboratory rat. The choice of 
species shall take into consideration such factors as the comparative 
metabolism of the chemical and species sensitivity to the toxic effects 
of the test substance, as evidenced by the results of other studies, the 
potential for combined studies, and the availability of other toxicity 
data for the species.

[[Page 218]]

    (ii) Age. Animals shall be young adults (150-200 gm for rats) at the 
start of exposure.
    (iii) Sex. Both sexes shall be used unless it is demonstrated that 
one sex is refractory to the effects.
    (2) Number of animals. A minimum of six animals per group shall be 
used. The tissues from each animal shall be examined separately. It is 
recomse (iv)mended that ten animals per group be used.
    (3) Control groups. (i) A concurrent control group(s) is (are) 
required. This group must be an untreated control group or, if a vehicle 
is used in administering the test substance, a vehicle control group. If 
the vehicle used has a known or potential toxic property, both untreated 
and vehicle control groups are required.
    (ii) A satellite group of animals may be treated with the high level 
for 90 days and observed for reversibility, persistence, or delayed 
occurrence of toxic effects for a post-treatment period of appropriate 
length; normally not less than 28 days.
    (4) Dose levels and dose selection. At least 3 doses, equally spaced 
on a log scale (e.g., \1/2\ log units) over a range of at least 1 log 
unit shall be used in addition to a zero dose or vehicle administration. 
The data should be sufficient to produce a dose-effect curve.
    (i) The highest dose shall produce (A) clear behavioral effects or 
(B) life-threatening toxicity.
    (ii) The data from the lower doses must show either (A) graded dose-
dependent effects at two dose levels or (B) no effects at two dose 
levels, respectively.
    (5) Duration of testing. The exposure duration will be specified in 
the test rule. This will generally be 90 days exposure.
    (6) Route of administration. The test substance shall be 
administered by a route specified in the test rule. This will generally 
be the route most closely approximating the route of human exposure. The 
exposure protocol shall conform to that outlined in the appropriate 
acute or subchronic toxicity guideline.
    (7) Combined protocol. The tests described herein may be combined 
with any other toxicity study, as long as none of the requirements of 
either are violated by the combination.
    (8) Study conduct--(i) Observation of animals. All toxicological 
(e.g., weight loss) and neurological signs (e.g., motor disturbance) 
shall be recorded frequently enough to observe any abnormality, and not 
less than weekly.
    (ii) Sacrifice of animals--(A) General. The goal of the techniques 
outlined for sacrifice of animals and preparation of tissues is 
preservation of tissues morphology to simulate the living state of the 
cell.
    (B) Perfusion technique. Animals shall be perfused in situ by a 
generally recognized technique. For fixation suitable for light or 
electronic microscopy, saline solution followed by buffered 2.5 percent 
glutaraldehyde or buffered 4.0 percent paraformaldehyde, is recommended. 
While some minor modifications or variations in procedures are used in 
different laboratories, a detailed and standard procedure for vascular 
perfusion may be found in the text by Zeman and Innes (1963) under 
paragraph (f)(7) of this section, Hayat (1970) under paragraph (f)(3) of 
this section, and by Spencer and Schaumburg (1980) under paragraph 
(f)(6) of this section. A more sophisticated technique is described by 
Palay and Chan-Palay (1974) under paragraph (f)(4) of this section.
    (C) Removal of brain and cord. After perfusion, the bonystructure 
(cranium and vertebral column) shall be exposed. Animals shall then be 
stored in fixative-filled bags at 4 [deg]C for 8-12 hours. The cranium 
and vertebral column shall be removed carefully by trained technicians 
without physical damage of the brain and cord. Detailed dissection 
procedures may be found in the text by Palay and Chan-Palay (1974) under 
paragraph (f)(4) of this section. After removal, simple measurement of 
the size (length and width) and weight of the whole brain (cerebrum, 
cerebellum, pons-medulla) shall be made. Any abnormal coloration or 
discoloration of the brain and cord shall also be noted and recorded.
    (D) Sampling. Unless a given test rule specifies otherwise, cross-
sections of the following areas shall be examined: The forebrain, the 
center of the cerebrum, the midbrain, the cerebellum

[[Page 219]]

and pons, and the medulla oblongata; the spinal cord at cervical and 
lumbar swelling (C3-C6 and L1-
L4); Gasserian ganglia, dorsal root ganglia (C3-
C6, L1-L4), dorsal and ventral root 
fibers (C3-C6, L\1\-L4), proximal 
sciatic nerve (mid-thigh and sciatic notch), sural nerve (at knee), and 
tibial nerve (at knee). Other sites and tissue elements (e.g., 
gastrocnemius muscle) should be examined if deemed necessary. Any 
observable gross changes shall be recorded.
    (iii) Specimen storage. Tissue samples from both the central and 
peripheral nervous system shall be further immersion fixed and stored in 
appropriate fixative (e.g., 10 percent buffered formalin for light 
microscopy; 2.5 percent buffered gluteraldehyde or 4.0 percent buffered 
paraformaldehyde for electron microscopy) for future examination. The 
volume of fixative versus the volume of tissues in a specimen jar shall 
be no less than 25:1. All stored tissues shall be washed with buffer for 
at least 2 hours prior to further tissue processing.
    (iv) Histopathology examination. (A) Fixation. Tissue specimens 
stored in 10 percent buffered formalin may be used for this purpose. All 
tissues must be immersion fixed in fixative for at least 48 hours prior 
to further tissue processing.
    (B) Dehydration. All tissue specimens shall be washed for at least 1 
hour with water or buffer, prior to dehydration. (A longer washing time 
is needed if the specimens have been stored in fixative for a prolonged 
period of time.) Dehydration can be performed with increasing 
concentration of graded ethanols up to absolute alcohol.
    (C) Clearing and embedding. After dehydration, tissue specimens 
shall be cleared with xylene and embedded in paraffin or paraplast. 
Multiple tissue specimens (e.g. brain, cord, ganglia) may be embedded 
together in one single block for sectioning. All tissue blocks shall be 
labelled showing at least the experiment number, animal number, and 
specimens embedded.
    (D) Sectioning. Tissue sections, 5 to 6 microns in thickness, shall 
be prepared from the tissue blocks and mounted on standard glass slides. 
It is recommended that several additional sections be made from each 
block at this time for possible future needs for special stainings. All 
tissue blocks and slides shall be filed and stored in properly labeled 
files or boxes.
    (E) Histopathological techniques. Although the information available 
for a given chemical substance may dictate test-rule specific changes, 
the following general testing sequence is proposed for gathering 
histopathological data:
    (1) General staining. A general staining procedure shall be 
performed on all tissue specimens in the highest treatment group. 
Hematoxylin and eosin (H&E) shall be used for this purpose. The staining 
shall be differentiated properly to achieve bluish nuclei with pinkish 
background.
    (2) Special stains. Based on the results of the general staining, 
selected sites and cellular components shall be further evaluated by the 
use of specific techniques. If H&E screening does not provide such 
information, a battery of stains shall be used to assess the following 
components in all appropriate required samples: neuronal body (e.g., 
Einarson's gallocyanin), axon (e.g., Bodian), myelin sheath (e.g., 
Kluver's Luxol Fast Blue) and neurofibrils (e.g., Bielchosky). In 
addition, peripheral nerve fiber teasing shall be used. Detailed 
staining methodology is available in standard histotechnological manuals 
such as AFIP (1968) under paragraph (f)(1) of this section, Ralis et al. 
(1973) under paragraph (f)(5) of this section, and Chang (1979) under 
paragraph (f)(2) of this section. The nerve fiber teasing technique is 
discussed in Spencer and Schaumberg (1980) under paragraph (f)(6) of 
this section. A section of normal tissue shall be included in each 
staining to assure that adequate staining has occurred. Any changes 
shall be noted and representative photographs shall be taken. If a 
lesion(s) is observed, the special techniques shall be repeated in the 
next lower treatment group until no further lesion is detectable.
    (3) Alternative technique. If the anatomical locus of expected 
neuro-pathology is well-defined, epoxy-embedded sections stained with 
toluidine blue may be used for small sized tissue samples. This 
technique obviates the need

[[Page 220]]

for special stains for cellular components. Detailed methodology is 
available in Spencer and Schaumberg (1980) under paragraph (f)(6) of 
this section.
    (4) Electron microscopy. Based on the results of light microscopic 
evaluation, specific tissue sites which reveal a lesion(s) shall be 
further evaluated by electron microscopy in the highest treatment group 
which does not reveal any light microscopic lesion. If a lesion is 
observed, the next lower treatment group shall be evaluated until no 
significant lesion is found. Detailed methodology is available in Hayat 
(1970) under paragraph (f)(3) of this section.
    (F) Examination--(1) General. All stained microscopic slides shall 
be examined with a standard research microscope. Examples of cellular 
alterations (e.g., neuronal vacuolation, degeneration, and necrosis) and 
tissue changes (e.g., gliosis, leukocytic infiltration, and cystic 
formation) shall be recorded and photographed.
    (2) Electron microscopy. Since the size of the tissue samples that 
can be examined is very small, at least 3 to 4 tissue blocks from each 
sampling site must be examined. Tissue sections must be examined with a 
transmission electron microscope. Three main categories of structural 
changes must be considered:
    (i) Neuronal body. The shape and position of the nucleus and 
nucleolus as well as any change in the chromatin patterns shall be 
noted. Within the neuronal cytoplasm, cytoplasmic organelles such as 
mitochondria, lysosomes, neurotubules, neurofilaments, microfilaments, 
endoplasmic reticulum and polyribosomes (Nissl substance), Golgi 
complex, and secretory granules shall be examined.
    (ii) Neuronal processes. The structural integrity or alterations of 
dendrites, axons (myelinated and unmyelinated), myelin sheaths, and 
synapses shall be noted.
    (iii) Supporting cells. Attention must also be paid to the number 
and structural integrity of the neuroglial elements (oligodendrocytes, 
astrocytes, and microglia) of the central nervous system, and the 
Schwann cells, satellite cells, and capsule cells of the peripheral 
nervous system. Any changes in the endothelial cells and ependymal 
lining cells shall also be noted whenever possible. The nature, 
severity, and frequency of each type of lesion in each specimen must be 
recorded. Representative lesions must be photographed and labeled 
appropriately.
    (e) Data collection, reporting, and evaluation. In addition to 
information meeting the requirements stated under 40 CFR part 792 
subpart J, the following specific information shall be reported:
    (1) Description of test system and test methods. A description of 
the general design of the experiment shall be provided. This shall 
include a short justification explaining any decisions where 
professional judgment is involved such as fixation technique and choice 
of stains.
    (2) Results. All observations shall be recorded and arranged by test 
groups. This data may be presented in the following recommended format:
    (i) Description of signs and lesions for each animal. For each 
animal, data must be submitted showing its identification (animal 
number, treatment, dose, duration), neurologic signs, location(s) nature 
of, frequency, and severity of lesion(s). A commonly-used scale such as 
1+, 2+, 3+, and 4+ for degree of severity ranging from very slight to 
extensive may be used. Any diagnoses derived from neurologic signs and 
lesions including naturally occurring diseases or conditions, should 
also be recorded.
    (ii) Counts and incidence of lesions, by test group. Data shall be 
tabulated to show:
    (A) The number of animals used in each group, the number of animals 
displaying specific neurologic signs, and the number of animals in which 
any lesion was found;
    (B) The number of animals affected by each different type of lesion, 
the average grade of each type of lesion, and the frequency of each 
different type and/or location of lesion.
    (iii) Evaluation of data. (A) An evaluation of the data based on 
gross necropsy findings and microscopic pathology observations shall be 
made and supplied. The evaluation shall include the relationship, if 
any, between the animal's exposure to the test substance and the 
frequency and severity of the lesions observed.

[[Page 221]]

    (B) The evaluation of dose-response, if existent, for various groups 
shall be given, and a description of statistical method must be 
presented. The evaluation of neuropathology data should include, where 
applicable, an assessment in conjunction with other neurotoxicity 
studies performed (eg. electrophysiological, behavioral, neurochemical).
    (f) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) AFIP. Manual of Histologic Staining Methods. (New York: McGraw-
Hill (1968).
    (2) Chang, L.W. A Color Atlas and Manual for Applied Histochemistry. 
(Springfield, IL: Charles C. Thomas, 1979).
    (3) Hayat, M.A. ``Vol. 1. Biological applications,'' Principles and 
techniques of electron microscopy. (New York: Van Nostrand Reinhold, 
1970)
    (4) Palay S.L., Chan-Palay, V. Cerebellar Cortex: Cytology and 
Organization. (New York: Springer-Verlag, 1974).
    (5) Ralis, H.M., Beesley, R.A., Ralis, Z.A. Techniques in 
Neurohistology. (London: Butterworths, 1973).
    (6) Spencer, P.S., Schaumburg, H.H. (eds). Experimental and Clinical 
Neurotoxicology. (Baltimore: Williams and Wilkins, 1980).
    (7) Zeman, W., JRM Innes, J.R.M. Craigie's Neuroanatomy of the Rat. 
(New York: Academic, 1963).

[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19082, May 20, 1987]



Sec. 798.6500  Schedule-controlled operant behavior.

    (a) Purpose. (1) In the assessment and evaluation of the potential 
human health effects of substances, it may be necessary to test for 
functional neurotoxic effects. Substances that have been observed to 
produce neurotoxic signs in other toxicity studies (e.g. CNS depression 
or stimulation), as well as substances with a structural similarity to 
known neurotoxicants should be evaluated for these effects.
    (2) This guideline defines procedures for conducting studies of 
schedule-controlled operant behavior, one way of evaluating functional 
neurotoxic effects (Dews, 1972 under paragraph (f)(1) of this section; 
NAS 1975, 1977, 1982 under paragraphs (f)(4), (5) and (6) of this 
section). Our purpose is to evaluate the effects of acute and repeated 
exposures on the rate and pattern of responding under schedules of 
reinforcement. Operant behavior tests may be used to evaluate many other 
aspects of behavior (Laties, 1978 under paragraph (f)(3) of this 
section). Additional tests may be necessary to completely assess the 
behavioral effects of any substance. Behavioral evaluation should be 
used in conjunction with neuropathologic evaluation and the evaluation 
of other toxic effects.
    (b) Definitions--(1) Neurotoxicity. Neurotoxicity or a neurotoxic 
effect is an adverse change in the structure or function of the nervous 
system following exposure to a chemical agent. Behavioral toxicity is an 
adverse change in the functioning of the organism with respect to its 
environment following exposure to a chemical agent.
    (2) Operant, operant behavior, operant conditioning. An operant is a 
class of behavioral responses which change or operates on the 
environment in the same way. Operant behavior is further distinguished 
as behavior which is modified by its consequences. Operant conditioning 
is the experimental procedure used to modify some class of behavior by 
reinforcement or punishment.
    (3) Schedule of reinforcement. A schedule of reinforcement specifies 
the relation between behavioral responses and the delivery of 
reinforcers, such as food or water (Ferster and Skinner, 1957 under 
paragraph (f)(2) of this section). For example, a fixed ratio (FR) 
schedule requires a fixed number of responses to produce a reinforcer 
(e.g. FR 30). On a fixed interval (FI) schedule, the first response 
after a fixed period of time is reinforced (e.g. FI 5 minutes).
    (c) Principle of the test method. Experimental animals are trained 
to perform under a schedule of reinforcement and measurements of their 
operant behavior are made. Several doses of the test substance are then 
administered according to the experimental design (between groups or 
within subjects) and the duration of exposure (acute or repeated). 
Measurements of the operant

[[Page 222]]

behavior are repeated. A descriptive and statistical evaluation of the 
data is made to evaluate the nature and extent of any changes in 
behavior in relation to exposures to the test substance. Comparisons are 
made between any exposures that influence the behavior and exposures 
that have neuropathological effects or effects on other targets of the 
chemical.
    (d) Test procedures--(1) Experimental design. These test procedures 
may be used to evaluate the behavior of experimental animals receiving 
either acute or repeated exposures. For acute exposure studies, either 
within-subject or between groups, experimental designs may be used. For 
repeated exposure studies, between groups designs should be used, but 
within subject comparisons (pre-exposure and post-exposure) are 
recommended and encouraged.
    (2) Animal selection--(i) Species. (A) For most studies, the 
laboratory mouse or rat is recommended. Standard strains should be used.
    (B) Under some circumstances other species may be recommended.
    (ii) Age. Experimental animals should be young adults. Rats or mice 
should be at least 14 and 6 weeks old, respectively, prior to exposure.
    (iii) Sex. (A) Approximately equal numbers of male and female 
animals are required for each dose level and control group.
    (B) Virgin females should be used.
    (iv) Experimental history. Animals should be experimentally and 
chemically naive.
    (3) Number of animals. Six to twelve animals should be exposed to 
each level of the test substance and/or control procedure. If post 
exposure effects are examined, a separate group, 6 to 12 additional 
animals not sacrificed for pathology, will required in subchronic 
studies.
    (4) Control groups--(i) Untreated controls. A concurrent ``sham'' 
exposure or vehicle control group or session (according to the design of 
the study) is required. The subjects should be treated similarly except 
that administration of the test substance is omitted.
    (ii) Positive controls. Positive control data is required to 
demonstrate that the experimental procedures, under the specific 
conditions in the testing laboratory, are sensitive to substances known 
to affect operant behavior. Both increases and decreases in response 
rate should be demonstrated. Data based on acute exposures will be 
adequate. Data should be collected according to the same experimental 
design as that proposed for the test substance. Historical data on the 
procedure collected in the same species and under the same conditions in 
the testing laboratory may be acceptable, but the presentation of 
concurrent control data is strongly encouraged since it provides 
evidence that the test has remained sensitive.
    (5) Dose levels and dose selection. At least 3 doses, equally spaced 
over a log scale (e.g., 10, 30, 100), over a range of at least 1 log 
unit shall be used in addition to a zero dose or vehicle administration. 
The data should be sufficient to produce a dose-effect curve.
    (i) The highest dose shall produce: (A) Clear behavioral effects; or 
(B) life-threatening toxicity.
    (ii) The data from the lower doses must show either: (A) Graded 
dose-dependent effects at 2 dose levels; or (B) no effects at 2 dose 
levels, respectively.
    (6) Duration of exposure. The duration and frequency of exposure 
will be specified in the test rule.
    (7) Route of Administration. The route of administration will also 
be specified in the test rule and will usually be identical to one of 
the anticipated or actual routes of human exposure. For some chemicals, 
another route (e.g. parenteral) may be justified. The exposure protocol 
should conform to that outlined in the appropriate acute or subchronic 
toxicity study guideline under subpart B or subpart C of this part.
    (8) Study conduct--(i) Apparatus. Behavioral responses and the 
delivery of reinforcers shall be controlled and monitored by automated 
equipment located so that its operation does not provide unintended cues 
or otherwise interfere with the ongoing behavior. Individual chambers 
should be sound attenuated to prevent disruptions of behavior by 
external noise. The response manipulanda, feeders, and any stimulus 
devices should be tested before each session; these devices should 
periodically be calibrated.

[[Page 223]]

    (ii) Chamber assignment. Concurrent treatment groups should be 
balanced across chambers. Each subject should be tested in the chamber 
to which it is initially assigned.
    (iii) Deprivation and training. (A) If a nonpreferred positive 
reinforcer is used, all subjects should be deprived of food until they 
reach a fixed percentage (e.g. 80 to 90 percent, commonly) of their ad 
libitum body weight or for a fixed period (e.g., 18 hours) prior to 
training. Deprivation should be kept constant throughout the study.
    (B) Subjects must be trained until they display demonstrable 
stability in performance across days prior to exposure. One simple and 
useful criterion is a minimum number of sessions on the schedule and no 
systematic trend during the 5 days before exposure.
    (C) Cumulative records of cumulative responding over time for each 
animal should be presented to demonstrate that the pattern of responding 
is representative of that generated by the schedule of reinforcement.
    (iv) Time, frequency, and duration of testing--(A) Time of testing. 
All experimental animals should be tested at the same time of day and 
with respect to the time of exposure. For acute studies, testing should 
be performed when effects are estimated to peak, usually shortly after 
exposure. For subchronic studies, subjects should be tested prior to 
daily exposure in order to assess cumulative effects.
    (B) Frequency of testing. The maintenance of stable operant behavior 
normally will require regular and frequent (e.g., 5 days a week) testing 
sessions. Animals should be weighed on each test day.
    (C) Duration of testing. (1) Experimental sessions should be long 
enough to reasonably see the effects of exposure, but brief enough to be 
practical. Under most circumstances, a session length of 30-40 minutes 
should be adequate.
    (2) If the nature or duration of effects following cessation of 
repeated exposure are a concern, animals from the high dose group should 
be tested following exposure for a suitable period of time.
    (v) Schedule selection. The schedule of reinforcement chosen should 
generate response rates that may increase or decrease as a function of 
exposure. Many schedules of reinforcement can do this: a single schedule 
maintaining a moderate response rate; fixed-interval schedules, which 
engender a variety of response rates in each interval; or multiple 
schedules, where different components may maintain high and low response 
rates.
    (e) Data reporting and evaluation. In addition to the reporting 
requirements specified under 40 CFR part 792, subpart J the final test 
report should contain the following information:
    (1) Description of system, test methods, experimental design, and 
control data. (i) A description of the experimental chamber, programming 
equipment, data collection devices, and environmental conditions.
    (ii) A description of the experimental design including 
counterbalancing procedures, and the stability criterion.
    (iii) A description and statistical evaluation of positive control 
and other control data, including standard measures of central tendency, 
variability, coefficient of variation of response rates, and the slope 
of the dose-effect curve.
    (2) Results. (i) Data for each animal should be arranged by test 
group in tabular form including the animal identification number, body 
weight, pre-exposure rate of responding, changes in response rate 
produced by the chemical, and group data for the same variables, 
including standard measures of central tendency, variability and 
coefficient of variation.
    (ii) A description and statistical evaluation of the test results: 
With particular reference to the overall statistical procedures (e.g., 
parametric or nonparametric) dose-effect curve, and calculation of 
slope. Presentation of calculations is encouraged.
    (f) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) Dews, P.B. ``Assessing the Effects of Drugs,'' Methods in 
Psychobiology, Vol. 2, Ed., R.D. Myers (New York: Academic Press, 1972) 
83-124.
    (2) Ferster, C.B. Skinner, B.F. Schedules of Reinforcement. (New 
York: Appleton-Century-Crofts, 1957).

[[Page 224]]

    (3) Laties, V.G. ``How Operant Conditioning can Contribute to 
Behavioral Toxicology,'' Environmental Health Perspectives, 28: 29-35 
(1978).
    (4) National Academy of Science. Principles for Evaluating Chemicals 
in the Environment. (Washington, DC: National Academy of Sciences, 
1975).
    (5) National Academy of Science. Principles and Procedures for 
Evaluating the Toxicity of Household Substances. (Washington, DC: 
National Academy of Sciences, 1977).
    (6) National Academy of Science. ``Strategies to determine needs and 
priorities for toxicity testing,'' Appendix 3B. Reference Protocol 
Guidelines For Neurobehavioral Toxicity Tests. 2: 123-129 (1982).



Sec. 798.6560  Subchronic delayed neuro-toxicity of organophosphorus 

substances.

    (a) Purpose. In the assessment and evaluation of the toxic 
characteristics of organophosphorus substances the determination of 
subchronic delayed neurotoxicity may be carried out, usually after 
initial information on delayed neurotoxicity has been obtained by acute 
testing or by the demonstration of inhibition and aging of neurotoxic 
esterase in hen neural tissue. The subchronic delayed neurotoxicity test 
provides information on possible health hazards likely to arise from 
repeated exposures over a limited period of time. It will provide 
information on dose response and can provide an estimate of a non-effect 
level which can be of use for establishing safety criteria for exposure.
    (b) Definitions. Subchronic delayed neurotoxicity is a prolonged, 
delayed-onset locomoter ataxia resulting from repeated daily 
administration of the test substance.
    (c) Principle of the test method. Multiple dose levels of the test 
substance are administered orally to domestic hens (Gallus gallus 
domesticus) for 90 days. The animals are observed at least daily for 
behavioral abnormalities, locomotor ataxia and paralysis. 
Histopathological examination of selected neural tissues is undertaken 
at the termination of the test period.
    (d) Test procedures--(1) Animal selection. The adult domestic laying 
hen, aged 8 to 14 months, is recommended. Standard size breeds and 
strains should be employed.
    (2) Number of animals. Ten hens should be used for each treatment 
and control group.
    (3) Control group--(i) General. A concurrent control group should be 
used. This group should be treated in a manner identical to the treated 
group, except that administration of the test substance is omitted.
    (ii) Reference substances. If a positive control is used, a 
substance which is known to produce delayed neurotoxicity should be 
employed. Examples of such substances are triorthocresyl phosphate 
(TOCP) and leptophos.
    (4) Housing and feeding conditions. Cages or enclosures which are 
large enough to permit free mobility of the hens and easy observation of 
gait should be used. Where the lighting is artificial, the sequence 
should be 12 hours light, 12 hours dark. Appropriate diets should be 
administered as well as an unlimited supply of drinking water.
    (5) Dose levels. At least three dose levels should be used in 
addition to the control group(s). The highest dose level should result 
in toxic effects, preferably delayed neurotoxicity, but not produce an 
incidence of fatalities which would prevent a meaningful evaluation. The 
lowest dose level should not produce any evidence of toxicity.
    (6) Route of administration. Oral dosing each day for at least 5 
days per week should be carried out, preferably by gavage or 
administration of gelatine capsules.
    (7) Study conduct--(i) General. Healthy young adult hens free from 
interfering viral diseases and medication and without abnormalities of 
gait should be acclimatized to the laboratory conditions for at least 5 
days prior to randomization and assignment to treatment and control 
groups. The test or control substance should be administered and 
observations begun. All hens should be carefully observed at least once 
daily throughout the test period. Signs of toxicity should be recorded, 
including the time of onset, degree and duration. Observations should

[[Page 225]]

include, but not be limited to, behavioral abnormality, locomotor ataxia 
and paralysis. At least once a week the hens should be taken outside the 
cages and subjected to a period of forced motor activity, such as ladder 
climbing, in order to enhance the observation of minimal responses. The 
hens should be weighed weekly. Any moribund hens should be removed and 
sacrificed.
    (ii) Pathology--(A) Gross necropsy. In the presence of clinical 
signs of delayed neurotoxicity useful information may be provided by 
gross necropsy.
    (B) Histopathology. Tissues from all animals should be fixed in 
situ, using perfusion techniques. Sections should include medulla 
oblongata, spinal cord and peripheral nerves. The spinal cord sections 
should be taken from the upper cervical bulb, the mid-thoracic and 
lumbosacral regions. Sections of the proximal region of the tibial nerve 
and its branches and of the sciatic nerve should be taken. Sections 
should be stained with appropriate myelin and axon-specific stains. 
Microscopic examination should be carried out on all hens in the control 
and high-dose groups. Microscopic examination should also be carried out 
on hens in the low and intermediate dose groups when there is evidence 
of effects in the high-dose group.
    (e) Data reporting and evaluation--(1) Test report. In addition to 
the reporting requirements specified under 40 CFR part 792, subpart J 
the final test report must include the following information:
    (i) Toxic response data by group with a description of clinical 
manifestations of nervous system damage; where a grading system is used 
the criteria should be defined.
    (ii) For each animal, time of death during the study or whether it 
survived to termination.
    (iii) The day of observation of each abnormal sign and its 
subsequent course.
    (iv) Body weight data.
    (v) Necropsy findings for each animal, when performed.
    (vi) A detailed description of all histopathological findings.
    (vii) Statistical treatment of results, where appropriate.
    (2) Treatment of results. (i) Data may be summarized in tabular 
form, showing for each test group the number of animals at the start of 
the test, the number of animals showing lesions or effects, the types of 
lesions or effects and the percentage of animals displaying each type of 
lesion or effect.
    (ii) All observed results should be evaluated by an appropriate 
statistical method. Any generally accepted statistical method may be 
used; the statistical methods should be selected during the design of 
the study.
    (3) Evaluation of results. The findings of a subchronic delayed 
neurotoxicity study should be evaluated in conjunction with the findings 
of preceding studies and considered in terms of the incidence and 
severity of observed neurotoxic effects and any other observed effects 
and histopathological findings in the treated and control groups. A 
properly conducted subchronic test should provide a satisfactory 
estimation of a no-effect level based on lack of clinical signs and 
histopathological changes.
    (f) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) Abou-Donia, M.B. ``Organophosphorus ester-induced delayed 
neurotoxicity'' Annual Review of Pharmacology and Toxicology, 21:511-548 
(1981).
    (2) Abou-Donia, M.B., Pressing, S.H. ``Delayed neurotoxicity from 
continuous low-dose oral administration of leptophos to hens.'' 
Toxicology and Applied Pharmacology, 38:595-608 (1976).
    (3) Baron, R.L. (ed). ``Pesticide Induced Delayed Neurotoxicity,'' 
Proceedings of a Conference, February 19-20, 1976, Washington, DC. U.S. 
Environmental Protection Agency. EPA Report No. 600/1-76-025, 
Washington, DC (1976).
    (4) Cavanaugh, J.B. ``Peripheral neuropathy caused by chemical 
agents'' Critical Reviews of Toxicity, 2:365-417 CRC Press, Inc. (1973).
    (5) Johannsen, F.R., Wright, P.L., Gordon, D.E., Levinskas, G.L., 
Radue, R.W., Graham, P.R. ``Evaluation of delayed neurotoxicity and 
dose-response relationship of phosphate esters in the

[[Page 226]]

adult hen,'' Toxicology and Applied Pharmacology, 41:291-304 (1977).
    (6) Johnson, M.K. ``Organophosphorus esters causing delayed 
neurotoxic effects: mechanism of action and structure/activity 
studies,'' Archives of Toxicology, 34:259-288 (1975).



PART 799_IDENTIFICATION OF SPECIFIC CHEMICAL SUBSTANCE AND MIXTURE TESTING 

REQUIREMENTS--Table of Contents




                      Subpart A_General Provisions

Sec.
799.1 Scope and purpose.
799.2 Applicability.
799.3 Definitions.
799.5 Submission of information.
799.10 Test standards.
799.11 Availability of test guidelines.
799.12 Test results.
799.17 Effects of non-compliance.
799.18 Chemicals subject of test rules or consent orders for which the 
          testing reimbursement period has passed.
799.19 Chemical imports and exports.

                 Subpart B_Specific Chemical Test Rules

799.1053 Trichlorobenzenes.
799.1560 Diethylene glycol butyl ether and diethylene glycol butyl ether 
          acetate.
799.1575 Diethylenetriamine (DETA).
799.1645 2-Ethylhexanol.
799.1700 Fluoroalkenes.
799.2155 Commercial hexane.
799.2325 Isopropanol.
799.2475 2-Mercaptobenzothiazole.
799.2700 Methyl ethyl ketoxime.
799.3300 Unsubstituted phenylenediamines.
799.4360 Tributyl phosphate.
799.4440 Triethylene glycol monomethyl ether.

                    Subpart C_Testing Consent Orders

799.5000 Testing consent orders for substances and mixtures with 
          Chemical Abstract Service Registry Numbers.
799.5025 Testing consent orders for mixtures without Chemical Abstracts 
          Service Registry Numbers.

                   Subpart D_Multichemical Test Rules

799.5055 Hazardous waste constituents subject to testing.
799.5075 Drinking water contaminants subject to testing.
799.5085 Chemical testing requirements for certain high production 
          volume chemicals.
799.5115 Chemical testing requirements for certain chemicals of interest 
          to the Occupational Safety and Health Administration.

              Subpart E_Product Properties Test Guidelines

799.6755 TSCA partition coefficient (n-octanol/water), shake flask 
          method.
799.6756 TSCA partition coefficient (n-octanol/water), generator column 
          method.
799.6784 TSCA water solubility: Column elution method; shake flask 
          method.
799.6786 TSCA water solubility: Generator column method.

Subparts F-G [Reserved]

                Subpart H_Health Effects Test Guidelines

799.9110 TSCA acute oral toxicity.
799.9120 TSCA acute dermal toxicity.
799.9130 TSCA acute inhalation toxicity.
799.9135 TSCA acute inhalation toxicity with histopathology.
799.9305 TSCA Repeated dose 28-day oral toxicity study in rodents.
799.9310 TSCA 90-day oral toxicity in rodents.
799.9325 TSCA 90-day dermal toxicity.
799.9346 TSCA 90-day inhalation toxicity.
799.9355 TSCA reproduction/developmental toxicity screening test.
799.9365 TSCA combined repeated dose toxicity study with the 
          reproduction/developmental toxicity screening test.
799.9370 TSCA prenatal developmental toxicity.
799.9380 TSCA reproduction and fertility effects.
799.9410 TSCA chronic toxicity.
799.9420 TSCA carcinogenicity.
799.9430 TSCA combined chronic toxicity/carcinogenicity.
799.9510 TSCA bacterial reverse mutation test.
799.9530 TSCA in vitro mammalian cell gene mutation test.
799.9537 TSCA in vitro mammalian chromosome aberration test.
799.9538 TSCA mammalian bone marrow chromosomal aberration test.
799.9539 TSCA mammalian erythrocyte micronucleus test.
799.9620 TSCA neurotoxicity screening battery.
799.9630 TSCA developmental neurotoxicity.
799.9748 TSCA metabolism and pharmacokinetics.
799.9780 TSCA immunotoxicity.

    Authority: 15 U.S.C. 2603, 2611, 2625.

    Source: 49 FR 39817, Oct. 10, 1984, unless otherwise noted.

[[Page 227]]



                      Subpart A_General Provisions



Sec. 799.1  Scope and purpose.

    (a) This part identifies the chemical substances, mixtures, and 
categories of substances and mixtures for which data are to be 
developed, specifies the persons required to test (manufacturers, 
including importers, and/or processors), specifies the test substance(s) 
in each case, prescribes the tests that are required including the test 
standards, and provides deadlines for the submission of reports and data 
to EPA.
    (b) This part requires manufacturers and/or processors of chemical 
substances or mixtures (``chemicals'') identified in subpart B to submit 
letters of intent to test, exemption applications, and study plans in 
accordance with EPA test rule development and exemption procedures 
contained in part 790 of this chapter and any modifications to such 
procedures contained in this part.
    (c) This part requires manufacturers and/or processors of chemicals 
identified in subpart B to conduct tests and submit data in accordance 
with the test standards contained in this part in order to develop data 
on the health and environmental effects and other characteristics of 
these chemicals. These data will be used to assess the risk of injury to 
human health or the environment presented by these chemicals.
    (d) This part contains certain TSCA test guidelines which are cross-
referenced in the test rules contained in this part.

[49 FR 39817, Oct. 10, 1984, as amended at 62 FR 43824, Aug. 15, 1997]



Sec. 799.2  Applicability.

    This part is applicable to each person who manufactures or intends 
to manufacture (including import) and/or to each person who processes or 
intends to process a chemical substance or mixture identified in subpart 
B for testing during the period commencing with the effective date of 
the specific chemical test rule until the end of the reimbursement 
period. Each set of testing requirements in subpart B specifies whether 
those requirements apply to manufacturers only, to processors only, or 
to both manufacturers and processors.



Sec. 799.3  Definitions.

    The definitions in section 3 of the Toxic Substances Control Act 
(TSCA) and the definitions of Sec. 790.3 of this chapter apply to this 
part.



Sec. 799.5  Submission of information.

    Information (letters, study plans, reports) submitted to EPA under 
this part must bear the Code of Federal Regulations section number of 
the subject chemical test rule (e.g., Sec. 799.1285 for Cumene) and 
must be addressed to the Document Control Office (DCO) (7407M), Office 
of Pollution Prevention and Toxics (OPPT), Environmental Protection 
Agency, 1200 Pennsylvania Ave., NW., Washington, DC 20460-0001.

[60 FR 34467, July 3, 1995, as amended at 71 FR 33642, June 12, 2006]



Sec. 799.10  Test standards.

    Testing required under subpart B must be performed using a study 
plan prepared according to the requirements of parts 790 and 792 of this 
chapter unless modified in specific chemical test rules in subpart B. 
All raw data, documentation, records, protocols, specimens and reports 
generated as a result of a study under subpart B must be developed, 
reported, and retained in accordance with TSCA Good Laboratory Practice 
Standards (GLP's) in part 792 of this chapter. These items must be made 
available during an inspection or submitted to EPA upon request by EPA 
or its authorized representative. Laboratories conducting testing for 
submission to the Agency in response to a test rule promulgated under 
section 4 of TSCA must adhere to the TSCA GLP's. Sponsors must notify 
the laboratory that the study is being conducted pursuant to TSCA 
section 4. Sponsors are also responsible for ensuring that laboratories 
conducting the test abide by the TSCA GLP standards. In accordance with 
Sec. 792.12 of this chapter, a certification concerning adherence to 
the TSCA GLP's must be submitted to EPA.

[[Page 228]]



Sec. 799.11  Availability of test guidelines.

    (a) The TSCA and FIFRA guidelines for the various study plans are 
available from the National Technical Information Service (NTIS). 
Address and telephone number: National Technical Information Service, 
5285 Port Royal Road, Springfield, VA 22161 (703-487-4650).
    (b) The OECD guidelines for the various study plans are available 
from the following address: OECD Publication and Information Center, 
1750 Pennsylvania Ave., NW., Washington, DC 20006 (202-724-1857).



Sec. 799.12  Test results.

    Except as set forth in specific chemical test rules in subpart B of 
this part, a positive or negative test result in any of the tests 
required under subpart B is defined in the TSCA test guidelines 
published by NTIS.



Sec. 799.17  Effects of non-compliance.

    Any person who fails or refuses to comply with any aspect of this 
part or part 790 is in violation of section 15 of TSCA. EPA will treat 
violations of Good Laboratory Practice Standards as indicated in Sec. 
792.17 of this chapter.



Sec. 799.18  Chemicals subject of test rules or consent orders for which the 

testing reimbursement period has passed.

    The following table lists substances and mixtures that have been the 
subjects of section 4 testing actions and for which the testing 
reimbursement period has terminated (sunset). The Federal Register 
citation in the table is for the final rule/consent order that includes 
the particular substance for which the sunset date listed in the table 
below applies. Section 12(b) export notification is no longer required 
for these substances and mixtures. Substances that are the subjects of 
two or more section 4 testing actions may have section 4 reimbursement 
or section 12(b) export notification requirements that have not sunset; 
see subparts B, C, and D of this part to determine if certain other 
section 4 testing requirements apply. Additionally, section 12(b) export 
notification may also be triggered by proposed or final action under 
TSCA section 5, 6, or 7 (in addition to final actions under section 4); 
see 40 CFR part 707, subpart D for further information regarding the 
TSCA section 12(b) export notification requirements.

----------------------------------------------------------------------------------------------------------------
  CAS No.                 Chemical Name                          FR cite                     Sunset dates
----------------------------------------------------------------------------------------------------------------
            C-9 Aromatic Hydrocarbon Fraction \1\     50 FR 20662, 5/17/85           Aug 13, 1994
   62-53-3  Aniline                                   53 FR 31804, 8/19/88           July 27, 1994
   71-55-6  1,1,1-Trichloroethane                     49 FR 39810, 10/10/84          June 29, 1992
   75-56-9  Propylene oxide                           50 FR 48762, 11/27/85          Dec,21, 1992
   78-87-5  1,2-Dichloropropane                       52 FR 37138, 10/5/87           April 17, 1995
   79-94-7  Tetrabromobisphenol-A                     52 FR 25219, 7/6/87            Aug 24, 1994
   80-05-7  Bisphenol A                               51 FR 33047, 9/18/86           April 6, 1993
   84-65-1  Anthraquinone                             52 FR 21018, 6/4/87            Aug 21, 1994
   87-61-6  1,2,3-trichlorobenzene                    51 FR 11728,4/7/86             Nov 13, 1993
   88-74-4  2-nitroaniline                            53 FR 31804, 8/19/88           Sept 19, 1994
   92-52-4  1,1-Biphenyl                              50 FR 37182, 9/12/85           March 15, 1994
   95-48-7  Ortho-cresols AKA 2-methylphenol          51 FR 15771, 4/28/86           Dec. 6, 1994
   95-50-1  1,2-dichlorobenzene                       51 FR 24657, 7/8/86            April 27, 1994
   95-51-2  2-chloroaniline                           53 FR 31804, 8/19/88           Sept 6, 1994
   95-76-1  3,4-dichloroaniline                       53 FR 31804, 8/19/88           Oct 2, 1994
   95-94-3  1,2,4,5-tetrachlorobenzene                51 FR 24657,7/8/86             April 27, 1994
   97-02-9  2,4-dinitroaniline                        53 FR 31804, 8/19/88           Oct 19, 1993
   98-82-8  Cumene                                    53 FR 28195, 7/27/88           March 11, 1995
   99-30-9  2,6-dichloro-4-nitroaniline               53 FR 31804, 8/19/88           Aug 6, 1994
  100-01-6  4-nitroaniline                            53 FR 31804, 8/19/88           Sept 19, 1994
  106-44-5  Para-cresols AKA 4-methylphenol           51 FR 15771, 4/28/86           Dec. 6, 1994
  106-46-7  1,4-dichlorobenzene                       51 FR 24657, 7/8/86            Jan 22, 1994
  106-47-8  4-chloroaniline                           53 FR 31804, 8/19/88           Oct 19, 1993
  108-39-4  Meta-cresols AKA 3-methylphenol           51 FR 15771, 4/28/86           Dec. 6, 1994
  108-90-7  Monochlorobenzene                         51 FR 24657, 7/8/86            Nov 13, 1991
  112-90-3  Oleylamine                                52 FR 31962, 8/24/87           Nov 28, 1994
  116-14-3  Tetrafluoroethene                         52 FR 21516, 6/8/87            May 19, 1993
  116-15-4  Hexafluoropropene                         52 FR 21516, 6/8/87            Jan 22, 1994
  123-31-9  Hydroquinone                              50 FR 53145, 12/30/85          Dec. 11, 1994
  149-57-5  2-Ethylhexanoic Acid                      51 FR 40318, 11/6/86           June 19, 1993
  328-84-7  3,4-Dichlorobenzotrifluoride              52 FR 23547, 6/23/87           Dec. 5, 1993

[[Page 229]]

 
25550-98-5  Diisodecyl Phenyl Phosphite               54 FR 8112, 2/24/89            May 21, 1995
----------------------------------------------------------------------------------------------------------------
\1\ Only substances obtained from the reforming of crude petroleum.


[60 FR 31923, June 19, 1995]



Sec. 799.19  Chemical imports and exports.

    Persons who export or who intend to export chemical substances or 
mixtures listed in subpart B, subpart C, or subpart D of this part are 
subject to the requirements of 40 CFR part 707.

[71 FR 66245, Nov. 14, 2006]



                 Subpart B_Specific Chemical Test Rules



Sec. 799.1053  Trichlorobenzenes.

    (a) Identification of testing substance. (1) 1,2,3- and 1,2,4-
trichlorobenzenes, CAS Numbers 87-61-6 and 120-82-1 respectively, shall 
be tested in accordance with this section.
    (2) The substances identified in paragraph (a)(1) of this section 
shall be 99 percent pure and shall be used as the test substances in 
each of the tests specified.
    (3) For health effects testing required under paragraph (e) of this 
section, the test substance shall not contain more than 0.05 percent 
benzene and 0.05 percent hexachlorobenzene.
    (b) Persons required to submit study plans, conduct tests, and 
submit data. (1) All persons who manufacture or process substances 
identified in paragraph (a)(1) of this section, other than an impurity, 
from May 21, 1986, to the end of the reimbursement period, shall submit 
a letter of intent to test or exemption applications and shall conduct 
tests, in accordance with part 792 of this chapter, and submit data as 
specified in this section, subpart A of this part and part 790 of this 
chapter for two-phase rulemaking.
    (2) Persons subject to this section are not subject to the 
requirements of Sec. 790.50(a) (2), (5), (6) and (b) and Sec. 
790.87(a)(1)(ii) of this chapter.
    (3) Persons who notify EPA of their intent to conduct tests in 
compliance with the requirements of this section must submit plans for 
those tests no later than 30 days before the initiation of each of those 
tests.
    (4) In addition to the requirements of Sec. 790.87(a)(2) and (3) of 
this chapter, EPA will conditionally approve exemption applications for 
this rule if EPA has received a letter of intent to conduct the testing 
from which exemption is sought and EPA has adopted test standards and 
schedules in a final Phase II test rule.
    (5) For health effects testing required under paragraph (e) of this 
section, all persons who manufacture (import) or process 1,2,4-
trichlorobenzene, other than as an impurity, after the effective date of 
this rule (August 21, 1986) to the end of the reimbursement period shall 
submit letters of intent to conduct testing or exemption applications, 
submit study plans, conduct tests, and submit data as specified in this 
section, subpart A of this part, and parts 790 and 792 of this chapter 
for single-phase rulemaking.
    (c) [Reserved]
    (d) Environmental effects testing. 1,2,3- and 1,2,4-
trichlorobenzenes shall be tested in accordance with this section.
    (1) Marine invertebrate acute toxicity testing--(i) Required 
testing. Testing using measured concentrations, flow through or static 
renewal systems, and systems that control for evaporation of the test 
substance, shall be conducted for 1,2,3- and 1,2,4-trichlorobenzenes. 
Testing shall be conducted with mysid shrimp (Mysidopis bahia) to 
develop data on the acute toxicity of the above chlorobenzene isomers to 
marine invertebrates.
    (ii) Test standards. The marine invertebrate (mysid shrimp, 
Mysidopis bahia) acute toxicity testing for 1,2,3- and 1,2,4-
trichlorobenzenes shall be conducted in accordance with Sec. 797.1930 
of this chapter.
    (iii) Reporting requirements. (A) The acute toxicity tests on marine 
invertebrates shall be completed and the final report submitted to EPA 
within 1 year

[[Page 230]]

of the effective date of the final Phase II test rule.
    (B) An interim progress report shall be submitted to the Agency 
within 6 months after the effective date of the final Phase II rule.
    (2) Marine fish acute toxicity testing--(i) Required testing. 
Testing using measured concentrations, flow through systems, and systems 
that control for evaporation of the test substance shall be conducted 
for 1,2,3-trichlorobenzene. Testing shall be conducted with Silversides 
(Menidia menidia) to develop data on the acute toxicity of 1,2,3-
trichlorobenzene to saltwater fish.
    (ii) Test standard. The marine fish (silverside minnow, Menida 
menidia) acute toxicity test shall be conducted for 1,2,3-
trichlorobenzene in accordance with Sec. 797.1400 of this chapter.
    (iii) Reporting requirements. (A) The marine fish (silversides 
minnow, Menidia menidia) acute toxicity test shall be completed and the 
final results submitted within 1 year of the effective date of the Phase 
II final test rule.
    (B) An interim progress report shall be submitted to EPA 6 months 
after the effective date of the final Phase II rule.
    (3) Freshwater fish acute toxicity testing--(i) Required testing. 
Testing using measured concentrations, flow through systems, and systems 
that control evaporation of the test substance shall be conducted for 
1,2,3-trichlorobenzene. A 96-hour LC50 test shall be conducted with the 
fathead minnow (Pimephales promelas) to develop data on the acute 
toxicity of 1,2,3-trichlorobenzene to freshwater fish.
    (ii) Test standard. The freshwater fish (fathead minnow, Pimephales 
promelas) acute toxicity test shall be conducted for 1,2,3-
trichlorobenzene in accordance with Sec. 797.1400 of this chapter.
    (iii) Reporting requirements. (A) The freshwater fish acute toxicity 
study shall be completed and the final report submitted to EPA within 1 
year of the effective date of the final Phase II test rule.
    (B) An interim progress report shall be submitted to EPA 6 months 
after the effective date of the final Phase II rule.
    (4) Freshwater invertebrate acute toxicity testing--(i) Required 
testing. Testing using measured concentrations, flow through or static 
renewal systems, and systems that control for evaporation of the test 
substance shall be conducted for 1,2,3-trichlorobenzene. A 96-hour EC50 
shall be conducted for one species of Grammarus to develop data on the 
acute toxicity of 1,2,3-trichlorobenzene to aquatic freshwater 
invertebrates.
    (ii) Test standard. The freshwater invertebrate (Gammarus sp.) acute 
toxicity test shall be conducted for 1,2,3-trichlorobenzene in 
accordance with Sec. 795.120 of this chapter.
    (iii) Reporting requirements. (A) The freshwater invertebrate acute 
toxicity test shall be completed and the final report submitted to EPA 
within 411 days of the effective date of the final Phase II rule.
    (B) An interim progress report shall be submitted to EPA 6 months 
after the effective date of the final Phase II rule.
    (5) Mysid shrimp chronic toxicity testing--(i) Required testing. 
Testing using measured concentrations, flow through or static renewal 
systems, and systems that control for evaporation of the test substance 
shall be conducted for 1,2,4-trichlorobenzene. Testing shall be 
conducted with mysid shrimp (Mysidopsis bahia) to develop data on the 
chronic toxicity of 1,2,3-trichlorobenzene, should the acute LC50 of 
this chemical to mysid shrimp be determined to be less than 1 ppm.
    (ii) Test standards. The mysid shrimp (Mysidopis bahia) chronic 
toxicity test shall be conducted for 1,2,4-trichlorobenzene in 
accordance with Sec. 797.1950 of this chapter. Testing shall also be 
conducted according to Sec. 797.1950 for 1,2,3-trichlorobenzene should 
the results of testing required by (d)(1)(ii) of this section yield an 
acute LC50 for this chemical substance of less than 1 ppm.
    (iii) Reporting requirements. (A) The mysid shrimp chronic toxicity 
test for 1,2,4-trichlorobenzene shall be completed and the final report 
submitted to EPA within 1 year of the effective date of the final Phase 
II rule. The mysid shrimp chronic toxicity test for 1,2,3-
trichlorobenzene, (required if the LC50 is less than 1 ppm), shall be 
completed and final report submitted to

[[Page 231]]

EPA within 15 months of the effective date of the final Phase II rule.
    (B) Progress reports shall be submitted to EPA at 6-month intervals, 
beginning 6 months after of the effective date of the final Phase II 
rule and until the final report is submitted to EPA.
    (e) Health effects testing--(1) Oncogenicity--(i) Required testing. 
(A) A test for oncogenic effects shall be conducted with 1,2,4-TCB in 
accordance with Sec. 798.3300 of this chapter.
    (B) The route of administration for the oncogenicity testing for 
1,2,4-TCB shall be via the animal feed.
    (C) Two rodent species shall be used and one shall be the Fischer-
344 rat.
    (ii) Reporting requirements. (A) The oncogenicity test shall be 
completed and the final results submitted to EPA by June 30, 1994.
    (B) Progress reports shall be submitted to the Agency every 6 months 
after the effective date of the final rule.
    (2) [Reserved]
    (f) [Reserved]
    (g) Effective date. (1) The effective date of the final phase II 
rule is August 14, 1987, except for paragraphs (d)(4)(iii)(A) and 
(e)(1)(ii)(A) of this section. The effective date for paragraph 
(d)(4)(iii)(A) of this section is March 1, 1990. The effective date for 
paragraph (e)(1)(ii)(A) of this section is June 12, 1992.
    (2) The guidelines and other test methods cited in this rule are 
referenced as they exist on the effective date of the final rule.

[51 FR 11737, Apr. 7, 1986; 51 FR 18444, May 20, 1986, as amended at 51 
FR 24667, July 8, 1986; 52 FR 24465, July 1, 1987; 55 FR 7327, Mar. 1, 
1990; 57 FR 24960, June 12, 1992; 57 FR 27845, June 22, 1992; 58 FR 
34205, June 23, 1993]



Sec. 799.1560  Diethylene glycol butyl ether and diethylene glycol butyl ether 

acetate.

    (a) Identification of test substances. (1) Diethylene glycol butyl 
ether (DGBE), CAS Number 112-34-5, and diethylene glycol butyl ether 
acetate (DGBA), CAS Number 124-17-4, shall be tested in accordance with 
this section.
    (2) DGBE of at least 95 percent purity and DGBA of at least 95 
percent purity shall be used as the test substances.
    (b) Persons required to submit study plans, conduct tests, and 
submit data. All persons who manufacture (including import) or process 
or intend to manufacture or process DGBE and/or DGBA, other than as an 
impurity, after April 11, 1988, to the end of the reimbursement period 
shall submit letters of intent to conduct testing, submit study plans 
and conduct tests, and submit data, or submit exemption applications as 
specified in this section, subpart A of this part, and parts 790 and 792 
of this chapter for single-phase rulemaking. Persons who manufacture or 
process DGBE are subject to the requirements to test DGBE in this 
section. Only persons who manufacture or process DGBA are subject to the 
requirements to test DGBA in this section.
    (c) Health effects testing--(1) Subchronic toxicity--(i) Required 
testing. (A) A 90-day subchronic toxicity test of DGBE shall be 
conducted in rats by dermal application in accordance with Sec. 
798.2250 of this chapter except for the provisions in paragraphs 
(e)(9)(iv), (10)(i)(A) and (ii)(B), (11) (ii) and (iii), and (12)(i) of 
Sec. 798.2250.
    (B) For the purpose of this section, the following provisions also 
apply:
    (1) A satellite group to evaluate fertility shall be established. 
Control males shall be cohabited with control females, and males and 
females administered the high dose shall be cohabited. Endpoints to be 
evaluated shall include percent mated; percent pregnant; length of 
gestation; litter size; viability at birth, on Day 4, and weaning, on 
Day 21; sex of the offspring; and litter weights at birth and Days 4, 7, 
14, and 21. Litters shall be standardized on day 4 in accordance with 
the reproductive and fertility effects guideline, Sec. 
798.4700(c)(6)(iv) of this chapter. Gross examinations shall be made at 
least once each day and physical or behavioral anomalies in the dam or 
offspring shall be recorded. At weaning, dams shall be sacrificed and 
examined for resorption sites indicative of post-implantation loss. An 
additional 20 males and 40 females will have to be added to the 
subchronic study for this test. If the animals in the high dose group 
exhibit marked toxicity (e.g. greater than 20 percent

[[Page 232]]

weight loss), then the fertility tests shall be conducted in the next 
highest dose group.
    (2) Cage-side observations shall include, but not be limited to, 
changes in skin and fur; eyes and mucous membranes; respiratory, 
circulatory autonomic, and central nervous systems; somatomotor 
activity; and behavior pattern. In addition a daily examination for 
hematuria shall be done.
    (3) Certain hematology determinations shall be carried out at least 
three times during the test period: Just prior to initiation of dosing 
(baseline data), after approximately 30 days on test, and just prior to 
terminal sacrifice at the end of the test period. Hematology 
determinations which are appropriate to all studies: Hematocrit, 
hemoglobin concentration, erythrocyte count, total and differential 
leucocyte count, mean corpuscular volume, and a platelet count.
    (4) Urinalyses shall be done at least three times during the test 
period: Just prior to initiation of dosing (baseline data), after 
approximately 30 days into the test, and just prior to terminal 
sacrifice at the end of the test period. The animals shall be kept in 
metabolism cages, and the urine shall be examined microscopically for 
the presence of erythrocytes and renal tubular cells, in addition to 
measurement of urine volume, specific gravity, glucose, protein/albumin, 
and blood.
    (5) The liver, kidney, adrenals, brain, gonads, prostate gland, 
epididymides, seminal vesicles, and pituitary gland shall be weighed 
wet, as soon as possible after dissection, to avoid drying.
    (6) The following organs and tissues, or representative samples 
thereof, shall be preserved in a suitable medium for possible future 
histopathological examination: All gross lesions; lungs--which should be 
removed intact, weighed, and treated with a suitable fixative to ensure 
that lung structure is maintained (perfusion with the fixative is 
considered to be an effective procedure); nasopharyngeal tissues; 
brain--including sections of medulla/pons, cerebellar cortex, and 
cerebral cortex; pituitary; thyroid/parathyroid; thymus; trachea; heart; 
sternum with bone marrow; salivary glands; liver; spleen; kidneys; 
adrenals; pancreas; gonads; uterus; oviducts; vagina; vas deferens; 
accessory genital organs (epididymis, prostate, and, if present, seminal 
vesicles); aorta; (skin); gall bladder (if present); esophagus; stomach; 
duodenum; jejunum; ileum; cecum; colon; rectum; urinary bladder; 
representative lymph node; (mammary gland); (thigh musculature); 
peripheral nerve; (eyes); (femur--including articular surface); (spinal 
cord at three levels--cervical, midthoracic, and lumbar); and (zymbal 
and exorbital lachrymal glands).
    (7) (i) Full histopathology on normal and treated skin and on organs 
and tissues listed in paragraph (c)(1)(i)(B)(6) of this section, as well 
as the accessory genital organs (epididymides, prostate, seminal 
vesicles) and the vagina, of all animals in the control and high dose 
groups.
    (ii) The integrity of the various cell stages of spermatogenesis 
shall be determined, with particular attention directed toward achieving 
optimal quality in the fixation and embedding; preparations of 
testicular and associated reproductive organ samples for histology 
should follow the recommendations of Lamb and Chapin (1985) under 
paragraph (d)(1) of this section, or an equivalent procedure. 
Histological analyses shall include evaluations of the spermatogenic 
cycle, i.e., the presence and integrity of the 14 cell stages. These 
evaluations should follow the guidance provided by Clermont and Perey 
(1957) under paragraph (d)(2) of this section. Information shall also be 
provided regarding the nature and level of lesions observed in control 
animals for comparative purposes.
    (iii) Data on female cyclicity shall be obtained by performing 
vaginal cytology over the last 2 weeks of dosing; the cell staging 
technique of Sadleir (1978) and the vaginal smear method in Hafez (1970) 
under paragraphs (d) (3) and (7) of this section or equivalent methods 
should be used. Data should be provided on whether the animal is cycling 
and the cycle length.
    (iv) The ovary shall be serially sectioned with a sufficient number 
of sections examined to adequately detail oocyte and follicular 
morphology. The methods of Mattison and Thorgiersson (1979) and Pederson 
and Peters (1968) under paragraphs (d) (4) and (5) of this

[[Page 233]]

section may provide guidance. The strategy for sectioning and evaluation 
is left to the discretion of the investigator, but shall be described in 
detail in the study plan and final report. The nature and background 
level of lesions in control tissue shall also be noted.
    (ii) Reporting requirements. (A) The subchronic test shall be 
completed and the final report submitted to EPA within 15 months of the 
effective date of the final test rule.
    (B) Progress reports shall be submitted to EPA every 6 months, 
beginning 6 months from the effective date of the final rule until 
submission of the final report to EPA.
    (2) Neurotoxicity/behavioral effects--(i) Required testing--(A) (1) 
Functional observational battery. A functional observational battery 
shall be performed in the rat by dermal application of DGBE for a period 
of 90 days according to Sec. 798.6050 of this chapter except for the 
provisions in paragraphs (b)(1), (d)(4)(ii), (5), and (8)(ii)(E) of 
Sec. 798.6050.
    (2) For the purpose of this section, the following provisions also 
apply:
    (i) Definition. Neurotoxicity is any adverse acute and/or lasting 
effect on the structure or function of the central and/or peripheral 
nervous system related to exposure to a chemical substance.
    (ii) Lower doses. The data from the lower doses shall show either 
graded dose-dependent effects in at least two of all the doses tested 
including the highest dose, or no neurotoxic (behavioral) effects at any 
dose tested.
    (iii) Duration and frequency of exposure. Animals shall be exposed 
for 6 hours/day, 5 days/week for a 90-day period.
    (iv) Sensory function. A simple assessment of sensory function 
(vision, audition, pain perception) shall be made. Marshall et al. 
(1971) in Sec. 798.6050(f)(8) of this chapter have described a 
neurologic exam for this purpose; these procedures are also discussed by 
Deuel (1977), under Sec. 798.6050(f)(4) of this chapter. Irwin (1968) 
under Sec. 798.6050(f)(7) of this chapter described a number of reflex 
tests intended to detect gross sensory deficits. Many procedures have 
been developed for assessing pain perception (e.g., Ankier (1974) under 
Sec. 798.6050(f)(1); D'Amour and Smith (1941) under Sec. 
798.6050(f)(3); and Evans (1971) under Sec. 798.6050(f)(6) of this 
chapter.
    (B)(1) Motor activity. A motor activity test shall be conducted in 
the rat by dermal application of DGBE for a period of 90 days according 
to Sec. 798.6200 of this chapter except for the provisions in 
paragraphs (c), (d)(3)(ii), (4)(ii), (5), (8)(i), and (iii) of Sec. 
798.6200.
    (2) For the purpose of this section, the following provisions also 
apply:
    (i) Principle of the test method. The test substance is administered 
to several groups of experimental animals, one dose being used per 
group. Measurements of motor activity are made. Where possible, the 
exposure levels at which significant changes in motor activity are 
produced are compared to those levels which produce toxic effects not 
originating in the central and/or peripheral nervous system.
    (ii) Positive control data. Positive control data are required to 
document the sensitivity of the activity measuring device and testing 
procedure. These data should demonstrate the ability to detect increases 
or decreases in activity and to generate a dose-effect curve or its 
equivalent using three values of the dose or equivalent independent 
variable. A single administration of the dose (or equivalent) is 
sufficient. It is recommended that chemical exposure be used to collect 
positive control data. Positive control data shall be collected at the 
time of the test study unless the laboratory can demonstrate the 
adequacy of historical data for this purpose.
    (iii) Lower doses. The data from the lower doses shall show either 
graded dose-dependent effects in at least two of all the doses tested 
including the highest dose, or no neurotoxic (behavioral) effects at any 
dose tested.
    (iv) Duration and frequency of exposure. Animals shall be exposed 
for 6 hours/day, 5 days/week for a 90-day period.
    (v) General. Motor activity shall be monitored by an automated 
activity recording apparatus. The device used shall be capable of 
detecting both increases and decreases in activity, i.e. baseline 
activity as measured by the

[[Page 234]]

device shall not be so low as to preclude decreases nor so high as to 
preclude increases. Each device shall be tested by a standard procedure 
to ensure, to the extent possible, reliability of operation across 
devices and across days for any one device. In addition, treatment 
groups shall be balanced across devices. Each animal shall be tested 
individually. The test session shall be long enough for motor activity 
to approach asymptotic levels by the last 20 percent of the session for 
most treatments and for the session control animals. All sessions should 
be of the same duration. Treatment groups shall be counter-balanced 
across test times. Effort should be made to ensure that variations in 
the test conditions are minimal and are not systematically related to 
treatment. Among the variables which can affect motor activity are sound 
level, size and shape of the test cage, temperature, relative humidity, 
lighting conditions, odors, use of home cage or novel test cage, and 
environmental distractions. Tests shall be executed by an appropriately 
trained individual.
    (vi) Subchronic. All animals shall be tested prior to initiation of 
exposure and at 30 4, 60 4, 
and 90 4 days during the exposure period. Testing 
shall occur prior to the daily exposure. Animals shall be weighed on 
each test day and at least once weekly during the exposure period.
    (C)(1) Neuropathology. A neuropathology test shall be conducted in 
the rat by dermal application of DGBE for a period of 90 days according 
to Sec. 798.6400 of this chapter except for the provisions in 
paragraphs (d)(4)(ii), (5), (8)(iv)(C), and (E)(2) of Sec. 798.6400.
    (2) For the purpose of this section, the following provisions also 
apply:
    (i) Lower doses. The data from the lower doses shall show either 
graded dose-dependent effects in at least two of all the doses tested 
including the highest dose, or no neurotoxic (behavioral) effects at any 
dose tested.
    (ii) Duration and frequency of exposure. Animals shall be exposed 
for 6 hours/day, 5 days/week for a 90-day period.
    (iii) Clearing and embedding. After dehydration, tissue specimens 
shall be cleared with xylene and embedded in paraffin or paraplast 
except for the sural nerve which should be embedded in plastic. Multiple 
tissue specimens (e.g. brain, cord, ganglia) may be embedded together in 
one single block for sectioning. All tissue blocks shall be labeled to 
provide unequivocal identification. A method for plastic embedding is 
described by Spencer et al. in paragraph (d)(6) of this section.
    (iv) Special stains. Based on the results of the general staining, 
selected sites and cellular components shall be further evaluated by the 
use of specific techniques. If hematoxylin and eosin screening does not 
provide such information, a battery of stains shall be used to assess 
the following components in all appropriate required samples: Neuronal 
body (e.g., Einarson's gallocyanin), axon (e.g., Bodian), myelin sheath 
(e.g., Kluver's Luxol Fast Blue), and neurofibrils (e.g., Bielchosky). 
In addition, peripheral nerve fiber teasing may be used. Detailed 
staining methodology is available in standard histotechnological manuals 
such as Armed Forces Institute of Pathology (AFIP) (1968) under Sec. 
798.6400(f)(1), Ralis et al. (1973) under Sec. 798.6400(f)(5), and 
Chang (1979) under Sec. 798.6400(f)(2) of this chapter. The nerve fiber 
teasing technique is discussed in Spencer and Schaumberg (1980) under 
Sec. 798.6400(f)(6) of this chapter. A section of normal tissue shall 
be included in each staining to assure that adequate staining has 
occurred. Any changes shall be noted and representative photographs 
shall be taken. If a lesion(s) is observed, the special techniques shall 
be repeated in the next lower treatment group until no further lesion is 
detectable.
    (ii) Reporting requirements. (A) The neurotoxicity/behavioral tests 
required under paragraph (c)(2) of this section shall be completed and 
the final reports submitted to EPA within 17 months of the effective 
date of the final rule.
    (B) Interim progress reports shall be submitted to EPA at 6-month 
intervals, beginning 6 months from the effective date of the final rule 
until submission of the applicable final report to EPA.
    (3) Developmental neurotoxicity--(i) Required testing. A 
developmental neurotoxicity test of DGBE shall be

[[Page 235]]

conducted after a public program review of the Tier I data from the 
functional observational battery, motor activity, and neuropathology 
tests in paragraph (c)(2) of this section, and the reproductive tests in 
paragraph (c)(1) of this section, and if EPA issues a Federal Register 
notice or sends a certified letter to the test sponsor specifying that 
the testing shall be initiated. The test shall be performed in rats in 
accordance with Sec. 795.250 of this chapter.
    (ii) Reporting requirements. (A) The developmental neurotoxicity 
test shall be completed and the final report submitted to EPA within 15 
months of EPA's notification of the test sponsor by certified letter or 
Federal Register notice under paragraph (c)(3)(i) of this section that 
the testing shall be initiated.
    (B) Progress reports shall be submitted to EPA every 6 months, 
beginning 6 months after the date of notification that the testing shall 
be initiated, until submission of the final report to EPA.
    (4) Pharmacokinetics--(i) Required testing. (A) Pharmacokinetics 
testing of DGBE and DGBA will be conducted in rats by the dermal route 
of administration in accordance with Sec. 795.225 of this chapter, 
except for the provisions in paragraphs (b) (1)(ii) and (3)(i) of Sec. 
795.225.
    (B) For the purpose of this section, the following provisions also 
apply:
    (1) Animals. Adult male and female Sprague Dawley rats shall be 
used. The rats shall be 7 to 8 weeks old and weigh 180 to 220 grams. 
Prior to testing, the animals shall be selected at random for each 
group. Animals showing signs of ill health shall not be used.
    (2) Observation of animals--Urinary and fecal excretion. The 
quantities of \14\C excreted in urine and feces by rats dosed as 
specified in paragraph (b)(2)(iv) of Sec. 795.225 shall be determined 
at 8, 24, 48, 72, and 96 hours after dosing, and if necessary, daily 
thereafter until at least 90 percent of the dose has been excreted or 
until 7 days after dosing (whichever occurs first). Four animals per sex 
per dose group shall be used for this purpose.
    (ii) Reporting requirements. (A) The pharmacokinetics tests shall be 
completed and the final reports submitted to EPA within 8 months of the 
effective date of the final amendment.
    (B) A progress report shall be submitted to EPA 6 months from the 
effective date of the final amendment.
    (d) References. For additional background information the following 
references should be consulted:
    (1) Lamb, J.C. and Chapin, R.E. ``Experimental models of male 
reproductive toxicology.'' In: ``Endocrine Toxicology.'' Thomas, J.A., 
Korach, K.S., and McLachlan, J.A., eds. New York, NY: Raven Press. pp. 
85-115. (1985).
    (2) Clermont, Y. and Perey, B. ``Quantitative study of the cell 
population of the seminiferous tubules in immature rats.'' American 
Journal of Anatomy. 100:241-267. (1957).
    (3) Sadleir, R.M.F.S. ``Cycles and seasons.'' In: ``Reproduction in 
Mammals: I. Germ Cells and Fertilization.'' Austin, C.R. and Short, 
R.V., eds. New York, NY: Cambridge Press. Chapter 4. (1978).
    (4) Mattison, D.R. and Thorgiersson, S.S. ``Ovarian aryl hydrocarbon 
hydroxylase activity and primordial oocyte toxicity of polycyclic 
aromatic hydrocarbons in mice.'' Cancer Research. 39:3471-3475. (1979).
    (5) Pederson, T. and Peters, H. ``Proposal for classification of 
oocytes and follicles in the mouse ovary. Journal of Reproduction and 
Fertility. 17:555-557. (1968).
    (6) Spencer, P.S., Bischoff, M.C., and Schaumburg, H.H. 
``Neuropathological methods for the detection of neurotoxic disease.'' 
In: ``Experimental and Clinical Neurotoxicology.'' Spencer, P.S. and 
Schaumburg, H.H., eds. Baltimore, MD: Williams & Wilkins, pp. 743-757. 
(1980).
    (7) Hafez, E.S., ed., ``Reproduction and Breeding Techniques for 
Laboratory Animals.'' Chapter 10. Philadelphia: Lea & Febiger (1970).
    (e) Effective date. (1) The effective date of the final rule is 
April 11, 1988, except for paragraph (c)(2)(ii)(A) of this section. The 
effective date for paragraph (c)(2)(ii)(A) of this section is March 1, 
1990. The effective date for paragraphs (c)(4)(ii)(A) and (c)(4)(ii)(B) 
of this section is November 27, 1989.

[[Page 236]]

    (2) The guidelines and other test methods cited in this rule are 
referenced as they exist on the effective date of the final rule.

[53 FR 5950, Feb. 26, 1988, as amended at 54 FR 27357, June 29, 1989; 54 
FR 41835, Oct. 12, 1989; 55 FR 7326, Mar. 1, 1990; 58 FR 34205, June 23, 
1993]



Sec. 799.1575  Diethylenetriamine (DETA).

    (a) Identification of chemical test substance. (1) 
Diethylenetriamine (CAS No. 111-40-0, also known as DETA) shall be 
tested in accordance with this part.
    (2) Diethylenetriamine of at least 99 percent purity shall be used 
as the test substances in all tests.
    (b) Persons required to submit study plans, conduct tests and submit 
data. All persons who manufacture or process diethylenetriamine from 
July 8, 1985, to the end of the reimbursement period shall submit 
letters of intent to test, exemption applications, and study plans and 
shall conduct tests and submit data as specified in this section, 
subpart A of this part and part 790 of this chapter (Test Rule 
Development and Exemption Procedures).
    (c) Health effects testing--(1) Mutagenic effects--Gene mutation--
(i) Required testing. (A) A sex-linked recessive lethal test in 
Drosophila melanogaster shall be conducted with DETA.
    (B) A mouse specific locus assay shall be conducted with DETA, if 
the sex-linked recessive lethal test in Drosophila melanogaster 
conducted pursuant to paragraph (c)(1)(i)(A) of this section produces a 
positive result.
    (ii) Test standards. (A) The testing for the sex-linked recessive 
lethal assay shall be conducted in accordance with the following revised 
EPA-approved modified study plan (June 19, 1986) originally submitted by 
the Diethylenetriamine Producers/Importers Alliance (DPIA): ``Sex-linked 
recessive lethal test in Drosophila melanogaster,'' with modifications 
as approved by EPA on March 9, 1987, and May 21, 1987.
    (B) The testing for the mouse visible specific locus assay shall be 
conducted in accordance with the following revised EPA-approved modified 
study plan (June 19, 1986) originally submitted by the 
Diethylenetriamine Producers/Importers Alliance (DPIA): ``Mouse specific 
locus test for visible markers.''
    (C) These revised EPA-approved modified study plans are available 
for inspection in the Non-Confidential Information Center (NCIC) (7407), 
Office of Pollution Prevention and Toxics, U.S. Environmental Protection 
Agency, Room B-607 NEM, 401 M St., SW., Washington, DC 20460, between 
the hours of 12 p.m. and 4 p.m. weekdays excluding legal holidays.
    (iii) Reporting requirements. (A) The sex-linked recessive lethal 
test of DETA in Drosophila melanogaster shall be completed and a final 
report submitted to the Agency within 14 months from the effective date 
of the final Phase II rule. Two interim progress reports shall be 
submitted at 6-month intervals, the first of which is due within 6 
months of the effective date of the final Phase II rule.
    (B) If required pursuant to paragraph (c)(1)(i)(B) of this section, 
the mouse specific locus test of DETA for visible markers shall be 
completed and a final report submitted to the Agency within 48 months 
from the designated date contained in EPA's notification of the test 
sponsor by certified letter or Federal Register notice that testing 
should be initiated. Seven interim progress reports shall be submitted 
at 6-month intervals, the first of which is due within 6 months of EPA's 
designated date.
    (2) Mutagenic effects--Chromosomal aberrations--(i) Required 
testing. (A) An in vitro cytogenetics test shall be conducted with DETA.
    (B) An in vivo cytogenetics test shall be conducted with DETA, if 
the in vitro cytogenetics test conducted pursuant to paragraph 
(c)(2)(i)(A) of this section produces a negative result.
    (C) A dominant lethal assay shall be conducted with DETA, if either 
the in vitro cytogenetics test conducted pursuant to paragraph 
(c)(2)(i)(A) of this section or the in vivo cytogenetics test conducted 
pursuant to paragraph (c)(2)(i)(B) of this section produces a positive 
result.
    (D) A heritable translocation assay shall be conducted with DETA, if 
the dominant lethal assay conducted pursuant to paragraph (c)(2)(i)(C) 
of this section produces a positive result.

[[Page 237]]

    (ii) Test standards. (A) The testing for cytogenetic effects shall 
be conducted in accordance with the following revised EPA-approved 
modified study plan (June 19, 1986) originally submitted by the 
Diethylenetriamine Producers/Importers Alliance (DPIA): ``In vitro 
cytogenetics test'' and ``In vivo cytogenetics test,'' with 
modifications as approved by EPA on March 9, 1987, and May 21, 1987.
    (B) Other testing for cytogenetic effects shall be conducted in 
accordance with the following revised EPA-approved modified study plans 
(June 19, 1986) originally submitted by the Diethylenetriamine 
Producers/Importers Alliance (DPIA): ``Dominant lethal assay of 
diethylenetriamine in CD rats,'' and ``Heritable translocation of 
diethylenetriamine in CD-1 mice.''
    (C) These revised EPA-approved modified study plans are available 
for inspection in the Non-Confidential Information Center (NCIC) (7407), 
Office of Pollution Prevention and Toxics, U.S. Environmental Protection 
Agency, Room B- 607 NEM, 401 M St., SW., Washington, DC 20460, between 
the hours of 12 p.m. and 4 p.m. weekdays excluding legal holidays.
    (iii) Reporting requirements. (A) The in vitro cytogenetics testing 
of DETA shall be completed and a final report submitted to the Agency 
within 6 months of the effective date of the final Phase II rule.
    (B) If required pursuant to paragraph (c)(2)(i)(B) of this section, 
the in vivo cytogenetics testing of DETA shall be completed and final 
report submitted to the Agency within 14 months of the effective date of 
the final Phase II rule. One interim progress report shall be submitted 
within 12 months of the final rule's effective date.
    (C) If required pursuant to paragraph (c)(2)(i)(C) of this section, 
the dominant lethal testing of DETA shall be completed and a final 
report submitted to the Agency within 20 months of the effective date of 
the final Phase II rule.
    (D) If required pursuant to paragraph (c)(2)(i)(D) of this section, 
the heritable translocation testing of DETA shall be completed and a 
final report submitted to the Agency within 18 months of the designated 
date contained in EPA's notification of the test sponsor by certified 
letter or Federal Register notice that testing should be initiated. Two 
interim progress reports shall be submitted at 6-month intervals, the 
first of which is due within 6 months of EPA's designated date.
    (3) Subchronic effects--(i) Required testing. A ninety-day oral 
subchronic toxicity test shall be conducted with DETA in at least one 
mammalian species.
    (ii) Test standard. The testing shall be conducted in accordance 
with the following revised EPA-approved modified study plans (June 19, 
1986) originally submitted by the Diethylenetriamine Producers/Importers 
Alliance (DPIA): ``Ninety-Day (subchronic) dietary toxicity study with 
diethylenetriamine in albino rats,'' with modifications approved by EPA 
on March 9, 1987, and May 21, 1987. This revised EPA-approved modified 
study plans is available for inspection in the Non-Confidential 
Information Center (NCIC) (7407), Office of Pollution Prevention and 
Toxics, U.S. Environmental Protection Agency, Room B-607 NEM, 401 M St., 
SW., Washington, DC 20460, between the hours of 12 p.m. and 4 p.m. 
weekdays excluding legal holidays.
    (iii) Reporting requirements. The testing shall be completed and a 
final report submitted to the Agency within 15 months of the effective 
date of the final Phase II rule. Two interim progress reports shall be 
submitted at 6-month intervals, the first of which is due within 6 
months of the effective date of the final Phase II rule.
    (d) Chemical fate testing--(1) Required testing. Testing to assess 
N-nitrosamine formation, resulting from aerobic biological and/or 
chemical transformation, shall be conducted with DETA using 
environmental samples of lake water, sewage, and soil.
    (2) Test standard. The testing shall be conducted in accordance with 
the following revised EPA-approved modified study plan (June 7, 1990) 
originally submitted by the Diethylenetriamine Producers/Importers 
Alliance (DPIA): ``Modified Final Copy (04-17-90); Diethylenetriamine: 
Environmental Fate in Sewage, Lake Water and Soil''. This revised EPA-
approved modified study plans are available for inspection in the Non-
Confidential Information

[[Page 238]]

Center (NCIC) (7407), Office of Pollution Prevention and Toxics, U.S. 
Environmental Protection Agency, Room B-607 NEM, 401 M St., SW., 
Washington, DC 20460, between the hours of 12 p.m. and 4 p.m. weekdays 
excluding legal holidays.
    (3) Reporting requirements. The testing shall be completed and a 
final report submitted to EPA within 20 months of the effective date of 
the final Phase II rule. Interim progress reports shall be submitted at 
6-month intervals, the first of which is due within 6 months of the 
effective date of the final Phase II rule.
    (e) Modifications. Persons subject to this section are not subject 
to the requirements of Sec. 790.50(a)(2)(ii) of this chapter.
    (f) Effective date. (1) The effective date of the final Phase II 
rule for diethylenetriamine is March 19, 1987, except for paragraphs 
(c)(4)(iii), (d)(2), and (d)(3) of this section. The effective date of 
paragraphs (c)(4)(iii), and (d)(3) of this section is March 1, 1990. The 
effective date for paragraph (d)(2) of this section is May 21, 1991.
    (2) The guidelines and other test methods cited in this rule are 
referenced as they exist on the effective date of the final rule.

[50 FR 21412, May 23, 1985; 50 FR 33543, Aug. 20, 1985; 51 FR 3468, Jan. 
28, 1986; 51 FR 4736, Feb. 7, 1986; 52 FR 3238, Feb. 3, 1987; 54 FR 
27356, June 29, 1989; 55 FR 3408, Feb. 1, 1990; 55 FR 7326, Mar. 1, 
1990; 56 FR 23230, May 21, 1991; 58 FR 34205, June 23, 1993; 60 FR 
34467, July 3, 1995]



Sec. 799.1645  2-Ethylhexanol.

    (a) Identification of test substance. (1) 2-Ethylhexanol (CAS No. 
104-76-7) shall be tested in accordance with this section.
    (2) 2-Ethylhexanol of at least 99.0-percent purity shall be used as 
the test substance.
    (b) Persons required to submit study plans, conduct tests, and 
submit data. All persons who manufacture or process, or intend to 
manufacture or process 2-ethylhexanol, other than as an impurity, from 
the effective date of this final rule to the end of the reimbursement 
period shall submit letters of intent to conduct testing, submit study 
plans, conduct tests, and submit data or exemption applications as 
specified in this section, subpart A of this part, and parts 790 and 792 
of this chapter for single-phase rulemaking.
    (c) Health effects--(1) Oncogenic effects--(i) Required testing. (A) 
Oncogenicity tests shall be conducted in Fisher 344 rats and B6C3Fl mice 
by the oral route with 2-ethylhexanol in accordance with Sec. 798.3300 
of this chapter, except for the provisions in Sec. 798.3300(b)(6).
    (B) For the purpose of this section, the following provisions also 
apply to the oncogenicity tests: (1) Administration of the test 
substance. 2-Ethylhexanol shall be administered either by 
microencapsulation before adding it to the diet or by gavage.
    (2) [Reserved]
    (ii) Reporting requirements. (A) The study plan for the oncogenicity 
test shall be submitted at least 45 days before the initiation of 
testing.
    (B) The oncogenicity testing shall be completed and final report 
submitted to the Agency within 53 months of the effective date of this 
final rule if 2-ethylhexanol is administered by gavage or within 56 
months of the effective date of this final rule if administered by 
microencapsulation.
    (C) Interim progress reports shall be submitted to EPA at 6-month 
intervals beginning 6 months after the effective date of the final rule, 
until the final report is submitted to EPA.
    (2) [Reserved]
    (d) Effective date. The effective date of this final rule requiring 
oncogenicity testing of 2-ethylhexanol is September 16, 1987.

[52 FR 28704, Aug. 3, 1987, as amended at 58 FR 34205, June 23, 1993]



Sec. 799.1700  Fluoroalkenes.

    (a) Identification of test substances. (1) Vinyl fluoride (VF; CAS 
No. 75-02-5), vinylidene fluoride (VDF; CAS No. 75-38-7), 
tetrafluoroethene (TFE; CAS No. 116-14-3), and hexafluoropropene (HFP; 
CAS No. 116-15-4) shall be tested in accordance with this section.
    (2) VF, VDF, TFE, and HFP of at least 99 percent purity shall be 
used as the test substances.
    (b) Persons required to submit study plans, conduct tests and submit 
data. All persons who manufacture VF, VDF,

[[Page 239]]

TFE, or HFP, other than as an impurity, from July 22, 1987 to the end of 
the reimbursement period shall submit letters of intent to conduct 
testing or exemption applications, submit study plans, conduct tests in 
accordance with the TSCA Good Laboratory Practice Standards (40 CFR part 
792), and submit data as specified in this section, subpart A of this 
part, and part 790 of this chapter for single-phase rulemaking, for the 
substances they manufacture.
    (c) Health effects testing--(1) Mutagenic effects--Gene mutation--
(i) Required testing. (A) (1) A detection of gene mutations in somatic 
cells in culture assay shall be conducted with TFE and HFP in accordance 
with Sec. 798.5300 of this chapter except for the provisions in 
paragraphs (c), (d)(3)(i), (4), (5) and (6) and (e).
    (2) For the purposes of this section, the following provisions also 
apply:
    (i) Reference substances. No reference substance is required.
    (ii) Test method--Type of cells used in the assay. Mutation 
induction at the HPRT locus shall be measured in Chinese hamster ovary 
(CHO) cells. Cells shall be checked for Mycoplasma contamination and may 
also be checked for karyotype stability.
    (iii) Test method--Metabolic activation. Cells shall be exposed to 
the test substance only in the presence of a metabolic activation system 
for TFE, and in both the presence and absence of a metabolic activation 
system for HFP. The metabolic activation system shall be derived from 
the post-mitochondrial fraction (S-9) of livers from rats pretreated 
with Aroclor 1254.
    (iv) Test method--Control groups. Positive and negative controls 
shall be included in each experiment. In assays with metabolic 
activation, the positive control substance shall be known to require 
such activation. Nitrogen shall serve as the negative control and 
diluting gas.
    (v) Test method--Test chemicals. The test should be designed to have 
a predetermined sensitivity and power. The number of cells, cultures, 
and concentrations of test substance used should reflect these defined 
parameters. The number of cells per culture is based on the expected 
background mutant frequency; a general guide is to use a number which is 
10 times the inverse of this frequency. Several concentrations (usually 
at least four) of the test substance shall be used. These shall yield a 
concentration-related toxic effect. The highest concentration shall 
produce a low level of survival (approximately 10 percent), and the 
survival in the lowest concentration shall approximate that of the 
negative control. Cytotoxicity shall be determined after treatment with 
the test substance both in the presence and in the absence of the 
metabolic activation system.
    (vi) Test performance. Cells in treatment medium with and without 
metabolic activation shall be exposed to varying concentrations of test 
gas-air mixtures by flushing treatment flasks (or chambers) with 10 
volumes of test gas-air mixture at a rate of 500 mL/min or that rate 
which will allow complete flushing within 1 minute. In the case of a 
test chamber volume of 1.67 L, a flow rate of 10 L/min is appropriate. 
Each flask shall be closed with a cap with a rubber septum. Headspace 
samples shall be taken at the beginning and end of the exposure period 
and analyzed to determine the amount of test gas in each flask. Flasks 
shall be incubated on a rocker panel at 37 [deg] C for 5 hours for tests 
with metabolic activation. For the non-activated portion of the test, 
the incubation time shall be 18 to 19 hours at 37 [deg] C. At the end of 
the exposure period, cells treated with metabolic activation shall be 
washed and incubated in culture medium for 21 to 26 hours prior to 
subculturing the viability and expression of mutant phenotype. Cells 
treated without metabolic activation shall be washed and subcultured 
immediately to determine viability and to allow for expression of mutant 
phenotype. Appropriate subculture schedules (generally twice during the 
expression period) shall be used. At the end of the expression period, 
which shall be sufficient to allow near optimal phenotypic expression of 
induced mutants (generally 7 days for this cell system), cells shall be 
grown in medium with and without selective agent

[[Page 240]]

for determination of numbers of mutants and cloning efficiency, 
respectively. This last growth period is generally 7 days at 37 [deg] C. 
Results of this test shall be confirmed in an independent experiment.
    (B)(1) A sex-linked recessive lethal test in Drosophila melanogaster 
shall be conducted with VDF and VF in accordance with Sec. 798.5275 of 
this chapter except for the provisions in paragraph (d)(5). This test 
shall also be performed with TFE or HFP if the somatic cells in culture 
assay conducted pursuant to paragraph (c)(1)(i)(A) of this section 
produces a positive result.
    (2) For the purposes of this section the following provisions also 
apply:
    (i) Test chemicals. It is sufficient to test a single dose of the 
test substance. This dose shall be the maximum tolerated dose or that 
which produces some indication of toxicity. Exposure shall be by 
inhalation.
    (ii) [Reserved]
    (C)(1) A mouse visible specific locus assay (MVSL) shall be 
conducted with VF, VDF, TFE, and HFP in accordance with Sec. 798.5200 
of this chapter, except for the provisions of paragraph (d)(5) of Sec. 
798.5200, or a mouse biochemical-specific locus assay (MBSL) shall be 
conducted with VF, VDF, TFE, and HFP in accordance with Sec. 798.5195 
of this chapter, except for the provisions of paragraph (d)(5) of Sec. 
798.5195, for whichever of these substances produces a positive test 
result in the sex-linked recessive lethal test in Drosophila 
melanogaster conducted pursuant to paragraph (c)(1)(i)(B) of this 
section if, after a public program review, EPA issues a Federal Register 
notice or sends a certified letter to the test sponsor specifying that 
the testing shall be initiated.
    (2) For the purposes of this section, the following provisions also 
apply:
    (i) Test chemicals. A minimum of two dose levels shall be tested. 
The highest dose tested shall be the highest dose tolerated without 
toxic effects, provided that any temporary sterility induced due to 
elimination of spermatagonia is of only moderate duration, as determined 
by a return of males to fertility within 80 days after treatment, or 
shall be the highest dose attainable. Animals shall be exposed to the 
test substance by inhalation. Exposure shall be for 6 hours a day. 
Duration of exposure shall be dependent upon accumulated total dose 
desired for each group.
    (ii) [Reserved]
    (ii) Reporting requirements. (A) Mutagenic effects-gene mutation 
tests shall be completed and the final reports shall be submitted to EPA 
as follows: Somatic cells in culture assay, within 6 months after the 
effective date of the final rule; Drosophila sex-linked recessive 
lethal, within 9 months (for VF and VDF) and within 15 months (for TFE 
and HFP) after the effective date of the final rule; MVSL or MBSL, 
within 51 months after the date of EPA's notification of the test 
sponsor by certified letter or Federal Register notice that testing 
shall be initiated.
    (B) Progress reports shall be submitted to the Agency every 6 months 
beginning 6 months after the effective date of the final rule or receipt 
of notice that testing shall be initiated.
    (2) Mutagenic effects--Chromosomal aberrations--(i) Required 
testing. (A)(1) A mouse micronucleus cytogenetics test shall be 
conducted with VDF and TFE in accordance with Sec. 798.5395 of this 
chapter except for the provisions in paragraphs (d)(5) (i), (ii), and 
(iii).
    (2) For the purposes of this section, the following provisions also 
apply:
    (i) Test method--Vehicle. No vehicle is required.
    (ii) Test method--Dose levels. Three dose levels shall be used. The 
highest dose tested shall be the maximum tolerated dose, that dose 
producing some indication of cytotoxicity (e.g., a change in the ratio 
of polychromatic to normochromatic erythrocytes, or the highest dose 
attainable).
    (iii) Test method--route of administration. Animals shall be exposed 
by inhalation with a single 6-hour exposure, with three sampling times 
between 20 and 72 hours.
    (B)(1) For each respective test substance, a dominant lethal assay 
shall be conducted with VF and HFP in accordance with Sec. 798.5450 of 
this chapter except for the provisions in paragraphs (d)(2)(i), (4) (i), 
(5) and (e). This test shall also be performed with TFE or VDF if the 
mouse micronucleus cytogenetics test conducted pursuant to

[[Page 241]]

paragraph (c)(2)(i)(A) of this section produces a positive result.
    (2) For the purposes of this section, the following provisions also 
apply:
    (i) Test method--Description. For this assay, the test substance 
shall be administered by inhalation for 5 consecutive days for 6 hours 
per day.
    (ii) Test method--Concurrent controls. Concurrent positive and 
negative (vehicle) controls shall be included in each experiment.
    (iii) Test method--Test chemicals. Exposure shall be by inhalation 
for 5 consecutive days for 6 hours per day. Three dose levels shall be 
used. The highest dose shall produce signs of toxicity (e.g., slightly 
reduced fertility) or shall be the highest attainable.
    (iv) Test performance. Individual males shall be mated sequentially 
to 1 or 2 virgin females. Females shall be left with the males for at 
least the duration of one estrus cycle or alternatively until mating has 
occurred as determined by the presence of sperm in the vagina or by the 
presence of a vaginal plug. In any event, females shall be left with the 
males for no longer than 7 days. The number of matings following 
treatment shall ensure that germ cell maturation is adequately covered. 
Mating shall continue for at least 6 weeks. Females shall be sacrificed 
in the second half of pregnancy, and uterine contents shall be examined 
to determine the number of implants and live and dead embryos. The 
examination of ovaries to determine the number of corpora lutea is left 
to the discretion of the investigator.
    (C)(1) A heritable translocation assay shall be conducted with VF, 
VDF, TFE, or HFP in accordance with Sec. 798.5460 of this chapter 
except for the provisions of paragraphs (d)(3)(i), (5), and (e)(1), if 
the dominant lethal assay conducted for that substance pursuant to 
paragraph (c)(2)(i)(B) of this section produces a positive result and 
if, after a public program review, EPA issues a Federal Register notice 
or sends a certified letter to the test sponsor specifying that the 
testing shall be initiated.
    (2) For the purposes of this section, the following provisions also 
apply:
    (i) Test method--Animal selection. The mouse shall be used as the 
test species.
    (ii) Test method. No vehicle is required. At least two dose levels 
shall be used. The highest dose level shall result in toxic effects 
(which shall not produce an incidence of fatalities which would preclude 
a meaningful evaluation) or shall be the highest dose attainable. 
Animals shall be exposed by inhalation.
    (iii) Test performance--Treatment and mating. The animals shall be 
dosed with the test substance 6 hours per day, 7 days per week over a 
period of 35 days. After treatment, each male shall be caged with 2 
untreated females for a period of 1 week. At the end of 1 week, females 
shall be separated from males and caged individually. When females give 
birth, the date of birth, litter size and sex of progeny shall be 
recorded. All male progeny shall be weaned and all female progeny shall 
be discarded.
    (ii) Reporting requirements. (A) Mutagenic effects-chromosomal 
aberration testing shall be completed and final results submitted to EPA 
after the effective date of the rule as follows: mouse micronucleus 
cytogenetics for VDF by November 22, 1988, and for TFE within 10 months 
after the effective date of the final rule; dominant lethal assay for VF 
and HFP by October 22, 1988, and for VDF and TFE within 19 months after 
the effective date of the rule; heritable translocation assay, within 25 
months after the date of EPA's notification of the test sponsor by 
certified letter or Federal Register notice that testing shall be 
initiated.
    (B) Progress reports shall be submitted to the Agency every 6 months 
beginning 6 months after the effective date of the final rule or receipt 
of notice that testing shall be initiated.
    (3) Subchronic toxicity--(i) Required Testing. (A) An inhalation 
subchronic toxicity test shall be conducted with HFP in accordance with 
the TSCA Test Guideline specified in Sec. 798.2450 of this chapter 
except for the provisions in paragraphs (d)(5), (10)(v), and 
(e)(3)(iv)(D).
    (B) For the purpose of this section the following provisions also 
apply:
    (1) Test procedures--Exposure conditions. The animals shall be 
exposed to the test substance 6 hours per day, 5 days per week for 90 
days.

[[Page 242]]

    (2) Test procedures--Observation of animals. Animals shall be 
weighted weekly, and food and water consumption shall also be measured 
weekly.
    (3) Test report--Individual animal data. Food and water consumption 
data shall be reported.
    (ii) Reporting requirements. (A) The required subchronic toxicity 
test shall be completed and final results submitted to the Agency within 
18 months after the effective date of the final rule.
    (B) Progress reports shall be submitted to the Agency every 6 months 
beginning 6 months after the effective date of the final rule.
    (4) Oncogenicity--(i) Required testing. (A) (1) Oncogenicity tests 
shall be conducted in both rats and mice by inhalation with VF in 
accordance with Sec. 798.3300 of this chapter, except for the 
provisions in paragraph (b)(7)(vi) of Sec. 798.3300.
    (2) For the purposes of this section, the following provisions also 
apply:
    (i) Test procedures--observations of animals. All mice of test 
groups in which survival is approximately 25 percent of mice at risk 
(approximately 25 percent of 70, or approximately 18 mice) will be 
sacrificed near the time that 25 percent survival is achieved. All mice 
surviving the 18-month test period will be sacrificed and necropsied. 
The order of sacrifice for mice at all pathological evaluations will be 
random among all exposure groups within a sex. Moribund animals should 
be removed and sacrificed when noticed.
    (ii) All rats of test groups in which survival is approximately 25 
percent of rats at risk (approximately 25 percent of 60, or 
approximately 15 rats) will be sacrificed near the time that 25 percent 
survival is achieved. All rats surviving the 24-month test period will 
be sacrificed and necropsied. The order of sacrifice for rats at all 
pathological evaluations will be random among all exposure groups within 
a sex. Moribund animals should be removed and sacrificed when noticed.
    (B) Oncogenicity testing shall be conducted in mice with VDF in 
accordance with Sec. 798.3300 of this chapter.
    (C) [Reserved]
    (D) Oncogenicity tests shall also be conducted by inhalation in both 
rats and mice with TFE in accordance with Sec. 798.3300 of this chapter 
if TFE yields a positive test result in any one of the following 
mutagenicity tests: The in vitro cytogenetics assay conducted pursuant 
to paragraph (c)(2)(i)(A) of this section, the mouse micronucleus 
cytogenetics assay conducted pursuant to paragraph (c)(2)(i)(B) of this 
section, the mammalian cells in culture assay conducted pursuant to 
paragraph (c)(1)(i)(A) of this section or the sex-linked recessive 
lethal assay in Drosophila melanogaster conducted pursuant to paragraph 
(c)(1)(i)(B) of this section if, after a public program review, EPA 
issues a Federal Register notice or sends a certified letter to the test 
sponsor specifying that the testing shall be initiated. Criteria for 
positive test results are established in 40 CFR 798.5375, 798.5385, 
798.5300 and 798.5275 of this chapter, respectively.
    (ii) Reporting requirements. (A) The oncogenicity testing for VDF 
shall be completed and the final results submitted to the Agency by 
March 23, 1992. The oncogenicity testing for VF shall be completed and 
the final results submitted to the Agency by July 22, 1992. For TFE and 
HFP, the oncogenicity testing shall be completed and the final results 
submitted to the Agency within 56 months after the date of EPA's 
notification of the test sponsor by certified letter or Federal Register 
notice that testing shall be initiated.
    (B) Progress reports shall be submitted every 6 months beginning 6 
months after the effective date of the final rule for VF and VDF and 
beginning 6 months after notification by certified letter or Federal 
Register notice that testing is to begin for TFE and HFP.
    (d) Effective date. (1) The effective date of the final rule is July 
22, 1987, except for paragraphs (c)(1)(i)(C)(1), (c)(1)(ii)(A), 
(c)(4)(i) and(c)(4)(ii)(A) of this section. The effective date of 
paragraphs (c)(1)(i)(C)(1) and (c)(1)(ii)(A) of this section is May 21, 
1990. The effective date of paragraphs (c)(4)(i)(A)(1) 
(c)(4)(i)(A)(2)(i), (c)(4)(i)(B) and (c)(4)(i)(D) of this section is May 
21, 1991. The effective date for paragraphs (c)(4)(i)(A)(2)(ii) and 
(c)(4)(i)(C) of this section is June 12, 1992. The effective

[[Page 243]]

date of paragraph (c)(4)(ii)(A) of this section is May 28, 1993.
    (2) The guidelines and other test methods cited in this rule are 
referenced as they exist on the effective date of the final rule.

[52 FR 21530, June 8, 1987, as amended at 52 FR 43762, Nov. 16, 1987; 54 
FR 27357, June 29, 1989; 54 FR 33148, Aug. 11, 1989; 55 FR 12643, Apr. 
5, 1990; 56 FR 23230, May 21, 1991; 57 FR 24960, June 12, 1992; 58 FR 
30992, May 28, 1993; 58 FR 34205, June 23, 1993]



Sec. 799.2155  Commercial hexane.

    (a) Identification of test substance. (1) ``Commercial hexane,'' for 
purposes of this section, is a product obtained from crude oil, natural 
gas liquids, or petroleum refinery processing in accordance with the 
American Society for Testing and Materials Designation D 1836-83 (ASTM D 
1836), consists primarily of six-carbon alkanes or cycloalkanes, and 
contains at least 40 liquid volume percent n-hexane (CAS No. 110-54-3) 
and at least 5 liquid volume percent methylcyclopentane (MCP; CAS No. 
96-37-7). ASTM D 1836, formally entitled ``Standard Specification for 
Commercial Hexanes,'' is published in 1986 Annual Book of ASTM 
Standards: Petroleum Products and Lubricants, ASTM D 1836-83, pp. 966-
967, 1986, is incorporated by reference, and is available for public 
inspection at the National Archives and Records Administration (NARA). 
For information on the availability of this material at NARA, call 202-
741-6030, or go to: http://www.archives.gov/federal--register/code--of--
federal--regulations/ibr--locations.html. This incorporation by 
reference was approved by the Director of the Office of the Federal 
Register in accordance with 5 U.S.C. 522(a) and 1 CFR part 51. This 
material is incorporated as it exists on the date of approval, and a 
notice of any change in this material will be published in the Federal 
Register. Copies of the incorporated material may be obtained from the 
Non-Confidential Information Center (NCIC) (7407), Office of Pollution 
Prevention and Toxics, U.S. Environmental Protection Agency, Room B-607 
NEM, 401 M St., SW., Washington, DC 20460, between the hours of 12 p.m. 
and 4 p.m. weekdays excluding legal holidays.
    (2) The commercial hexane test substance, for purposes of this 
section, is a product which conforms to the specifications of ASTM D1836 
and contains at least 40 liquid volume percent but no more than 55 
liquid volume percent n-hexane and no less than 10 liquid volume percent 
MCP.
    (b) Persons required to submit study plans, conduct tests, and 
submit data. All persons who manufacture (including import) or process 
or intend to manufacture or process commercial hexane, as defined in 
paragraph (a)(1) of this section and other than as an impurity, from the 
effective date of the final rule to the end of the reimbursement period 
shall submit letters of intent to conduct testing, submit study plans, 
conduct tests in accordance with part 792 of this chapter, and submit 
data, or submit exemption applications, as specified in this section, 
subpart A of this part, and part 790 of this chapter for single-phase 
rulemaking. Persons who manufacture commercial hexane as a byproduct are 
covered by the requirements of this section. Notwithstanding Sec. 
790.50(a)(1) of this chapter, persons who notify EPA of their intent to 
conduct neurotoxicity testing in compliance with paragraph (c)(7) of 
this section may submit study plans for those tests less than 45 days 
before beginning testing provided that EPA receives the study plans 
before this testing begins.
    (c) Health effects testing--(1) Subchronic inhalation toxicity--(i) 
Required testing. (A) A subchronic inhalation toxicity test shall be 
conducted with commercial hexane in accordance with Sec. 798.2450 of 
this chapter except for the provisions in paragraphs (d)(4)(ii) and (5) 
of Sec. 798.2450.
    (B) For the purposes of this section, the following provisions also 
apply:
    (1) High dose level. The highest concentration should result in 
toxic effects but neither produce an incidence of fatalities which would 
prevent a meaningful evaluation nor exceed the lower explosive limit of 
commercial hexane.
    (2) Exposure conditions. Animals shall be dosed for 6 hours/day, 5 
days/week for 90 days.
    (ii) Reporting requirements. (A) The subchronic inhalation toxicity 
test

[[Page 244]]

shall be completed and the final report submitted to EPA within 15 
months of the effective date of the final rule.
    (B) Interim progress reports shall be submitted to EPA for the 
subchronic inhalation toxcity test at 6-month intervals beginning 6 
months after the effective date of the final rule, until the final 
report is submitted to EPA.
    (2) Oncogenicity--(i) Required testing. (A) An oncogenicity test 
shall be conducted with commercial hexane in accordance with Sec. 
798.3300 of this chapter except for the provisions in paragraphs 
(b)(3)(ii) and (6) of Sec. 798.3300.
    (B) For the purposes of this section, the following provisions also 
apply:
    (1) High dose level. The high dose level should elicit signs of 
minimal toxicity without substantially altering the normal life span and 
should not exceed the lower explosive limit of commercial hexane.
    (2) Administration of test substance. Animals shall be exposed to 
commercial hexane by inhalation.
    (ii) Reporting requirements. (A) The oncogenicity test shall be 
completed and the final report submitted to EPA within 53 months of the 
effective date of the final rule. The mouse portion of the oncogenicity 
study shall be submitted by June 5, 1993.
    (B) Interim progress reports shall be submitted to EPA for the 
oncogenicity test at 6-month intervals beginning 6 months after the 
effective date of the final rule, until the final report is submitted to 
EPA.
    (3) Reproduction and fertility effects--(i) Required testing. (A) A 
reproduction and fertility effects test shall be conducted with 
commercial hexane in accordance with Sec. 798.4700 of this chapter 
except for the provisions in paragraphs (c)(3)(ii) and (5) of Sec. 
798.4700.
    (B) For the purposes of this section, the following provisions also 
apply:
    (1) High dose level. The highest dose level should induce toxicity 
but not high levels of mortality in the parental (P) animals. In 
addition, the highest dose level should not exceed the lower explosive 
limit of commercial hexane.
    (2) Administration of test substance. Animals shall be exposed to 
commercial hexane by inhalation.
    (ii) Reporting requirements. (A) The reproduction and fertility 
effects test shall be completed and the final report submitted to EPA 
within 29 months of the effective date of the final rule.
    (B) Interim progress reports shall be submitted to EPA for the 
reproduction and fertility effects test at 6-month intervals beginning 6 
months after the effective date of the final rule, until the final 
report is submitted to EPA.
    (4) Inhalation developmental toxicity--(i) Required testing. (A) An 
inhalation developmental toxicity test shall be conducted with 
commercial hexane in accordance with Sec. 795.4350 of this chapter 
except for the provisions in paragraph (e)(3)(iv) of Sec. 798.4350.
    (B) For the purposes of this section, the following provisions also 
apply:
    (1) High dose level. Unless limited by the physical/chemical nature 
or biological properties of the test substance, the highest 
concentration level shall induce some overt maternal toxicity such as 
reduced body weight or body weight gain, but not more than 10 percent 
maternal deaths. In addition, the highest dose level should not exceed 
the lower explosive limit of commercial hexane.
    (2) [Reserved]
    (ii) Reporting requirements. (A) The inhalation developmental 
toxicity test shall be completed and the final report submitted to EPA 
within 12 months of the effective date of the final rule.
    (B) Interim progress reports shall be submitted to EPA for the 
inhalation developmental toxicity test at 6-month intervals beginning 6 
months after the effective date of the final rule, until the final 
report is submitted to EPA.
    (5) Mutagenic effects--gene mutations--(i) Required testing. (A)(1) 
A Salmonella typhimurium reverse mutation assay shall be conducted with 
commercial hexane in accordance with Sec. 798.5265 of this chapter 
except for the provisions in paragraphs (d)(4) and (e) of Sec. 
798.5265.
    (2) For the purposes of this section, the following provisions also 
apply:
    (i) Metabolic activation. Bacteria shall be exposed to commercial 
hexane both in the presence and absence of an appropriate metabolic 
activation system.
    (ii) Test performance. The assay shall be performed using the 
desiccator method described as follows: The agar overlay plates shall be 
placed uncovered in a 9-liter desiccator. A volume of

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the liquid test substance shall be added to the glass Petri dish 
suspended beneath the porcelain shelf of the desiccator. The highest 
exposure concentration should not result in a vapor concentration which 
exceeds the lower explosive limit of commerical hexane. A magnetic 
stirring bar to serve as a fan to assure rapid and even distribution of 
the vapor shall be placed on the bottom of the inside of the desiccator. 
The desiccator shall be placed on a magnetic stirrer within a 37[deg] C 
room or chamber for 7 to 10 hours. The plates shall then be removed, 
their lids replaced, followed by incubation for an additional 40 hours 
at 37[deg] C before counting. An appropriate selective medium with an 
adequate overlay agar shall be used. All plating should be done in at 
least triplicate.
    (B)(1) A gene mutation test in mammalian cells shall be conducted 
with commercial hexane in accordance with Sec. 798.5300 of this chapter 
except for the provisions in paragraphs (d)(3)(ii) and (4) of Sec. 
798.5300 if the results from the Salmonella typhimurium test conducted 
pursuant to paragraph (c)(5)(i)(A) of this section are negative.
    (2) For the purposes of this section, the following provisions also 
apply:
    (i) Cell growth and maintenance. Appropriate culture media and 
incubation conditions (culture vessels, CO2 concentrations, 
temperature, and humidity) shall be used. The cell culture shall be 
directly dosed by pipetting liquid commercial hexane mixed with liquid 
DMSO into the culture medium. Cells shall be exposed to test substance 
both in the presence and absence of an appropriate metabolic activation 
system.
    (ii) [Reserved]
    (C)(1) A sex-linked recessive lethal test in Drosophila melanogaster 
shall be conducted with commercial hexane in accordance with Sec. 
798.5275 of this chapter except for the provisions in paragraphs (d)(5) 
(ii) and (iii) of Sec. 798.5275, unless the results of both the 
Salmonella typhimurium test conducted pursuant to paragraph (c)(5)(i)(A) 
of this section and the mammalian cells in the culture gene mutation 
test conducted pursuant to paragraph (c)(5)(i)(B) of this section, if 
required, are negative.
    (2) For the purposes of this section, the following provisions also 
apply:
    (i) Dose levels. For the initial assessment of mutagenicity, it is 
sufficient to test a single dose of the test substance for screening 
purposes. This dose should be the maximum tolerated dose, or that which 
produces some indication of toxicity or shall be the highest dose 
attainable and should not exceed the lower explosive limit of commercial 
hexane. For dose-response purposes, at least three additional dose 
levels should be used.
    (ii) Route of administration. The route of administration shall be 
by exposure to commercial hexane vapors.
    (D)(1) Unless the results of the sex-linked recessive lethal test in 
Drosophila melanogaster conducted with commercial hexane pursuant to 
paragraph (c)(5)(i)(C) of this section are negative, EPA shall conduct a 
public program review of all of the mutagenicity data available for this 
substance. If, after this review, EPA decides that testing of commercial 
hexane for causing heritable gene mutations in mammals is necessary, it 
shall notify the test sponsor by certified letter or Federal Register 
notice that testing shall be initiated in either the mouse visible 
specific locus test or the mouse biochemical specific locus test. The 
mouse visible specific locus test, if conducted, shall be performed for 
commercial hexane in accordance with Sec. 798.5200 of this chapter 
except for the provisions in paragraphs (d)(5)(ii) and (d)(5)(iii) of 
Sec. 798.5200. The mouse biochemical specific locus test, if conducted, 
shall be performed for commercial hexane in accordance with Sec. 
798.5195 of this chapter except for the provisions in paragraphs 
(d)(5)(ii) and (d)(5)(iii) of Sec. 798.5195.
    (2) For the purposes of this section, the following provisions also 
apply:
    (i) Dose levels. A minimum of two dose levels shall be tested. The 
highest dose tested shall be the highest dose tolerated without toxic 
effects, provided that any temporary sterility induced due to 
elimination of spermatogonia is of only moderate duration, as determined 
by a return of males to fertility within 80 days of treatment, or shall 
be the highest dose attainable below the lower explosive

[[Page 246]]

limit concentration of commercial hexane. Exposure shall be for 6 hours 
a day. Duration of exposure shall be dependent upon the accumulated 
total dose desired for each group.
    (ii) Route of administration. Animals shall be exposed to commercial 
hexane by inhalation.
    (ii) Reporting requirements. (A) The gene mutation tests shall be 
completed and final reports submitted to EPA as follows:
    (1) The Salmonella typhimurium reverse mutation assay within 8 
months of the effective date of the final rule.
    (2) The gene mutation in mammalian cells assay within 17 months of 
the effective date of the final rule.
    (3) The sex-linked recessive-lethal test in Drosophila melanogaster 
within 24 months of the effective date of the final rule.
    (4) The mouse visible specific locus test or the mouse biochemical 
specific locus test shall be completed and a final report shall be 
submitted to EPA within 51 months of the date on which the test sponsor 
is notified by EPA by certified letter or Federal Register notice that 
testing shall be initiated.
    (B) Interim progress reports for each test shall be submitted to EPA 
for the gene mutation in mammalian cells assay and Drosophila sex-linked 
recessive lethal test at 6-month intervals beginning 6 months after the 
effective date of the final rule, until the applicable final report is 
submitted to EPA.
    (C) Interim progress reports for either the mouse visible specific 
locus test or the mouse biochemical specific locus test shall be 
submitted to EPA at 6-month intervals, beginning 6 months after EPA's 
notification of the test sponsor that testing should be initiated, until 
the applicable final report is submitted to EPA.
    (6) Mutagenic effects--chromosomal aberrations--(i) Required 
testing. (A)(1) An in vitro cytogenetics test shall be conducted with 
commercial hexane in accordance with Sec. 798.5375 of this chapter 
except for the provisions in paragraph (e)(3) of Sec. 798.5375.
    (2) For the purposes of this section, the following provisions also 
apply:
    (i) Treatment with test substance. The test substance shall be added 
in liquid form mixed with DMSO to the treatment vessels.
    (ii) [Reserved]
    (B)(1) An in vivo cytogenetics test shall be conducted with 
commercial hexane in accordance with Sec. 798.5385 of this chapter 
except for the provisions in paragraphs (d)(5) (ii), (iii) and (iv) of 
Sec. 798.5385, if the in vitro test conducted pursuant to paragraph 
(c)(6)(i)(A) of this section is negative.
    (2) For the purposes of this section, the following provisions also 
apply:
    (i) Dose levels. For an initial assessment, one dose level of the 
test substance may be used, the dose being the maximum tolerated dose 
(to a maximum of 5,000 mg/kg), or that producing some indication of 
cytotoxicity (e.g., partial inhibition of mitosis), or shall be the 
highest dose attainable (to a maximum of 5,000 mg/kg) and should not 
exceed the lower explosive limit of commercial hexane. Additional dose 
levels may be used. For determination of dose-response, at least three 
dose levels should be used.
    (ii) Route of administration. Animals shall be exposed to commercial 
hexane by inhalation.
    (iii) Treatment schedule. The duration of exposure shall be for 6 
hours per day for 5 consecutive days.
    (C)(1) A dominant lethal assay shall be conducted with commercial 
hexane in accordance with Sec. 798.5450 of this chapter except for the 
provisions in paragraphs (d)(5) (ii) and (iii) of Sec. 798.5450, unless 
both the in vitro and in vivo cytogenetics tests conducted pursuant to 
paragraphs (c)(6)(i) (A) and (B) of this section are negative.
    (2) For the purposes of this section, the following provisions also 
apply:
    (i) Dose levels. Normally, three dose levels shall be used. The 
highest dose shall produce signs of toxicity (e.g., slightly reduced 
fertility and slightly reduced body weight). The highest dose should not 
exceed the lower explosive limit of commercial hexane. However, in an 
initial assessment of dominant lethality, a single high dose may be 
sufficient. Nontoxic substances shall be tested at 5 g/kg or, if this is 
not practicable, then at the highest dose attainable.

[[Page 247]]

    (ii) Route of administration. Animals shall be exposed to commercial 
hexane by inhalation.
    (iii) Treatment schedule. The duration of exposure shall be for 6 
hours per day for 5 consecutive days.
    (D)(1) A heritable translocation test shall be conducted with 
commercial hexane in accordance with Sec. 798.5460 of this chapter 
except for the provisions in paragraphs (d)(5) (ii) and (iii) of Sec. 
798.5460, if the results of the dominant lethal assay conducted pursuant 
to paragraph (c)(6)(i)(C) of this section are positive and if, after a 
public program review, EPA issues a Federal Register notice or sends a 
certified letter to the test sponsor specifying that the testing shall 
be initiated.
    (2) For the purposes of this section, the following provisions also 
apply:
    (i) Dose levels. At least two dose levels shall be used. The highest 
dose level shall result in toxic effects (which shall not produce an 
incidence of fatalities which would prevent a meaningful evaluation) or 
shall be the highest dose attainable or 5 g/kg body weight and should 
not exceed the lower explosive limit of commercial hexane.
    (ii) Route of administration. Animals shall be exposed to commercial 
hexane by inhalation.
    (iii) Reporting requirements. (A) The chromosomal aberration tests 
shall be completed and the final reports submitted to EPA as follows:
    (1) The in vitro cytogenetics test within 15 months of the effective 
date of the final rule.
    (2) The in vivo cytogenetics test within 19 months of the effective 
date of the final rule.
    (3) The dominant lethal assay within 28 months of the effective date 
of the final rule.
    (4) The heritable translocation test within 25 months of the date of 
EPA's notification of the test sponsor by certified letter or Federal 
Register notice that testing shall be initiated.
    (B) Interim progress reports for each test shall be submitted to EPA 
for the in vivo cytogenetics and the dominant lethal assays at 6-month 
intervals beginning 6 months after the effective date of the final rule, 
until the applicable final report is submitted to EPA.
    (C) Interim progress reports shall be submitted to EPA for the 
heritable translocation assay at 6-month intervals beginning 6 months 
after the date of EPA's notification of the test sponsor that testing 
shall be initiated, until the final report is submitted to EPA.
    (7) Neutrotoxicity--(i) Required testing. (A)(1) A schedule-
controlled operant behavior test shall be conducted with commercial 
hexane in accordance with Sec. 798.6500 of this chapter except for the 
provisions in paragraphs (d)(5)(i), (6) and (7) of Sec. 798.6500.
    (2) For the purposes of this section, the following provisions also 
apply:
    (i) High dose level. The highest dose shall produce clear behavioral 
effects or life-threatening toxicity. In addition, the highest dose 
should not exceed the lower explosive limit of commercial hexane.
    (ii) Duration and frequency of exposure. Animals shall be dosed once 
for 4 to 6 hours.
    (iii) Route of administration. Animals shall be exposed to 
commercial hexane by inhalation.
    (B)(1) A functional observation battery shall be conducted with 
commercial hexane in accordance with Sec. 798.6050 of this chapter 
except for the provisions in paragraphs (d)(4)(i), (5), and (6) of Sec. 
798.6050.
    (2) For the purposes of this section, the following provisions also 
apply:
    (i) High dose level. The highest dose shall produce clear behavioral 
effects or life-threatening toxicity. In addition, the highest dose 
should not exceed the lower explosive limit of commercial hexane.
    (ii) Duration and frequency of exposure. Animals shall be dosed for 
6 hours/day, 5 days/week for 90 days.
    (iii) Route of exposure. Animals shall be exposed to commercial 
hexane by inhalation.
    (C)(1) A motor activity test shall be conducted with commercial 
hexane in accordance with Sec. 798.6200 of this chapter except for the 
provisions in paragraphs (d)(4)(i), (5), and (6) of Sec. 798.6200.
    (2) For the purposes of this section, the following provisions also 
apply:
    (i) High dose level. The highest dose shall produce clear effects on 
motor activity of life-threatening toxicity. In addition, the highest 
dose should not

[[Page 248]]

exceed the lower explosive limit of commercial hexane.
    (ii) Duration and frequency of exposure. Animals shall be dosed for 
6 hours/day, 5 days/week for 90 days.
    (iii) Route of exposure. Animals shall be exposed to commercial 
hexane by inhalation.
    (D)(1) A neuropathology test shall be conducted with commercial 
hexane in accordance with Sec. 798.6400 of this chapter except for the 
provisions in paragraphs (d)(4)(i), (5), and (6) of Sec. 798.6400.
    (2) For the purposes of this section, the following provisions also 
apply:
    (i) High dose level. The highest dose shall produce clear behavior 
effects or life-threatening toxicity. In addition, the highest dose 
should not exceed the lower explosive limit of commercial hexane.
    (ii) Duration and frequency of exposure. Animals shall be dosed for 
6 hours/day, 5 days/week for 90 days.
    (iii) Route of exposure. Animals shall be exposed to commercial 
hexane by inhalation.
    (ii) Reporting requirements. (A) The schedule-controlled operant 
behavior, functional observation battery, motor activity, and 
neuropathology tests shall be completed and the final reports submitted 
to EPA within 15 months of the effective date of the final rule.
    (B) Interim progress reports for each test shall be submitted to EPA 
for the schedule-controlled operant behavior, functional observation 
battery, motor activity, and neuropathology tests at 6-month intervals 
beginning 6 months after the effective date of the applicable final 
rule, until the applicable final report is submitted to EPA.
    (8) Pharmacokinetics--(i) Required testing. (A) Pharmacokinetics 
testing shall be conducted in rats in accordance with Sec. 795.232 of 
this chapter, except for paragraph (c)(1)(ii) of Sec. 795.232.
    (B) For the purposes of this section, the following provisions also 
apply:
    (1) Test animals. Adult male and female rats shall be used for 
testing. The rats shall be 9 to 11 weeks old and their weight range 
should be comparable from group to group. The animals shall be purchased 
from a reputable dealer and shall be permanently identified upon 
arrival. The animals shall be selected at random for the testing groups, 
and any animal showing signs of ill health shall not be used.
    (2) Species and strain. The rat strain used shall be the same as the 
strain used in the subchronic and chronic tests required under Sec. 
798.2450(d)(1)(i) and Sec. 798.3300(b)(1)(i).
    (ii) Reporting requirements. (A) The inhalation and dermal 
pharmacokinetics tests shall be completed and the final report submitted 
to EPA by August 21, 1992.
    (B) Interim progress reports shall be submitted to EPA for the 
inhalation and dermal pharmacokinetics tests at 6-month intervals, 
beginning 6 months after the effective date specified in paragraph 
(d)(1) of this section, until the final report is submitted to EPA.
    (d) Effective date. (1) The effective date of this final rule is 
November 17, 1988, except for the provisions of paragraphs 
(c)(2)(ii)(A), (c)(5)(i)(D), (c)(5)(ii)(A)(4), (c)(5)(ii)(C), (c)(8)(i) 
and (c)(8)(ii)(A) of this section. The effective date for paragraphs 
(c)(5)(i)(D), (c)(5)(ii)(A)(4) and (c)(5)(ii)(C) of this section is May 
21, 1990. The effective date for paragraphs (c)(8)(i) and (c)(8)(ii)(A) 
of this section is June 12, 1992. The effective date of paragraph 
(c)(2)(ii)(A) is September 8, 1994.
    (2) The guidelines and other test methods cited in this rule are 
referenced as they exist on the effective date of the final rule.

[53 FR 3392, Feb. 5, 1988, as amended at 53 FR 38953, Oct. 4, 1988; 55 
FR 634, Jan. 8, 1990; 55 FR 7325, Mar. 1, 1990; 55 FR 12643, Apr. 5, 
1990; 57 FR 24961, June 12, 1992; 58 FR 34205, June 23, 1993; 59 FR 
46357, Sept. 8, 1994; 60 FR 34467, July 3, 1995; 69 FR 18803, Apr. 9, 
2004]



Sec. 799.2325  Isopropanol.

    (a) Identification of test substance. (1) Isopropanol (CAS No. 67-
63-0) shall be tested in accordance with this section.
    (2) Isopropanol of at least 99.8 percent purity shall be used as the 
test substance.
    (b) Persons required to submit study plans, conduct tests, and 
submit data. All persons who manufacture (including import or byproduct 
manufacture) or intend to manufacture or process isopropanol, from the 
effective date of

[[Page 249]]

this rule to the end of the reimbursement period, shall submit letters 
of intent to conduct testing, submit study plans, conduct tests, and 
submit data or submit exemption applications as specified in this 
section, subpart A of this part, and parts 790 and 792 of this chapter 
for single-phase rulemaking.
    (c) Health effects testing--(1) Subchronic inhalation toxicity--(i) 
Required testing. A subchronic inhalation toxicity test shall be 
conducted with isopropanol in accordance with Sec. 798.2450 of this 
chapter.
    (ii) Reporting requirements. (A) The subchronic inhalation toxicity 
test shall be completed and the final report submitted to EPA within 15 
months of the date specified in paragraph (d) of this section.
    (B) Progress reports shall be submitted to EPA for the subchronic 
inhalation toxicity test at 6-month intervals beginning 6 months after 
the date specified in paragraph (d)(1) of this section until submission 
of the final report.
    (2) Reproduction and fertility effects--(i) Required testing. A 
reproduction and fertility effects test shall be conducted by gavage 
with isopropanol in accordance with Sec. 798.4700 of this chapter.
    (ii) Reporting requirements. (A) The reproduction and fertility 
effects test shall be completed and the final report submitted to EPA 
within 29 months of the date specified in paragraph (d)(1) of this 
section.
    (B) Progress reports shall be submitted at 6-month intervals 
beginning 6 months after the date specified in paragraph (d)(1) of this 
section until submission of the final report.
    (3) Developmental toxicity--(i) Required testing. A developmental 
toxicity test shall be conducted in two mammalian species by gavage with 
isopropanol in accordance with Sec. 798.4900 of this chapter.
    (ii) Reporting requirements. (A) The developmental toxicity test 
shall be completed and the final report submitted to EPA within 12 
months of the date specified in paragraph (d)(1) of this section.
    (B) A progress report shall be submitted 6 months after the date 
specified in paragraph (d)(1) of this section.
    (4) Mutagenic effects--gene mutations--(i) Required testing. (A) A 
gene mutation test in mammalian cells shall be conducted with 
isopropanol in accordance with Sec. 798.5300 of this chapter.
    (B)(1) A sex-linked recessive lethal test in Drosophila melanogaster 
shall be conducted with isopropanol in accordance with Sec. 798.5275 of 
this chapter, except for the provisions in paragraphs (d)(5)(ii) and 
(iii) of Sec. 798.5275, unless the results of the mammalian cells in 
the culture gene mutation test conducted pursuant to paragraph 
(c)(5)(i)(A) of this section are negative.
    (2) For the purpose of this section, the following provisions also 
apply:
    (i) Route of administration. The route of administration shall be by 
exposure to isopropanol vapors or by injection of isopropanol.
    (ii) [Reserved]
    (C)(1) The mouse visible specific locus (MVSL) test shall be 
conducted with isopropanol by inhalation in accordance with Sec. 
798.5200, except for the provisions in paragraphs (d)(5)(ii) and (iii) 
of Sec. 798.5200, if the results of the sex-linked recessive lethal 
test conducted pursuant to paragraph (c)(4)(i)(B) of this section are 
positive and if, after a public program review, EPA issues a Federal 
Register notice or sends a certified letter to the test sponsor 
specifying that the testing shall be initiated.
    (2) For the purpose of this section, the following provisions also 
apply:
    (i) Dose levels and duration of exposure. A minimum of 2 dose levels 
shall be tested. The duration of exposure shall be for 6 hours per day. 
Duration of exposure shall be dependent upon accumulated total dose 
desired for each group.
    (ii) Route of administration. Animals shall be exposed to 
isopropanol by inhalation.
    (ii) Reporting requirements. (A) The gene mutation tests shall be 
completed and final report submitted to EPA as follows:
    (1) The gene mutation in mammalian cells assay within 6 months of 
the date specified in paragraph (d)(1) of this section.
    (2) The sex-linked recessive-lethal test in Drosophila melanogaster 
within

[[Page 250]]

18 months of the date specified in paragraph (d)(1) of this section.
    (3) The mouse visible specific-locus test within 51 months of the 
date of EPA's notification of the test sponsor by certified letter or 
Federal Register notice under paragraph (c)(4)(i)(C) of this section 
that testing shall be initiated.
    (B) Progress reports shall be submitted to EPA for the Drosophila 
sex-linked recessive lethal test at 6-month intervals beginning 6 months 
after the date specified in paragraph (d)(1) of this section until the 
submission of the final report.
    (C) Progress reports shall be submitted to EPA for the mouse visible 
specific locus test at 6-month intervals beginning 6 months after the 
date of EPA's notification of the test sponsor that testing shall be 
initiated until submission of the final report.
    (5) Mutagenic effects--chromosomal aberrations--(i) Required 
testing. (A)(1) The micronucleus test shall be conducted with 
isopropanol in accordance with Sec. 798.5395 of this chapter.
    (2) For the purpose of this section, the following provisions also 
apply:
    (i) Route of administration. Animals shall be exposed to isopropanol 
by either inhalation or oral gavage or inperitoneally (IP).
    (ii) Duration of exposure. For inhalation, the duration of exposure 
shall be for 6 hours per day for 5 consecutive days with one sacrifice 
time or for 6 hours for 1 day with three sacrifice times.
    (B)(1) A dominant lethal assay shall be conducted with isopropanol 
in accordance with Sec. 798.5450 of this chapter, except for the 
provisions in paragraphs (d)(5)(ii) and (iii) of Sec. 798.5450, unless 
the micronucleus test conducted pursuant to paragraphs (c)(5)(i)(A) of 
this section is negative.
    (2) For the purpose of this section, the following provisions also 
apply:
    (i) Route of administration. Animals shall be exposed to isopropanol 
by inhalation.
    (ii) Duration of exposure. The duration of exposure shall be for 6 
hours per day for 5 consecutive days.
    (C)(1) The mouse visible specific locus test (MVSL) shall be 
conducted with isopropanol by inhalation in accordance with Sec. 
798.5200 of this chapter, except for the provisions in paragraphs 
(d)(5)(ii) and (d)(5)(iii) of Sec. 798.5200, or a mouse biochemical 
specific locus test (MBSL) shall be conducted with isopropanol by 
inhalation in accordance with Sec. 798.5195 of this chapter, except for 
the provisions in paragraphs (d)(5)(ii) and (d)(5)(iii) of Sec. 
798.5195, if the results of the sex-linked recessive lethal test 
conducted pursuant to paragraph (c)(4)(i)(B) of this section are 
positive and if, after a public program review, EPA issues a Federal 
Register notice or sends a certified letter to the test sponsor 
specifying that the testing shall be initiated.
    (2) For the purpose of this section, the following provisions also 
apply:
    (i) Route of administration. Animals shall be exposed to isopropanol 
by inhalation.
    (ii) [Reserved]
    (ii) Reporting requirements. (A) The chromosomal aberration tests 
shall be completed and the final reports submitted to EPA as follows:
    (1) The micronucleus test within 15 months of the date specified in 
paragraph (d)(1) of this section.
    (2) The dominant lethal assay within 27 months of the date specified 
in paragraph (d)(1) of this section.
    (3) The MVSL or MBSL test within 51 months of the date of EPA's 
notification of the test sponsor by certified letter or Federal Register 
notice under paragraph (c)(4)(i)(C) of this section that testing shall 
be initiated.
    (B) Progress reports shall be submitted to EPA for the micronucleus 
and the dominant lethal assays at 6-month intervals beginning 6 months 
after the date specified in paragraph (d)(1) of this section until 
submission of the final report.
    (C) Progress reports shall be submitted to EPA for the heritable 
translocation assay at 6-month intervals beginning 6 months after the 
date of EPA's notification of the test sponsor that testing shall be 
initiated until submission of the final report.
    (6) Neurotoxicity--(i) Required testing. (A)(1) A functional 
observation battery shall be conducted with isopropanol in accordance 
with Sec. 798.6050 of this chapter except for the provisions in 
paragraphs (d)(5) and (6) of Sec. 798.6050.

[[Page 251]]

    (2) For the purpose of this section, the following provisions also 
apply:
    (i) Duration and frequency of exposure. For subchronic study, 
animals shall be dosed for 6 hours per day, 5 days per week for 90 days. 
For acute study, animals shall be dosed for 4 to 6 hours once.
    (ii) Route of exposure. Animals shall be exposed to isopropanol by 
inhalation.
    (B)(1) A motor activity test shall be conducted with isopropanol in 
accordance with Sec. 798.6200 of this chapter except for the provisions 
in paragraphs (d)(5) and (6) of Sec. 798.6200.
    (2) For the purpose of this section, the following provisions also 
apply:
    (i) Duration of exposure. For subchronic study, animals shall be 
dosed for 6 hours per day, 5 days per week for 90 days. For acute study, 
animals shall be dosed for 4 to 6 hours once.
    (ii) Route of exposure. Animals shall be exposed to isopropanol by 
inhalation.
    (C)(1) A neuropathology test shall be conducted with isopropanol in 
accordance with Sec. 798.6400 of this chapter except for the provisions 
in paragraphs (d)(5) and (6) of Sec. 798.6400.
    (2) For the purpose of this section, the following provisions also 
apply:
    (i) Duration of exposure. Animals shall be dosed for 6 hours per 
day, 5 days per week for 90 days.
    (ii) Route of exposure. Animals shall be exposed to isopropanol by 
inhalation.
    (D) The developmental neurotoxicity test shall be conducted with 
isopropanol in accordance with Sec. 795.250 of this chapter, except for 
paragraph (c)(1)(iv).
    (1) For purposes of this section, the following provisions also 
apply:
    (i) Numbers of animals. The objective is for a sufficient number of 
pregnant rats to be exposed to ensure that an adequate number of 
offspring are produced for neurotoxicity evaluation. At least 24 litters 
shall be used at each dose level.
    (ii) [Reserved]
    (2) [Reserved]
    (ii) Reporting requirements. (A) The acute functional observation 
battery and motor activity tests shall be completed and the final report 
submitted to EPA within 15 months of the date specified in paragraph 
(d)(1) of this section. The subchronic functional observation battery, 
motor activity, and neuropathology tests shall be completed and the 
final reports submitted to EPA within 18 months of the date specified in 
paragraph (d)(1) of this section. The developmental neurotoxicity test 
shall be completed and the final report submitted to EPA within 21 
months of the date specified in paragraph (d)(1) of this section.
    (B) Progress reports shall be submitted to EPA for the functional 
observation battery, motor activity, neuropathology, and developmental 
neurotoxicity tests at 6-month intervals beginning 6 months after the 
date specified in paragraph (d)(1) of this section until submission of 
the applicable final report.
    (7) Pharmacokinetics studies--(i) Required testing. An oral and 
inhalation pharmacokinetics test shall be conducted with isopropanol in 
accordance with Sec. 795.231 of this chapter.
    (ii) Reporting requirements. (A) The pharmacokinetic test shall be 
completed and the final report submitted to EPA within 15 months of the 
date specified in paragraph (d)(1) of this section.
    (B) Progress reports shall be submitted to EPA for the 
pharmacokinetics test at 6-month intervals beginning 6 months after the 
date specified in paragraph (d)(1) of this section until submission of 
the final report.
    (8) Oncogenicity--(i) Required testing. An oncogenicity test shall 
be conducted by inhalation with isopropanol in accordance with Sec. 
798.3300 of this chapter.
    (ii) Reporting requirements. (A) The oncogenicity test shall be 
completed and the final report submitted to EPA by July 5, 1994.
    (B) Progress reports shall be submitted at 6-month intervals 
beginning 6 months after the date specified in paragraph (d)(1) of this 
section until submission of the final report.
    (d) Effective date. (1) The effective date of this final rule is 
December 4, 1989, except for the provisions of paragraphs 
(c)(5)(i)(C)(1), (c)(5)(ii)(A)(3), (c)(6)(i)(D), and (c)(8)(ii)(A), of 
this section. The effective date for paragraphs

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(c)(5)(i)(C)(1), and (c)(5)(ii)(A)(3) of this section is May 21, 1990. 
The effective date for paragraphs (c)(6)(i)(D) of this section is May 
21, 1991. The effective date of paragraph (c)(8)(ii)(A) is September 29, 
1995.
    (2) The guidelines and other test methods cited in this rule are 
references as they exist on the effective date of the final rule.

[54 FR 43262, Oct. 23, 1989, as amended at 55 FR 12644, Apr. 5, 1990; 56 
FR 23231, May 21, 1990; 58 FR 34205, June 23, 1993; 60 FR 56956, Nov. 
13, 1995]



Sec. 799.2475  2-Mercaptobenzothiazole.

    (a) Identification of test substance. (1) 2-Mercaptobenzothiazole 
(MBT, CAS No. 149-30-4) shall be tested in accordance with this section.
    (2) MBT of at least 97.6 percent purity (plus or minus 1.5 percent) 
shall be used as the test substance.
    (b) Persons required to submit study plans, conduct tests, and 
submit data. All persons who manufacture (including byproduct 
manufacture, and import of MBT and MBT-containing articles) or process 
or intend to manufacture or process MBT, other than as an impurity, 
after October 21, 1988, to the end of the reimbursement period shall 
submit letters of intent to conduct testing, submit study plans, conduct 
tests, and submit data, or submit exemption applications as specified in 
this section, subpart A of this part, and parts 790 and 792 of this 
chapter for single-phase rulemaking.
    (c) Chemical fate--(1) Aerobic aquatic biodegradation--(i) Required 
testing. Aerobic aquatic biodegradation testing shall be conducted with 
MBT in accordance with Sec. 796.3100 of this chapter.
    (ii) Reporting requirements. (A) The aerobic aquatic biodegradation 
test shall be completed and the final report submitted to EPA within 12 
months of the effective date of the final rule.
    (B) An interim progress report shall be submitted to EPA 6 months 
after the effective date of the final rule.
    (2) Indirect photolysis-screening level test--(i) Required testing. 
Indirect photolysis testing shall be conducted with MBT in accordance 
with Sec. 795.70 of this chapter.
    (ii) Reporting requirements. (A) The indirect photolysis test shall 
be completed and the final report submitted to EPA within 12 months of 
the effective date of the final rule.
    (B) An interim progress report shall be submitted to EPA 6 months 
after the effective date of the final rule.
    (3) Chemical mobility--(i) Required testing. Chemical mobility 
testing shall be conducted with MBT in accordance with Sec. 796.2750 of 
this chapter.
    (ii) Reporting requirements. (A) The chemical mobility test shall be 
completed and the final report submitted to EPA within 12 months of the 
effective date of the final rule.
    (B) An interim progress report shall be submitted to EPA 6 months 
after the effective date of this final rule.
    (d) Environmental effects--(1) Fish chronic toxicity--(i) Required 
testing. (A) Chronic toxicity testing of MBT shall be conducted using 
rainbow trout (Salmo gairdneri.) according to Sec. 797.1600 of this 
chapter, except for paragraphs (c)(4)(iv)(A), (c)(4)(x)(E) and 
(c)(4)(x)(F), (c)(6)(iv)(A), (d)(2)(vii)(A)(2), and (d)(3)(iv) of Sec. 
797.1600.
    (B) For the purpose of this section, the following provisions also 
apply:
    (1) The first feeding for the fathead and sheepshead minnow fry 
shall begin shortly after transfer of the fry from the embryo cups to 
the test chambers. Silversides are fed the first day after hatch. Trout 
species initiate feeding at swim-up. The trout fry shall be fed trout 
starter mash or live newly-hatched brine shrimp nauplii (Artemia salina) 
three times a day ad libitum, with excess food siphoned off daily. The 
minnow fry shall be fed live newly-hatched brine shrimp nauplii (Artemia 
salina) at least three times a day.
    (2) All physical abnormalities (e.g., stunted bodies, scoliosis, 
etc.) shall be photographed and preserved.
    (3) At termination, all surviving fish shall be measured for growth. 
Total length measurements should be used except in cases where fin 
erosion occurs, then the use of standard length measurements shall be 
permitted. Standard length measurements should be made directly with a 
caliper, but may be measured photographically. Measurements shall be 
made to the nearest millimeter (0.1 mm is desirable). Weight 
measurements shall also

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be made for each fish alive at termination (wet, blotted dry, and to the 
nearest 0.01 g for the minnows and 0.1 g for the trout). If the fish 
exposed to the toxicant appear to be edematous compared to control fish, 
determination of dry, rather than wet, weight is recommended.
    (4)(i) Test substance measurement. Prior to addition of the test 
substance to the dilution water, it is recommended that the test 
substance stock solution be analyzed to verify the concentration. After 
addition of the test substance, the concentration of test substance 
shall be measured in the test substance delivery chamber prior to 
beginning, and during, the test. The concentration of test substance 
should also be measured at the beginning of the test in each test 
concentration (including both replicates) and control(s), and at least 
once a week thereafter. Equal aliquots of test solution may be removed 
from each replicate chamber and pooled for analysis. If a malfunction in 
the delivery system is discovered, water samples shall be taken from the 
affected test chambers immediately and analyzed.
    (ii) pH. It is recommended that a pH of 7 be maintained in the test 
chambers.
    (iii) Reporting. An analysis of the stability of the stock solution 
for the duration of the test shall be reported.
    (5) [Reserved]
    (6) For brook and rainbow trout, a 16-hour light and 8-hour dark 
photoperiod shall be provided.
    (ii) Reporting requirements. (A) The fish chronic toxicity test 
shall be completed and the final report submitted to EPA within 12 
months of the effective date of the final rule.
    (B) An interim progress report shall be submitted to EPA 6 months 
after the effective date of the final rule.
    (2) Daphnid chronic toxicity--(i) Required testing. (A) Daphnid 
chronic toxicity testing shall be conducted with MBT using Daphnia magna 
according to Sec. 797.1330 of this chapter.
    (B) For the purposes of this section, the following provisions also 
apply:
    (1) Test substance measurement. Test substance concentration shall 
be measured in the test substance delivery chamber prior to beginning, 
and during, the test.
    (2) pH. It is recommended that a pH of 7 be maintained in the test 
chambers.
    (3) Reporting. An analysis of the stability of the stock solution 
for the duration of the test shall be reported and data comparing trout 
starter mash with A. salina for supporting trout growth should be 
submitted with the final report.
    (ii) Reporting requirements. (A) The daphnid chronic toxicity test 
shall be completed and the final report submitted to EPA within 12 
months of the effective date of the final rule.
    (B) An interim progress report shall be submitted to EPA 6 months 
after the effective date of the final rule.
    (e) Health effects--(1) Developmental toxicity testing--(i) Required 
testing. Developmental toxicity testing shall be conducted in two 
mammalian species with MBT in accordance with Sec. 798.4900 of this 
chapter, using the oral route of administration.
    (ii) Reporting requirements. (A) The developmental toxicity test 
shall be completed and the final report submitted to EPA within 12 
months of the effective date of the final rule.
    (B) An interim progress report shall be submitted to EPA 6 months 
after the effective date of the final rule.
    (2) Reproductive toxicity--(i) Required testing. Reproductive 
toxicity testing shall be conducted with MBT in accordance with Sec. 
798.4700 of this chapter, using the oral route of administration.
    (ii) Reporting requirements. (A) The reproductive test shall be 
completed and the final report submitted to EPA within 29 months of the 
effective date of the final rule.
    (B) Progress reports shall be submitted to EPA at 6-month intervals 
beginning 6 months after the effective date of the final rule until 
submission of the final report.
    (3) Neurotoxicity--(i) Required testing. (A)(1) An acute and 
subchronic functional observation battery shall be conducted with MBT in 
accordance with Sec. 798.6050 of this chapter except for the provisions 
in paragraphs (d)(5) and (6) of Sec. 798.6050.
    (2) For the purpose of this section, the following provisions also 
apply:

[[Page 254]]

    (i) Duration and frequency of exposure. For acute study, animals 
shall be administered MBT over a period not to exceed 24 hours. For 
subchronic study, animals shall be dosed daily for at least 90 days.
    (ii) Route of exposure. Animals shall be exposed to MBT orally.
    (B)(1) An acute and subchronic motor activity test shall be 
conducted with MBT in accordance with Sec. 798.6200 of this chapter 
except for the provisions in paragraphs (d)(5) and (6) of Sec. 
798.6200.
    (2) For the purpose of this section the following provisions also 
apply:
    (i) Duration and frequency of exposure. For acute study, animals 
shall be administered over a period not to exceed 24 hours. For 
subchronic study, animals shall be dosed daily for at least 90 days.
    (ii) Route of exposure. Animals shall be exposed to MBT orally.
    (C)(1) A subchronic neuropathology test shall be conducted with MBT 
in accordance with Sec. 798.6400 of this chapter except for the 
provisions in paragraphs (d)(5) and (6) of Sec. 798.6400.
    (2) For the purpose of this section, the following provisions also 
apply:
    (i) Duration and frequency of exposure. Animals shall be dosed daily 
for at least 90 days.
    (ii) Route of exposure. Animals shall be exposed to MBT orally.
    (ii) Reporting requirements. (A) The functional observation battery, 
motor activity, and neuropathology tests shall be completed and the 
final reports for each test submitted to EPA within 18 months of the 
effective date of the final rule.
    (B) A progress report shall be submitted to EPA for the functional 
observation battery, motor activity, and neuropathology tests, 
respectively, 6 months after the effective date of the final rule.
    (4) Mutagenic effects--Chromosomal aberrations--(i) Required 
testing. (A) A dominant lethal assay shall be conducted with MBT in 
accordance with Sec. 798.5450 of this chapter, using the oral route of 
administration.
    (B) A heritable translocation assay shall be conducted with MBT in 
accordance with the test guideline specified in Sec. 798.5460 of this 
chapter if MBT produces a positive result in the dominant lethal assay 
conducted pursuant to paragraph (e)(4)(i)(A) of this section and if, 
after a public program review, EPA issues a Federal Register notice or 
sends a certified letter to the test sponsor specifying that the testing 
shall be initiated.
    (ii) Reporting requirements. (A) Mutagenic effects--Chromosomal 
aberration testing of MBT shall be completed and the final report 
submitted to EPA as follows: Dominant lethal assay, within 12 months 
after the effective date of this rule; heritable translocation assay, 
within 24 months after notification under paragraph (e)(4)(i)(B) of this 
section that the testing shall be initiated.
    (B) For the dominant lethal assay, an interim progress report shall 
be submitted to EPA 6 months after the effective date of the final rule; 
for the heritable translocation assay, progress reports shall be 
submitted to EPA at 6-month intervals beginning 6 months after the date 
of EPA's notification of the test sponsor that testing shall be 
initiated until submission of the final report.
    (f) Effective date. (1) The effective date of this final rule is 
October 21, 1988, except for paragraphs (a)(2), (d)(1)(i), 
(d)(2)(i)(B)(3), and (e)(3)(ii)(A) of this section. The effective date 
for paragraphs (a)(2), (d)(1)(i), (d)(2)(i)(B)(3), and (e)(3)(ii)(A) of 
this section is March 1, 1990.
    (2) The guidelines and other test methods cited in this rule are 
referenced as they exist on the effective date of the final rule.

[53 FR 34530, Sept. 7, 1988; 53 FR 37393, Sept. 26, 1988, as amended at 
55 FR 7326, Mar. 1, 1990; 58 FR 34205, June 23, 1993]



Sec. 799.2700  Methyl ethyl ketoxime.

    (a) Identification of test substance. (1) Methyl ethyl ketoxime 
(MEKO, CAS No. 96-29-7) shall be tested in accordance with this section.
    (2) MEKO of at least 99 percent purity shall be used as the test 
substance.
    (b) Persons required to submit study plans, conduct tests, and 
submit data. All persons who manufacture (including import) or process 
or intend to manufacture or process MEKO, including persons who 
manufacture or process or intend to manufacture or process

[[Page 255]]

MEKO as a byproduct, or who import or intend to import products which 
contain MEKO, after the date specified in paragraph (e) of this section 
to the end of the reimbursement period, shall submit letters of intent 
to conduct testing, submit study plans, conduct tests and submit data, 
or submit exemption applications, as specified in this section, subpart 
A of this part, and parts 790 and 792 of this chapter for single-phase 
rulemaking. Persons who manufacture, import, or process MEKO only as an 
impurity are not subject to these requirements.
    (c) Health effects testing--(1) Pharmacokinetics testing--(i) 
Required testing. Pharmacokinetics testing shall be conducted with MEKO 
in accordance with paragraph (c)(1)(ii) of this section.
    (ii) [Reserved]
    (2) Oncogenicity--(i) Required testing. Oncogenicity testing shall 
be conducted in accordance with Sec. 798.3300 of this chapter.
    (ii) Route of administration. MEKO shall be administered either 
orally or by inhalation.
    (iii) Reporting requirements. (A) Oncogenicity testing shall be 
completed and a final report submitted to EPA within 53 months of the 
date specified in paragraph (e) of this section.
    (B) Interim progress reports shall be submitted to EPA at 6-month 
intervals, beginning 6 months after the date specified in paragraph (e) 
of this section, until submission of the final report to EPA.
    (3) Developmental toxicity--(i) Required testing. Developmental 
toxicity testing shall be conducted in a rodent and a nonrodent 
mammalian species in accordance with Sec. 798.4900 of this chapter.
    (ii) Route of administration. MEKO shall be administered orally.
    (iii) Reporting requirements. (A) Developmental toxicity testing 
shall be completed and a final report submitted to EPA within 15 months 
of the date specified in paragraph (e) of this section.
    (B) Interim progress reports shall be submitted to EPA at 6-month 
intervals, beginning 6 months after the date specified in paragraph (e) 
of this section.
    (4) Reproductive toxicity--(i) Required testing. (A) Reproductive 
toxicity testing shall be conducted orally in accordance with Sec. 
798.4700 of this chapter except for the provisions in paragraphs (c) 
(8)(iii) and (9)(i) of Sec. 798.4700.
    (B) For the purpose of this section, the following provisions also 
apply:
    (1) The following organs and tissues, or representative samples 
thereof, shall be preserved in a suitable medium for possible future 
histopathological examination: Vagina, uterus, oviducts, ovaries, 
testes, epididymides, vas deferens, seminal vesicles, prostate, 
pituitary gland, and, target organ(s) of all P and F1 animals 
selected for mating.
    (2)(i) Full histopathology shall be conducted on the organs and 
tissues listed in paragraph (c)(4)(i)(B)(1) of this section for all high 
dose and control P and F1 animals selected for mating.
    (ii) The integrity of the various cell stages of spermatogenesis 
shall be determined, with particular attention directed toward achieving 
optimal quality in the fixation and embedding. Preparations of 
testicular and associated reproductive organ samples for histology 
should follow the recommendations of Lamb and Chapin (1985) under 
paragraph (d)(1) of this section, or an equivalent procedure. 
Histopathology of the testes shall be conducted on all P and 
F1 adult males at the time of sacrifice, and histological 
analyses shall include evaluations of the spermatogenic cycle, i.e., the 
presence and integrity of the 14 cell stages. These evaluations should 
follow the guidance provided by Clermont and Percy (1957) under 
paragraph (d)(2) of this section. Information shall also be provided 
regarding the nature and level of lesions observed in control animals 
for comparative purposes.
    (iii) Data on female cyclicity shall be obtained by conducting 
vaginal cytology in P and F1 females over the last 3 weeks 
prior to mating; the cell staging technique of Sadleir (1978) and the 
vaginal smear method in Hafez (1978) under paragraphs (d)(3) and (d)(7) 
of this section, respectively, or equivalent methods should be used. 
Data shall be provided on whether the animal is cycling and the cycle 
length.
    (iv) P and F1 females shall continue to be exposed to 
MEKO for at least an additional 2 weeks following weaning of offspring 
to permit them to begin cycling once again. They shall then be

[[Page 256]]

sacrificed and their ovaries shall be serially sectioned with a 
sufficient number of sections examined to adequately detail oocyte and 
follicular morphology. The methods of Mattison and Thorgiersson (1979) 
and Pederson and Peters (1968) under paragraphs (d) (4) and (5) of this 
section, respectively, may provide guidance. The strategy for sectioning 
and evaluation is left to the discretion of the investigators, but shall 
be described in detail in the study plan and final report. The nature 
and background level of lesions in control tissue shall also be noted.
    (v) Gross and histopathologic evaluations shall be conducted on the 
mammary glands in F1 females and F2 pups 
sacrificed at weaning and in adult F1 females at the 
termination of the study. Any abnormalities shall be described in the 
final report.
    (ii) Reporting requirements. (A) Reproductive toxicity testing shall 
be completed and a final report submitted to EPA within 29 months of the 
date specified in paragraph (e) of this section.
    (B) Interim progress reports shall be submitted to EPA at 6-month 
intervals, beginning six months after the date specified in paragraph 
(e) of this section until submission of the final report to EPA.
    (5) Mutagenic effects--gene mutations--(i) Required testing. The 
sex-linked recessive lethal assay in Drosophila shall be conducted with 
MEKO in accordance with Sec. 798.5275 of this chapter.
    (ii) Reporting requirements. (A) The sex-linked recessive lethal 
assay in Drosophila shall be completed and a final report submitted to 
EPA within 18 months of the date specified in paragraph (e) of this 
section.
    (B) Interim progress reports shall be submitted to EPA at 6-month 
intervals beginning 6 months after the date specified in paragraph (e) 
of this section.
    (6) Mutagenic effects--chromosomal aberrations--(i) Required 
testing. (A) An in vivo mammalian bone marrow cytogenetics test shall be 
conducted with MEKO in accordance with either Sec. 798.5385 
(chromosomal analysis) of this chapter, or Sec. 798.5395 (micronucleus 
assay) of this chapter except for the provisions in paragraphs (d)(5) 
(ii), (iii), and (iv) of Sec. Sec. 798.5385 and 798.5395.
    (B) For the purpose of this section, the following provisions also 
apply if Sec. 798.5385 of this chapter is used in conducting the test:
    (1) Dose levels and duration of exposure. At least three dose levels 
shall be tested. The highest dose tested shall be the maximum tolerated 
dose or that dose producing some signs of cytotoxicity (e.g., partial 
inhibition of mitosis) or shall be the highest dose attainable. Under 
oral administration, animals shall be exposed once per day for 5 
consecutive days. Under administration by inhalation, animals shall be 
exposed 6 hours per day for 5 consecutive days.
    (2) Route of administration. Animals shall be exposed to MEKO either 
orally or by inhalation.
    (C) For the purpose of this section, the following provisions also 
apply if Sec. 798.5395 of this chapter is used in conducting the test:
    (1) Dose levels and duration of exposure. At least three-dose levels 
shall be tested. The highest dose tested shall be the maximum tolerated 
dose or that dose producing some signs of cytotoxicity (e.g., a change 
in the ratio of polychromatic to normochromatic erythrocytes) or shall 
be the highest dose attainable. Under oral administration animals shall 
be exposed once per day for 5 consecutive days. Under administration by 
inhalation, animals shall be exposed 6 hours per day for 5 consecutive 
days.
    (2) Route of administration. Animals shall be exposed to MEKO either 
orally or by inhalation.
    (ii) Reporting requirements. (A) The oral in vivo mammalian 
cytogenetics test shall be completed and a final report submitted to EPA 
within 14 months of the date specified in paragraph (e) of this section. 
The inhalation in vivo mammalian cytogenetics test shall be completed 
and a final report submitted to EPA within 17 months of the date 
specified in paragraph (e) of this section.
    (B) Interim progress reports shall be submitted to EPA at 6-month 
intervals, beginning 6 months after the date specified in paragraph (e) 
of this section.
    (7) Neurotoxicity--(i) Required testing--(A) Functional 
observational battery. (1)

[[Page 257]]

A functional observational battery shall be conducted with MEKO in 
accordance with Sec. 798.6050 of this chapter except for the provisions 
in paragraphs (d) (4)(ii), (5), and (6) of Sec. 798.6050.
    (2) For the purpose of this section, the following provisions also 
apply:
    (i) Route of exposure. Animals shall be exposed either orally or by 
inhalation.
    (ii) Lower doses. The data from the lower doses shall show either 
graded dose-dependent effects in at least two of all the doses tested, 
including the highest dose, or no neurotoxic (behavioral) effects at any 
dose tested.
    (iii) Duration and frequency of exposure. For the oral acute 
testing, animals shall be exposed once. For the oral subchronic testing, 
animals shall be exposed once per day 5 days per week for a 90-day 
period. For the inhalation acute testing, animals shall be exposed for 6 
hours for 1 day. For the inhalation subchronic testing, animals shall be 
exposed 6 hours per day 5 days per week for a 90-day period.
    (B) Motor activity. (1) A motor activity test shall be conducted 
with MEKO in accordance with Sec. 798.6200 of this chapter except for 
provisions in paragraphs (d) (4)(ii), (5), and (6) of Sec. 798.6200.
    (2) For the purpose of this section, the following provisions also 
apply:
    (i) Route of exposure. Animals shall be exposed either orally or by 
inhalation.
    (ii) Lower doses. The data from the lower doses shall show either 
graded dose-dependent effects in at least two of all the doses tested 
including the highest dose, or no neurotoxic (behavioral) effects at any 
dose tested.
    (iii) Duration and frequency of exposure. For the acute oral 
testing, animals shall be exposed once. For the oral subchronic testing, 
animals shall be exposed once per day 5 days per week for a 90-day 
period. For the acute inhalation testing, animals shall be exposed for 6 
hours for 1 day. For the inhalation subchronic testing, the animals 
shall be exposed for 6 hours per day 5 days per week for a 90-day 
period.
    (C) Neuropathology. (1) A neuropathology test shall be conducted 
with MEKO in accordance with Sec. 798.6400 of this chapter except for 
the provisions in paragraphs (d) (4)(ii), (5), (6), and (8)(iv)(C) of 
Sec. 798.6400.
    (2) For the purpose of this section, the following provisions also 
apply:
    (i) Route of exposure. Animals shall be exposed either orally or by 
inhalation.
    (ii) Lower doses. The data from the lower doses shall show either 
graded dose-dependent effects in at least two of all the doses tested 
including the highest dose, or no neurotoxic (behavioral) effects at any 
dose tested.
    (iii) Duration and frequency of exposure. Animals shall be exposed 
orally once per day 5 days per week for a 90-day period; or if exposed 
by inhalation, for 6 hours per day 5 days per week for a 90-day period.
    (iv) Clearing and embedding. After dehydration, tissue specimens 
shall be cleared with xylene and embedded in paraffin or paraplast 
except for the sural nerve which should be embedded in plastic. Multiple 
tissue specimens (e.g., brain, cord, ganglia) may be embedded together 
in one single block for sectioning. All tissue blocks shall be labeled 
to provide unequivocal identification. A suggested method for plastic 
embedding is described by Spencer et al. in paragraph (d)(6) of this 
section.
    (ii) Reporting requirements. (A) The neurotoxicity tests required 
under this paragraph (c)(7) and administered orally shall be completed 
and the final results submitted to EPA within 18 months of the date 
specified in paragraph (e) of this section. The neurotoxicity tests 
required under this paragraph (c)(7) and administered by inhalation 
shall be completed and the final results submitted to EPA within 21 
months of the date specified in paragraph (e) of this section.
    (B) Interim progress reports shall be submitted to EPA at 6-month 
intervals beginning 6 months after the date specified in paragraph (e) 
of this section until submission of the final report to EPA.
    (d) References. For additional background information, the following 
references should be consulted.
    (1) Lamb, J. and Chapin, R.E. ``Experimental models of male 
reproductive toxicology.'' In: ``Endocrine Toxicity.'' Thomas, J.A., 
Korach, K.S., and McLachlan, J.A., eds. New York, NY: Raven Press. pp. 
85-115. (1985).

[[Page 258]]

    (2) Clermont, Y. and Percey, B. ``Quantitative study of the cell 
population of the seminiferous tubules in immature rats.'' ``American 
Journal of Anatomy.'' 100:241-267. (1957).
    (3) Sadleir, R.M.F.S. ``Cycles and seasons.'' In: ``Reproduction in 
Mammals: I. Germ Cells and Fertilization.'' Austin, R. and Short R.V., 
eds. New York, NY: Cambridge Press. Chapter 4. (1978).
    (4) Mattison, D.R. and Thorgiersson, S.S. ``Ovarian aryl hydrocarbon 
hydroxylase activity and primordial oocyte toxicity of polycyclic 
aromatic hydrocarbons in mice.'' ``Cancer Research.'' 39:3471-3475. 
(1979).
    (5) Pederson, T. and Peters, H. ``Proposal for classification of 
oocytes and follicles in the mouse ovary.'' ``Journal of Reproduction 
and Fertility.'' 17:555-557. (1968).
    (6) Spencer, P.S., Bischoff, M., and Schaumburg, H.H. 
``Neuropathological methods for the detection of neurotoxic disease.'' 
In: ``Experimental and Clinical Neurotoxicology.'' Spencer, P.S. and 
Schaumburg, H.H., eds. Baltimore, MD: Williams and Wilkins, pp. 743-757 
(1980).
    (7) Hafez, E.S., ed., ``Reproduction and Breeding Techniques for 
Laboratory Animals.'' Chapter 10. Philadelphia: Lea and Febiger. (1970).
    (e) Effective dates. (1) The effective date of this final rule is 
October 27, 1989.
    (2) The guidelines and other test methods cited in this section are 
referenced here as they exist on October 27, 1989.

[54 FR 37808, Sept. 13, 1989, as amended at 58 FR 34205, June 23, 1993]



Sec. 799.3300  Unsubstituted phenylenediamines.

    (a) Identification of test substance. (1) The unsubstituted 
phenylenediamines (pda's), para-phenylenediamine (p-pda, CAS No. 106-50-
3), or its sulfate salt (p-pda.H2SO4, CAS No. 
1624-57-75), meta-phenylenediamine (m-pda, CAS No. 108-45-2), or its 
sulfate salt (m-pda.H2SO4, CAS No. 54-17-08), and 
ortho-phenylenediamine (o-pda, CAS No. 95-54-5) shall be tested in 
accordance with this section.
    (2) p-Pda, m-pda, and o-pda of at least 98 percent purity shall be 
used as the test substances. Either the hydrochloride or sulfate salt of 
m-pda shall be used as the test substances. Either the hydrochloride or 
sulfate salt of m-pda shall be used as a test substance in the 
oncogenicity test in paragraph (c)(2) of this section if the free base 
proves to be unstable under the conditions of this study. Either the 
hydrochloride or sulfate salt of o-pda, p-pda, or m-pda shall be used as 
a test substance in the 90-day subchronic neurotoxicity studies in 
paragraph (c)(3)(B) of this section if the free base proves to be 
unstable under the conditions of these studies. The salt(s) shall be of 
at least 98 percent purity.
    (b) Persons required to submit study plans, conduct tests, and 
submit data. (1) All persons who manufacture (including import or by-
product manufacture) or process m-pda or m-
pda.H2SO4, or intend to manufacture or process m-
pda or m-pda.H2SO4, after the effective date of 
this rule to the end of the reimbursement period shall submit letters of 
intent to test, submit study plans, conduct tests, and submit data, or 
submit exemption applications as specified in paragraphs (c), (d), and 
(e) of this section, subpart A of this part, and parts 790 and 792 of 
this chapter for single-phase rulemaking.
    (2) All persons who manufacture (including import or by-product 
manufacture) or process p-pda, or p-pda.H2SO4, or 
intend to manufacture or process p-pda, or p-pda 
H2SO4, after the effective date of this rule to 
the end of the reimbursement period shall submit letters of intent to 
test, submit study plans, conduct tests, and submit data, or submit 
exemption applications as specified in paragraphs (c)(3), (d), and (e) 
of this section, subpart A of this part and parts 790 and 792 of this 
chapter for single-phase rulemaking.
    (3) All persons who manufacture (including import or by-product 
manufacture) or process o-pda, or intend to manufacture or process o-pda 
after the effective date of this rule to the end of the reimbursement 
period shall submit letters of intent to test, submit study plans, 
conduct tests, and submit data, or submit exemption applications as 
specified in paragraphs (c)(3), (d), and (e) of this section, subpart A 
of this part, and parts 790 and 792 of this chapter for single-phase 
rulemaking.

[[Page 259]]

    (c) Health effects testing--(1) Mutagenicity testing--(i) Required 
testing. (A) The sex-linked recessive lethal (SLRL) assay shall be 
conducted, by injection, in Drosophila melanogaster with m-pda in 
accordance with Sec. 798.5275 of this chapter.
    (B) If the SLRL assay conducted pursuant to paragraph (c)(1)(i)(A) 
of this section is positive, either the mouse visible specific locus 
test (MVSL) or the mouse biochemical specific locus test (MBSL) shall be 
conducted for m-pda by gavage in accordance with Sec. Sec. 798.5200 or 
798.5195 of this chapter, if after public program review, EPA issues a 
Federal Register notice or sends a certified letter to the test 
sponsor(s) specifying that testing shall be initiated. The test sponsor 
shall notify EPA of its choice in writing in its first interim report.
    (C) The mouse bone marrow cytogenetics: micronucleus (MBMC) assay 
shall be conducted on m-pda in accordance with Sec. 798.5395 of this 
chapter.
    (D) If the MBMC assay conducted pursuant to paragraph (c)(1)(i)(C) 
of this section is positive, the dominant lethal assay (DL) in mice 
shall be conducted on m-pda pursuant to Sec. 798.5450 of this chapter.
    (E) If the DL conducted pursuant to paragraph (c)(1)(i)(D) of this 
section is positive, heritable translocation (HT) testing in the mouse 
on m-pda shall be conducted pursuant to Sec. 798.5460 of this chapter, 
if after a public program review, EPA issues a Federal Register notice 
or sends a certified letter to the test sponsor(s) specifying that 
testing shall be initiated.
    (ii) Reporting requirements. (A) The tests shall be completed and 
the final reports for the MBMC assay shall be submitted to the EPA no 
later than January 16, 1991. The final report for the SLRL in Drosophila 
melanogaster shall be submitted no later than April 15, 1991.
    (B) If required, the DL test shall be completed and the final report 
shall be received by EPA no later than 24 months after the effective 
date of this final rule.
    (C) If required, the MVSL or the MBSL shall be completed and the 
final report shall be received by EPA no later than 51 months after EPA 
issues a Federal Register Notice or sends a certified letter to the test 
sponsor(s) identified under paragraph (c)(1)(i)(B) of this section 
specifying that testing shall be initiated.
    (D) If required, the HT test shall be completed and the final report 
shall be submitted to EPA not later than 36 months after the date on 
which EPA notifies the test sponsor under paragraph (c)(1)(i)(E) of this 
section to begin testing.
    (E) Interim reports for the SLRL assay and MBMC are required at 6-
month intervals beginning 6 months after the effective date of this 
section. If the DL is triggered, interim reports are required at 6 month 
intervals beginning with the date of initiation of the study.
    (F) Interim reports for the HT and either the MBSL or MVSL are 
required at 6-month intervals beginning 6 months after the date of 
notification by EPA that testing shall be initiated, and ending when the 
final report is submitted.
    (2) Oncogenicity--(i) Required testing. A 2-year dermal oncogenicity 
bioassay shall be conducted with m-pda if, after public program review, 
EPA issues a Federal Register notice specifying that the testing shall 
be initiated.
    (ii) [Reserved]
    (iii) Reporting requirements. (A) The final results and final report 
for the oncogenicity bioassay shall be submitted to EPA no later than 53 
months after EPA issues a Federal Register notice or sends a certified 
letter to the test sponsor under paragraph (c)(2)(i) of this section 
specifying that the testing shall be initiated.
    (B) Interim reports for the oncogenicity study are required at 6-
month intervals beginning 6 months after the date of notification by EPA 
that testing shall be initiated and ending when the final report is 
submitted.
    (3) Neurotoxicity--(i) Required testing. (A) Acute neurotoxicity 
testing in the neurotoxicity functional observational battery (FOB) in 
accordance with Sec. 798.6050 of this chapter, and the motor activity 
test (MAT) in accordance with Sec. 798.6200 of this chapter, shall be 
conducted for o-, m-, and p-pda.

[[Page 260]]

The test chemicals shall be administered in a single oral dose. Clinical 
observations shall be made at a minimum of 1, 4, 24, and 48 hours and at 
7 days after dosing.
    (B) If neurotoxic effects are observed at 24 hours, or longer, 
during the testing conducted pursuant to paragraph (c)(3)(i)(A) of this 
section, then 90-day subchronic neurotoxic FOB and MAT tests shall be 
conducted in accordance with Sec. Sec. 798.6050 and 798.6200 of this 
chapter, respectively, for each isomer showing such effects. At the end 
of these tests, the animals shall be sacrificed and the nervous tissue 
preserved and examined as described in the neuropathology test standard, 
Sec. 798.6400 of this chapter.
    (ii) Reporting requirements. (A) The acute neurotoxicity tests shall 
be completed and the final report submitted to EPA no later than 
September 15, 1990. If triggered, the final report of the subchronic 
neurotoxicity testing and the neuropathological examination shall be 
submitted to EPA on the following schedules. If one isomer is triggered, 
the reporting deadline is July 15, 1990. If two isomers are triggered, 
the reporting deadline is January 15, 1992. If three isomers are 
triggered, the reporting deadline is July 15, 1992.
    (B) [Reserved]
    (d) Chemical fate testing--(1) Indirect photolysis testing--(i) 
Required testing. Indirect photolysis studies shall be conducted with p-
, m-, and o-pda to determine the half-life in water of each of the three 
unsubstituted pda's in accordance with Sec. 795.70 of this chapter.
    (ii) Reporting requirements. (A) The final report shall be submitted 
to EPA no later than 8 months after the effective date of the final 
rule.
    (B) The final report shall include a calculation of the predicted 
environmental concentration (PEC), 100xPEC, and 1,000xPEC for each 
isomer. PEC shall be calculated by using results from the indirect 
photolysis studies and solving the following equations for the 
appropriate isomer: o-pda: PECo = 0.3629 + 1.0468 log t 1/2; m-pda: PECm 
= 0.6830 + 1.9702 log t 1/2; p-pda: PECp = 0.0085 + 0.0024 log t 1/2, 
where PEC is the predicted concentration in ppb and t 1/2 is the half-
life for oxidation (i.e., indirect photolysis) expressed in minutes. 
PEC, 100xPEC, and 1,000xPEC shall be used in the decision logic 
described in paragraph (e) of this section.
    (2) [Reserved]
    (e) Environmental effects testing--(1) Acute toxicity testing--(i) 
Required testing. (A) Flow-through fish acute toxicity tests in the 
rainbow trout (Salmo gairdneri) shall be conducted with o-, m-, and p-
pda in accordance with Sec. 797.1400 of this chapter.
    (B) Acute flow-through studies on the freshwater invertebrate 
Gammarus shall be conducted with o-, m-, and p-pda in accordance with 
Sec. 795.120 of this chapter.
    (C) If the concentration affecting 50 percent of the population 
(LC50 or EC50) for any study conducted pursuant to 
paragraphs (e)(1)(i)(A) and (B) of this section is less than or equal to 
100xPEC, less than or equal to 1 milligram/liter (mg/L), or less than or 
equal to 100 mg/L and shows indications of chronicity, chronic toxicity 
testing shall be conducted pursuant to paragraph (e)(2) of this section. 
Indications of chronicity shall be the following: for fish or aquatic 
invertebrates, the ratio of 24 hour/96 hour LC50s is greater 
than or equal to 2; for gammarids, the ratio of 24 hour/48 hour 
EC50s is greater than or equal to 2.
    (ii) Reporting requirements. The final reports for acute toxicity 
testing shall be submitted as follows:
    (A) Testing on the rainbow trout shall be completed and submitted to 
EPA 9 months after the effective date of the final rule for o-pda and p-
pda. Testing for m-pda shall be completed and submitted by January 15, 
1991.
    (B) The acute toxicity testing in freshwater Gammarus shall be 
completed and submitted no later than January 15, 1991.
    (2) Chronic toxicity testing--(i) Required testing. (A) A fish 
partial life-cycle flow-through test shall be conducted in the more 
sensitive fish species, either Pimephales promelas or Salmo gairdneri, 
with each isomer, o-, m-, and p-pda, demonstrating an LC50, 
determined by testing of fish pursuant to paragraph (e)(1)(i)(A) of this 
section, equal to or less than 100xPEC; or less than 1 mg/L; or less 
than 100 mg/L with indications of chronicity. Chronicity indicators are 
defined in paragraph

[[Page 261]]

(e)(1)(i)(C) of this section. Testing shall be conducted in accordance 
with Sec. 797.1600 of this chapter.
    (B) An invertebrate life-cycle flow-through toxicity test shall be 
conducted in Daphnia magna for o- and p-pda in accordance with Sec. 
797.1330 of this chapter.
    (ii) Reporting requirements. (A) The fish partial life-cycle flow-
through test shall be completed and final results shall be submitted to 
EPA no later than December 1, 1992.
    (B) The invertebrate life-cycle flow-through toxicity test shall be 
completed and the final report submitted to EPA no later than January 
15, 1993.
    (C) Progress reports shall be submitted at 6 month intervals after 
the effective date of the final rule.
    (f) Effective dates. (1) The effective date of this final rule is 
January 16, 1990, except for paragraphs (c)(1)(i)(B), (c)(1)(ii)(A), 
(c)(1)(ii)(C), (c)(1)(ii)(F), (c)(3)(ii)(A), (e)(1)(ii), (e)(2)(ii)(A), 
and (e)(2)(ii)(B) of this section. The effective date for paragraphs 
(c)(1)(i)(B), (c)(1)(ii)(C), and (c)(1)(ii)(F) of this section is May 
21, 1990. The effective date for paragraphs (c)(1)(ii)(A), 
(c)(3)(ii)(A), and (e)(1)(ii), of this section is May 21, 1991. The 
effective date for paragraph (e)(2)(ii)(A) is June 12, 1992. The 
effective date for paragraph (e)(2)(ii)(B) is May 28, 1993.
    (2) The guidelines and other test methods cited in this rule are 
referenced as they exist on the effective date of the final rule.

[54 FR 49294, Nov. 30, 1989, as amended at 55 FR 12644, Apr. 5, 1990; 56 
FR 23231, May 21, 1991; 57 FR 24961, June 12, 1992; 58 FR 30992, May 28, 
1993; 58 FR 34205, June 23, 1993]



Sec. 799.4360  Tributyl phosphate.

    (a) Identification of test substance. (1) Tributyl phosphate (TBP, 
CAS No. 126-73-8) shall be tested in accordance with this section.
    (2) TBP of at least 99 percent purity shall be used as the test 
substance.
    (b) Persons required to submit study plans, conduct tests, and 
submit data. All persons who manufacture (including import and byproduct 
manufacture) or process or intend to manufacture or process TBP, other 
than as an impurity, from the effective date of the final rule to the 
end of the reimbursement period shall submit letters of intent to 
conduct testing, submit study plans, conduct tests, and submit data, or 
submit exemption applications as specified in this section, subpart A of 
this part, and part 790 of this chapter for single-phase rulemaking.
    (c) Health effects testing--(1) Neurotoxicity--(i) Required testing. 
(A)(1) An acute and subchronic functional observational battery shall be 
conducted with TBP in accordance with Sec. 798.6050 of this chapter 
except for the provisions of paragraphs (d) (5) and (6) of Sec. 
798.6050.
    (2) For the purpose of this section, the following provisions also 
apply:
    (i) Animal selection. Testing shall be performed in laboratory rats.
    (ii) Duration of testing. For the acute testing, the substance shall 
be administered over a period not to exceed 24 hours; for the subchronic 
testing, test species shall be exposed daily for at least 90 days.
    (iii) Route of exposure. Animals shall be exposed to TBP orally.
    (B)(1) An acute and subchronic motor activity test shall be 
conducted with TBP in accordance with Sec. 798.6200 of this chapter 
except for the provisions of paragraphs (d) (5) and (6) of Sec. 
798.6200.
    (2) For the purpose of this section, the following provisions also 
apply:
    (i) Animal selection. Testing shall be performed in laboratory rats.
    (ii) Duration of testing. For the acute testing, the substance shall 
be administered over a period not to exceed 24 hours; for the subchronic 
testing, test species shall be exposed daily for at least 90 days.
    (iii) Route of administration. Animals shall be exposed to TBP 
orally.
    (C)(1) A neuropathology test shall be conducted with TBP in 
accordance with Sec. 798.6400 of this chapter except for the provision 
of paragraphs (d)(1)(i) (5) and (6) of Sec. 798.6400.
    (2) For the purpose of this section, the following provisions also 
apply:
    (i) Animal selection. Testing shall be performed in laboratory rats.
    (ii) Duration of testing. Animals shall be exposed for at least a 
90-day period.
    (iii) Route of administration. Animals shall be exposed to TBP 
orally.
    (ii) Reporting requirements--(A) The neurotoxicity tests required 
under

[[Page 262]]

paragraph (c)(1)(i) (A), (B), and (C) of this section shall be completed 
and final reports submitted to EPA within 18 months of the effective 
date of the final rule.
    (B) An interim progress report for these neurotoxicity tests shall 
be submitted to EPA 6 months after the effective date of the final rule.
    (2) Developmental toxicity--(i) Required testing. (A) A 
developmental toxicity study shall be conducted with TBP in accordance 
with Sec. 798.4900 of this chapter, except for the provisions of 
paragraph (e)(5) of Sec. 798.4900.
    (B) for the purpose of this section, the following provision also 
applies:
    (1) Route of administration. The animals shall be exposed to TBP by 
gavage.
    (2) [Reserved]
    (ii) Reporting requirements. (A) The developmental toxicity study 
required under paragraph (c)(2) of this section shall be completed and a 
final report submitted to EPA by January 27, 1991.
    (B) An interim progress report shall be submitted to EPA 6 months 
after the effective date of the final rule.
    (3) Reproductive and fertility--(i) Required testing. (A) A 
reproduction and fertility study shall be conducted with TBP in 
accordance with Sec. 798.4700 of this chapter, except for the 
provisions of paragraph (c)(5)(i)(A) of Sec. 798.4700.
    (B) for the purpose of this section, the following provisions also 
apply:
    (1) Route of administration. Animals should be exposed to TBP by 
gavage.
    (2) [Reserved]
    (ii) Reporting requirements. (A) The reproduction and fertility 
effects study required under paragraph (c)(3) of this section shall be 
completed and a final report submitted to EPA by August 17, 1992.
    (B) Interim program reports shall be submitted to EPA at 6 month 
intervals, beginning 6 months after the effective date of the final 
rule, until the final report is submitted to EPA.
    (4) Mutagenic effects--Gene mutation--(i) Required testing. (A) A 
detection of gene mutation in somatic cells in culture test shall be 
conducted with TBP in accordance with Sec. 798.5300 of this chapter.
    (B)(1) If TBP produces a positive result in the assay conducted 
pursuant to paragraph (c)(4)(i)(A) of this section, a sex-linked 
recessive lethal test in Drosophila melanogaster shall be conducted with 
TBP in accordance with Sec. 798.5275 of this chapter, except for the 
provisions of paragraph (d)(5)(iii) of Sec. 798.5275.
    (2) For the purpose of this section, the following provisions also 
apply:
    (i) Route of administration. Animals shall be exposed to TBP orally.
    (ii) [Reserved]
    (iii) Reporting requirements. (A) The somatic cells in culture assay 
shall be completed and the final report submitted to EPA, within 10 
months after the effective date of the final rule. If required, the 
Drosophila sex-linked recessive lethal assay shall be completed and the 
final report submitted to EPA within 22 months after the effective date 
of the final rule.
    (B) Interim progress reports shall be submitted to EPA at 6 month 
intervals beginning 6 months after initiation of the sex-linked 
recessive lethal test in Drosophila until the applicable final reports 
are submitted to EPA.
    (5) Mutagenic effects--Chromosomal aberration--(i) Required testing. 
(A) An in vitro mammalian cytogenetics test shall be conducted with TBP 
in accordance with Sec. 798.5375 of this chapter.
    (B)(1) If TBP produces a negative result in the in vitro 
cytogenetics test conducted pursuant to paragraph (c)(5)(i)(A) of this 
section, an in vivo mammalian bone marrow cytogenetics test shall be 
conducted with TBP in accordance with Sec. 798.5385 of this chapter, 
except for the provisions of paragraph (d)(5)(iii) of Sec. 798.5385.
    (2) For the purpose of this section, the following provisions also 
apply:
    (i) Route of administration. Animals shall be exposed to TBP orally.
    (ii) [Reserved]
    (C)(1) If TBP produces a positive result in either the in vitro or 
the in vivo cytogenetics test conducted pursuant to paragraphs (c)(5)(i) 
(A) and (B) of this section, a rodent dominant-lethal assay shall be 
conducted with TBP in accordance with Sec. 798.5450 of this chapter, 
except for the provisions of paragraph (d)(5)(iii) of Sec. 798.5450.
    (2) For the purpose of this section, the following provisions also 
apply:

[[Page 263]]

    (i) Route of administration. Animals shall be exposed orally to TBP.
    (ii) [Reserved]
    (D)(1) A rodent heritable trans- location assay shall be conducted 
with TBP if the dominant-lethal assay conducted for TBP pursuant to 
paragraph (c)(5)(i)(C) of this section produces a positive result, and 
if, after a public program review, EPA issues a Federal Register notice 
or sends a certified letter to the test sponsor specifying that the 
testing shall be initiated. This test shall be conducted in accordance 
with Sec. 798.5460 of this chapter except for the provisions of 
paragraph (d)(5)(iii) of Sec. 798.5460.
    (2) For the purpose of this section, the following provisions also 
apply:
    (i) Route of administration. Animals shall be exposed to TBP orally.
    (ii) [Reserved]
    (ii) Reporting requirements. (A)(1) The in vitro mammalian 
cytogenetics test shall be completed and the final report submitted to 
EPA within 10 months after the effective date of the final rule.
    (2) If required, the in vivo mammalian bone-marrow cytogenetics test 
shall be completed and the final report submitted to EPA within 24 
months after the effective date of the final rule.
    (3) If required, the dominant lethal assay shall be completed and 
the final report submitted to EPA within 36 months after the effective 
date of the final rule.
    (4) If required, the heritable translocation assay shall be 
completed and the final report submitted to EPA within 25 months after 
the date of EPA's notification of the test sponsor under paragraph 
(c)(5)(i)(D) of this section that testing shall be initiated.
    (B) Interim progress reports shall be submitted to EPA at 6 month 
intervals beginning 6 months after initiation of the rodent dominant 
lethal assay and the rodent heritable translocation assay respectively, 
if required, until the applicable final reports are submitted to EPA.
    (6) Oncogenicity--(i) Required testing. (A) An oncogenicity test 
shall be conducted with TBP in accordance with Sec. 798.3300 of this 
chapter except for the provisions of paragraphs (b)(1)(i), (b)(6)(i) and 
(b)(9), of Sec. 798.3300.
    (B) For the purpose of this section, the following provisions also 
apply:
    (1) Animal selection. TBP shall be tested in Sprague-Dawley rats and 
in mice.
    (2) Route of administration. Animals shall be exposed to TBP orally.
    (3) Clinical examinations. At 12 months, 18 months and during month 
24, a blood smear shall be obtained from all animals. A differential 
blood count shall be performed on blood smears from those animals in the 
highest dosage group and the controls. If these data, or data from the 
pathological examination indicate a need, then the 12- and 18-month 
blood smears from other dose levels shall also be examined. Differential 
blood counts shall be performed for the next lower group(s) if there is 
a major discrepancy between the highest group and the controls. If 
clinical observations suggest a deterioration in health of the animals 
during the study, a differential blood count of the affected animals 
shall be performed.
    (ii) Reporting requirements. (A) The oncogenicity test required 
under paragraph (c)(6) of this section shall be completed and a final 
report submitted to EPA within 53 months of the effective date of the 
final rule.
    (B) Interim progress reports shall be submitted to EPA at 6 month 
intervals beginning 6 months after the effective date of the final rule, 
until the final report is submitted to EPA.
    (7) Dermal sensitization--(i) Required testing. A dermal 
sensitization test shall be conducted with TBP in accordance with Sec. 
798.4100 for this chapter.
    (ii) Reporting requirements. The dermal sensitization test shall be 
completed and the final report submitted to EPA within 6 months of the 
effective date of the final rule.
    (8) Oral/Dermal Pharmacokinetics--(i) Required testing. (A) A 
pharmaco- kinetics test shall be conducted with TBP in accordance with 
Sec. 795.228 of this chapter, except for the provisions of paragraphs 
(c)(1)(iii)(B), (c)(2)(ii)(C)(1) and (c)(2)(ii)(C)(2) of Sec. 795.228.
    (B) For the purposes of this section, the following provisions also 
apply:
    (1) Animal care. During the acclimatization period, the animals 
shall be

[[Page 264]]

housed in suitable cages. All animals shall be provided with certified 
feed and tap water ad libitum.
    (2) Dermal treatment. For dermal treatment, two doses, comparable to 
the low and high oral doses, shall be dissolved in a suitable vehicle 
and applied in volumes adequate to deliver comparable doses. The backs 
of the animals should be lightly clipped with an electric clipper 24 
hours before treatment. The test substance shall be applied to the 
intact clipped skin (approximately 2 cm\2\ for rats, 40 cm\2\ for mini-
pigs). The dosed areas shall be protected with a suitable porous 
covering which is secured in place, and the animals shall be housed 
separately.
    (ii) Reporting requirements. (A) The pharmacokinetics test required 
in paragraph (c)(8)(i) of this section shall be completed and the final 
report submitted to EPA by December 26, 1992.
    (B) Interim 6 month progress reports shall be submitted to EPA 
beginning at 6 months after the effective date of the final rule and 
continuing until submission of the final report.
    (d) Environmental effects testing--(1) Algal acute toxicity--(i) 
Required testing. (A) Algal acute toxicity testing shall be conducted 
with TBP using Selenastrum capricornutum in accordance with Sec. 
797.1050 of this chapter except for the provisions of paragraphs 
(c)(6)(i)(A),(B), and (ii) of Sec. 797.1050.
    (B) For the purpose of this section, the following provisions also 
apply:
    (1) Summary of the test. The algal cells at the end of 24, 48, and 
72 hours shall be enumerated.
    (2) Chemical measurement. The final separation of the algal cells 
from the test solution shall be done using an ultrafiltration (e.g., 
0.45 micrometer pore size) technique. The total and dissolved (e.g., 
filtered) concentrations of the test substance shall be measured in each 
test chamber and the delivery chamber before the test and in each test 
chamber at 0 and 96 hours.
    (ii) Reporting requirements. The algal acute toxicity test required 
in paragraph (d)(1) of this section shall be completed and the final 
report submitted to EPA within 9 months of effective date of the final 
rule.
    (2) Fish acute toxicity--(i) Required testing. (A) Fish acute 
toxicity testing shall be conducted with TBP using Salmo gairdneri 
(rainbow trout) in accordance with Sec. 797.1400 of this chapter.
    (B) For the purpose of this section, the following provisions also 
apply:
    (1) Chemical measurement. The total and dissolved (e.g., filtered) 
concentrations of the test substance shall be measured in each test 
chamber delivery chamber before the test. If the dissolved test 
substance concentration is greater than 80 percent of total test 
substance concentration, then only total or dissolved test concentration 
shall be measured in each chamber at 0, 48, and 96 hours. If the 
dissolved test substance concentration is less than or equal to 80 
percent of total test substance, then total and dissolved test substance 
concentration shall be measured at 0, 48 and 96 hours.
    (2) Test procedures. The test shall be performed under flow-through 
conditions.
    (ii) Reporting requirements. The fish acute toxicity test shall be 
completed and the final report submitted to EPA within 9 months of the 
effective date of the final rule.
    (3) Daphnid acute toxicity--(i) Required testing. (A) Daphnid acute 
toxicity testing shall be conducted with TBP using Daphnia magna or D. 
pulex in accordance with Sec. 797.1300 of this chapter.
    (B) For the purpose of this section, the following provisions also 
apply:
    (1) Chemical measurement. The total and dissolved (e.g., filtered) 
concentrations of the test substance shall be measured in each test 
chamber and the delivery chamber before the test. If the dissolved test 
substance concentration is greater than 80 percent of total test 
substance concentration, then only total or dissolved test concentration 
shall be measured in each chamber at 0, 24, and 48 hours. If the 
dissolved test substance concentration is less than or equal to 80 
percent of total test substance, then total and dissolved test substance 
concentration shall be measured at 0, 29, and 48 hours.
    (2) Test procedures. The test shall be performed under flow-through 
conditions.
    (ii) Reporting requirements. The daphnid acute toxicity test shall 
be

[[Page 265]]

completed and the final report submitted to EPA within 9 months of the 
effective date of the final rule.
    (4) Gammarid acute toxicity--(i) Required testing. (A) Gammarid 
acute toxicity testing shall be conducted with TBP using Gammarus 
lacustris, G. fasciatus, or G. pseudolimnaeus in accordance with Sec. 
795.120 of this chapter.
    (B) For the purpose of this section, the following provisons also 
apply:
    (1) Chemical measurement. The total and dissolved (e.g., filtered) 
concentrations of the test substance shall be measured in each test 
chamber and the delivery chamber before the test. If the dissolved test 
substance concentration is greater than 80 percent of total test 
substance concentration, then only total or dissolved test concentration 
shall be measured in each chamber at 0, 48, and 96 hours. If the 
dissolved test substance concentration is less than or equal to 80 
percent of total test substance, then total and dissolved test substance 
concentration shall be measured at 0, 48, and 96 hours.
    (2) Test procedures. The test shall be performed under flow-through 
conditions.
    (ii) Reporting requirements. The Gammarid acute toxicity test shall 
be completed and the final report submitted to EPA within 9 months of 
the effective date of the final rule.
    (5) Daphnid chronic toxicity--(i) Required testing. (A) Daphnid 
chronic toxicity testing shall be conducted with TBP using Daphnia magna 
or D. pulex in accordance with Sec. 797.1330 of this chapter, if the 
algal EC50, the rainbow trout LC50, the daphnid EC50, or the gammarid 
LC50 determined in accordance with paragraphs (d)(1), (2), (3) and (4) 
of this section satisfy the following criteria: Any such value is <= 1 
mg/L; or any fish or aquatic invertebrate EC50 or LC50 is <= 100 mg/L 
and either the rainbow trout or gammarid 24-hour to 96-hour LC50 ratio 
= 2, or the daphnid 24-hour to 48-hour EC50 or LC50 ratio is 
= 2.
    (B) For the purpose of this section, the following provisions also 
apply:
    (1) Chemical measurement. The total and dissolved (e.g., filtered) 
concentrations of the test substance shall be measured in each test 
chamber and the delivery chamber before the test. If the dissolved test 
substance concentration is greater than 80 percent of total test 
substance concentration, then only total or dissolved test substance 
concentration shall be measured in each test chamber at 0, 7, 14, and 21 
days. If the dissolved test substance concentration is less than or 
equal to 80 percent of total test substance concentration, then total 
and dissovled test substance concentration shall be measured at 0, 7, 
14, and 21 days.
    (2) Test procedures. The test shall be performed under flow-through 
conditions.
    (ii) Reporting requirements. (A) The daphnid chronic toxicity test, 
if required, shall be completed and the final report submitted to EPA by 
September 27, 1991.
    (B) An interim progress report shall be submitted to EPA 6 months 
after the initiation of the test.
    (6) Fish early-life stage toxicity--(i) Required testing. A fish 
early-life stage toxicity test shall be conducted with TBP in accordance 
with Sec. 797.1600 of this chapter, using the fish with the lower LC50 
value (either the rainbow trout (Salmo gairdneri) or the fathead minnow 
(Pimephales promelas)), if the algal EC50, the rainbow trout LC50, the 
gammarid LC50 or the daphnid EC50 determined in accordance with 
paragraphs (d)(1), (2), (3), and (4) of this section satisfy the 
following criteria: Any such value is <= 1 mg/L; or any fish or aquatic 
invertebrate EC50 or LC50 is <= 100 mg/L and either the rainbow trout or 
gammarid 24 hour to 96 hour LC50 ratio = 2, or the daphnid 
24-hour to 48-hour EC50 or LC50 ratio is = 2.
    (ii) Reporting requirements. (A) The fish early-life stage flow-
through toxicity test shall be completed and the final report submitted 
to EPA by December 27, 1991.
    (B) An interim progress report shall be submitted to EPA 6 months 
after the initiation of the test.
    (7) Benthic sediment invertebrate bioassay--(i) Required testing. 
(A) A benthic sediment invertebrate bioassay shall be conducted on TBP 
with the midge (Chironomus tentans) if chronic toxicity testing is 
required pursuant to paragraph (d)(5) of this section and if the log Koc 
calculated according to paragraph (e)(2)(B)(1) of this section is

[[Page 266]]

greater than or equal to 3.5 but less than or equal to 6.5. The total 
aqueous sediment concentrations and interstitial water concentrations of 
the test substance shall be measured in each test chamber at 0, 4, 7, 
10, and 14 days. The aqueous concentrations of the test substance in the 
delivery chamber shall be measured at 0, 4, 7, 10, and 14 days. TBP-
spiked clean freshwater sediments containing low, medium, and high 
organic carbon content shall be used.
    (B) The benthic sediment invertebrate bioassay shall be conducted 
according to the test procedure specified in the American Society for 
Testing and Materials, Special Technical Publication 854 (ASTM STP 854) 
entitled, ``Aquatic Safety Assessment of Chemicals Sorbed to 
Sediments,'' by W.J. Adams, R.A. Kimerle, and R.G. Mosher, published in 
Aquatic Toxicity and Hazard Assessment: Seventh Symposium, ASTM STP 854, 
pp. 429-453, R.D. Caldwell, R. Purdy, and R.C. Bahner, Eds., 1985 which 
is incorporated by reference. This published procedure is available for 
public inspection at the National Archives and Records Administration 
(NARA). For information on the availability of this material at NARA, 
call 202-741-6030, or go to: http://www.archives.gov/federal--register/
code--of--federal--regulations/ibr--locations.html. Copies may be 
obtained from the Non-Confidential Information Center (NCIC) (7407), 
Office of Pollution Prevention and Toxics, U.S. Environmental Protection 
Agency, Room B-607 NEM, 401 M St., SW., Washington, DC 20460, between 
the hours of 12 p.m. and 4 p.m. weekdays excluding legal holidays. This 
incorporation by reference was approved by the Director of the Federal 
Register in accordance with 5 U.S.C. 522(a) and 1 CFR part 51. The 
method is incorporated as it exists on the effective date of this rule 
and a notice of any change to the method will be published in the 
Federal Register.
    (ii) Reporting requirements. (A) The benthic sediment invertebrate 
bioassay, if required, shall be completed and the final report submitted 
to EPA within 21 months of the effective date of the final rule.
    (B) An interim progress report shall be submitted to EPA for the 
benthic sediment invertebrate bioassy 6 months after the initiation of 
the test.
    (e) Chemical fate testing--(1) Vapor pressure--(i) Required testing. 
Vapor pressure testing shall be conducted with TBP in accordance with 
Sec. 796.1950 of this chapter.
    (ii) Reporting requirements. The vapor pressure test required in 
paragraph (d)(1) of this section shall be completed and the final report 
submitted to EPA by September 27, 1990.
    (2) Sediment and soil adsorption isotherm--(i) Required testing. 
Sediment and soil absorption isotherm testing shall be conducted with 
TBP in accordance with Sec. 796.2750 of this chapter and EPA will 
provide two soil and two sediment samples.
    (ii) Reporting requirements. (A) The sediment and soil absorption 
isotherm test required under paragraph (d)(2) of this section shall be 
completed and the final report submitted to EPA by September 27, 1990.
    (B) For the purpose of this section, the following provisions also 
apply:
    (1) A Koc value shall be calculated for each test sediment using the 
equation Koc=K/ (percent of organic carbon in test sediment).
    (2) [Reserved]
    (3) Hydrolysis as a function of pH at 25 [deg]C--(i) Required 
testing. Hydrolysis testing shall be completed with TBP in accordance 
with Sec. 796.3500 of this chapter.
    (ii) Reporting requirements. The hydrolysis test required under 
paragraph (e)(3)(i) of this section shall be completed and the final 
report submitted to EPA by September 27, 1990.
    (f) Effective date. (1) The effective date of this final rule is 
September 27, 1989, except for paragraphs (c)(2)(ii)(A), (c)(3)(ii)(A), 
(c)(6)(i)(A), (c)(6)(i)(B)(3), (c)(8)(i), (c)(8)(ii)(A), (d)(5)(ii)(A), 
(d)(6)(ii)(A), (e)(1)(ii), (e)(2)(ii)(A), and (e)(3)(ii) of this 
section. The effective date for paragraphs (c)(2)(ii)(A), (c)(3)(ii)(A), 
(c)(8)(i), (e)(1)(ii), (e)(2)(ii)(A), and (e)(3)(ii) of this section is 
May 21, 1991. The effective date for (c)(8)(ii)(A), (d)(5)(ii)(A), and 
(d)(6)(ii)(A) of this section is June 12, 1992. The effective date for 
(c)(6)(i)(A), (c)(6)(i)(B)(3), and (c)(8)(ii)(A) is May 28, 1993.

[[Page 267]]

    (2) The guidelines and other test methods cited in this rule are 
referenced as they exist on the effective date of the final rule.

[54 FR 33413, Aug. 14, 1989; 56 FR 23231, May 21, 1991, as amended at 57 
FR 24961, June 12, 1992; 58 FR 30992, May 28, 1993; 58 FR 34205, June 
23, 1993; 60 FR 34467, July 3, 1995; 69 FR 18803, Apr. 9, 2004]



Sec. 799.4440  Triethylene glycol monomethyl ether.

    (a) Identification of test substance. (1) Triethylene glycol 
monomethyl ether (TGME, CAS No. 112-35-6) shall be tested in accordance 
with this section.
    (2) TGME of at least 90 percent purity shall be used as the test 
substance.
    (b) Persons required to submit study plans, conduct tests, and 
submit data. All persons who manufacture or process TGME, other than as 
an impurity, after May 17, 1989, to the end of the reimbursement period 
shall submit letters of intent to conduct testing, submit study plans, 
conduct tests and submit data, or submit exemption applications as 
specified in this section, subpart A of this part, and parts 790 and 792 
of this chapter for single-phase rulemaking.
    (c) Developmental neurotoxicity--(1) Required testing. Developmental 
neurotoxicity testing shall be performed in the Sprague-Dawley rat by 
gavage in accordance with Sec. 795.250 of this chapter except for the 
provision in paragraph (c)(3)(iii) of Sec. 795.250.
    (2) For the purpose of this section, the following provisions also 
apply:
    (i) Number of animals. The objective is for a sufficient number of 
pregnant rats to be exposed to ensure that an adequate number of 
offspring are produced for neurotoxicity evaluation. At least 24 litters 
are recommended at each dose level.
    (ii) Dose levels and dose selection. In the absence of developmental 
toxicity or maternal toxicity the maximum dose shall be 5 grams/
kilogram.
    (3) Reporting requirements--(i) The developmental neurotoxicity test 
shall be completed and the final report submitted to EPA within 21 
months of the initiation of the test.
    (ii) Progress reports shall be submitted to EPA at 6- month 
intervals, beginning six months after the initiation of the test.
    (d) Effective date. (1) The effective date of this final rule is May 
17, 1989, except for paragraph (c)(2)(i) and (c)(3)(i) of this section. 
The effective date for paragraph (c)(2)(ii) and (c)(3)(i) of this 
section is May 21, 1991.
    (2) The guidelines and other test methods cited in this rule are 
referenced as they exist on the effective date of the final rule.

[54 FR 13477, Apr. 3, 1989; 56 FR 23232, May 21, 1991, as amended at 58 
FR 34205, June 23, 1993]



                    Subpart C_Testing Consent Orders



Sec. 799.5000  Testing consent orders for substances and mixtures with 

Chemical Abstract Service Registry Numbers.

    This section sets forth a list of substances and mixtures which are 
the subject of testing consent orders adopted under 40 CFR part 790. 
Listed below in Chemical Abstract Service (CAS) Registry Number order 
are the substances and mixtures which are the subject of these orders 
and the Federal Register citations providing public notice of such 
orders.

------------------------------------------------------------------------
                   Substance or                          FR Publication
  CAS Number       mixture name          Testing              Date
------------------------------------------------------------------------
       67-64-1  Acetone...........  Health effects...  January 23, 1995.
       71-55-6  1,1,1-              Health effects...  August 23, 1989.
                 Trichloroethane.
       78-83-1  Isobutyl alcohol..  Health effects...  January 23, 1995.
       79-10-7  Acrylic Acid......  Health effects...  March 4, 1992.
       84-74-2  Di-n-butyl          Environmental      January 9, 1989.
                 phthalate.          effects.
       84-75-3  Di-n-hexyl          Environmental      January 9, 1989.
                 phthalate.          effects.
                ..................  Chemical fate....  January 9, 1989.
      100-40-3  4-Vinylcyclohexene  Health effects...  September 23,
                                                        1991.
                ..................  Chemical fate....  September 23,
                                                        1991.
      106-91-2  Glycidyl            Health effects...  January 26, 1995.
                 methacrylate.
      108-10-1  Methyl isobutyl     Health effects...  January 23, 1995.
                 ketone.
      109-99-9  Tetrahydrofuran...  Health effects...  January 23, 1995.

[[Page 268]]

 
      110-82-7  Cyclohexane.......  Health Effects     November 18,
                                     and                1994.
                                     Environmental
                                     Releases Report.
      112-35-6  Triethylene glycol  Health effects...  April 3, 1989.
                 monomethyl ether.
      112-50-5  Triethylene glycol  Health effects...  April 3, 1989.
                 monoethyl ether.
      117-81-7  Di-2-ethylhexyl     Chemical fate....  January 9, 1989.
                 phthalate.
      119-06-2  Ditridecyl          Chemical fate....  January 9, 1989.
                 phthalate.
      123-86-4  N-butyl acetate...  Health effects...  January 23, 1995.
      131-11-3  Dimethly phthalate  Environmental      January 9, 1989.
                                     effects.
      141-78-6  Ethyl acetate.....  Health effects...  January 23, 1995.
      141-79-7  Mesityl oxide.....  Health effects...  September 5,
                                                        1991.
      143-22-6  Triethylene glycol  Health effects...  January 9, 1989.
                 monobutyl ether.
      143-33-9  Sodium cyanide....  Chemical fate....  December 17,
                                                        1991.
                ..................  Terrestrial        December 17,
                                     effects.           1991.
      556-67-2  Octamethylcyclo-    Chemical fate....  January 10, 1989.
                 tetrasiloxane.
                ..................  Environmental      January 10, 1989.
                                     effects.
      628-63-7  N-amyl acetate....  Health effects...  January 23, 1995.
      872-50-4  N-                  Health effects...  November 23,
                 methylpyrrolidone.                     1993.
      994-05-8  Tertiary-amyl       Health effects...  March 21, 1995.
                 methyl ether.
     1634-04-4  Methyl tert-butyl   Health effects...  March 31, 1988.
                 ether.
     2461-18-9  Lauryl glycidyl     Health effects...  June 11, 1996.
                 ether \1\.
     3618-72-2  C.I. Disperse Blue  Health effects...  November 21,
                 79:1 Acetamide,N-                      1989.
                 [5-[bis[2-
                 (acetyloxy)
                 ethyl]amino]-2-
                 [(2-bromo-4, 6-
                 dinitrophenyl)
                 azo]-4-
                 methoxyphenyl]-.
                ..................  Environmental      November 21,
                                     effects.           1989.
     3648-20-2  Diundecyl           Environmental      January 9, 1989.
                 phthalate.          effects.
     4170-30-3  Crotonaldehyde....  Environmental      November 9, 1989.
                                     effects.
                ..................  Chemical fate....  November 9, 1989.
     4675-54-3  Bisphenol A         Health effects...  August 1, 1994.
                 diglycidyl ether.  Exposure
                                     evaluation.
    15965-99-8  Hexadecyl glycidyl  Health effects...  June 11, 1996.
                 ether \1\.
    16245-97-9  n-Octadecyl         Health effects...  June 11, 1996.
                 glycidyl ether
                 \1\.
    26761-40-0  Diisodecyl          Chemical fate....  January 9, 1989.
                 phthalate.
    38954-75-5  Tetradecyl          Health effects...  June 11, 1996.
                 glycidyl ether
                 \1\.
    68081-84-5  Alkyl (C10-C16)     Health effects...  June 11, 1996.
                 glycidyl ether
                 \1\.
    68515-47-9  Ditridecyl          Chemical fate....  January 9, 1989.
                 phthalate (mixed
                 isomers).
    68515-49-1  Diisodecyl          Chemical fate....  January 9, 1989.
                 phthalate (mixed
                 isomers).
    68515-50-4  Dihexyl phthalate   Environmental      January 9, 1989.
                 (mixed isomers).    effects.
                ..................  Chemical fate....  January 9, 1989.
    68609-97-2  Alkyl (C12-C14)     Health effects...  June 11, 1996.
                 glycidyl ether
                 \1\.
   84852-15-3*  4-Nonylphenol,      Environmental      February 21,
                 branched.           effects.           1990.
                ..................  Chemical fate....  February 21,
                                                        1990.
   120547-52-6  Alkyl (C12-C13)     Health effects...  March 22, 1996.
                 glycidyl ether.
   142844-00-6  Refractory ceramic  Exposure           May 14, 1993.
                 fibers.             monitoring.
------------------------------------------------------------------------
\1\ As represented by alkyl (C12-C13) glycidyl ether (CAS No. 120547-52-
  6)


[57 FR 18829, May 1, 1992, as amended at 57 FR 24961, June 12, 1992; 58 
FR 28520, May 14, 1993; 58 FR 34205, June 23, 1993; 58 FR 61816, Nov. 
23, 1993; 59 FR 38920, Aug. 1, 1994; 59 FR 59663, Nov. 18, 1994; 60 FR 
4519, Jan. 23, 1995; 60 FR 5140, Jan. 26, 1995; 60 FR 14911, Mar. 21, 
1995; 60 FR 31924, June 19, 1995; 61 FR 11742, Mar. 22, 1996; 61 FR 
29487, June 11, 1996]



Sec. 799.5025  Testing consent orders for mixtures without Chemical Abstracts 

Service Registry Numbers.

    This section sets forth a list of mixtures (with no Chemical 
Abstracts Service Registry Numbers) which are the subject of testing 
consent orders adopted under 40 CFR part 790. Listed below are the 
mixtures which are the subject of these orders and the Federal Register 
citations providing public notice of such orders.

------------------------------------------------------------------------
        Mixture/substance             Required test       FR citation
------------------------------------------------------------------------
Di(heptyl, nonyl, undecyl)
 phthalate (D711P) as a mixture
 of the following six substances:
  (1) diheptyl phthalate           Environmental       January 9, 1989.
   (branched and linear isomers),   effects.
   CAS No. 68515-44-6

[[Page 269]]

 
  (2) dinonyl phthalate (branched  ......do..........  Do.
   and linear isomers), CAS No.
   68515-45-7
  (3) di(heptyl, nonyl) phthalate  ......do..........  Do.
   (branched and linear isomers),
   CAS No. 111381-89-6
  (4) diundecyl phthalate          ......do..........  Do.
   (branched and linear isomers),
   CAS No. 3648-20-2
  (5) di(heptyl, undecyl)          ......do..........  Do.
   phthalate (branched and linear
   isomers), CAS No., 111381-90-9
  (6) di(nonyl, undecyl)           ......do..........  Do.
   phthalate (branched and linear
   isomers), CAS No. 111381-91-0)
Fluoropolymer composite
 substance:
  (1) For Dry Non-Melt Resin                           .................
   containing the following
   chemical substances as
   specified in the ECA:
  (i) Ethene, tetrafluoro-,        Environmental       July 8, 2005.
   homopolymer, CAS No. 9002-84-0   effects.
  (ii) Polytetrafluoroethylene,    ......do..........  Do.
   Document Control Number (DCN)
   63040000018A
  (iii) Propane, 1,1,1,2,2,3,3-    ......do..........  Do.
   heptafluoro-3-
   [(trifluoroethenyl)oxy]-,
   polymer with
   tetrafluoroethene, CAS No.
   26655-00-5
  (2) For Dry Melt Fluoropolymer
   Resin containing the following
   chemical substances as
   specified in the ECA:
  (i) 1-Propene, 1,1,2,3,3,3-      ......do..........  Do.
   hexafluoro-, polymer with
   tetrafluoroethene, CAS No.
   25067-11-2
  (ii) Propane, 1,1,1,2,2,3,3-     ......do..........  Do.
   heptafluoro-3-
   [(trifluoroethenyl)oxy]-,
   polymer with
   tetrafluoroethene, CAS No.
   26655-00-5
  (iii) Ethene, tetrafluoro-,      ......do..........  Do.
   polymer with
   trifluoro(pentafluoroethoxy)et
   hene, CAS No. 31784-04-0
  (iv) 1-Propene, 1,1,2,3,3,3-     ......do..........  Do.
   hexafluoro-, polymer with 1,1-
   difluoroethene and
   tetrafluoroethene, CAS No.
   25190-89-0
  (v) ETFE, DCN 63040000026        ......do..........  Do.
  (vi) 1-Propene, 1,1,2,3,3,3-     ......do..........  Do.
   hexafluoro-, polymer with
   ethene and tetrafluoroethene,
   CAS No. 35560-16-8
  (3) For Dry Non-Melt
   Fluoroelastomer Resin/Gum
   containing the following
   chemical substances as
   specified in the ECA:
  (i) 1-Propene, 1,1,2,3,3,3-      ......do..........  Do.
   hexafluoro-, polymer with 1,1-
   difluoroethene, CAS No. 9011-
   17-0
  (ii) 1-Propene, 1,1,2,3,3,3-     ......do..........  Do.
   hexafluoro-, polymer with 1,1-
   difluoroethene and
   tetrafluoroethene, CAS No.
   25190-89-0
  (iii) 1-Propene, polymer with    ......do..........  Do.
   1,1- difluoroethene and
   tetrafluoroethene, CAS No.
   54675-89-7
  (iv) 1-Propene, polymer with     ......do..........  Do.
   tetrafluoroethene, CAS No.
   27029-05-6
  (v) Ethene, tetrafluoro-,        ......do..........  Do.
   polymer with
   trifluoro(trifluoromethoxy)
   ethene, CAS No. 26425-79-6
  (vi) Ethene, chlorotrifluoro-,   ......do..........  Do.
   polymer with 1,1-
   difluoroethene, CAS No. 9010-
   75-7
  (vii) Fluoroelastomer, DCN No.   ......do..........  Do.
   63040000018C
  (viii) Fluoroelastomer DCN       ......do..........  Do.
   63040000018D
  (ix) A low temperature           ......do..........  Do.
   fluoroelastomer, ACC No.
   137678
  (4) For Aqueous Fluoropolymer
   Dispersions containing the
   following chemical substances
   as specified in the ECA:
  (i) Ethene, tetrafluoro-,        ......do..........  Do.
   homopolymer, CAS No. 9002-84-0
  (ii) 1-Propene, 1,1,2,3,3,3-     ......do..........  Do.
   hexafluoro-, polymer with
   tetrafluoroethene, CAS No.
   25067-11-2
  (iii) Propane, 1,1,1,2,2,3,3-    ......do..........  Do.
   heptafluoro-3-
   [(trifluoroethenyl)oxy]-,
   polymer with
   tetrafluoroethene, CAS No.
   26655-00-5
  (iv) 1-Propene, 1,1,2,3,3,3-     ......do..........  Do.
   hexafluoro-, polymer with 1,1-
   difluoroethene and
   tetrafluoroethene, CAS No.
   25190-89-0
  (v) Polytetrafluoroethylene,     ......do..........  Do.
   DCN No. 63040000018B
Fluorotelomer-based composite
 substance:
  (1) For Paper containing three
   of the following chemical
   substances as specified in the
   ECA:

[[Page 270]]

 
  (i) Perfluoroalkylethyl          Environmental       July 8, 2005.
   acrylate copolymer, EPA-         effects.
   designated accession number
   (ACC) 171790
  (ii) Perfluoroalkyl acrylate     ......do..........  Do.
   copolymer, ACC 158022
  (iii) Perfluoroalkyl             ......do..........  Do.
   methacrylate polymer, EPA
   document control number (DCN)
   63040000037A
  (iv) Substituted methacrylate,   ......do..........  Do.
   propenoic acid, perfluoroalkyl
   esters, DCN 63040000033B
  (v) Perfluoroalkyl acrylic       ......do..........  Do.
   polymer, DCN 63040000037C
  (vi) Poly-.beta.-                ......do..........  Do.
   fluoroalkylethyl acrylate and
   alkyl acrylate, ACC 174993
  (vii) Poly(.beta.-               ......do..........  Do.
   fluoroalkylethyl acrylate and
   alkyl acrylate), ACC 70430
  (viii) Polysubstituted acrylic   ......do..........  Do.
   copolymer, ACC 157381
  (ix) Perfluoroalkyl acrylate     ......do..........  Do.
   copolymer latex, ACC No. 70907
  (2) For Textile containing six
   of the following chemical
   substances as specified in the
   ECA:
  (i) Perfluoroalkylethyl          ......do..........  Do.
   acrylate copolymer, EPA-
   designated accession number
   (ACC) 171790
  (ii) Perfluoroalkyl acrylate     ......do..........  Do.
   copolymer, ACC 158022
  (iii) Perfluoroalkyl             ......do..........  Do.
   methacrylate polymer, EPA
   document control number (DCN)
   63040000037A
  (iv) Substituted methacrylate,   ......do..........  Do.
   propenoic acid, perfluoroalkyl
   esters, DCN 63040000033B
  (v) Perfluoroalkyl acrylic       ......do..........  Do.
   polymer, DCN 63040000037C
  (vi) Poly-.beta.-                ......do..........  Do.
   fluoroalkylethyl acrylate and
   alkyl acrylate, ACC 174993
  (vii) Poly(.beta.-               ......do..........  Do.
   fluoroalkylethyl acrylate and
   alkyl acrylate), ACC 70430
  (viii) Polysubstituted acrylic   ......do..........  Do.
   copolymer, ACC 157381
  (ix) Perfluoroalkyl acrylate     ......do..........  Do.
   copolymer latex, ACC 70907
------------------------------------------------------------------------


[55 FR 3059, Jan. 30, 1990, as amended at 70 FR 39629, 39636, July 8, 
2005]



                   Subpart D_Multichemical Test Rules



Sec. 799.5055  Hazardous waste constituents subject to testing.

    (a) Identification of test substances. (1) The table in paragraph 
(c) of this section identifies those chemical substances that shall be 
tested in accordance with this section.
    (2) Substances of at least 98-percent purity shall be used as the 
test substances.
    (b) Persons required to submit study plans, conduct tests, and 
submit data. All persons who manufacuture (including import or 
manufacture as a byproduct) or process or intend to manufacture or 
process one or more of the substances in paragraph (c) of this section, 
other than as an impurity, after July 29, 1988, to the end of the 
reimbursement period shall submit letters of intent to conduct testing, 
submit study plans, conduct tests, and submit data, or submit exemption 
applications for those substances they manufacture or process, or intend 
to manufacture or process, as specified in this section, subpart A of 
this part, and parts 790 and 792 of this chapter for single-phase 
rulemaking.
    (c) Designation of testing. The substances identified in the 
following table by name and CAS number shall be tested in accordance 
with the designated requirements under paragraphs (d) and (e) of this 
section. The paragraph numbers listed for a substance refer to the 
specific testing and reporting requirements specified in paragraphs (d) 
and (e) of this section.

------------------------------------------------------------------------
                                                  Required testing under
           Chemical name               CAS No.    paragraphs (d) and (e)
                                                      of this section
------------------------------------------------------------------------
Acetamide, 2-fluoro................     640-19-7  (e)(1)
Bis(2-chloroethoxy)methane.........     111-91-1  (d)(2), (e)(1)
Bis(2-chloroisopropyl)ether........     108-60-1  (d)(2)
4-Bromobenzyl cyanide..............   16532-79-9  (d)(1), (2), (e)(1)
Bromoform..........................      75-25-2  (d)(2)
4-Chlorobenzo-trichloride..........    5216-25-1  (e)(1)
2,4-D..............................      94-75-7  (d)(2)
Dibromomethane 74-95-3 (d)(2)......
1,2-Dichlorobenzene................      95-50-1  (d)(2)
1,1-Dichloroethane.................      75-34-3  (d)(2)
1,3-Dichloropropanol...............      96-23-1  (d)(1), (e)(1)
Dihydrosafrole.....................      94-58-6  (d)(2)
Endrin.............................      72-20-8  (d)(2)
Ethyl methacrylate.................      97-63-2  (d)(2)

[[Page 271]]

 
Maleic hydrazide...................     123-33-1  (d)(1), (2)
Malononitrile......................     109-77-3  (d)(1), (e)(1)
Methanethiol.......................      74-93-1  (d)(1)
Methyl chloride....................      74-87-3  (d)(2)
p- Nitrophenol.....................     100-02-7  (e)(1)
Pentachlorobenzene.................     608-93-5  (d)(2)
Pentachloroethane..................      76-01-7  (d)(2)
1,2,4,5-Tetrachlorobenzene.........      95-94-3  (d)(2)
Trichloromethanethiol..............      75-70-7  (d)(1), (2), (e)(1)
------------------------------------------------------------------------

    (d) Chemical fate testing--(1) Soil adsorption--(i) Required 
testing. A soil adsorption isotherm test shall be conducted with the 
substances designated in paragraph (c) of this section in accordance 
with Sec. 796.2750 of this chapter except that the provisions of Sec. 
796.2750 (b)(1)(vii)(A) shall not apply to 1,3-Dichloropropanol.
    (ii) Reporting requirements. The sediment and soil adsorption 
isotherm tests shall be completed and the final results submitted to EPA 
within 9 months of the effective date of the final rule except that 
final results for testing of 1,3-Dichloropropanol and Methanethiol shall 
be completed and submitted to EPA within 11 months and 15 months, 
respectively, of the effective date of the final rule.
    (2) Hydrolysis--(i) Required testing. A test of hydrolysis as a 
function of pH at 25 [deg]C shall be conducted with the substances 
designated in paragraph (c) of this section in accordance with Sec. 
796.3500 of this chapter.
    (ii) Reporting requirements. The hydrolysis tests with the 
substances designated in paragraph (c) of this section shall be 
completed and the final results submitted to EPA within 6 months of the 
effective date of the final rule except that hydrolysis tests for 
Dibromomethane, Dihydrosafrole, Ethyl methacrylate, and Methyl chloride 
shall be completed and the final results submitted to EPA within 12 
months of the effective date of the final rule; and hydrolysis tests for 
1,2-Dichlorobenzene and 1,2,4,5-Tetrachlorobenzene shall be completed 
and final results submitted to EPA within 9 months of the effective date 
of the final rule.
    (e) Health effects testing--(1) Subchronic toxicity--(i) Required 
test. (A) An oral gavage subchronic toxicity test shall be conducted in 
the rat with the substances designated in paragraph (c) of this section 
except for bis(2-chloroethoxy) methane (CAS No. 111-91-1) in accordance 
with Sec. 798.2650 of this chapter.
    (B) For Bis(2-chloroethoxy)methane, an oral gavage subchronic 
toxicity test shall be conducted in the rat in accordance with Sec. 
798.2650 of this chapter except for the provisions in paragraphs 
(e)(9)(i)(A) and (e)(9)(i)(B). For Bis(2-chloroethoxy)methane, the 
following provisions also apply:
    (1) Hematology determinations shall be carried out at least two 
times during the test period: Just after dosing on day 30 and just prior 
to terminal sacrifice. Hematology determinations which are appropriate 
to all studies are: Hematocrit, hemoglobin concentration, erythrocyte 
count, total and differential leukocyte count, and a measure of clotting 
potential such as clotting time, prothrombin time, thromboplastin time, 
or platelet count.
    (2) Certain clinical biochemistry determinations on blood shall be 
carried out at least two times: Just after dosing on day 30 and just 
prior to terminal sacrifice. Test areas which are considered appropriate 
to all studies are: Electrolyte balance, carbohydrate metabolism, and 
liver and kidney function. The selection of specific tests will be 
influenced by observations on the mode of action of the substance. 
Suggested determinations are: Calcium, phosphorus, chloride, sodium, 
potassium, fasting glucose (with the period of fasting appropriate to 
the species), serum glutamic oxaloacetic transaminase (now known as 
serum aspartate aminotransferase), ornithine decarboxylase, gamma 
glutamyl transpeptidase, urea nitrogen, albumen blood creatinine, total 
bilirubin and total serum protein measurements. Other determinations 
which may be necessary for an adequate toxicological evaluation include: 
Analysis of lipids, hormones, acid/base balance, methemoglobin, and 
cholinesterase activity. Additional clinical biochemistry may be 
employed, where necessary, to extend the investigation of observed 
effects.
    (ii) Reporting requirements. (A) The oral gavage subchronic tests 
with the

[[Page 272]]

substances designated in paragraph (c) of this section shall be 
completed and submitted to EPA within 12 months of the effective date of 
the final rule except that the tests with Bis(2-chloroethoxy)methane, 
1,3-Dichloropropanol, and Malononitrile shall be completed and the 
results submitted to EPA within 15 months of the effective date of the 
final rule.
    (B) Progress reports for each test shall be submitted to the Agency 
6 months after the effective date of the final rule.
    (2) [Reserved]
    (f) Effective date. (1) The effective date of the final rule is July 
29, 1988, except for paragraphs (d)(1)(i), (d)(1)(ii), (d)(2)(ii), 
(e)(1)(i), and (e)(1)(ii)(A) of this section. The effective date of 
paragraphs (d)(1)(i), (d)(1)(ii), (d)(2)(ii), (e)(1)(i)(B) and 
(e)(1)(ii)(A) of this section is March 1, 1990. The effective date of 
paragraph (e)(1)(i)(A), is May 21, 1991.
    (2) The guidelines and other test methods cited here are referenced 
as they exist on the effective date of the final rule.

[53 FR 22324, June 15, 1988; 53 FR 48645, Dec. 2, 1988, as amended at 54 
FR 49760, Dec. 1, 1989; 55 FR 7324, Mar. 1, 1990; 56 FR 23232, May 21, 
1991; 58 FR 34205, June 23, 1993]



Sec. 799.5075  Drinking water contaminants subject to testing.

    (a) Identification of test substance. (1) 1,1,2,2-tetrachloroethane 
(CAS No. 79-34-5), and 1,3,5-trimethylbenzene (CAS No. 108-67-8) shall 
be tested as appropriate in accordance with this section.
    (2) A test substance of at least 99 percent purity shall be used for 
Chloroethane, 1,1-dichloroethane, and 1,3,5-trimethylbenzene. A test 
substance of at least 98 percent purity shall be used for 1,1,2,2-
tetrachloroethane.
    (b) Persons required to submit study plans, conduct tests, and 
submit data. All persons who manufacture (including import and by-
product manufacture) or process, or who intend to manufacture or 
process, the substances listed in paragraph (a) of this section after 
the effective date of this section to the end of the reimbursement 
period shall submit letters of intent to test, submit study plans, 
conduct tests, and submit data, or submit exemption applications as 
specified in this section, subpart A of this part, and parts 790 and 792 
of this chapter for single-phase rulemaking, for the substances they 
manufacture subject to exclusions contained in Sec. 790.42(a)(2), 
(a)(4) and (a)(5). These sections provide that processors, persons who 
manufacture less than 500 kg (1,100 lbs) annually, or persons who 
manufacture small quantities of the chemical solely for research and 
development as defined in Sec. 790.42(a)(5) shall not be required to 
submit study plans, conduct tests and submit data, or submit exemption 
applications as specified in this section unless directed to do so in a 
subsequent notice as set forth in Sec. 790.48(b).
    (c) Health effects testing--(1) Subacute toxicity--(i) Required 
testing. (A) An oral 14-day repeated dose toxicity test shall be 
conducted with 1,1,2,2-tetrachloroethane, and 1,3,5-trimethylbenzene in 
accordance with Sec. 798.2650 of this chapter except for the provisions 
in Sec. 798.2650 (a), (b)(1), (c), (e)(3), (e)(4)(i), (e)(5), (e)(6), 
(e)(7)(i), (e)(7)(iv), (e)(7)(v), (e)(8)(vii), (e)(9)(i)(A), 
(e)(9)(i)(B), (e)(11)(v), and (f)(2)(i). Each substance shall be tested 
in one mammalian species, preferably a rodent, but a non-rodent may be 
used. The species and strain of animals used in this test should be the 
same as those used in the 90-day subchronic test required in paragraph 
(c)(2)(i) of this section. The tests shall be performed using drinking 
water. However, if, due to poor stability or palatability, a drinking 
water test is not feasible for a given substance, that substance shall 
be administered either by oral gavage, in the diet, or in capsules.
    (B) For the purpose of this section, the following provisions also 
apply:
    (1) Purpose. To assess and evaluate the toxic characteristics of a 
substance, the determination of subacute toxicity should be carried out 
after initial information on toxicity has been obtained by acute 
testing. The 14-day repeated dose oral study provides information on the 
health hazard likely to arise from repeated short-term exposure by the 
oral route over a very limited period of time. It has been designed to 
permit the determination of the no-observed-adverse-effect level

[[Page 273]]

and toxic effects associated with continuous or repeated exposure to a 
test substance for 14 days and to evaluate reversibility, persistence, 
and delayed occurrence of toxic effects during a 14-day follow-up 
recovery period. The test is not capable of determining those effects 
that have a long latency period for development (e.g., carcinogenicity 
and life shortening). It will provide information on target organs and 
the possibility of accumulation, and can be used in selecting dose 
levels for subchronic studies and for establishing safety criteria for 
short-term human exposure.
    (2) Definitions. Subacute oral toxicity is the manifestation of 
adverse effect(s) occurring as a result of the repeated daily exposure 
of experimental animals to a substance by the oral route for 14 days.
    (3) Principle of the test method. The test substance is administered 
orally in graduated daily doses to several groups of experimental 
animals, one dose level per group, for a period of 14 days. During the 
period of administration the animals are observed daily to detect signs 
of toxicity. Animals which die during the period of administration are 
necropsied. At the conclusion of the test, all animals, except the 
satellite group, are necropsied and histopathological examinations are 
carried out. The satellite group is necropsied after the 14-day recovery 
period.
    (4) Satellite group (Rodent only). A satellite group of 20 animals 
(10 animals per sex) shall be treated with the high dose level for 14 
days and observed for reversibility, persistence, and delayed occurrence 
of toxic effects for a post-treatment recovery period of at least 14 
days.
    (5) Dose levels and dose selection. In subacute toxicity tests, it 
is desirable to have a dose response relationship as well as a NOAEL. 
Therefore, at least 3 dose levels with a control and, where appropriate, 
a vehicle control (corresponding to the concentration of vehicle at the 
highest exposure level) shall be used. Doses shall be spaced 
appropriately to produce test groups with a range of toxic effects. The 
data should be sufficient to produce a dose-response curve.
    (6) Exposure conditions. The animals are dosed with the test 
substance every day for 14 days.
    (7) Observation period. All animals shall be observed daily during 
the 14-day exposure period.
    (8) Observation period of satellite group. Animals in the satellite 
group scheduled for follow-up observations shall be kept for at least 14 
days further without treatment to detect recovery from, or persistence 
of, and delayed onset of toxic effects and shall be observed daily.
    (9) Administration of test substance. For substances of low 
toxicity, it is important to ensure that when administered in the 
drinking water, by gavage, in the diet, or in capsules, the quantities 
of the test substance involved do not interfere with normal nutrition. 
When the test substance is administered in the diet, either a constant 
dietary concentration (ppm) or a constant dose level in terms of the 
animals' body weight shall be used; the alternative used shall be 
specified in the final test report.
    (10) Time of administration of test substance. For a substance 
administered by gavage or capsule, the dose shall be given at 
approximately the same time each day, and adjusted on day 7 to maintain 
a constant dose level in terms of animal body weight.
    (11) Observation of animals. At the end of the 14-day exposure 
period, all survivors, except those in the satellite group, shall be 
necropsied. All survivors in the satellite group shall be necropsied 
after a recovery period of at least 14 days.
    (12) Hematology determinations. Certain hematology determinations 
shall be carried out at least two times during the test period: Just 
prior to initiation of dosing if adequate historical baseline data are 
not available (baseline data) and just prior to terminal sacrifice at 
the end of the test period. Hematology determinations which are 
appropriate to all studies are: Hematocrit, hemoglobin concentration, 
erythrocyte count, total and differential leukocyte count, and a measure 
of clotting potential such as clotting time, prothrombin time, 
thromboplastin time, or platelet count.

[[Page 274]]

    (13) Clinical biochemical determinations. Certain clinical 
biochemistry determinations on blood should be carried out at least two 
times: Just prior to initiation of dosing (if adequate historical 
baseline data are not available) and just prior to terminal sacrifice at 
the end of the test period. Test areas which are considered appropriate 
to all studies are: Electrolyte balance, carbohydrate metabolism, and 
liver and kidney function. The selection of specific tests will be 
influenced by observations on the mode of action of the substance. 
Suggested determinations are: Calcium, phosphorus, chloride, sodium, 
potassium, fasting glucose (with the period of fasting appropriate to 
the species), serum alanine aminotransferase, serum aspartate 
aminotransferase, gamma glutamyl transpeptidase, urea nitrogen, albumin, 
blood creatinine, and total serum protein measurements. Other 
determinations which may be necessary for an adequate toxicological 
evaluation include: analyses of lipids, hormones, acid/base balance, 
methemoglobin, and cholinesterase activity. Additional clinical 
biochemistry may be employed, where necessary, to extend the 
investigation of observed effects.
    (14) Histopathology. Histopathology of the lungs of all animals 
shall be performed. Special attention to examination of the lungs of 
rodents shall be made for evidence of infection since this provides a 
convenient assessment of the state of health of the animals.
    (15) Evaluation of the study results. The findings of a subacute 
oral toxicity study should be evaluated in conjunction with the findings 
of preceding studies and considered in terms of the toxic effects and 
the necropsy and histopathological findings. The evaluation will include 
the relationship between the dose of the test substance and the presence 
or absence, the incidence and severity, of abnormalities, including 
behavioral and clinical abnormalities, gross lesions, identified target 
organs, body weight changes, effects on mortality and any other general 
or specific toxic effects. A properly conducted subacute test should 
provide a satisfactory estimation of a NOAEL.
    (ii) Reporting requirements. (A) Each subacute test shall be 
completed and the final report submitted to EPA within 12 months of the 
date specified in paragraph (d)(1) of this section, except for 1,1,2,2-
tetrachloroethane. The subacute testing for 1,1,2,2-tetrachloroethane. 
The subacute testing for 1,1,2,2-tetrachloroethane shall be completed 
and the final report submitted to EPA by February 15, 1996.
    (B) Except for 1,3,5-trimethylbenzene, a progress report shall be 
submitted to EPA for each test beginning 6 months after the date 
specified in paragraph (d)(1) of this section and at 6-month intervals 
thereafter until the final report is submitted to EPA . The progress 
report for 1,3,5-trimethylbenzene shall be submitted to EPA by April 10, 
1995.
    (2) Subchronic toxicity--(i) Required testing. (A) An oral 90-day 
subchronic toxicity test shall be conducted with 1,3,5-trimethylbenzene 
in accordance with Sec. 798.2650 of this chapter except for the 
provisions in Sec. 798.2650 (e)(3), (e)(7)(i), and (e)(11)(v). The 
tests shall be performed using drinking water. However, if, due to poor 
stability or palatability, a drinking water test is not feasible for a 
given substance, that substance shall be administered either by oral 
gavage, in the diet, or in capsules.
    (B) For the purpose of this section, the following provisions also 
apply:
    (1) Satellite group (Rodent only). A satellite group of 20 animals 
(10 animals per sex) shall be treated with the high dose level for 90 
days and observed for reversibility, persistence, and delayed occurrence 
of toxic effects for a post-treatment period of appropriate length, 
normally not less than 28 days.
    (2) Histopathology. Histopathology of the lungs of all animals shall 
be performed. Special attention to examination of the lungs of rodents 
shall be made for evidence of infection since this provides a convenient 
assessment of the state of health of the animals.
    (ii) Reporting requirements. (A) The subchronic testing for 
chloroethane shall be completed and the final report submitted to EPA by 
June 27, 1995. The subchronic testing for 1,1-dichloroethane and 
1,1,2,2-tetrachlorethane shall be completed and the final report 
submitted to EPA by August 27, 1995. The subchronic testing for 1,3,5-
trimethylbenzene shall be

[[Page 275]]

completed and the final report submitted to EPA by April 10, 1995.
    (B) For each test, a progress report shall be submitted to EPA 
beginning 9 months after the date specified in paragraph (d)(1) of this 
section and at 6-month intervals thereafter until the final report is 
submitted to EPA.
    (d) Effective date. (1) This section is effective on December 27, 
1993, except for paragraphs (a)(1), (a)(2), (c)(1)(i)(A), (c)(1)(ii)(A), 
(c)(1)(ii)(B), (c)(2)(i)(A), and (c)(2)(ii)(A). The effective date for 
paragraphs (a)(2), (c)(1)(ii)(B), and (c)(2)(ii)(A) is September 29, 
1995. The effective date for paragraphs (a)(1), (c)(1)(i)(A), and 
(c)(2)(i)(A) is February 27, 1996. The effective date for paragraph 
(c)(1)(ii)(A) is June 30, 1997.
    (2) The guidelines and other test methods cited in this section are 
referenced as they exist on the effective date of the final rule.

[58 FR 59681, Nov. 10, 1993; 58 FR 1992, Jan. 13, 1994, as amended at 60 
FR 56956, Nov. 13, 1995; 61 FR 7223, Feb. 27, 1996; 62 FR 35105, June 
30, 1997]



Sec. 799.5085  Chemical testing requirements for certain high production 

volume chemicals.

    (a) What substances will be tested under this section? Table 2 in 
paragraph (j) of this section identifies the chemical substances that 
must be tested under this section. For the chemical substances 
identified as ``Class 1'' substances in Table 2 in paragraph (j) of this 
section, the purity of each chemical substance must be 99% or greater, 
except for 1,3-propanediol, 2,2-bis[(nitrooxy)methyl]-, dinitrate 
(ester) (CAS No. 78-11-5), also known as pentaerythritol tetranitrate 
(PETN). PETN cannot be tested at 99% purity because of its explosive 
properties. It must be diluted in water or tested as a stabilized 
mixture with an appropriate stabilizer (e.g., D-lactose monohydrate is 
the stabilizer in PETN, NF which is a mixture of 20% by weight PETN and 
80% by weight D-lactose monohydrate). The stabilizer used must be tested 
as a control. For the chemical substances identified as ``Class 2'' 
substances in Table 2 in paragraph (j), a representative form of each 
chemical substance must be tested. The representative form selected for 
a given Class 2 chemical substance should meet industry or consensus 
standards where they exist.
    (b) Am I subject to this section? (1) If you manufacture (including 
import) or intend to manufacture, or process or intend to process, any 
chemical substance listed in Table 2 in paragraph (j) of this section at 
any time from April 17, 2006 to the end of the test data reimbursement 
period as defined in 40 CFR 791.3(h), you are subject to this section 
with respect to that chemical substance.
    (2) If you do not know or cannot reasonably ascertain that you 
manufacture or process a chemical substance listed in Table 2 in 
paragraph (j) of this section during the time period described in 
paragraph (b)(1) of this section (based on all information in your 
possession or control, as well as all information that a reasonable 
person similarly situated might be expected to possess, control, or 
know, or could obtain without an unreasonable burden), you are not 
subject to this section with respect to that chemical substance.
    (c) If I am subject to this section, when must I comply with it? 
(1)(i) Persons subject to this section are divided into two groups, as 
set forth in Table 1 of this paragraph: Tier 1 (persons initially 
required to comply) and Tier 2 (persons not initially required to 
comply). If you are subject to this section, you must determine if you 
fall within Tier 1 or Tier 2, based on Table 1 of this paragraph.


[[Page 276]]



   Table 1--Persons Subject to the Rule: Persons in Tier 1 and Tier 2
------------------------------------------------------------------------
                                          Persons not initially required
  Persons initially required to comply     to comply with this section
       with this section (Tier 1)                    (Tier 2)
------------------------------------------------------------------------
Persons not otherwise specified in       A. Persons who manufacture (as
 column 2 of this table that              defined at TSCA section 3(7))
 manufacture (as defined at TSCA          or intend to manufacture a
 section 3(7)) or intend to manufacture   chemical substance included in
 a chemical substance included in this    this section solely as one or
 section.                                 more of the following:
                                         --As a byproduct (as defined at
                                          40 CFR 791.3(c));
                                         --As an impurity (as defined at
                                          40 CFR 790.3);
                                         --As a naturally occurring
                                          substance (as defined at 40
                                          CFR 710.4(b));
                                         --As a non-isolated
                                          intermediate (as defined at 40
                                          CFR 704.3);
                                         --As a component of a Class 2
                                          substance (as described at 40
                                          CFR 720.45(a)(1)(i));
                                         --In amounts of less than 500
                                          kg (1,100 lbs.) annually (as
                                          described at 40 CFR
                                          790.42(a)(4)); or
                                         --For R & D (as described at 40
                                          CFR 790.42(a)(5)).
                                         B. Persons who process (as
                                          defined at TSCA section 3(10))
                                          or intend to process a
                                          chemical substance included in
                                          this section (see 40 CFR
                                          790.42(a)(2)).
------------------------------------------------------------------------


    (ii) Table 1 of paragraph (c)(1)(i) of this section expands the list 
of persons specified in Sec. 790.42(a)(2), (a)(4), and (a)(5) of this 
chapter, who, while legally subject to this section, must comply with 
the requirements of this section only if directed to do so by EPA under 
the circumstances set forth in paragraphs (c)(5) and (c)(8) of this 
section.
    (2) If you are in Tier 1 with respect to a chemical substance listed 
in Table 2 in paragraph (j) of this section, you must, for each test 
required under this section for that chemical substance, either submit 
to EPA a letter of intent to test or apply to EPA for an exemption from 
testing. The letter of intent to test or the exemption application must 
be received by EPA no later than May 15, 2006.
    (3) If you are in Tier 2 with respect to a chemical substance listed 
in Table 2 in paragraph (j) of this section, you are considered to have 
an automatic conditional exemption and you will be required to comply 
with this section with regard to that chemical substance only if 
directed to do so by EPA under paragraphs (c)(5) or (c)(8) of this 
section.
    (4) If no person in Tier 1 has notified EPA of its intent to conduct 
one or more of the tests required by this section on any chemical 
substance listed in Table 2 in paragraph (j) of this section by May 15, 
2006, EPA will publish a Federal Register document that will specify the 
test(s) and the chemical substance(s) for which no letter of intent has 
been submitted, and notify manufacturers and processors in Tier 2 of 
their obligation to submit a letter of intent to test or to apply for an 
exemption from testing.
    (5) If you are in Tier 2 with respect to a chemical substance listed 
in Table 2 in paragraph (j) of this section, and if you manufacture or 
process this chemical substance as of April 17, 2006, or within 30 days 
after publication of the Federal Register document described in 
paragraph (c)(4) of this section, you must, for each test specified for 
that chemical substance in the document described in paragraph (c)(4) of 
this section, either submit to EPA a letter of intent to test or apply 
to EPA for an exemption from testing. The letter of intent to test or 
the exemption application must be received by EPA no later than 30 days 
after publication of the document described in paragraph (c)(4) of this 
section.
    (6) If no manufacturer or processor has notified EPA of its intent 
to conduct one or more of the tests required by this section for any of 
the chemical substances listed in Table 2 in paragraph (j) of this 
section within 30 days after the publication of the Federal Register 
document described in paragraph (c)(4) of this section, EPA will notify 
all manufacturers and processors of those chemical substances of this 
fact by certified letter or by publishing a Federal Register document 
specifying the test(s) for which no letter of intent has been submitted. 
This letter or Federal Register document will additionally notify all 
manufacturers and processors that all exemption applications concerning 
the test(s) have been denied, and will give the manufacturers and 
processors of the chemical substance(s) an opportunity to take 
corrective action.
    (7) If no manufacturer or processor has notified EPA of its intent 
to conduct one or more of the tests required

[[Page 277]]

by this section for any of the chemical substances listed in Table 2 in 
paragraph (j) of this section within 30 days after receipt of the 
certified letter or publication of the Federal Register document 
described in paragraph (c)(6) of this section, all manufacturers and 
processors subject to this section with respect to that chemical 
substance who are not already in violation of this section will be in 
violation of this section.
    (8) If a problem occurs with the initiation, conduct, or completion 
of the required testing or the submission of the required data with 
respect to a chemical substance listed in Table 2 in paragraph (j) of 
this section, under the procedures in Sec. Sec. 790.93 and 790.97 of 
this chapter, EPA may initiate termination proceedings for all testing 
exemptions with respect to that chemical substance and may notify 
persons in Tier 1 and Tier 2 that they are required to submit letters of 
intent to test or exemption applications within a specified period of 
time.
    (9) If you are required to comply with this section, but your 
manufacturing or processing of a chemical substance listed in Table 2 in 
paragraph (j) of this section begins after the applicable compliance 
date referred to in paragraphs (c)(2), (c)(5), or (c)(8) of this 
section, you must either submit a letter of intent to test or apply to 
EPA for an exemption. The letter of intent to test or the exemption 
application must be received by EPA no later than the day you begin 
manufacturing or processing.
    (d) What must I do to comply with this section? (1) To comply with 
this section you must either submit to EPA a letter of intent to test, 
or apply to and obtain from EPA an exemption from testing.
    (2) For each test with respect to which you submit to EPA a letter 
of intent to test, you must conduct the testing specified in paragraph 
(h) of this section and submit the test data to EPA.
    (3) You must also comply with the procedures governing test rule 
requirements in part 790 of this chapter, as modified by this section, 
including the submission of letters of intent to test or exemption 
applications, the conduct of testing, and the submission of data; Part 
792--Good Laboratory Practice Standards of this chapter; and this 
section. The following provisions of 40 CFR part 790 do not apply to 
this section: Paragraphs (a), (d), (e), and (f) of Sec. 790.45; 
paragraph (a)(2) and paragraph (b) of Sec. Sec. 790.80; 790.82(e)(1); 
790.85; and 790.48.
    (e) If I do not comply with this section, when will I be considered 
in violation of it? You will be considered in violation of this section 
as of 1 day after the date by which you are required to comply with this 
section.
    (f) How are EPA's data reimbursement procedures affected for 
purposes of this section? If persons subject to this section are unable 
to agree on the amount or method of reimbursement for test data 
development for one or more chemical substances included in this 
section, any person may request a hearing as described in 40 CFR part 
791. In the determination of fair reimbursement shares under this 
section, if the hearing officer chooses to use a formula based on 
production volume, the total production volume amount will include 
amounts of a chemical substance produced as an impurity.
    (g) Who must comply with the export notification requirements? Any 
person who exports, or intends to export, a chemical substance listed in 
Table 2 in paragraph (j) of this section is subject to part 707, subpart 
D, of this chapter.
    (h) How must I conduct my testing? (1) The tests that are required 
for each chemical substance are indicated in Table 2 in paragraph (j) of 
this section. The test methods that must be followed are provided in 
Table 3 in paragraph (j) of this section. You must proceed in accordance 
with these test methods as required according to Table 3 in paragraph 
(j) of this section, or as appropriate if more than one alternative is 
allowed according to Table 3 in paragraph (j) of this section. Included 
in Table 3 in paragraph (j) of this section are the following 11 methods 
which are incorporated by reference:
    (i) Standard Test Method for Relative Initial and Final Melting 
Points and the Melting Range of Organic Chemicals, ASTM E 324-99.
    (ii) Standard Test Method for Partition Coefficient (N-Octanol/
Water) Estimation by Liquid Chromatography, ASTM E 1147-92. (Reapproved 
1997)

[[Page 278]]

    (iii) Standard Guide for Conducting Acute Toxicity Tests on Test 
Materials with Fishes, Macroinvertebrates, and Amphibians, ASTM E 729-
96. (Reapproved 2002)
    (iv) Standard Test Method for Measurements of Aqueous Solubility, 
ASTM E 1148-02.
    (v) Standard Test Method for Estimating Acute Oral Toxicity in Rats, 
ASTM E 1163-98. (Reapproved 2002)
    (vi) Standard Guide for Conducting Daphnia Magna Life-Cycle Toxicity 
Tests, ASTM E 1193-97. (Reapproved 2004)
    (vii) Standard Guide for Conducting Static Toxicity Tests with 
Microalgae, ASTM E 1218-04.
    (viii) Standard Test Method for Determining Biodegradability of 
Organic Chemicals in Semi-Continuous Activated Sludge (SCAS), ASTM E 
1625-94. (Reapproved 2001)
    (ix) Standard Test Method for Vapor Pressure of Liquids by 
Ebulliometry, ASTM E 1719-97.
    (x) Standard Test Method for Determining Vapor Pressure by Thermal 
Analysis, ASTM E 1782-03.
    (xi) Water Quality--Evaluation of Ultimate Aerobic Biodegradability 
of Organic Compounds in Aqueous Medium--Static Test (Zahn-Wellens 
Method), Second Edition, June 1, 1999, ISO 9888-99.
    (2) The Director of the Federal Register approved this incorporation 
by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. You 
may obtain copies of the ASTM guidelines from the American Society for 
Testing and Materials, 100 Bar Harbor Dr., West Conshohocken, PA 19428-
2959, and a copy of the ISO guideline from the International 
Organization for Standardization, Case Postale, 56 CH-1211 Geneve 20 
Switzerland. You may inspect each test method at the EPA Docket Center, 
EPA West, Rm. B102, 1301 Constitution Ave., NW., Washington, DC or at 
the National Archives and Records Administration (NARA). For information 
on the availability of this material at NARA, call (202) 741-6030, or go 
to: http://www.archives.gov/federal--register/code--of--federal--
regulations/ibr--locations.html.
    (i) Reporting requirements. A final report for each specific test 
for each subject chemical substance must be received by EPA by May 17, 
2007, unless an extension is granted in writing pursuant to 40 CFR 
790.55. A robust summary of the final report for each specific test 
should be submitted in addition to and at the same time as the final 
report. The term ``robust summary'' is used to describe the technical 
information necessary to adequately describe an experiment or study and 
includes the objectives, methods, results, and conclusions of the full 
study report which can be either an experiment or in some cases an 
estimation or prediction method. Guidance for the compilation of robust 
summaries is described in a document entitled Draft Guidance on 
Developing Robust Summaries which is available at: http://www.epa.gov/
chemrtk/robsumgd.htm.
    (j) Designation of specific chemical substances and testing 
requirements. The chemical substances identified by chemical name, 
Chemical Abstract Service Number (CAS No.), and class in Table 2 of this 
paragraph must be tested in accordance with the requirements designated 
in Tables 2 and 3 of this paragraph, and the requirements described in 
40 CFR Part 792--Good Laboratory Practice Standards:


                              Table 2--Chemical Substances And Testing Requirements
----------------------------------------------------------------------------------------------------------------
                                                                                     Required tests/(See Table 3
                 CAS No.                          Chemical name            Class           of this section)
----------------------------------------------------------------------------------------------------------------
74-95-3                                   Methane, dibromo-                  1       A, C1, E2, F2
----------------------------------------------------------------------------------------------------------------
75-36-5                                   Acetyl chloride                    1       A, B, C2, E2, F1
----------------------------------------------------------------------------------------------------------------
78-11-5                                   1,3-Propanediol, 2,2-              1       A4, A5, B, C6, F2
                                           bis[(nitrooxy)methyl]-,
                                           dinitrate (ester)
----------------------------------------------------------------------------------------------------------------
84-65-1                                   9,10-Anthracenedione               1       A, F2
----------------------------------------------------------------------------------------------------------------
108-19-0                                  Imidodicarbonic diamide            1       A, B, C1, D, E1, E2, F1
----------------------------------------------------------------------------------------------------------------

[[Page 279]]

 
110-44-1                                  2,4-Hexadienoic acid,              1       A, C4
                                           (2E,4E)-
----------------------------------------------------------------------------------------------------------------
112-52-7                                  Dodecane, 1-chloro                 1       A, B, C3, D, E1, E2, F1
----------------------------------------------------------------------------------------------------------------
118-82-1                                  Phenol, 4,4'-                      1       A, B, D, E1, E2, F2
                                           methylenebis[2,6-bis(1,1-
                                           dimethylethyl)]-
----------------------------------------------------------------------------------------------------------------
149-44-0                                  Methanesulfinic acid,              1       A, B, C1, E2, F1
                                           hydroxy-, monosodium salt
----------------------------------------------------------------------------------------------------------------
409-02-9                                  Heptenone, methyl-                 2       A, B, C1, D, E1, E2, F1
----------------------------------------------------------------------------------------------------------------
594-42-3                                  Methanesulfenyl chloride,          1       A, B, C1, E1, E2, F2
                                           trichloro-
----------------------------------------------------------------------------------------------------------------
624-83-9                                  Methane, isocyanato-               1       A, C1
----------------------------------------------------------------------------------------------------------------
1324-76-1                                 Benzenesulfonic acid, [[4-         2       A, B, C1, D, E1, E2, F1
                                           [[4-(phenylamino)phenyl][4-
                                           (phenylimino)-2,5-
                                           cyclohexadien-1-
                                           ylidene]methyl]phenyl]amino
                                           ]-
----------------------------------------------------------------------------------------------------------------
2941-64-2                                 Carbonochloridothioic acid,        1       A, B, C1, E2, F1
                                           S-ethyl ester
----------------------------------------------------------------------------------------------------------------
8005-02-5                                 C.I. Solvent Black 7               2       A, B, C1, D, E2, F1
----------------------------------------------------------------------------------------------------------------
68611-64-3                                Urea, reaction products with       2       A, B, C1, D, E1, E2, F1
                                           formaldehyde
----------------------------------------------------------------------------------------------------------------



       Table 3--Key to the Test Requirements Denoted by Alphanumeric Symbols in Table 2 of This Paragraph
----------------------------------------------------------------------------------------------------------------
                                                                 Test requirements and
           Testing category                  Test symbol               references           Special conditions
----------------------------------------------------------------------------------------------------------------
Physical/chemical properties           A                        1. Melting Point: ASTM   n-Octanol/water
                                                                 E 324 (capillary tube)   Partition Coefficient
                                                                2. Boiling Point: ASTM    or log Kow:
                                                                 E 1719 (ebulliometry).  Which method is
                                                                3. Vapor Pressure: ASTM   required, if any, is
                                                                 E 1782 (thermal          determined by the test
                                                                 analysis).               substance's estimated
                                                                4. n-Octanol/Water        \1\ log Kow as
                                                                 Partition Coefficient    follows:
                                                                 (log 10 basis) or log   log Kow <0: no testing
                                                                 Kow: (See special        required.
                                                                 conditions for the log  log Kow range 0-1:
                                                                 Kow test requirement     Method A or B.
                                                                 and select the          log Kow range >1-4:
                                                                 appropriate method to    Method A or B or C.
                                                                 use, if any, from       log Kow range >4-6:
                                                                 those listed in this     Method B or C.
                                                                 column.).               log Kow >6: Method C.
                                                                 Method A: 40 CFR        Test sponsors are
                                                                 799.6755 (shake flask).  required to provide in
                                                                 Method B: ASTM E 1147    the final study report
                                                                 (liquid                  the underlying
                                                                 chromatography).         rationale for the
                                                                 Method C: 40 CFR         method selected. In
                                                                 799.6756 (generator      order to ensure
                                                                 column).                 environmental
                                                                5. Water Solubility:      relevance, EPA highly
                                                                 (See special             recommends that the
                                                                 conditions for the       selected study be
                                                                 water solubility test    conducted at pH 7.
                                                                 requirement and select  Water Solubility:
                                                                 the appropriate method  Which method is
                                                                 to use, if any, from     required, if any, is
                                                                 those listed in this     determined by the test
                                                                 column.).                substance's estimated
                                                                 Method A: ASTM E 1148    \2\ water solubility.
                                                                 (shake flask).           Test sponsors are
                                                                 Method B: 40 CFR         required to provide in
                                                                 799.6784 (shake flask).  the final study report
                                                                 Method C: 40 CFR         the underlying
                                                                 799.6784 (column         rationale for the
                                                                 elution).                method selected. In
                                                                 Method D: 40 CFR         order to ensure
                                                                 799.6786 (generator      environmental
                                                                 column).                 relevance, EPA highly
                                                                                          recommends that the
                                                                                          selected study be
                                                                                          conducted at pH 7.
                                                                                         5,000 mg/L:
                                                                                          Method A or B.
                                                                                         10 mg/L --
                                                                                          5,000 mg/L: Method A,
                                                                                          B, C, or D.
                                                                                          0.001 mg/L--
                                                                                          10 mg/L: Method C or
                                                                                          D.
                                                                                         <=0.001 mg/L: No
                                                                                          testing required.
----------------------------------------------------------------------------------------------------------------
Environmental fate and pathways--      B                        For B, choose either of  None
 Inherent biodegradation                                         the methods listed in
                                                                 this column:
                                                                1. ASTM 1625
                                                                 (semicontinuous
                                                                 activated sludge test)
                                                                 OR.
                                                                2. ISO 9888 (Zahn-
                                                                 Wellens method).
----------------------------------------------------------------------------------------------------------------

[[Page 280]]

 
Aquatic toxicity                       C1                       For C1, Test Group 1 or  The following are the
                                                                 Test Group 2 listed in   special conditions for
                                                                 this column must be      C1, C2, C3, C4, C5,
                                                                 used to fulfill the      and C7 testing; there
                                                                 testing requirements--   are no special
                                                                 See special              conditions for C6.
                                                                 conditions.              If log Kow <4.2: Test
                                                                Test Group 1 for C1:...   Group 1 is required
                                                                1. Acute Toxicity to     If log Kow >= 4.2: Test
                                                                 Fish: ASTM E 729.        Group 2 is required
                                                                2. Acute Toxicity to     Which test group is
                                                                 Daphnia: ASTM E 729.     required is determined
                                                                3. Toxicity to Plants     by the test
                                                                 (Algae): ASTM E 1218.    substance's measured
                                                                Test Group 2 for C1:...   log Kow as obtained
                                                                1. Chronic Toxicity to    under A\3\.
                                                                 Daphnia: ASTM E 1193.
                                                                2. Toxicity to Plants
                                                                 (Algae): ASTM E 1218.
                                      --------------------------------------------------
                                       C2                       For C2, Test Group 1 or
                                                                 Test Group 2 listed in
                                                                 this column must be
                                                                 used to fulfill the
                                                                 testing requirements--
                                                                 See special
                                                                 conditions.
                                                                Test Group 1 for C2:...
                                                                1. Acute Toxicity to
                                                                 Daphnia: ASTM E 729.
                                                                2. Toxicity to Plants
                                                                 (Algae): ASTM E 1218.
                                                                Test Group 2 for C2:...
                                                                1. Chronic Toxicity to
                                                                 Daphnia: ASTM E 1193.
                                                                2. Toxicity to Plants
                                                                 (Algae): ASTM E 1218.
                                      --------------------------------------------------
                                       C3                       For C3, Test Group 1 or
                                                                 Test Group 2 listed in
                                                                 this column must be
                                                                 used to fulfill the
                                                                 testing requirements--
                                                                 See special
                                                                 conditions.
                                                                Test Group 1 for C3:...
                                                                1. Acute Toxicity to
                                                                 Fish: ASTM E 729.
                                                                2. Toxicity to Plants
                                                                 (Algae): ASTM E 1218.
                                                                Test Group 2 for C3:...
                                                                1. Chronic Toxicity to
                                                                 Daphnia: ASTM E 1193.
                                                                2. Toxicity to Plants
                                                                 (Algae): ASTM E 1218.
                                      --------------------------------------------------
                                       C4                       For C4, Test Group 1 or
                                                                 Test Group 2 listed in
                                                                 this column must be
                                                                 used to fulfill the
                                                                 testing requirements--
                                                                 See special
                                                                 conditions.
                                                                Test Group 1 for C4:...
                                                                1. Acute Toxicity to
                                                                 Fish: ASTM E 729.
                                                                2. Acute Toxicity to
                                                                 Daphnia: ASTM E 729.
                                                                Test Group 2 for C4:...
                                                                1. Chronic Toxicity to
                                                                 Daphnia: ASTM E 1193.
                                      --------------------------------------------------
                                       C5                       For C5, Test Group 1 or
                                                                 Test Group 2 below
                                                                 must be used to
                                                                 fulfill the testing
                                                                 requirements--See
                                                                 special conditions.
                                                                Test Group 1 for C5:...
                                                                1. Acute Toxicity to
                                                                 Daphnia: ASTM E 729.
                                                                Test Group 2 for C5:...
                                                                1. Chronic Toxicity to
                                                                 Daphnia: ASTM E 1193.
                                      --------------------------------------------------
                                       C6                       Toxicity to Plants
                                                                 (Algae): ASTM E 1218
                                      --------------------------------------------------
                                       C7                       For C7, Test Group 1 or  .......................
                                                                 Test Group 2 of this
                                                                 column must be used to
                                                                 fulfill the testing
                                                                 requirements--See
                                                                 special conditions.
                                                                Test Group 1 for C7:...
                                                                1. Acute Toxicity to
                                                                 Fish: ASTM E 729.
                                                                Test Group 2 for C7:...
                                                                1. Chronic Toxicity to
                                                                 Daphnia: ASTM E 1193.
----------------------------------------------------------------------------------------------------------------

[[Page 281]]

 
Mammalian toxicity--Acute              D                        See special conditions   Which testing method is
                                                                 for this test            required is determined
                                                                 requirement and select   by the test
                                                                 the method that must     substance's physical
                                                                 be used from those       state at room
                                                                 listed in this column.   temperature (25
                                                                Method A: Acute           [deg]C). For those
                                                                 Inhalation Toxicity      test substances that
                                                                 (rat): 40 CFR 799.9130.  are gases at room
                                                                Method B: EITHER:......   temperature, Method A
                                                                1. Acute (Up/Down) Oral   is required;
                                                                 Toxicity (rat): ASTM E   otherwise, use either
                                                                 1163 OR.                 of the two methods
                                                                 2. Acute (Up/Down)       listed under Method B.
                                                                 Oral Toxicity (rat):    In Method B, 40 CFR
                                                                 40 CFR                   799.9110(d)(1)(i)(A)
                                                                 799.9110(d)(1)(i)(A).    refers to the OECD 425
                                                                                          Up/Down Procedure \4\.
                                                                                         Estimating starting
                                                                                          dose for Method B:
                                                                                          Data from the neutral
                                                                                          red uptake basal
                                                                                          cytotoxicity assay\5\
                                                                                          using normal human
                                                                                          keratinocytes or mouse
                                                                                          BALB/c 3T3 cells may
                                                                                          be used to estimate
                                                                                          the starting dose.
----------------------------------------------------------------------------------------------------------------
Mammalian toxicity--Genotoxicity       E1                       Bacterial Reverse        None
                                                                 Mutation Test (in
                                                                 vitro): 40 CFR
                                                                 799.9510
                                      --------------------------------------------------------------------------
                                       E2                       Conduct any one of the   Persons required to
                                                                 following three tests    conduct testing for
                                                                 for chromosomal          chromosomal damage are
                                                                 damage:                  encouraged to use the
                                                                In vitro Mammalian        in vitro Mammalian
                                                                 Chromosome Aberration    Chromosome Aberration
                                                                 Test: 40 CFR 799.9537    Test (40 CFR 799.9537)
                                                                 OR.                      to generate the needed
                                                                Mammalian Bone Marrow     data unless known
                                                                 Chromosomal Aberration   chemical properties
                                                                 Test (in vivo in         (e.g., physical/
                                                                 rodents: mouse           chemical properties,
                                                                 (preferred species),     chemical class
                                                                 rat, or Chinese          characteristics)
                                                                 hamster): 40 CFR         preclude its use. A
                                                                 799.9538 OR.             subject person who
                                                                Mammalian Erythrocyte     uses one of the in
                                                                 Micronucleus Test        vivo methods instead
                                                                 [sampled in bone         of the in vitro method
                                                                 marrow] (in vivo in      to address a
                                                                 rodents: Mouse           chromosomal damage
                                                                 (preferred species),     test requirement must
                                                                 rat, or Chinese          submit to EPA a
                                                                 hamster): 40 CFR         rationale for
                                                                 799.9539.                conducting that
                                                                                          alternate test in the
                                                                                          final study report.
                                      --------------------------------------------------------------------------
 Mammalian toxicity--Repeated dose/    F1                       Combined Repeated Dose   Where F1 is required,
 reproduction/ developmental                                     Toxicity Study with      EPA recommends use of
                                                                 the Reproduction/        the Combined Repeated
                                                                 Developmental Toxicity   Dose Toxicity Study
                                                                 Screening Test: 40 CFR   with the Reproduction/
                                                                 799.9365 OR              Developmental Toxicity
                                                                Reproduction/             Screening Test (40 CFR
                                                                 Developmental Toxicity   799.9365). However,
                                                                 Screening Test: 40 CFR   there may be valid
                                                                 799.9355 AND.            reasons to test a
                                                                Repeated Dose 28-Day      particular chemical
                                                                 Oral Toxicity Study in   using both 40 CFR
                                                                 rodents: 40 CFR          799.9355 and 40 CFR
                                                                 799.9305.                799.9305 to fill
                                                                                          Mammalian Toxicity--
                                                                                          Repeated Dose/
                                                                                          Reproduction/
                                                                                          Developmental data
                                                                                          needs. A subject
                                                                                          person who uses the
                                                                                          combination of 40 CFR
                                                                                          799.9355 and 40 CFR
                                                                                          799.9305 in place of
                                                                                          40 CFR 799.9365 must
                                                                                          submit to EPA a
                                                                                          rationale for
                                                                                          conducting these
                                                                                          alternate tests in the
                                                                                          final study reports.
                                                                                          Where F2 or F3 is
                                                                                          required, no rationale
                                                                                          for conducting the
                                                                                          required test need be
                                                                                          provided in the final
                                                                                          study report.
                                      --------------------------------------------------
                                       F2                       Reproduction/
                                                                 Developmental Toxicity
                                                                 Screening Test: 40 CFR
                                                                 799.9355
                                      --------------------------------------------------
                                       F3                       Repeated Dose 28-Day
                                                                 Oral Toxicity Study in
                                                                 rodents: 40 CFR
                                                                 799.9305
----------------------------------------------------------------------------------------------------------------
\1\ EPA recommends, but does not require, that log Kow be quantitatively estimated prior to initiating this
  study. One method, among many similar methods, for estimating log Kow is described in the article entitled
  Atom/Fragment Contribution Method for Estimating Octanol-Water Partition Coefficients) by W.M. Meylan and P.H.
  Howard in the Journal of Pharmaceutical Sciences. 84(1):83-92. January 1992. This reference is available under
  docket ID number EPA-HQ-OPPT-2005-0033 at the EPA Docket Center, Rm. B102, 1301 Constitution Ave., NW.,
  Washington, DC, from 8:30 a.m. to 4:30 p.m., Monday through Friday, excluding legal holidays.
\2\ EPA recommends, but does not require, that water solubility be quantitatively estimated prior to initiating
  this study. One method, among many similar methods, for estimating water solubility is described in the
  article entitled Improved Method for Estimating Water Solubility From Octanol/Water Partition Coefficient by
  W.M. Meylan, P.H. Howard, and R.S. Boethling in Environmental Toxicology and Chemistry. 15(2):100-106. 1996.
  This reference is available under docket ID number EPA-HQ-OPPT-2005-0033 at the EPA Docket Center, Rm. B102,
  1301 Constitution Ave., NW., Washington, DC, from 8:30 a.m. to 4:30 p.m., Monday through Friday, excluding
  legal holidays.
\3\ Chemical substances that are dispersible in water may have log Kow values greater than 4.2 and may still be
  acutely toxic to aquatic organisms. EPA recommends, but does not require, that test sponsors who wish to
  conduct Test Group 1 studies on such chemicals to submit to EPA for approval a written request to conduct Test
  Group 1 studies 90 days prior to conducting such studies. The written request should include the rationale for
  conducting Test Group 1 studies.

[[Page 282]]

 
\4\ The OECD 425 Up/Down Procedure, revised by OECD in December 2001, is available under docket ID number EPA-HQ-
  OPPT-2005-0033 at the EPA Docket Center, Rm. B102, 1301 Constitution Ave., NW., Washington, DC, from 8:30 a.m.
  to 4:30 p.m., Monday through Friday, excluding legal holidays.
\5\ The neutral red uptake basal cytotoxicity assay, which may be used to estimate the starting dose for the
  mammalian toxicity-acute endpoint, is available under docket ID number EPA-HQ-OPPT-2005-0033 at the EPA Docket
  Center, Rm. B102, 1301 Constitution Ave., NW., Washington, DC, from 8:30 a.m. to 4:30 p.m., Monday through
  Friday, excluding legal holidays.

    (k) Effective date. This section is effective on April 17, 2006.

[71 FR 13730, Mar. 16, 2006, as amended at 71 FR 71062, Dec. 8, 2006]



Sec. 799.5115  Chemical testing requirements for certain chemicals of interest 

to the Occupational Safety and Health Administration.

    (a) What substances will be tested under this section? Table 2 in 
paragraph (j) of this section identifies the chemical substances that 
must be tested under this section. For the chemical substances 
identified as ``Class 1'' substances in Table 2 in paragraph (j) of this 
section, the purity of each chemical substance must be 99% or greater, 
unless otherwise specified in this section. For the chemical substances 
identified as ``Class 2'' substances in Table 2 in paragraph (j) of this 
section, a representative form of each chemical substance must be 
tested.
    (b) Am I subject to this section? (1) If you manufacture (including 
import) or intend to manufacture, or process or intend to process, any 
chemical substance listed in Table 2 in paragraph (j) of this section at 
any time from May 26, 2004, to the end of the test data reimbursement 
period as defined in 40 CFR 791.3(h), you are subject to this section 
with respect to that chemical substance.
    (2) If you do not know or cannot reasonably ascertain that you 
manufacture or process a chemical substance listed in Table 2 in 
paragraph (j) of this section during the time period described in 
paragraph (b)(1) of this section (based on all information in your 
possession or control, as well as all information that a reasonable 
person similarly situated might be expected to possess, control, or 
know, or could obtain without an unreasonable burden), you are not 
subject to this section with respect to that chemical substance.
    (c) If I am subject to this section, when must I comply with it? 
(1)(i) Persons subject to this section are divided into two groups, as 
set forth in Table 1 of this paragraph: Tier 1 (persons initially 
required to comply) and Tier 2 (persons not initially required to 
comply). If you are subject to this section, you must determine if you 
fall within Tier 1 or Tier 2, based on Table 1 of this paragraph.

   Table 1--Persons Subject to the Rule: Persons in Tier 1 and Tier 2
------------------------------------------------------------------------
                                          Persons not initially required
  Persons initially required to comply     to comply with this section
       with this section (Tier 1)                    (Tier 2)
------------------------------------------------------------------------
Persons not otherwise specified in       A. Persons who manufacture (as
 column 2 of this table that              defined at TSCA section 3(7))
 manufacture (as defined at TSCA          or intend to manufacture a
 section 3(7)) or intend to manufacture   chemical substance included in
 a chemical substance included in this    this section solely as one or
 section.                                 more of the following:
                                         --As a byproduct (as defined at
                                          40 CFR 791.3(c));
                                         --As an impurity (as defined at
                                          40 CFR 790.3);
                                         --As a naturally occurring
                                          substance (as defined at 40
                                          CFR 710.4(b));
                                         --As a non-isolated
                                          intermediate (as defined at 40
                                          CFR 704.3);
                                         --As a component of a Class 2
                                          substance (as described at 40
                                          CFR 720.45(a)(1)(i));
                                         --In amounts of less than 500
                                          kilograms (kg) (1,100 lbs)
                                          annually (as described at 40
                                          CFR 790.42(a)(4)); or
                                         --For research and development
                                          (as described at 40 CFR
                                          790.42(a)(5)).
                                         B. Persons who process (as
                                          defined at TSCA section 3(10))
                                          or intend to process a
                                          chemical substance included in
                                          this section (see 40 CFR
                                          790.42(a)(2)).
------------------------------------------------------------------------

    (ii) Table 1 in paragraph (c)(1)(i) of this section expands the list 
of persons specified in Sec. 790.42(a)(2), (a)(4), and (a)(5) of this 
chapter, who, while legally subject to this section, must comply with 
the requirements of this section only if directed to do so by EPA under 
the circumstances set forth in paragraphs (c)(4) through (c)(7) and 
(c)(10) of this section.
    (2) If you are in Tier 1 with respect to a chemical substance listed 
in Table 2 in paragraph (j) of this section, you must, for each test 
required under this section for that chemical substance, either submit 
to EPA a letter of intent

[[Page 283]]

to test or apply to EPA for an exemption from testing. The letter of 
intent to test or the exemption application must be received by EPA no 
later than June 25, 2004.
    (3) If you are in Tier 2 with respect to a chemical substance listed 
in Table 2 in paragraph (j) of this section, you are considered to have 
an automatic conditional exemption and you will be required to comply 
with this section with regard to that chemical substance only if 
directed to do so by EPA under paragraphs (c)(5), (c)(7), or (c)(10) of 
this section.
    (4) If no person in Tier 1 has notified EPA of its intent to conduct 
one or more of the tests required by this section on any chemical 
substance listed in Table 2 in paragraph (j) of this section by June 25, 
2004, EPA will publish a Federal Register document that would specify 
the test(s) and the chemical substance(s) for which no letter of intent 
has been submitted, and notify manufacturers in Tier 2A of their 
obligation to submit a letter of intent to test or to apply for an 
exemption from testing.
    (5) If you are in Tier 2A with respect to a chemical substance 
listed in Table 2 in paragraph (j) of this section, and if you 
manufacture this chemical substance as of May 26, 2004, or within 30 
days after publication of the Federal Register document described in 
paragraph (c)(4) of this section, you must, for each test specified for 
that chemical substance in the document described in paragraph (c)(4) of 
this section, either submit to EPA a letter of intent to test or apply 
to EPA for an exemption from testing. The letter of intent to test or 
the exemption application must be received by EPA no later than 30 days 
after publication of the document described in paragraph (c)(4) of this 
section.
    (6) If no manufacturer in Tier 1 or Tier 2A has notified EPA of its 
intent to conduct one or more of the tests required by this section on 
any chemical substance listed in Table 2 in paragraph (j) of this 
section within 30 days after the publication of the Federal Register 
document described in paragraph (c)(4) of this section, EPA will publish 
another Federal Register document that would specify the test(s) and the 
chemical substance(s) for which no letter of intent has been submitted, 
and notify processors in Tier 2B of their obligation to submit a letter 
of intent to test or to apply for an exemption from testing.
    (7) If you are in Tier 2B with respect to a chemical substance 
listed in Table 2 in paragraph (j) of this section, and if you process 
this chemical substance as of May 26, 2004, or within 30 days after 
publication of the Federal Register document described in paragraph 
(c)(6) of this section, you must, for each test specified for that 
chemical substance in the document described in paragraph (c)(6) of this 
section, either submit to EPA a letter of intent to test or apply to EPA 
for an exemption from testing. The letter of intent to test or the 
exemption application must be received by EPA no later than 30 days 
after publication of the document described in paragraph (c)(6) of this 
section.
    (8) If no manufacturer or processor has notified EPA of its intent 
to conduct one or more of the tests required by this section for any of 
the chemical substances listed in Table 2 in paragraph (j) of this 
section within 30 days after the publication of the Federal Register 
document described in paragraph (c)(6) of this section, EPA will notify 
all manufacturers and processors of those chemical substances of this 
fact by certified letter or by publishing a Federal Register document 
specifying the test(s) for which no letter of intent has been submitted. 
This letter or Federal Register document will additionally notify all 
manufacturers and processors that all exemption applications concerning 
the test(s) have been denied, and will give the manufacturers and 
processors of the chemical substance(s) an opportunity to take 
corrective action.
    (9) If no manufacturer or processor has notified EPA of its intent 
to conduct one or more of the tests required by this section for any of 
the chemical substances listed in Table 2 in paragraph (j) of this 
section within 30 days after receipt of the certified letter or 
publication of the Federal Register document described in paragraph 
(c)(8) of this section, all manufacturers and processors subject to this 
section with respect to that chemical substance who

[[Page 284]]

are not already in violation of this section will be in violation of 
this section.
    (10) If a problem occurs with the initiation, conduct, or completion 
of the required testing or the submission of the required data with 
respect to a chemical substance listed in Table 2 in paragraph (j) of 
this section, under the procedures in Sec. Sec. 790.93 and 790.97 of 
this chapter, EPA may initiate termination proceedings for all testing 
exemptions with respect to that chemical substance and may notify 
persons in Tier 1 and Tier 2 that they are required to submit letters of 
intent to test or exemption applications within a specified period of 
time.
    (11) If you are required to comply with this section, but your 
manufacturing or processing of a chemical substance listed in Table 2 in 
paragraph (j) of this section begins after the applicable compliance 
date referred to in paragraphs (c)(2), (c)(5), (c)(7), or (c)(10) of 
this section, you must either submit a letter of intent to test or apply 
to EPA for an exemption. The letter of intent to test or the exemption 
application must be received by EPA no later than the day you begin 
manufacturing or processing.
    (d) What must I do to comply with this section? (1) To comply with 
this section you must either submit to EPA a letter of intent to test, 
or apply to and obtain from EPA an exemption from testing.
    (2) For each test with respect to which you submit to EPA a letter 
of intent to test, you must conduct the testing specified in paragraph 
(h) of this section and submit the test data to EPA.
    (3) You must also comply with the procedures governing test rule 
requirements in part 790 of this chapter, as modified by this section, 
including the submission of letters of intent to test or exemption 
applications, the conduct of testing, and the submission of data; Part 
792--Good Laboratory Practice Standards of this chapter; and this 
section. The following provisions of 40 CFR part 790 do not apply to 
this section: Paragraphs (a), (d), (e), and (f) of Sec. 790.45; 
paragraph (a)(2) and paragraph (b) of Sec. 790.80; and Sec. 790.48.
    (e) If I do not comply with this section, when will I be considered 
in violation of it? You will be considered in violation of this section 
as of 1 day after the date by which you are required to comply with this 
section.
    (f) How are EPA's data reimbursement procedures affected for 
purposes of this section? If persons subject to this section are unable 
to agree on the amount or method of reimbursement for test data 
development for one or more chemical substances included in this 
section, any person may request a hearing as described in 40 CFR part 
791. In the determination of fair reimbursement shares under this 
section, if the hearing officer chooses to use a formula based on 
production volume, the total production volume amount will include 
amounts of a chemical substance produced as an impurity.
    (g) Who must comply with the export notification requirements? Any 
person who exports, or intends to export, a chemical substance listed in 
Table 2 in paragraph (j) of this section is subject to part 707, subpart 
D, of this chapter.
    (h) How must I conduct my testing? The chemical substances 
identified by Chemical Abstract Service Registry Number (CAS No.) and 
chemical name in Table 2 in paragraph (j) of this section must be tested 
as follows:
    (1) Applicability. This in vitro dermal absorption rate test 
standard must be used for all testing conducted under this section. In 
certain instances, modifications to the test standard may be considered. 
The procedures for applying for a modification to the test standard are 
specified in 40 CFR 790.55.
    (2) Source. The test standard is based on the Protocol for In Vitro 
Percutaneous Absorption Rate Studies, referenced in paragraph (h)(8)(v) 
of this section.
    (3) Purpose. In the assessment and evaluation of the characteristics 
of a chemical substance or mixture for which testing is required under 
this section (test substance), it is important to determine the rate of 
absorption of the test substance in cases where dermal exposure to the 
test substance in the workplace may result in systemic toxicity. This 
test standard is designed to develop data that describe the rate at 
which test substances are absorbed through the skin so that the

[[Page 285]]

body burden of a test substance resulting from dermal exposure in the 
workplace can be better evaluated.
    (4) Principles of the test standard. This test standard describes 
procedures for measuring a permeability constant (Kp) and two short-term 
dermal absorption rates for test substances in liquid form. The test 
standard utilizes in vitro diffusion cell techniques which allow 
absorption studies to be conducted with human cadaver skin. In vitro 
diffusion studies are necessary for measuring a Kp. This test standard 
specifies the use of static or flow-through diffusion cells and non-
viable human cadaver skin. It also requires the use of radiolabeled test 
substances unless it can be demonstrated that procedures utilizing a 
non-radiolabeled test substance are able to measure the test substance 
with a sensitivity equivalent to the radiolabeled method.
    (5) Test procedure--(i) Choice of membrane--(A) Skin selection. 
Human cadaver skin must be used in all testing conducted under this test 
standard. This test standard does not require use of live skin, or the 
maintenance of skin viability during the course of the experiment. 
However, the time elapsed between death and harvest of tissue must be 
reported.
    (B) Number of skin samples. Data for the determination of a Kp must 
be obtained from a minimum of six skin samples and the skin samples must 
come from at least three different human subjects (two skin samples from 
each subject) in order to allow for biological variation between 
subjects. Data for the determination of each short-term (i.e., 10 minute 
and 60 minute) absorption rate must be obtained from a minimum of six 
skin samples and the skin samples must come from at least three 
different human subjects (two skin samples from each subject).
    (C) Anatomical region. In order to minimize the variability in skin 
absorption measurements for these tests, samples of human cadaver skin 
must be obtained from the abdominal region of human subjects of known 
source and disease state.
    (D) Validation of human cadaver skin barrier. Prior to conducting an 
experiment with the test substance, barrier properties of human cadaver 
skin must be pretested either by:
    (1) Measuring the absorption of a standard compound such as 
tritiated water as discussed, for example, in the reference in paragraph 
(h)(8)(i) of this section;
    (2) Determining an electrical resistance to an alternating current, 
at up to two volts; or
    (3) Measuring trans-epidermal water loss from the stratum corneum.
    (ii) Preparation of membrane. Full thickness skin must not be used. 
A suitable membrane must be prepared from skin either with a dermatome 
at a thickness of 200 to 500 micrometers ([micro]m), or with heat 
separation by treating the skin at 60 [deg]C for 45 seconds to 2 minutes 
after which the epidermis can be peeled from the dermis. These epidermal 
membranes can be stored frozen (-20 [deg]C) for up to 3 months, if 
necessary, if they are frozen quickly and the barrier properties of the 
samples are confirmed immediately prior to commencement of the 
experiment.
    (iii) Diffusion cell design. Either static or flow-through diffusion 
cells must be used in these studies. To ensure that an increase in 
concentration of the test substance in the receptor fluid does not alter 
penetration rate, the testing laboratory must verify that the 
concentration of the test substance in the receptor fluid is less than 
10% of the initial concentration in the donor chamber. Concentration of 
the neat (i.e., undiluted) liquid must be taken as the density of the 
test substance.
    (iv) Temperature. Skin must be maintained at a physiological 
temperature of 32 [deg]C during the test.
    (v) Testing hydrophobic chemicals. When testing hydrophobic 
chemicals, polyethoxyoleate (polyethylene glycol (PEG) 20 oleyl ether) 
must be added to the receptor fluid at a concentration of 6%.
    (vi) Vehicle. If the test substance is a liquid at room temperature 
and does not damage the skin during the determination of Kp, it must be 
applied neat. If the test substance cannot be applied neat because it is 
a solid at room temperature or because it damages the skin when applied 
neat, it

[[Page 286]]

must be dissolved in water. If the concentration of a hydrophobic test 
substance in water is not high enough so that a steady-state absorption 
can be obtained, the test substance must be dissolved in isopropyl 
myristate. A sufficient volume of liquid must be used to completely 
cover the skin and provide the amount of test substance as described in 
paragraph (h)(5)(vii) of this section.
    (vii) Dose--(A) Kp. A Kp must be determined for each test chemical, 
except for methyl isoamyl ketone (MIAK; CAS No.: 110-12-3, Chemical 
Abstracts (CA) Index Name: 2-Hexanone, 5-methoxy-) and dipropylene 
glycol methyl ether (DPGME; CAS No.: 34590-94-8, CA Index Name: 
Propanol, 1(or 2)-(2-methoxymethylethoxy)-). An ``infinite dose'' of the 
test substance must be applied to the skin to achieve the steady-state 
rate of absorption necessary for calculation of a Kp. Infinite dose is 
defined as the concentration of a test substance required to give an 
undepletable reservoir on the surface of the skin. The actual 
concentration required to give an undepletable reservoir on the surface 
of the skin depends on the rate of penetration of the test substance. 
Preliminary studies may be necessary to determine this concentration. 
Percutaneous absorption must be determined under occluded (i.e., 
covered) conditions unless it is demonstrated that such conditions cause 
leakage of material or damage to the skin membrane as a result of 
unrealistically high pressures or excessive hydration. Skin barrier 
integrity must be verified at the end of the experiment by the methods 
discussed in paragraph (h)(5)(i)(D) of this section.
    (B) Short-term absorption rates. Short-term absorption rates must be 
determined for all test chemicals. The dose of test chemical applied to 
the skin must be sufficient to completely cover the exposed skin 
surface. A minimum of four diffusion cells must be set up using skin 
from a single subject. Two diffusion cells must be terminated at 10 
minutes. The remaining two diffusion cells must be terminated at 60 
minutes. Skin absorption at each sampling time is the sum of the 
receptor fluid levels and the absorbed test substance that remains in 
the skin, as discussed, for example, in the reference in paragraph 
(h)(8)(iii) of this section. Unabsorbed chemical must be removed from 
the skin surface by washing gently with soap and water. This experiment 
must be repeated with skin from two additional subjects. In order to 
ensure reliable short-term absorption rates, percutaneous absorption 
must be determined under occluded conditions unless it is demonstrated 
that such conditions cause leakage of material or damage to the skin 
membrane as a result of unrealistically high pressures or excessive 
hydration.
    (viii) Study duration--(A) Kp. The in vitro dermal absorption rate 
test must be performed until at least four absorption measurements per 
diffusion cell experiment are obtained during the steady-state 
absorption portion of the experiment. A preliminary study may be useful 
to establish time points for sampling. The required absorption 
measurements can be accomplished in an hour or two with fast-penetrating 
chemicals but may require 24 hours or longer for slow-penetrating 
chemicals. Unabsorbed test substance need not be removed from the 
surface of the skin after each experiment.
    (B) Short-term absorption rates. The test substance must be applied 
to skin for durations of 10 and 60 minutes. At the end of the study, the 
unabsorbed test substance must be removed from the surface of the skin 
with soap and water and the amount absorbed into the skin and receptor 
fluid must be determined, as discussed, for example, in the reference in 
paragraph (h)(8)(iii) of this section.
    (6) Results--(i) Kp. The Kp must be calculated by dividing the 
steady-state rate of absorption (measured in micrograms ([micro]g) x 
hr-1 x centimeters (cm)-2) by the concentration of 
the test substance (measured in [micro]g x cm-3) applied to 
the skin. (For example, if the steady-state rate is 1 microgram x 
hr-1 x cm-2 and the concentration applied to the 
skin is 1,000 micrograms x cm-3, then the Kp value is 
calculated to be 0.001 cm x hr-1.) The mean and standard 
deviation of the calculated Kp values for all diffusion cell experiments 
must be determined.
    (ii) Short-term absorption rate. The absorption rates ([micro]g x 
hr-1 x cm-2) must

[[Page 287]]

be determined from the total amount of test substance found in the 
receptor fluid and skin after the 10-minute and 60-minute exposures for 
each diffusion cell experiment. The mean and standard deviation of 10-
minute short-term absorption rates from all experiments must be 
calculated. The mean and standard deviation of 60-minute short-term 
absorption rates from all experiments must also be calculated.
    (7) Test report. In addition to compliance with the TSCA Good 
Laboratory Practice Standards (GLPS) at 40 CFR part 792, the following 
specific information must be collected and reported by the date in 
paragraph (i) of this section:
    (i) Test systems and test methods. (A) A description of the date, 
time, and location of the test, the name(s) of the person(s) conducting 
the test, the location of records pertaining to the test, as well as a 
GLPS statement. These statements must be certified by the signatures of 
the individuals performing the work and their supervisors.
    (B) A description of the source, identity, and purity of the test 
substance and the source, identity, and handling of the test skin. There 
must be a detailed description of the test procedure and all materials, 
devices used and doses tested, as well as a detailed description and 
illustration of static or flow-through cell design. There must also be a 
description of the skin preparation method, including measurements of 
the skin membrane thickness.
    (C) A description of the analytical techniques to be used, including 
their accuracy, precision, and detection limits (in particular for non-
radiolabeled tests), and, if a radiolabel is used, there must be a 
description of the radiolabel (e.g., type, location of, and 
radiochemical purity of the label).
    (D) All data must be clearly identified as to dose and specimen. 
Derived values (means, permeability coefficient, graphs, charts, etc.) 
are not sufficient.
    (ii) Conduct of study. Data must be collected and reported on the 
following:
    (A) Monitoring of testing parameters.
    (B) Temperature of chamber.
    (C) Receptor fluid pH.
    (D) Barrier property validation.
    (E) Analysis of receptor fluid for radioactivity or test chemical
    (iii) Results. The mean Kp and mean short-term absorption rates must 
be presented along with their standard deviations and the number of 
diffusion cell experiments. In addition, all raw data from each 
individual diffusion cell must be retained to support the calculations 
of permeability constants and short-term absorption rates. When a 
radiolabeled test substance is used, a full balance of the radioactivity 
must be presented, including cell rinsing and stability of the test 
substance in the donor compartment.
    (8) References. For background information on this test standard, 
the following references may be consulted. These references are 
available under docket ID number OPPT-2003-0006 at the EPA Docket 
Center, Rm. B102-Reading Room, EPA West, 1301 Constitution Ave., NW., 
Washington, DC, from 8:30 a.m. to 4:30 p.m., Monday through Friday, 
excluding legal holidays.
    (i) Bronaugh, R.L., Stewart, R.F., and Simon, M. Methods for In 
Vitro Percutaneous Absorption Studies VII: Use of Excised Human Skin. 
Journal of Pharmaceutical Sciences. 75:1094-1097. 1986.
    (ii) Bronaugh, R.L. and Stewart, R.F. Methods for In Vitro 
Percutaneous Absorption Studies IV: The Flow-Through Diffusion Cell. 
Journal of Pharmaceutical Sciences. 74:64-67. 1985.
    (iii) Bronaugh, R.L., Stewart, R.F., and Storm, J.E. Extent of 
Cutaneous Metabolism During Percutaneous Absorption of Xenobiotics. 
Toxicology and Applied Pharmacology. 99:534-543. 1989.
    (iv) Walker, J.D., Whittaker, C. and McDougal, J.N. Role of the TSCA 
Interagency Testing Committee in Meeting the U.S. Government Data Needs: 
Designating Chemicals for Percutaneous Absorption Rate Testing. 
Dermatotoxicology. F. Marzulli and H. Maibach, Eds. Taylor & Francis, 
Washington, DC. pp. 371-381. 1996.
    (v) Bronaugh, R.L., and Collier, S.W. Protocol for In Vitro 
Percutaneous Absorption Studies. In Vitro Percutaneous Absorption: 
Principles, Fundamentals, and Applications. R.L. Bronaugh and H.I. 
Maibach, Eds. CRC Press, Boca Raton, FL. pp. 237-241. 1991.

[[Page 288]]

    (i) Reporting requirements. The reports submitted under this section 
must include the information specified in paragraph (h)(7) of this 
section. A final report for each chemical substance must be received by 
EPA by June 27, 2005, unless an extension is granted in writing pursuant 
to 40 CFR 790.55.
    (j) Designation of specific chemical substances for testing. The 
chemical substances identified by chemical name, CAS No., and class in 
Table 2 of this paragraph must be tested in accordance with the testing 
requirements in paragraph (h) of this section and the requirements 
described in 40 CFR part 792.

           Table 2--Chemical Substances Designated For Testing
------------------------------------------------------------------------
               CAS No.                       Chemical name         Class
------------------------------------------------------------------------
75-05-8..............................  Acetonitrile.............       1
75-15-0..............................  Carbon disulfide.........       1
75-35-4..............................  Vinylidene chloride......       1
77-73-6..............................  Dicyclopentadiene........       1
78-59-1..............................  Isophorone...............       1
78-87-5..............................  Propylene dichloride.....       1
79-20-9..............................  Methyl acetate...........       1
79-46-9..............................  2-Nitropropane...........       1
91-20-3..............................  Naphthalene..............       1
92-52-4..............................  Biphenyl.................       1
98-29-3..............................  tert-Butylcatechol.......       1
100-00-5.............................  p-Nitrochlorobenzene.....       1
100-01-6.............................  p-Nitroaniline...........       1
100-44-7.............................  Benzyl chloride..........       1
106-42-3.............................  p-Xylene.................       1
106-46-7.............................  p-Dichlorobenzene........       1
107-06-2.............................  Ethylene dichloride......       1
107-31-3.............................  Methyl formate...........       1
108-03-2.............................  1-Nitropropane...........       1
108-90-7.............................  Chlorobenzene............       1
108-93-0.............................  Cyclohexanol.............       1
109-66-0.............................  Pentane..................       1
109-99-9.............................  Tetrahydrofuran..........       1
110-12-3.............................  Methyl isoamyl ketone....       1
111-84-2.............................  Nonane...................       1
120-80-9.............................  Catechol.................       1
122-39-4.............................  Diphenylamine............       1
123-42-2.............................  Diacetone alcohol........       1
127-19-5.............................  Dimethyl acetamide.......       1
142-82-5.............................  n-Heptane................       1
150-76-5.............................  p-Methoxyphenol..........       1
25013-15-4...........................  Vinyl toluene............       2
34590-94-8...........................  Dipropylene glycol methyl       2
                                        ether.
------------------------------------------------------------------------

    (k) Effective date This section is effective on May 26, 2004.

[69 FR 22436, Apr. 26, 2004, as amended at 71 FR 18654, Apr. 12, 2006]



              Subpart E_Product Properties Test Guidelines

    Source: 65 FR 78751, Dec. 15, 2000, unless otherwise noted.



Sec. 799.6755  TSCA partition coefficient (n-octanol/water), shake flask 

method.

    (a) Scope--(1) Applicability. This section is intended to meet the 
testing requirements of the Toxic Substances Control Act (TSCA) (15 
U.S.C. 2601).
    (2) Source. The source material used in developing this TSCA test 
guideline is the Office of Prevention, Pesticides and Toxics (OPPTS) 
harmonized test guideline 830.7550 (August 1996, final guideline). The 
source is available at the address in paragraph (f) of this section.
    (b) Introductory information--(1) Prerequisites. Suitable analytical 
method, dissociation constant, water solubility, and hydrolysis 
(preliminary test).
    (2) Coefficient of variation. The coefficient of variation on the 
mean values reported by the participants of the Organization for 
Economic Coopertion and Development (OECD) Laboratory Intercomparison 
Testing, Part I, 1979, appeared to be dependent on the chemicals tested; 
it ranges from 0.17 to 1.03.
    (3) Qualifying statements. This method applies only to pure, water 
soluble substances which do not dissociate or associate, and which are 
not surface active. In order to use the partition coefficient (P) as a 
screening test for bioaccumulation, it should be ascertained that the 
impurities in the commercial product are of minor importance. Testing of 
P (n-octanol/water) cannot be used as a screening test in the case of 
organometallic compounds.
    (4) Alternative methods. High-pressure liquid chromatography (HPLC) 
methods described in the references in paragraphs (f)(3), (f)(4), and 
(f)(5) of this section may be considered as an alternative test method.
    (c) Method--(1) Introduction, purpose, scope, relevance, 
application, and limits of test. The P of a substance between water and 
a lipophilic solvent (n-octanol) is one model variable which may be used 
to describe the transfer of a substance from the aquatic environment 
into an organism and the potential bioaccumulation of the substance. 
Studies show a highly significant relationship between the P of 
different substances in the system water/n-octanol

[[Page 289]]

and their bioaccumulation in fish described in paragraph (f)(1) of this 
section.
    (2) Definitions--Partition coefficient (P) is defined as the ratio 
of the equilibrium concentrations (Ci) of a dissolved 
substance in a two-phase system consisting of two largely immiscible 
solvents. The P therefore is the quotient of two concentrations and is 
usually given in the form of its logarithm to base 10 (log P). In this 
case n-octanol and water:

                               Equation 1:
[GRAPHIC] [TIFF OMITTED] TR15DE00.039

    (3) Reference substances. The reference substances need not be 
employed in all cases when investigating a new substance. They are 
provided primarily so that calibration of the method may be performed 
from time to time and to offer the chance to compare the results when 
another method is applied. The values presented in table 1 of this 
section are not necessarily representative of the results which can be 
obtained with this test method as they have been derived from an earlier 
version of the test guideline.

                 Table 1--Data for Reference Substances
------------------------------------------------------------------------
          Tested substance \1\                       Pow \2\
------------------------------------------------------------------------
Di(2-ethylhexyl)phthalate (OECD).......   1.3 x 10\5\ (4.6 x 10\4\ - 2.8
                                          x 10\5\)
Hexachlorobenzene (OECD)...............   3.6 x 10\5\ (1.1 x 10\5\ - 8.3
                                          x 10\5\)
o-Dichlorobenzene European Economic       5.1 x 10\3\ (1.5 x 10\3\ - 2.3
 Community (EEC).                         x 10\4\)
Dibutyl phthalate (EEC)................   1.3 x 10\4\ (1.7 x 10\3\ - 2.8
                                          x 10\4\)
Trichloroethylene (OECD)...............   2.0 x 10\3\ (5.2x10\2\-
                                          3.7x10\3\)
Urea (OECD)............................   6.2x10-2 (2.0x10-2--2.4x10-1)
------------------------------------------------------------------------
\1\ Substances not tested: Ethyl acetate, 4-methyl-2,4-pentanediol.
\2\ Total, mean, and range of mean values (in parentheses) submitted by
  the participants of the OECD or EEC Laboratory Intercomparison
  Testing.

    (4) Principle of the test method. In order to determine a P, 
equilibrium between all interacting components of the system must be 
achieved, and the concentrations of the substances dissolved in the two 
phases must be determined. A study of the literature on this subject 
indicates that there are many different techniques which can be used to 
solve this problem, i.e. the thorough mixing of the two phases followed 
by their separation in order to determine the equilibrium concentration 
for the substance being examined.
    (5) Quality criteria--(i) Repeatability. In order to assure the 
precision of the P, duplicate determinations are to be made under three 
different test conditions, whereby the quantity of substance specified 
as well as the ratio of the solvent volumes may be varied. The 
determined values of the P expressed as their common logarithms should 
fall within a range of  0.3 log units.
    (ii) Sensitivity. The sensitivity of the method is determined by the 
sensitivity of the analytical procedure. This should be sufficient to 
permit the assessment of values of Pow up to 105 when the 
concentration of the solute in either phase is not more than 0.01 mol/
Liter (L). The substance being tested must not be water insoluble (mass 
concentration [rho] <= 10-6 gram (g)/L.
    (iii) Specificity. The Nernst Partition Law applies only at constant 
temperature, pressure, and pH for dilute solutions. It strictly applies 
to a pure substance dispersed between two pure solvents. If several 
different solutes occur in one or both phases at the same time, this may 
affect the results. Dissociation or association of the dissolved 
molecules result in deviations from the Nernst Partition Law. Such 
deviations are indicated by the fact that the P becomes dependent upon 
the concentration of the solution. Because of the multiple equilibria 
involved, this test guideline should not be applied to ionizable 
compounds without corrections being made. The use of buffer solutions in 
place of water should be considered for such compounds.

[[Page 290]]

    (iv) Possibility of standardization. This method can be 
standardized.
    (d) Description of the test procedure--(1) Preparations: Preliminary 
estimate of the P. The size of the P can be estimated either by means of 
calculation or by use of published solubilities of the test substance in 
the pure solvents. Alternatively, it may be roughly determined by 
performing a simplified preliminary test. For this:

                               Equation 2:
[GRAPHIC] [TIFF OMITTED] TR15DE00.040

    (2) Preparation of the solvents--(i) n-Octanol. The determination of 
the P should be carried out with analytical grade n-octanol. Inorganic 
contaminants can be removed from commercial n-octanol by washing with 
acid and base, drying, and distilling. More sophisticated methods will 
be required to separate the n-octanol from organic contaminants with 
similar vapor pressure if they are present.
    (ii) Water. Distilled water or water twice-distilled from glass or 
quartz apparatus should be employed. Water taken directly from an ion 
exchanger should not be used.
    (iii) Presaturation of the solvents. Before a P is determined, the 
phases of the solvent system are mutually saturated by shaking at the 
temperature of the experiment. For doing this, it is practical to shake 
two large stock bottles of purified n-octanol or distilled water each 
with a sufficient quantity of the other solvent for 24 hours on a 
mechanical shaker, and then to let them stand long enough to allow the 
phases to separate and to achieve a saturation state.
    (3) Preparation for the test. The entire volume of the two-phase 
system should nearly fill the test vessel. This will help prevent loss 
of material due to volatilization. The volume ratio and quantities of 
substance to be used are fixed by the following:
    (i) The preliminary assessment of the P as discussed in paragraph 
(d)(1) of this section).
    (ii) The minimum quantity of test substance required for the 
analytical procedure.
    (iii) The limitation of a maximum concentration in either phase of 
0.01 mol/L.
    (iv) Three tests are carried out. In the first, the calculated 
volume ratio is added; in the second, twice the volume of n-octanol is 
added; and in the third, half the volume of n-octanol is added.
    (4) Test substance. The test substance should be the purest 
available. For a material balance during the test a stock solution is 
prepared in n-octanol with a mass concentration between 1 and 100 
milligram/milliliter (mg/mL). The actual mass concentration of this 
stock solution should be precisely determined before it is employed in 
the determination of the P. This solution should be stored under stable 
conditions.
    (5) Test conditions. The test temperature should be kept constant 
( 1 [deg]C) and lie in the range of 20-25 [deg]C.
    (6) Performance of the test--(i) Establishment of the partition 
equilibrium. Duplicate test vessels containing the required, accurately 
measured amounts of the two solvents together with the necessary 
quantity of the stock solution should be prepared for each of the test 
conditions. The n-octanol parts should be measured by volume. The test 
vessels should either be placed in a suitable shaker or shaken by hand. 
A recommended method is to rotate the centrifuge tube quickly through 
180[deg] about its transverse axis so that any trapped air rises through 
the two phases. Experience has shown that 50 such rotations are usually 
sufficient for the establishment of the partition equilibrium. To be 
certain, 100 rotations in 5 minutes are recommended.
    (ii) Phase separation. In order to separate the phases, 
centrifugation of the mixture should be carried out. This

[[Page 291]]

should be done in a laboratory centrifuge maintained at room 
temperature, or, if a non-temperature-controlled centrifuge is used, the 
centrifuge tubes should be reequilibrated at the test temperature for at 
least 1 hour before analysis.
    (7) Analysis. (i) For the determination of the P, it is necessary to 
analyze the concentrations of the test substance in both phases. This 
may be done by taking an aliquot of each of the two phases from each 
tube for each test condition and analyzing them by the chosen procedure. 
The total quantity of substances present in both phases should be 
calculated and compared with the quantity of the substance originally 
introduced.
    (ii) The aqueous phase should be sampled by the following procedure 
to minimize the risk of including traces of the n-octanol: A glass 
syringe with a removable needle should be used to sample the water 
phase. The syringe should initially be partially filled with air. Air 
should be gently expelled while inserting the needle through the n-
octanol layer. An adequate volume of aqueous phase is withdrawn into the 
syringe. The syringe is quickly removed from the solution and the needle 
detached. The contents of the syringe may then be used as the aqueous 
sample.
    (iii) The concentration in the two-separated phases should 
preferably be determined by a substance-specific method. Examples of 
physical-chemical determinations which may be appropriate are:
    (A) Photometric methods.
    (B) Gas chromatography.
    (C) HPLC.
    (D) Back-extraction of the aqueous phase and subsequent gas 
chromatography.
    (e) Data and reporting--(1) Treatment of results. The reliability of 
the determined values of P can be tested by comparison of the means of 
the duplicate determinations with the overall mean.
    (2) Test report. The following should be included in the report:
    (i) Name of the substance, including its purity.
    (ii) Temperature of the determination.
    (iii) The preliminary estimate of the P and its manner of 
determination.
    (iv) Data on the analytical procedures used in determining 
concentrations.
    (v) The measured concentrations in both phases for each 
determination. This means that a total of 12 concentrations must be 
reported.
    (vi) The weight of the test substance, the volume of each phase 
employed in each test vessel, and the total calculated amount of test 
substance present in each phase after equilibration.
    (vii) The calculated values of the P and the mean should be reported 
for each set of test conditions as should the mean for all 
determinations. If there is a suggestion of concentration dependency of 
the P, this should be noted in the report.
    (viii) The standard deviation of individual P values about their 
mean should be reported.
    (ix) The mean P from all determinations should also be expressed as 
its logarithm (base 10).
    (f) References. For additional background information on this test 
guideline, the following references should be consulted. These 
references are available from the TSCA Nonconfidential Information 
Center, Rm. NE-B607, Environmental Protection Agency, 401 M St., SW., 
Washington, DC, 12 noon to 4 p.m., Monday through Friday, excluding 
legal holidays.
    (1) Neely, W.B. et al. Partition Coefficients to Measure 
Bioconcentration Potential of Organic Chemicals in Fish. Environmental 
Science and Technology 8:1113 (1974).
    (2) Leo, A. et al. Partition Coefficients and Their Uses. Chemical 
Reviews 71:525 (1971).
    (3) Miyake, K. and H. Terada, Direct measurements of partition 
coefficients in an octanol-water system. Journal of Chromatography 
157:386 (1978).
    (4) Veith G.D. and R.T. Morris, A Rapid Method for Estimating Log P 
for Organic Chemicals, EPA-600/3-78-049 (1978).
    (5) Mirrless, M.S. et al., Direct measurement of octanol-water 
partition coefficient by high pressure liquid chromatography. Journal of 
Medicinal Chemistry 19:615 (1976).

[[Page 292]]

    (6) EPA Draft Guidance of September 8, 1978 (F-16).
    (7) Konemann H. et al. Determination of log Poct values 
of chlorosubstituted benzenes, toluenes, and anilines by high 
performance liquid chromatography on ODS silica, Journal of 
Chromatography 178:559 (1979).
    (8) Organization for Economic Cooperation and Development, 
Guidelines for The Testing of Chemicals, OECD 107, Partition Coefficient 
(n-octanol/water) (Shake Flask Method, Adopted 27 July 1995), OECD, 
Paris, France.



Sec. 799.6756  TSCA partition coefficient (n-octanol/water), generator column 

method.

    (a) Scope--(1) Applicability. This section is intended to meet the 
testing requirements of the Toxic Substances Control Act (TSCA) (15 
U.S.C. 2601).
    (2) Source. The source material used in developing this TSCA test 
guideline is the Office of Pollution Prevention, Pesticides and Toxic 
Substances (OPPTS) harmonized test guideline 830.7560 (August 1996, 
final guideline). This source is available at the address in paragraph 
(e) of this section.
    (b)(1) Purpose. (i) The measurement and estimation of the n-octanol/
water partition coefficient (Kow), has become the cornerstone 
of a myriad of structure-activity relationships (SAR) property. The 
coefficient has been used extensively for correlating structural changes 
in drugs with changes observed in biological, biochemical, or toxic 
effects. These correlations are then used to predict the effect of a new 
drug for which a Kow could be measured.
    (ii) In the study of the environmental fate of organic chemicals, 
the Kow has become a key parameter. Kow is 
correlated to water solubility, soil/sediment sorption coefficient, and 
bioconcentration and is important to SAR.
    (iii) Of the three properties that can be estimated from 
Kow, water solubility is the most important because it 
affects both the fate and transport of chemicals. For example, highly 
soluble chemicals become quickly distributed by the hydrologic cycle, 
have low-sorption coefficients for soils and sediments, and tend to be 
more easily degraded by microorganisms. In addition, chemical 
transformation processes such as hydrolysis, direct photolysis, and 
indirect photolysis (oxidation) tend to occur more readily if a compound 
is soluble.
    (iv) Direct correlations between Kow and both the soil/
sediment sorption coefficient and the bioconcentration factor are to be 
expected. In these cases, compounds that are more soluble in n-octanol 
(more hydrophobic and lipophilic) would be expected to partition out of 
the water and into the organic portion of soils/sediments and into 
lipophilic tissue. The relationship between Kow and the 
bioconcentration factor, are the principal means of estimating 
bioconcentration factors. This relationship is discussed in the 
reference listed in paragraph (e)(14) of this section. These factors are 
then used to predict the potential for a chemical to accumulate in 
living tissue.
    (v) This section describes a method for determining the 
Kow based on the dynamic coupled column liquid 
chromatographic (DCCLC) technique, a technique commonly referred to as 
the generator column method. The method described herein can be used in 
place of the standard shake-flask method specified in Sec. 799.6755 for 
compounds with a log10Kow greater than 1.0.
    (2) Definitions. The following definitions apply to this section.
    Extractor column is used to extract the solute from the aqueous 
solution produced by the generator column. After extraction onto a 
bonded chromatographic support, the solute is eluted with a solvent/
water mixture and subsequently analyzed by high-performance liquid 
chromatography (HPLC), gas chromatography (GC), or any other analytical 
procedure. A detailed description of the preparation of the extractor 
column is given in paragraph (c)(1)(i) of this section.
    Generator column is used to partition the test substance between the 
n-octanol and water phases. The column in figure 1 in paragraph 
(c)(1)(i)(A)(2) of this section is packed with a solid support and is 
coated with the test substance at a fixed concentration in n-octanol. 
The test substance is eluted from the column with water and the aqueous 
solution leaving the column

[[Page 293]]

represents the equilibrium concentration of the test substance that has 
partitioned from the n-octanol phase into the water phase. Preparation 
of the generator column is described in paragraph (c)(1)(i) of this 
section.
    n-Octanol/water partition coefficient (Kow) is defined as 
the ratio of the molar concentrations of a chemical in n-octanol and 
water, in dilute solution. The coefficient Kow is a constant 
for a given chemical at a given temperature. Since Kow is the 
ratio of two molar concentrations, it is a dimensionless quantity. 
Sometimes Kow is reported as the decadic logarithm 
(log10Kow). In this equation, Coctanol 
and Cwater are the molar concentration of the solute in n-
octanol and water, respectively, at a given temperature. This test 
procedure determines Kow at 25 0.05 
[deg]C. The mathematical statement of Kow is:

                               Equation 1:
[GRAPHIC] [TIFF OMITTED] TR15DE00.041

    Response factor (RF) is the solute concentration required to give a 
one unit area chromatographic peak or one unit output from the HPLC 
recording integrator at a particular recorder and detector attenuation. 
The factor is required to convert from units of area to units of 
concentration. The determination of the RF is given in paragraph 
(c)(3)(iii)(C)(2) of this section.
    Sample loop is a \1/16\ inch (in) outside diameter (O.D.) (1.6 
millimeter (mm)) stainless steel tube with an internal volume between 20 
and 50 [micro]L. The loop is attached to the sample injection valve of 
the HPLC and is used to inject standard solutions into the mobile phase 
of the HPLC when determining the RF for the recording integrator. The 
exact volume of the loop must be determined as described in paragraph 
(c)(3)(iii)(C)(1) of this section when the HPLC method is used.
    (3) Principle of the test method. (i) This test method is based on 
the DCCLC technique for determining the aqueous solubility of organic 
compounds. The development of this test method is described in the 
references listed in paragraphs (e)(6), (e)(12), and (e)(19) of this 
section. The DCCLC technique utilizes a generator column, extractor 
column, and HPLC coupled or interconnected to provide a continuous 
closed-flow system. Aqueous solutions of the test compound are produced 
by pumping water through the generator column that is packed with a 
solid support coated with an approximately 1.0% weight/weight (w/w) 
solution of the compound in n-octanol. The aqueous solution leaving the 
column represents the equilibrium concentration of the test chemical 
which has partitioned from the n-octanol phase into the water phase. The 
compound is extracted from the aqueous solution onto an extractor 
column, then eluted from the extractor column with a solvent/water 
mixture and subsequently analyzed by HPLC using a variable wavelength 
ultraviolet (UV) absorption detector operating at a suitable wavelength. 
Chromatogram peaks are recorded and integrated using a recording 
integrator. The concentration of the compound in the effluent from the 
generator column is determined from the mass of the compound (solute) 
extracted from a measured volume of water (solvent). The Kow 
is calculated from the ratio of the molar concentration of the solute in 
the 1.0% (w/w) n-octanol and molar concentration of the solute in water 
as determined using the generator column technique.
    (ii) Since the HPLC method is only applicable to compounds that 
absorb in the UV, an alternate GC method, or any other reliable 
quantitative procedure must be used for those compounds that do not 
absorb in the UV. In the GC method the saturated solutions produced in 
the generator column are extracted using an appropriate organic solvent 
that is subsequently injected into the GC, or any other suitable 
analytical device, for analysis of the test compound.
    (4) Reference chemicals. (i) Columns 2, 3, 4, and 5 of table 1 in 
paragraph (b)(4)(ii) of this section list the experimental values of the 
decadic logarithm of the n-octanol/water partition coefficient 
(log10Kow) at 25 [deg]C for a number of organic 
chemicals as obtained from the scientific literature. These values were 
obtained by any one of the following experimental methods: Shake-flask; 
generator column; reverse-phase HPLC; or reverse-phase thin-layer 
chromatography, as indicated in the footnotes

[[Page 294]]

following each literature citation. The estimation method of Hawker and 
Connell as described in paragraph (e)(8) of this section, correlates 
log10Kow with the total surface area of the 
molecule and was used to estimate log10Kow for 
biphenyl and the chlorinated biphenyls. These estimated values are 
listed in column 7 of table 1 in paragraph (b)(4)(ii) of this section. 
Recommended values of log10Kow were obtained by 
critically analyzing the available experimental and estimated values and 
averaging the best data. These recommended values are listed in column 8 
of table 1 in paragraph (b)(4)(ii) of this section.
    (ii) The recommended values listed in table 1 of this section have 
been provided primarily so that the generator column method can be 
calibrated and to allow the chemical laboratory the opportunity to 
compare its results with these values. The testing laboratory has the 
option of choosing its reference chemicals, but references must be given 
to establish the validity of the measured values of 
log10Kow.

                                Table 1--n-Octanol/Water Partition Coefficient at 25 [deg]C for Some Reference Compounds
--------------------------------------------------------------------------------------------------------------------------------------------------------
                                                                                 Experimental log10Kow             Estimated log10Kow
                                                                    ------------------------------------------------------------------
                                                                                                                              Hawker      Recommended
                              Chemical                                Hansch and   Generator   Banerjee    Other    Hansch      and         log10Kow
                                                                       Leo \1\       Column       \2\     values    and Leo   Connell
                                                                                     Method                           \3\       \4\
--------------------------------------------------------------------------------------------------------------------------------------------------------
Ethyl acetate......................................................   0.73, 0.66    \5\0.68       --        --       0.671      --         \17\0.685
1-Butanol..........................................................  0.88, 0.89,    \5\0.785      --        --       0.823      --         \23\0.852
                                                                      0.32, 0.88
1-Pentanol.........................................................   1.28, 1.40    \5\1,53       --        --       1.35       --          \17\1.39
Nitrobenzene.......................................................  1.85, 1.88,    \5\1.85      1.83     \6\1.82    1.89       --          \17\1.84
                                                                         1.79
Benzene............................................................   2.15, 2.13       --        2.12       --       2.14       --          \17\2.14
Trichloroethylene..................................................      2.29       \5\2.53      2.42       --       2.27       --          \17\2.38
Chlorobenzene......................................................   2.84, 2.46    \7\2.98       --      \8\2.84    2.86       --          \18\2.80
o-Dichlorobenzene..................................................      3.38       \7\3.38      3.40     \8\3.38    3.57       --          \17\3.42
n-Propylbenzene....................................................  3.66, 3.66,    \5\3.69       --        --       3.85       --          \17\3.69
                                                                      3.68, 3.57
Biphenyl...........................................................  3.95, 4.17,    \7\3.67,     4.04     \6\3.75    4.03      4.09         \17\3.96
                                                                      4.09, 4.04    \9\3.89,
                                                                                    \10\3.79
2-Chlorobiphenyl...................................................       --        \7\4.50,      --     \10\3.90     --       4.99         \19\4.49
                                                                                    \9\4.38                  ,
                                                                                                         \11\3.75
                                                                                                             ,
                                                                                                         \12\4.59
                                                                                                             ,
                                                                                                         \13\4.54
1,2,3,5-Tetrachlorobenzene.........................................       --        \7\4.65      4.46       --       4.99       --          \17\4.70
2,2'-Dichlorobiphenyl..............................................       --        \9\4.90       --     \9\4.90,     --       4.65         \20\4.80
                                                                                                         \10\3.63
                                                                                                             ,
                                                                                                         \11\3.55
                                                                                                             ,
                                                                                                         \14\4.51
                                                                                                             ,
                                                                                                         \15\5.02
Pentachlorobenzene.................................................       --        \7\5.03      4.94       --       5.71       --          \24\4.99
2,4,5-Trichlorobiphenyl............................................       --        \7\5.51,      --     \10\5.67     --       5.60         \17\5.70
                                                                                    \9\5.81                  ,
                                                                                                         \10\5.86
                                                                                                             ,
                                                                                                         \15\5.77
2,3,4,5-Tetrachlorobiphenyl........................................       --        \4\6.18,      --        --        --       6.04         \17\5.98
                                                                                    \7\5.72
2,2',4,5,5'-Pentachlorobi-phenyl...................................      6.11       \9\6.50,      --     \13\6.11     --       6.38         \17\6.31
                                                                                    \7\5.92                  ,
                                                                                                         \12\6.85
2,2',3,3',6,6'-Hexachloro-biphenyl.................................       --        \4\5.76,      --        --        --       6.22         \17\6.36
                                                                                    \7\6.63,
                                                                                    \9\6.81
2,2',3,3',4,4',6-Heptachlorobiphenyl...............................       --        \7\6.68       --        --        --       7.11         \17\6.90
2,2',3,3',5,5',6,6'-Octachlorobiphenyl.............................       --        \7\7.11,      --     \12\8.42     --       7.24         \21\7.16
                                                                                    \9\7.14
2,2',3,3',4, 4',5,6,6'-Nona-chlorobiphenyl.........................       --        \4\7.52       --        --        --       7.74         \17\7.63
2,2',3,3',4, 5,5'6,6'-Nona-chlorobiphenyl..........................       --        \7\8.16       --        --        --       7.71         \17\7.94
Decachlorobiphenyl.................................................       --        \7\8.26,      --     \12\9.60     --       8.18        \22\8.21
                                                                                    \9\8.20
--------------------------------------------------------------------------------------------------------------------------------------------------------
\1\ Hansch and Leo (1979). Shake-flask method in paragraph (e)(8) of this section.
\2\ Banerjee, Yalkowski, and Valvani (1980). Shake-flask method in paragraph (e)(1) of this section.
\3\ Hansch and Leo (1984). Estimates log10Kow using the CLogP3 computer program in paragraph (e)(9) of this section.
\4\ Hawker and Connell (1988). Generator column method and an estimation method correlating log10Kow with the total surface area of the molecule in
  paragraph (e)(8) of this section.
\5\ Tewari et al. (1982). Generator column method in paragraph (e)(14) of this section.

[[Page 295]]

 
\6\ Veith, Austin, and Morris (1979). Reverse-phase HPLC method in paragraph (e)(16) of this section.
\7\ Miller et al. (1984). Generator column method in paragraph (e)(11) of this section.
\8\ Chiou and Schmedding (1982). Shake-flask method in paragraph (e)(4) of this section.
\9\ Woodburn, Doucette, and Andren (1984). Generator column method in paragraph (e)(19) of this section.
\10\ Rapaport and Eisenreich (1984). Reverse-phase HPLC method in paragraph (e)(13) of this section.
\11\ Woodburn (1982). Reverse-phase HPLC method in paragraph (e)(18) of this section.
\12\ Bruggemann, Van der Steen, and Hutzinger (1978). Shake-flask method in paragraph (e)(2) of this section.
\13\ Tulp and Hutzinger (1978). Shake-flask method in paragraph (e)(15) of this section.
\14\ Chiou, Porter, and Schmedding (1983). Shake-flask method in paragraph (e)(5) of this section.
\15\ Bruggemann, Van Der Steen , and Hutzinger (1982). Reverse-phase thin-layer chromatography in paragraph (e)(2) of this section.
\16\ Chiou et al. (1977). Shake-flask method in paragraph (e)(3) of this section.
\17\ Average value using all the data.
\18\ Average value using all the data except the datum point 2.46.
\19\ Average value using all the data except the data points 3.90 and 3.75.
\20\ Average value using all the data except the data points 3.63 and 3.55.
\21\ Average value using all the data except the datum point 8.42.
\22\ Average value using all the data except the datum point 9.60.
\23\ Average value using all the data except the datum point 0.32.
\24\ Average value using all the data excluding the estimated datum point 5.71.

    (5) Applicability and specificity. The test guideline is designed to 
determine the Kow of solid or liquid organic chemicals in the 
range log10Kow 1.0 to <=6.0 (10 to <=10\6\).
    (c) Test procedure--(1) Test conditions--(i) Special laboratory 
equipment--(A)(1) Generator column. Either of two different methods for 
connecting to the generator column shall be used depending on whether 
the eluted aqueous phase is analyzed by HPLC (Procedure A, as described 
in paragraph (c)(3)(iii) of this section) or by solvent extraction 
followed by GC analysis, or any other reliable method of solvent extract 
(Procedure B, as described in paragraph (c)(3)(iv) of this section).
    (2)(i) The design of the generator column is shown in the following 
figure 1:
[GRAPHIC] [TIFF OMITTED] TR15DE00.042

    (ii) The column consists of a 6 mm (\1/4\ in) O.D. pyrex tube joined 
to a short enlarged section of 9 mm pyrex tubing which in turn is 
connected to another section of 6 mm (\1/4\ in) O.D. pyrex tubing. 
Connections to the inlet teflon

[[Page 296]]

tubing (\1/8\ in O.D.) and to the outlet stainless steel tubing (\1/16\ 
in O.D.) are made by means of stainless steel fittings with teflon 
ferrules. The column is enclosed in a water jacket for temperature 
control as shown in the following figure 2:

Figure 2--Setup Showing Generator Column Enclosed in a Water Jacket and 
         Overall Arrangement of the Apparatus Used in GC Method
[GRAPHIC] [TIFF OMITTED] TR15DE00.043

    (B) Constant temperature bath with circulation pump-bath and capable 
of controlling temperature to 25 0.05 [deg]C. 
(Procedures A and B, as described in paragraphs (c)(3)(iii) and 
(c)(3)(iv) of this section, respectively).
    (C) HPLC equipped with a variable wavelength UV absorption detector 
operating at a suitable wavelength and a recording integrator (Procedure 
A, as described in paragraph (c)(3)(iii) of this section).
    (D) Extractor column--6.6 x 0.6 centimeter (cm) stainless steel tube 
with end fittings containing 5 micron frits filled with a superficially 
porous phase packing (such as Bondapack C18 Corasil: Waters 
Associates) (Procedure A, as described in paragraph (c)(3)(iii) of this 
section).
    (E) Two 6-port high-pressure rotary switching valves (Procedure A, 
as described in paragraph (c)(3)(iii) of this section).
    (F) Collection vessel--8 x \3/4\ in section of pyrex tubing with a 
flat bottom connected to a short section of \3/8\ in O.D. borosilicate 
glass tubing. The collecting vessel is sealed with a \3/8\ in teflon cap 
fitting (Procedure B, as described in paragraph (c)(3)(iv) of this 
section).
    (G) GC, or any other reliable analytic equipment, equipped with a 
detector sensitive to the solute of interest (Procedure B, as described 
in paragraph (c)(3)(iv) of this section).
    (ii) Purity of n-octanol and water. Purified n-octanol, described in 
paragraph (c)(2)(i) of this section, and water meeting appropriate 
American Society for Testing and Materials Type II standards, or an 
equivalent grade, are recommended to minimize the effects of dissolved 
salts and other impurities. An ASTM Type II water standard is presented 
in the reference listed in paragraph (e)(20) of this section).
    (iii) Purity of solvents. It is important that all solvents used in 
this method be reagent or HPLC grade and contain no impurities which 
could interfere with the determination of the test compound.
    (iv) Reference compounds. In order to ensure that the HPLC system is 
working properly, at least two of the reference compounds listed in 
table 1 in paragraph (b)(4)(ii) of this section should be run. Reference 
compounds shall be reagent or HPLC grade to avoid interference by 
impurities.
    (2) Preparation of reagents and solutions--(i) n-Octanol and water. 
Very pure n-octanol can be obtained as follows: Wash pure n-octanol 
(minimum 98% pure) sequentially with 0.1N H2SO4, with 0.1N 
NaOH, then with distilled water until neutral. Dry the n-octanol with 
magnesium sulfate and distill twice in a good distillation column under 
reduced pressure [b.p. about 80 [deg]C at 0.27 kPa (2 torr)]. The n-
octanol produced should be at least 99.9% pure. Alternatively, a grade 
equivalent to Fisher Scientific Co. No. A-402 ``Certified Octanol-1'' 
can be used. Reagent-grade water shall be used throughout the test 
procedure, such as ASTM Type II

[[Page 297]]

water, or an equivalent grade, as described in paragraph (c)(1)(ii) of 
this section.
    (ii) Presaturated water. Prepare presaturated water with n-octanol 
to minimize the depletion of n-octanol from the column when measuring 
the Kowof a test chemical. This is very important when the 
test chemical is lipophilic and the log10Kow <=4.
    (3) Performance of the test. Initially, an approximately 1.0% (w/w) 
solution of the test substance in n-octanol is prepared. Precise 
measurement of the solute concentration in this solution is required for 
the Kowcalculation. Subsequently, the 1.0% (w/w) solution is 
coated on the generator column and using either Procedure A or Procedure 
B as described in paragraphs (c)(3)(iii) and (c)(3)(iv) of this section, 
the molar concentration of the test substance in reagent-grade water is 
determined.
    (i) Test solution. The test solution consists of an approximately 
1.0% (w/w) solution of the test substance in n-octanol. A sufficient 
quantity (about 10-20 milliliter (mL)) of the test solution should be 
prepared to coat the generator column. The solution is prepared by 
accurately weighing out, using a tared bottle, quantities of both the 
test substance and n-octanol required to make a 1.0% (w/w) solution. 
When the weights are measured precisely (to the nearest 0.1 milligram 
(mg)), knowing the density of n-octanol (0.827 gram (g)/mL at 25 
[deg]C), then the molar concentration of the test substance in the n-
octanol is sufficiently accurate for the purposes of the test procedure. 
If desired, however, a separate analytical determination (e.g., by GC, 
or any other reliable analytical method) may be used to check the 
concentration in the test solution. If storage is required, the test 
solution should be kept stoppered to prevent volatilization of the test 
chemical.
    (ii) Test procedures. Prior to the determination of the 
Kow of the test chemical, two procedures shall be followed:
    (A) The saturated aqueous solution leaving the generator column 
shall be tested for the presence of an emulsion, using a Tyndall 
procedure (i.e. light scattering). If colloids are present, they must be 
removed prior to injection into the extractor column by lowering the 
flow rate of water.
    (B) The efficiency of removal of the solute (the test chemical) by 
solvent extraction from the extractor column shall be determined and 
used in the determination of the Kow of the test chemical.
    (iii) Procedure A--HPLC method. (A) Procedure A covers the 
determination of the aqueous solubility of compounds which absorb in the 
UV. Two reciprocating piston pumps deliver the mobile phase (water or 
solvent/water mixture) through two 6-port high-pressure rotary valves 
and a 30x0.6 cm C18 analytical column to a UV absorption 
detector operating at a suitable wavelength. Chromatogram peaks are 
recorded and integrated with a recording integrator. One of the 6-port 
valves is the sample injection valve used for injecting samples of 
standard solutions of the solute in an appropriate concentration for 
determining RFs or standard solutions of basic chromate for determining 
the sample-loop volume. The other 6-port valve in the system serves as a 
switching valve for the extractor column which is used to remove solute 
from the aqueous solutions. The HPLC analytical system is shown 
schematically in the following figure 3:

        Figure 3--Schematic of HPLC--Generator Column Flow System

[[Page 298]]

[GRAPHIC] [TIFF OMITTED] TR15DE00.044

    (B) The general procedure for analyzing the aqueous phase after 
equilibration is as follows; a detailed procedure is given in paragraph 
(c)(3)(iii)(C)(4) of this section:
    (1) Direct the aqueous solution from the generator column to 
``Waste'' in figure 3 in paragraph (c)(3)(iii)(A) of this section with 
the switching valve in the inject position in order to equilibrate 
internal surfaces with the solution, thus insuring that the analyzed 
sample would not be depleted by solute adsorption on surfaces upstream 
from the valve.
    (2) At the same time, water is pumped from the HPLC pumps in order 
to displace the solvent from the extractor column.
    (3) The switching valve is next changed to the load position to 
divert a sample of the solution from the generator column through the 
extractor column, and the liquid leaving the extractor column is 
collected in a tared weighing bottle. During this extraction step, the 
HPLC mobile phase is changed to a solvent/water mixture to condition the 
analytical column.
    (4) After the desired volume of sample is extracted, the switching 
valve is returned to the inject position for elution from the extractor 
column and analysis. Assuming that all of the solute was adsorbed by the 
extractor column during the extraction step, the chromatographic peak 
represents all of the solute in the extracted sample, provided that the 
extraction efficiency is 100%. If the extraction efficiency is less than 
100%, then the extraction efficiency shall be measured and used to 
determine the actual amount of the solute extracted.
    (5) The solute concentration in the aqueous phase is calculated from 
the peak area, the weight of the extracted liquid collected in the 
weighing bottle, the extraction efficiency, and the RF.
    (C)(1) Determination of the sample-loop volume. Accurate measurement 
of the sample loop may be accomplished by using a spectrophotometric 
method such as the one described in the reference listed in paragraph 
(e)(6) of this section. For this method, measure absorbance, 
Aloop, at 373 nanometers (nm) for at least three solutions, 
each of which is prepared by collecting from the sample valve an 
appropriate number, n, of loopfuls of an aqueous stock solution of 
K2CrO4 (1.3% by weight) and diluting to 50 mL with 
0.2% KOH. (For a 20 [micro]L loop, use n = 5; for a 50 [micro]L loop, 
use n = 2.) Also measure the absorbance, Astock, of the same 
stock solution after diluting 1:500 with 0.2% KOH. Calculate the loop 
volume to the nearest 0.1 [micro]L using the relation:

                               Equation 2:
[GRAPHIC] [TIFF OMITTED] TR15DE00.045

    (2) Determination of the RF. (i) For all determinations adjust the 
mobile phase solvent/water ratio and flow rate to obtain a reasonable 
retention time on the HPLC column. For example, typical concentrations 
of organic solvent in the mobile phase range from 50

[[Page 299]]

to 100% while flow rates range from 1 to 3 mL/minutes (min); these 
conditions often give a 3 to 5 min retention time.
    (ii) Prepare standard solutions of known concentrations of the 
solute in a suitable solvent. Concentrations must give a recorder 
response within the maximum response of the detector. Inject samples of 
each standard solution into the HPLC system using the calibrated sample 
loop. Obtain an average peak area from at least three injections of each 
standard sample at a set detector absorbance unit full scale (AUFS), 
i.e., at the same absorbance scale attenuation setting.
    (iii) Calculate the RF from the following equation:

                               Equation 3:
[GRAPHIC] [TIFF OMITTED] TR15DE00.046

    (3) Loading of the generator column. (i) The design of the generator 
column was described in paragraph (c)(1)(i) of this section and is shown 
in figure 1 in paragraph (c)(1)(i)(A)(2)(i) of this section. To pack the 
column, a plug of silanized glass wool is inserted into one end of the 6 
mm pyrex tubing. Silanized diatomaceous silica support (about 0.5g of 
100-120 mesh Chromosorb W chromatographic support material) is poured 
into the tube with tapping and retained with a second plug of silanized 
glass wool.
    (ii) The column is loaded by pulling the test solution through the 
dry support with gentle suction and then allowing the excess solution to 
drain out. After loading the column, draw water up through the column to 
remove any entrapped air.
    (4) Analysis of the solute. Use the following procedure to collect 
and analyze the solute:
    (i) With the switching valve in figure 3 in paragraph (c)(3)(iii)(A) 
of this section in the inject position (i.e., water to waste), pump 
water through the generator column at a flow rate of approximately 1 mL/
min for approximately 15 min to bring the system into equilibrium. Pump 
water to the generator column by means of a minipump or pressurized 
water reservoir as shown in the following figure 4:

                 Figure 4--Water Reservoir for GC Method


    [GRAPHIC] [TIFF OMITTED] TR15DE00.047
    
    (ii) Flush out the organic solvent that remains in the system from 
previous runs by changing the mobile phase to 100% H2O and 
allowing the water to reach the HPLC detector, as indicated by a 
negative reading. As soon as this occurs, place a 25 mL weighing bottle 
(weighed to the nearest mg) at the waste position and immediately turn 
the switching valve to the load position.
    (iii) Collect an amount of water from the generator column (as 
determined

[[Page 300]]

by trial and error) in the weighing bottle, corresponding to the amount 
of solute adsorbed by the extractor column that gives a reasonable 
detector response. During this extraction step, switch back to the 
original HPLC mobile phase composition, i.e., solvent/water mixture, to 
condition the HPLC analytical column.
    (iv) After the desired volume of sample has been extracted, turn the 
switching valve back to the inject position in figure 3 in paragraph 
(c)(3)(iii)(A) of this section. As soon as the switching valve is turned 
to the inject position, remove the weighing bottle, cap it and replace 
it with the waste container; at the same time turn on the recording 
integrator. The solvent/water mobile phase will elute the solute from 
the extractor column and transfer the solute to the HPLC analytical 
column.
    (v) Determine the weight of water collected to the nearest mg and 
record the corresponding peak area. Using the same AUFS setting repeat 
the analysis of the solute at least two more times and determine the 
average ratio of peak area to grams of water collected. In this 
equation, S = solubility (M), RF = response factor, Vloop = 
sample-loop volume (L), and R = ratio of area to grams of water. 
Calculate the solute solubility in water using the following equation:

                               Equation 4:
[GRAPHIC] [TIFF OMITTED] TR15DE00.048

    (iv) Procedure B--GC Method. In the GC method, or any other reliable 
quantitative method, aqueous solutions from the generator column enter a 
collecting vessel in figure 2 in paragraph (c)(1)(i)(A)(2)(ii) of this 
section containing a known weight of extracting solvent which is 
immiscible in water. The outlet of the generator column is positioned 
such that the aqueous phase always enters below the extracting solvent. 
After the aqueous phase is collected, the collecting vessel is stoppered 
and the quantity of aqueous phase is determined by weighing. The solvent 
and the aqueous phase are equilibrated by slowly rotating the collecting 
vessel. A small amount of the extracting solvent is then removed and 
injected into a GC equipped with an appropriate detector. The solute 
concentration in the aqueous phase is determined from a calibration 
curve constructed using known concentrations of the solute. The 
extraction efficiency of the solvent shall be determined in a separate 
set of experiments.
    (A) Determination of calibration curve. (1) Prepare solute standard 
solutions of concentrations covering the expected range of the solute 
solubility. Select a column and optimum GC operating conditions for 
resolution between the solute and solvent and the solute and extracting 
solvent. Inject a known volume of each standard solution into the 
injection port of the GC. For each standard solution determine the 
average of the ratio R of peak area to volume (in [micro]L) for the 
chromatographic peak of interest from at least three separate 
injections.
    (2) After running all the standard solutions, determine the 
coefficients, a and b, using linear regression analysis on the equation 
of concentration (C) vs. R in the form:

                               Equation 5:
[GRAPHIC] [TIFF OMITTED] TR15DE00.049

    (B) Loading of the generator column. The generator column is packed 
and loaded with solute in the same manner as for the HPLC method in 
paragraph (c)(3)(iii) of this section. As shown in figure 2 in paragraph 
(c)(1)(i)(A)(2)(ii) of this section, attach approximately 20 cm of 
straight stainless steel tubing to the bottom of the generator column. 
Connect the top of the generator column to a water reservoir in figure 4 
in paragraph (c)(3)(iii)(C)(4)(i) of this section using teflon tubing. 
Use air or nitrogen pressure (5 PSI) from an air or nitrogen cylinder to 
force water from the reservoir through the column. Collect water in an 
Erlenmeyer flask for approximately 15 min while the solute concentration 
in water equilibrates; longer time may be required for less soluble 
compounds.
    (C) Collection and extraction of the solute. During the 
equilibration time, add a known weight of extracting solvent to a 
collection vessel which can be capped. The extracting solvent should

[[Page 301]]

cover the bottom of the collection vessel to a depth sufficient to 
submerge the collecting tube but still maintain 100:1 water/solvent 
ratio. Record the weight (to the nearest mg) of a collection vessel with 
cap and extracting solvent. Place the collection vessel under the 
generator column so that water from the collecting tube enters below the 
level of the extracting solvent in figure 2 in paragraph 
(c)(1)(i)(A)(2)(ii) of this section. When the collection vessel is 
filled, remove it from under the generator column, replace cap, and 
weigh the filled vessel. Determine the weight of water collected. Before 
analyzing for the solute, gently rotate the collection vessel contents 
for approximately 30 min, controlling the rate of rotation so as not to 
form an emulsion; rotating the flask end over end five times per minute 
is sufficient. The extraction efficiency of the solvent shall be 
determined in a separate set of experiments.
    (D) Analysis of the solute. (1) After rotating, allow the collection 
vessel to stand for approximately 30 min; then remove a known volume of 
the extracting solvent from the vessel using a microliter syringe and 
inject it into the GC. Record the ratio of peak area to volume injected 
and, from the regression equation of the calibration line, determine the 
concentration of solute in the extracting solvent. If the extraction 
efficiency is not 100%, the measured extraction efficiency shall be used 
to obtain the correct concentration of solute extracted. In this 
equation, Ces is the molar concentration of solute in 
extracting solvent, dH2O and des are 
the densities in grams per milliliter of water and extracting solvent, 
respectively, and ges and gH2O are the 
grams of extracting solvent and water, respectively, contained in the 
collection vessels. The molar concentration of solute in water C(M) is 
determined from the following equation:

                               Equation 6:
[GRAPHIC] [TIFF OMITTED] TR15DE00.050

    (2) Make replicate injections from each collecting vessel to 
determine the average solute concentration in water for each vessel. To 
make sure the generator column has reached equilibrium, run at least two 
additional (for a total of three) collection vessels and analyze the 
extracted solute as described in paragraph (c)(3)(iv)(D)(1) of this 
section. Calculate C(M) from the average solute concentration in the 
three vessels.
    (3) If another analytical method is used in place of the GC, then 
Procedure B, as described in paragraph (c)(3)(iv) of this section, shall 
be modified and the new analytical procedure shall be used to determine 
quantitatively the amount of solute extracted in the extraction solvent.
    (v) Analysis of reference compounds. Prior to analyzing the test 
solution, make duplicate runs on at least two of the reference compounds 
listed in table 1 in paragraph (b)(4)(ii) of this section. When using 
the reference compounds, follow the same procedure previously described 
for preparing the test solution and running the test. If the average 
value obtained for each compound is within 0.1 log unit of the reference 
value, then the test procedure and HPLC system are functioning properly; 
if not a thorough checking over of the HPLC and careful adherence to the 
test procedures should be done to correct the discrepancy.
    (vi) Modification of procedures for potential problems--
Decomposition of the test compound. If the test compound decomposes in 
one or more of the aqueous solvents required during the period of the 
test at a rate such that an accurate value for water solubility cannot 
be obtained, then it will be necessary to carry out detailed 
transformation studies, such as hydrolysis studies. If decomposition is 
due to aqueous photolysis, then it will be necessary to carry out the 
studies in the dark, under red or yellow lights, or by any other 
suitable method to eliminate this transformation process.
    (d) Data and reporting--(1) Test report. (i) For the test solution, 
report the weights to the nearest 0.1 mg of the test substance and n-
octanol. Also report the weight percent and molar concentration of the 
test substance in the n-octanol; the density of n-octanol at 25 [deg]C 
is 0.827 grams per milliliter (gm)/mL.
    (ii) For each run provide the molar concentration of the test 
substance in

[[Page 302]]

water for each of three determinations, the mean value, and the standard 
deviation.
    (iii) For each of the three determinations calculate the 
Kow as the ratio of the molar concentration of the test 
substance in n-octanol to the molar concentration in water. Also 
calculate and report the mean Kow and its standard deviation. 
Values of Kow shall be reported as their logarithms 
(log10Kow).
    (iv) Report the temperature (0.05 [deg]C) at 
which the generator column was controlled during the test.
    (v) For each reference compound report the individual values of 
log10Kow and the average of the two runs.
    (vi) For compounds that decompose at a rate such that a precise 
value for the solubility cannot be obtained, provide a statement to that 
effect.
    (2) Specific analytical, calibration, and recovery procedures. (i) 
For the HPLC method describe and/or report:
    (A) The method used to determine the sample-loop volume and the 
average and standard deviation of that volume.
    (B) The average and standard deviation of the RF.
    (C) The extraction solvent and the extraction efficiency used.
    (D) Any changes made or problems encountered in the test procedures.
    (ii) For the GC method report:
    (A) The column and GC operating conditions of temperature and flow 
rate.
    (B) The average and standard deviation of the average area per 
microliter obtained for each of the standard solutions.
    (C) The form of the regression equation obtained in the calibration 
procedure.
    (D) The extracting solvent and extraction efficiency used.
    (E) The average and standard deviation of solute concentration in 
each collection vessel.
    (F) Any changes made or problems encountered in the test procedure.
    (iii) If another approved analytical method is used to determine the 
concentration of the test chemical in water, then all the important test 
conditions shall be reported.
    (iv) If the concentration of the test substance in n-octanol is 
determined by an independent analytical method such as GC, provide a 
complete description of the method.
    (e) References. For additional background information on this test 
guideline, the following references should be consulted. These 
references are available from the TSCA Nonconfidential Information 
Center, Rm. NE-B607, Environmental Protection Agency, 401 M St., SW., 
Washington, DC, 12 noon to 4 p.m., Monday through Friday, excluding 
legal holidays.
    (1) Banerjee, S. et al., Water solubility and octanol/water 
partition coefficient of organics. Limitation of the solubility-
partition coefficient correlation. Environmental Science and Technology 
14:1227-1229 (1980).
    (2) Bruggemann W.A. et al., Reversed-phase thin-layer chromatography 
of polynuclear aromatic hydrocarbons and chlorinated biphenyls. 
Relationship with hydrophobicity as measured by aqueous solubility and 
octanol/water partition coefficient. Journal of Chromatography 238: 335-
346 (1982).
    (3) Chiou, C.T. et al. Partition coefficient and bioaccumulation of 
selected organic chemicals. Environmental Science and Technology 11:475-
478 (1977).
    (4) Chiou, C.T. and Schmedding, D.W., Partitioning of organic 
compounds in octanol/water systems. Environmental Science and Technology 
16:4-10 (1982).
    (5) Chiou, C.T et al., Partition equilibria of nonionic organic 
compounds between soil, organic matter, and water. Environmental Science 
and Technology 17:227-231 (1983).
    (6) DeVoe, H. et al. ``Generator Columns and High Pressure Liquid 
Chromatography for Determining Aqueous Solubilities and Octanol-Water 
Partition Coefficients of Hydrophobic Substances,'' Journal of Research 
of the National Bureau of Standards, 86:361-366 (1981).
    (7) Fujita, T. et al. ``A New Substituent Constant, Derived from 
Partition Coefficients.'' Journal of the American Chemical Society, 
86:5175 (1964).
    (8) Hansch, C. and Leo, A. 1985 MEDCHEM Project, version 26. Pomona 
College, Claremont, CA. USA.

[[Page 303]]

    (9) Hansch, C. and Leo, A. Medchem Software Manual. CLOGP3 Users 
Guide. Release 3.32. December 1984. Medicinal Chemistry Project, Pomona 
College, Claremont, CA.
    (10) Hawker, D.W. and Connell, D.W. Octanol-water partition 
coefficients of polychlorinated biphenyl congeners. Environmental 
Science and Technology 22:382-387 (1988).
    (11) May, W.E. et al. ``Determination of the aqueous solubility of 
polynuclear aromatic hydrocarbons by a coupled column liquid 
chromatographic technique,'' Analytical Chemistry, 50:175-179 (1978).
    (12) May, W.E. et al. ``Determination of the Solubility Behavior of 
Some Polycyclic Aromatic Hydrocarbons in Water,'' Analytical Chemistry 
50:997-1000 (1978).
    (13) Miller, M.M. et al. Aqueous solubilities, octanol/water 
partition coefficients and entropies of melting of chlorinated benzenes 
and biphenyls. Journal of Chemical and Engineering Data 29:184-190 
(1984).
    (14) Neely, W.B. et al. Partition Coefficient to Measure 
Bioconcentration Potential of Organic Chemicals in Fish, Environmental 
Science Technology, 8:113-115 (1974).
    (15) Rappaport, R.A. and Eisenrich, S.J. Chromatographic 
determination of octanol-water partition coefficients (Kow's) 
for 58 polychlorinated biphenyl congeners. Environmental Science and 
Technology 18:163-170 (1984).
    (16) Tewari, Y.B. et al. Aqueous solubility and octanol/water 
partition coefficients of organic compounds at 25 [deg]C. Journal of 
Chemical and Engineering Data 27:451-454 (1982).
    (17) Tulp, M.T.M. and Hutzinger, O. Some thoughts on aqueous 
solubilities and partition coefficients of PCB, and the mathematical 
correlation between bioaccumulation and physio-chemical properties. 
Chemosphere 10:849-860 (1978).
    (18) Veith, G.D. et al. A rapid method for estimating 
log10 P for organic chemicals, Water Research 13:43-47 
(1979).
    (19) Wasik, S.P. et al. Octanol/water partition coefficient and 
aqueous solubilities of organic compounds, Report NBSIR 81-2406 (1981). 
National Bureau of Standards, U.S. Department of Commerce, Washington, 
DC.
    (20) Woodburn, K.B. Measurement and application of the octanol/water 
partition coefficients for selected polychlorinated biphenyls. Master's 
Thesis (1982), University of Wisconsin at Madison, Madison, WI.
    (21) Woodburn, K.B. et al. Generator column determination of 
octanol/water partition coefficients for selected polychlorinated 
biphenyl congeners. Environmental Science and Technology 18:457-459 
(1984).
    (22) ASTM D 1193-91 (Approved Sep 15, 1991), ``Standard 
Specification for Reagent Water.'' American Society for Testing and 
Materials (ASTM), 1916 Race St., Philadelphia, PA 19103.



Sec. 799.6784  TSCA water solubility: Column elution method; shake flask 

method.

    (a) Scope--(1) Applicability. This section is intended to meet the 
testing requirements of the Toxic Substances Control Act (TSCA) (15 
U.S.C. 2601).
    (2) Source. The source material used in developing this TSCA test 
guideline is the Office of Pollution Prevention, Pesticides and Toxics 
(OPPTS) harmonized test guideline 830.7840 (March 1998, revised final 
guideline). This source is available at the address in paragraph (f) of 
this section.
    (b) Introductory information--(1) Prerequisites. Suitable analytical 
method, structural formula, vapor pressure curve, dissociation constant, 
and hydrolysis independence of pH (preliminary test).
    (2) Coefficient of variation. The coefficient of variation on the 
mean values reported by the participants of the Organization for 
Economic Cooperation and Development (OECD) Laboratory Intercomparison 
Testing, Part I, 1979, appeared to be dependent on the chemicals tested 
and the test temperatures; it ranges from 0.05 to 0.34 for the column 
elution method, and from 0.03 to 1.12 for the flask method.
    (3) Qualifying statements. (i) The method is not applicable to 
volatile substances. Care should be taken that the substances examined 
are as pure as possible and stable in water. It must be ascertained that 
the identity of the

[[Page 304]]

substance is not changed during the procedure.
    (ii) The column elution method is not suitable for volatile 
substances. The carrier material used here may not yet be optimal. This 
method is intended for material with solubilities below approximately 
10-2 gram/Liter (g/L).
    (iii) The flask method is intended for materials with solubility 
above 10-2 g/L. It is not applicable to volatile substances; 
this method may pose difficulties in the case of surface-active 
materials.
    (c) Method--(1) Introduction, purpose, scope, relevance, 
application, and limits of test. (i) A solution is a homogeneous mixture 
of different substances in a solvent. The particle sizes of the 
dispersed substances are of the same magnitude as molecules and ions; 
therefore, the smallest volumes which can be obtained from a solution 
are always of uniform composition.
    (ii) Solubility in water is a significant parameter because:
    (A) The spatial and temporal movement (mobility) of a substance is 
largely determined by its solubility in water.
    (B) Water soluble substances gain ready access to humans and other 
living organisms.
    (C) The knowledge of the solubility in water is a prerequisite for 
testing biological degradation and bioaccumulation in water and for 
other tests.
    (iii) No single method is available to cover the whole range of 
solubilities in water, from relatively soluble to very low-soluble 
chemicals. A general test guideline for the determination of the 
solubility in water must include methods which cover the whole range of 
water soluble substances. Therefore, this section includes two methods:
    (A) One which applies to substances with low solubilities 
(<10-2 g/L), referred to as the ``column elution method.''
    (B) The other which applies to substances with higher solubilities 
(<=10-2 g/L), referred to as the ``flask method.''
    (2) Definition. The solubility in water of a substance is specified 
by the saturation mass concentration of the substance in water and is a 
function of temperature. The solubility in water is specified in units 
of weight per volume of solution. The SI-unit is killogram/meter (kg/
m)\3\; g/L may also be used.
    (3) Reference substances. The reference substances need not be 
employed in all cases when investigating a new substance. They are 
provided primarily so that calibration of the method may be performed 
from time to time and to offer the chance to compare the results when 
another method is applied. The values presented in table 1 of this 
section are not necessarily representative of the results which can be 
obtained with this test method as they have been derived from an earlier 
version of the test method.

                                     Table 1--Data for Reference Substances
----------------------------------------------------------------------------------------------------------------
                                                   T,     Mean (milligram                                 No. of
                     Method                      [deg]C       (mg)/L)               Range (mg/L)           labs
----------------------------------------------------------------------------------------------------------------
Fluoranthene
        Elution method.........................    15          0.275               0.104 to 0.920            6
                                                   25          0.373               0.198 to 1.050            7
Hexachlorobenzene
        Elution method.........................    15       9.21 x 10-3       2.06 x 10-3 to 2.16 x 10-2     6
                                                   25       9.96 x 10-3       1.19 x 10-3 to 2.31 x 10-2     7
[gamma]-Hexachlorocyclohexane
        Elution method.........................    15           6.50                4.43 to 10.5             6
                                                   25           9.20                6.64 to 14.5             7
2,4-Dichlorophenoxyacetic acid
        Flask method...........................    15          0.633               0.380 to 0.764            5
                                                   25          0.812               0.655 to 0.927            5
Mercury(II) chloride:
        Flask method...........................    15           53.0                47.7 to 56.5             4
                                                   25           66.4                58.3 to 70.4             4
4-Nitrophenol:
        Flask method...........................    15           9.95                8.88 to 10.9             6
                                                   25           14.8                13.8 to 15.9             6
----------------------------------------------------------------------------------------------------------------


[[Page 305]]

    (4) Principle of the test methods. The approximate amount of the 
sample and the time necessary to achieve the saturation mass 
concentration should be determined in a simple preliminary test.
    (i) Column elution method. This method is based on the elution of a 
test substance with water from a microcolumn which is charged with an 
inert carrier material such as glass beads, silica gel, or sand, and an 
excess of test substance. The water solubility is determined when the 
mass concentration of the eluate is constant. This is shown by a 
concentration plateau as a function of time in the following figure 1:

     Figure 1--Concentration versus Time of Substance in the Eluate
[GRAPHIC] [TIFF OMITTED] TR15DE00.051

    (ii) Flask method. In this method, the substance (solids must be 
pulverized) is dissolved in water at a temperature somewhat above the 
test temperature. When saturation is achieved, the mixture is cooled and 
kept at the test temperature, stirring as long as necessary to reach 
equilibrium. Such a procedure is described in the reference listed in 
paragraph (f)(2) of this section. Subsequently, the mass concentration 
of the substance in the aqueous solution, which must not contain any 
undissolved particles, is determined by a suitable analytical method.
    (5) Quality criteria--(i) Repeatability. For the column elution 
method <30% is acceptable; for the flask method <15% should be observed.
    (ii) Sensitivity. This depends upon the method of analysis, but mass 
concentration determinations down to at least 10-6 g/L can be 
determined.
    (iii) Specificity. These methods should only be applied to:
    (A) Pure substance.
    (B) Substances that are stable in water.
    (C) Slightly soluble substances, i.e. <10-2 g/L for the 
column elution method.
    (D) Organic substances for the column elution method.
    (iv) Possibility of standardization. These methods can be 
standardized.
    (d) Description of the test procedures--(1) Preparations--(i) 
Apparatus--(A) Column elution method. (1) The schematic arrangement of 
the system is presented in the following figure 2:

[[Page 306]]

                  Figure 2--Schematic Test Arrangement
[GRAPHIC] [TIFF OMITTED] TR15DE00.052

    (2) Although any size is acceptable, provided it meets the criteria 
for reproducibility and sensitivity. The column should provide for a 
head space of at least five bed-volumes of water and a minimum of five 
samples. Alternatively, the size can be reduced if make-up solvent is 
employed to replace the initial five bed-volumes removed with 
impurities. A suitable microcolumn is shown in the following figure 3:

[[Page 307]]

          Figure 3--Microcolumn (all dimensions in millimeters)
[GRAPHIC] [TIFF OMITTED] TR15DE00.053

    (3) The column should be connected to a recycling pump capable of 
controlling flows of approximately 25 mL/hours (h). The pump is 
connected with polytetrafluoroethylene and/or glass connections. The 
column and pump, when assembled, should have provision for sampling the 
effluent and equilibrating the head space at atmospheric pressure. The 
column material is supported with a small (5 millimeter (mm)) plug of 
glass wool, which must also serve to filter particles.
    (B) Flask method. For the flask method, the following material is 
needed:
    (1) Normal laboratory glassware and instrumentation.
    (2) A device suitable for the agitation of solutions under 
controlled constant temperatures.
    (3) A centrifuge (preferably thermostatted), if required with 
emulsions.
    (4) Equipment for analytical determinations.
    (2) Reagents. The substance to be tested should be as pure as 
possible, particularly in the flask method where purification is not 
provided. The carrier material for the column elution

[[Page 308]]

method should be inert. Possible materials which can be employed are 
glass beads and silica. A suitable volatile solvent of analytical 
reaction quality should be used to apply the test substance to the 
carrier material. Double distilled water from glass or quartz apparatus 
should be employed as the eluent or solvent. Water directly from an ion 
exchanger must not be used.
    (3) Test conditions. The test is preferably run at 20  0.5 [deg]C (293 [deg]K). If temperature dependence is 
suspected in the solubility (<= 3%/ [deg]C), two other temperatures 
should also be used--both differing from each other and the initially 
chosen temperature by 10 [deg]C. In this case the temperature control 
should be  0.1 [deg]C. One of these additional 
temperatures should be below the initial temperature. The chosen 
temperature(s) should be kept constant in all parts of the equipment 
(including the leveling vessel).
    (4) Performance of the tests--(i) Preliminary test. (A) To 
approximately 0.1 g of the sample (solid substances must be pulverized) 
in a glass-stoppered 10 milliliter (mL) graduated cylinder, increasing 
volumes of distilled water at room temperature are added according to 
the steps shown in Table 2 of this section:

                                      Table 2--Determination of Solubility
----------------------------------------------------------------------------------------------------------------
                 Solubility data                    step 1   step 2  step 3  step 4  step 5  step 6     step 7
----------------------------------------------------------------------------------------------------------------
Total volume H2O added (mL)......................     0.1      0.5      1       2      10      100      <=100
Approximate solubility (g/L).....................   <=1,000    200     100     50      10       1         <1
----------------------------------------------------------------------------------------------------------------

    (B) After each addition of water to give the indicated total volume, 
the mixture is shaken vigorously for 10 min and is visually checked for 
any undissolved parts of the sample. If, after a total of 10 mL of water 
has been added (step 5), the sample or parts of it remain undissolved, 
the contents of the measuring cylinder is transferred to a 100 mL 
measuring cylinder which is then filled up with water to 100 mL (step 6) 
and shaken. At lower solubilities the time required to dissolve a 
substance can be considerably long (24 h should be allowed). The 
approximate solubility is given in the table under that volume of added 
water in which complete dissolution of the sample occurs. If the 
substance is still apparently insoluble, further dilution should be 
undertaken to ascertain whether the column elution or flask solubility 
method should be used.
    (ii) Column elution--(A) Apparatus. (1) The equipment is arranged as 
shown in figures 2 and 3 in paragraphs (d)(1)(i)(A)(1) and 
(d)(1)(i)(A)(2) of this section. Approximately 600 milligrams (mg) of 
carrier material is weighed and transferred to a 50 mL round-bottom 
flask. A suitable, weighed amount of test substance is dissolved in the 
chosen solvent, and an appropriate amount of the test substance solution 
is added to the carrier material. The solvent must be completely 
evaporated, e.g. in a rotary evaporator; otherwise water saturation of 
the carrier is not achieved due to partition effects on the surface of 
the carrier.
    (2) The loading of carrier material may cause problems (erroneous 
results) if the test substance is deposited as an oil or a different 
crystal phase. The problem should be examined experimentally.
    (3) The loaded carrier material is allowed to soak for about 2 h in 
approximately 5 mL of water, and then the suspension is added to the 
microcolumn. Alternatively, dry loaded carrier material may be poured in 
the microcolumn, which has been filled with water and then equilibrated 
for approximately 2 h.
    (B) Test procedure. The elution of the substance from the carrier 
material can be carried out in two different ways: Leveling vessel or 
circulating pump. The two principles should be used alternatively.
    (1) Leveling vessel, see figure 3 in paragraph (d)(1)(i)(A)(2) and 
figure 4 in paragraph (d)(4)(iii) of this section.
    (i) The connection to the leveling vessel is made by using a ground 
glass joint which is connected by teflon tubing. It is recommended that 
a flow rate

[[Page 309]]

of approximately 25 mL/h be used. Successive eluate fractions should be 
collected and analyzed by the chosen method.
    (ii) Fractions from the middle eluate range where the concentrations 
are constant (30%) in at least five consecutive 
fractions are used to determine the solubility in water.
    (iii) A second run is to be performed at half the flow rate of the 
first. If the results of the two runs are in agreement, the test is 
satisfactory; if there is a higher apparent solubility with the lower 
flow rate, then the halving of the flow rate must continue until two 
successive runs give the same solubility.
    (2) Circulating pump, see figures 2 and 3 in paragraphs 
(d)(1)(i)(A)(1) and (d)(1)(i)(A)(2) of this section.
    (i) With this apparatus, the microcolumn must be modified. A 
stopcock with 2-way action must be used, see figure 3 in paragraph 
(d)(1)(i)(A)(2) of this section). The circulating pump can be, e.g. a 
peristaltic pump (be careful that no contamination and/or adsorption 
occurs with the tube material) or a membrane pump.
    (ii) The flow through the column is started. It is recommended that 
a flow rate of approximately 25 mL/h be used (approximately 10 bed 
volumes per h for the described column). The first five-bed volumes 
(minimum) are discarded to remove water soluble impurities.
    (iii) Following this, the recycling pump is connected and the 
apparatus allowed to run until equilibration is established, as defined 
by five successive samples whose concentrations do not differ by more 
than 30% in a random fashion (see paragraph (f)(2) of this section). 
These samples should be separated from each other by time intervals 
corresponding to the passage of at least 10 bed-volumes of the eluent.
    (3) In both cases (using a circulation pump or a leveling vessel) 
the fractions should be checked for the presence of colloidal matter by 
examination for the Tyndall effect (light scattering). Presence of such 
particles invalidates the results, and the test should be repeated with 
improvements in the filtering action of the column. The pH of each 
sample should be recorded. A second run should be performed at the same 
temperature.
    (iii) Flask method: Test procedure. The quantity of material 
necessary to saturate the desired volume of water is estimated from the 
preliminary test. The volume of water required will depend on the 
analytical method and the solubility range. About five times the 
quantity of material determined in paragraph (d)(4)(i)(A) of this 
section is weighed into each of three glass vessels fitted with glass 
stoppers (e.g. centrifuge tubes, flasks). The chosen volume of water is 
added to each vessel, and the vessels are tightly stoppered. The closed 
vessels are then agitated at 30 [deg]C. (A shaking or stirring device 
capable of operating at constant temperature should be used, e.g. 
magnetic stirring in a thermostatically controlled water bath.) After 1 
day, one of the vessels is removed and re-equilibrated for 24 h at the 
test temperature with occasional shaking. The contents of the vessel are 
then centrifuged at the test temperature, and the concentration of 
compound in the clear aqueous phase is determined by a suitable 
analytical method. The other two flasks are treated similarly after 
initial equilibration at 30 [deg]C for 2 and 3 days, respectively. If 
the concentration results from at least the last two vessels agree with 
the required reproducibility, the test is satisfactory. The whole test 
should be repeated, using longer equilibration times if the results from 
vessels one, two, and three show a tendency to increasing values. The 
arrangement of the apparatus is shown in the following figure 4:

[[Page 310]]

Figure 4--Test Arrangement for the Determination of Solubility in Water 
         of Slightly Soluble, Low Volatility Organic Substances
[GRAPHIC] [TIFF OMITTED] TR15DE00.054


1 = Leveling vessel (e.g. 2.5 L chemical flask)

2 = Column (see figure 3 in paragraph (d)(1)(i)(A)(2) of this section)

3 = Fraction accumulator

4 = Thermostat

5 = Teflon tubing

6 = Glass stopper

7 = Water line (between thermostat and column, inner diameter: 
approximately 8 mm)
    (iv) Analysis. A substance-specific analytical method is required 
for these determinations, since small amounts of soluble impurities can 
cause large errors in the measured solubility. Examples of such methods 
are gas or liquid chromatography, titration methods, photometric 
methods, and polarographic methods.
    (e) Data and reporting--(1) Column elution method--(i) Treatment of 
results. The mean value from at least five consecutive samples taken 
from the saturation plateau (figure 1 in paragraph (c)(4)(i) of this 
section) should be determined for each run, as should the standard 
deviation. A comparison should be made between the two means to ensure 
that they agree with a repeatability of less than 30%.
    (ii) Test report. The report should contain an indication of the 
results of the preliminary test plus the following information:
    (A) The individual concentrations, flow rates and pHs of each 
samples.
    (B) The means and standard deviations from at least five samples 
from the saturation plateau of each run.
    (C) The average of the two successive, acceptable runs.
    (D) The temperature of the runs.
    (E) The method of analysis employed.
    (F) The nature of the carrier material employed.
    (G) Loading of carrier material.
    (H) Solvent used.

[[Page 311]]

    (I) Statement that the identity of the substance in the saturated 
solution has been proved.
    (2) Flask method--(i) Treatment of results. The individual results 
should be given for each of the three flasks and those results deemed to 
be constant (repeatability <15%) should be averaged and given in units 
of mass per volume of solution. This may require the conversion of mass 
units to volume units, using the density when the solubility is very 
high (100 g/L).
    (ii) Test report. The report should include the following 
information:
    (A) The individual analytical determinations and the average where 
more than one value was determined for each flask.
    (B) The average of the value for the different flasks which were in 
agreement.
    (C) The test temperature.
    (D) The analytical method employed.
    (f) References. For additional information on this test guideline, 
the following references should be consulted. These references are 
available from the TSCA Nonconfidential Information Center, Rm. NE-B607, 
Environmental Protection Agency, 401 M St., SW., Washington, DC, 12 noon 
to 4 p.m., Monday through Friday, excluding legal holidays.
    (1) Veith, G.D. and V.M. Comstock. Apparatus for continuously 
saturating water with hydrophobic organic chemicals. Journal of the 
Fishing Research Board of Canada 32:1849-1851 (1975).
    (2) Organization for Economic Cooperation and Development, 
Guidelines for The Testing of Chemicals, OECD 105, Water Solubility 
(Column Elution Method--Shake Flask Method), OECD, Paris, France (1981).



Sec. 799.6786  TSCA water solubility: Generator column method.

    (a) Scope--(1) Applicability. This section is intended to meet the 
testing requirements of the Toxic Substances Control Act (TSCA) (15 
U.S.C. 2601).
    (2) Source. The source material used in developing this TSCA test 
guideline is the Office of Pollution Prevention, Pesticides and Toxics 
(OPPTS) harmonized test guideline 830.7860 (March 1998, revised final 
guideline). The source is available at the address in paragraph (e) of 
this section.
    (b) Introduction--(1) Purpose. (i) The water solubility of a 
chemical is defined as the equilibrium concentration of the chemical in 
a saturated aqueous solution at a given temperature and pressure. The 
aqueous phase solubility is an important factor in governing the 
movement, distribution, and rate of degradation of chemicals in the 
environment. Substances that are relatively water soluble are more 
likely to be widely distributed by the hydrologic cycle than those which 
are relatively insoluble. Furthermore, substances with higher water 
solubility are more likely to undergo microbial or chemical degradation 
in the environment because dissolution makes them ``available'' to 
interact and, therefore, react with other chemicals and microorganisms. 
Both the extent and rate of degradation via hydrolysis, photolysis, 
oxidation, reduction, and biodegradation depend on a chemical being 
soluble in water (i.e., homogeneous kinetics).
    (ii) Water provides the medium in which many organisms live, and 
water is a major component of the internal environment of all living 
organisms (except for dormant stages of certain life forms). Even 
organisms which are adapted to life in a gaseous environment require 
water for normal functioning. Water is thus the medium through which 
most other chemicals are transported to and into living cells. As a 
result, the extent to which chemicals dissolve in water will be a major 
determinant for movement through the environment and entry into living 
systems.
    (iii) The water solubility of a chemical also has an effect on its 
sorption into and desorption from soils and sediments, and on 
volatilization from aqueous media. The more soluble a chemical substance 
is, the less likely it is to sorb to soils and sediments and the less 
likely it is to volatilize from water. Finally, the design of most 
chemical tests and many ecological and health tests requires precise 
knowledge of the water solubility of the chemical to be tested.
    (2) Definitions. The following definitions apply to this section.

[[Page 312]]

    Concentration (C) of a solution is the amount of solute in a given 
amount of solvent or solution and can be expressed as a weight/weight or 
weight/volume relationship. The conversion from a weight relationship to 
one of volume incorporates density as a factor. For dilute aqueous 
solutions, the density of the solvent is approximately equal to the 
density of the solution; thus, concentrations expressed in milligrams 
per liter (mg/L) are approximately equal to 10-3 g/10\3\ g or 
parts per million (ppm); those expressed in micrograms per liter 
([micro]g/L) are approximately equal to 10-6 g/10\3\ g or 
parts per billion (ppb). In addition, concentration can be expressed in 
terms of molarity, normality, molality, and mole fraction. For example, 
to convert from weight/volume to molarity molecular mass is incorporated 
as a factor.
    Density is the mass of a unit volume of a material. It is a function 
of temperature, hence the temperature at which it is measured should be 
specified. For a solid, it is the density of the impermeable portion 
rather than the bulk density. For solids and liquids, suitable units of 
measurement are grams per cubic centimeter (g/cm\3\). The density of a 
solution is the mass of a unit volume of the solution and suitable units 
of measurement are g/cm\3\.
    Extractor column is used to extract the solute from the saturated 
solutions produced by the generator column. After extraction onto a 
chromatographic support, the solute is eluted with a solvent/water 
mixture and subsequently analyzed by high-pressure liquid chromatography 
(HPLC), gas chromatography (GC), or any other suitable analytical 
procedure. A detailed description of the preparation of the extractor 
column is given in paragraph (c)(1)(i)(D) of this section.
    Generator column is used to produce or generate saturated solutions 
of a solute in a solvent. The column, see figure 1 in paragraph 
(c)(1)(i)(A) of this section, is packed with a solid support coated with 
the solute, i.e., the organic compound whose solubility is to be 
determined. When water (the solvent) is pumped through the column, 
saturated solutions of the solute are generated. Preparation of the 
generator column is described in paragraph (c)(1)(i)(A) of this section.
    Response factor (RF) is the solute concentration required to give a 
1 unit area chromatographic peak or 1 unit output from the HPLC 
recording integrator at a particular recorder attenuation. The factor is 
required to convert from units of area to units of concentration. The 
determination of the RF is given in paragraph (c)(3)(ii)(B)(2) of this 
section.
    Sample loop is a \1/16\ inch (in) outer diameter (O.D.) (1.6 
millimeter (mm)) stainless steel tube with an internal volume between 20 
and 50 [micro]L. The loop is attached to the sample injection valve of 
the HPLC and is used to inject standard solutions into the mobile phase 
of the HPLC when determining the RF for the recording integrator. The 
exact volume of the loop must be determined as described in paragraph 
(c)(3)(ii)(B)(1) of this section when the HPLC method is used.
    Saturated solution is a solution in which the dissolved solute is in 
equilibrium with an excess of undissolved solute; or a solution in 
equilibrium such that at a fixed temperature and pressure, the 
concentration of the solute in the solution is at its maximum value and 
will not change even in the presence of an excess of solute.
    Solution is a homogeneous mixture of two or more substances 
constituting a single phase.
    (3) Principle of the test method. (i) This test method is based on 
the dynamic coupled column liquid chromatographic (DCCLC) technique for 
determining the aqueous solubility of organic compounds that was 
initially developed by May et al. (as described in the references listed 
in paragraphs (e)(5) and (e)(6) of this section), modified by DeVoe et 
al. (as described in the reference listed in paragraph (e)(1) of this 
section), and finalized by Wasik et al. (as described in the reference 
listed in paragraph (e)(11) of this section). The DCCLC technique 
utilizes a generator column, extractor column and HPLC coupled or 
interconnected to provide a continuous closed flow system. Saturated 
aqueous solutions of the test compound are produced by pumping water 
through the generator column that is packed with a

[[Page 313]]

solid support coated with the compound. The compound is extracted from 
the saturated solution onto an extractor column, then eluted from the 
extractor column with a solvent/water mixture and subsequently analyzed 
by HPLC using a variable wavelength ultraviolet (UV) detector operating 
at a suitable wavelength. Chromatogram peaks are recorded and integrated 
using a recording integrator. The concentration of the compound in the 
effluent from the generator column, i.e., the water solubility of the 
compound, is determined from the mass of the compound (solute) extracted 
from a measured volume of water (solvent).
    (ii) Since the HPLC method is only applicable to compounds that 
absorb in the UV, an alternate GC method, or any other reliable 
procedure (which must be approved by OPPTS), can be used for those 
compounds that do not absorb in the UV. In the GC method the saturated 
solutions produced in the generator column are extracted using an 
appropriate organic solvent that is subsequently injected into the GC, 
or any other suitable analytical device, for analysis of the test 
compound.
    (4) Reference chemicals. Table 1 of this section lists the water 
solubilities at 25 [deg]C for a number of reference chemicals as 
obtained from the scientific literature. The data from Wasik et al. (as 
described in the reference listed in paragraph (e)(11) of this section), 
Miller et al. and Tewari et al. (as described in the references listed 
in paragraphs (e)(7) and (e)(10) of this section, respectively) were 
obtained from the generator column method. The water solubilities data 
were also obtained from Mackay et al. and Yalkowski et al. (as described 
in the references listed in paragraphs (e)(4) and (e)(12) of this 
section, respectively) and other scientists by the conventional shake 
flask method. These data have been provided primarily so that the 
generator column method can be calibrated from time to time and to allow 
the chemical testing laboratory an opportunity to compare its results 
with those listed in table 1 of this section. The water solubility 
values at 25 [deg]C reported by Yalkowski et al. are their preferred 
values and, in general, represent the best available water solubility 
data at 25 [deg]C. The testing laboratory has the option of choosing its 
own reference chemicals, but references must be given to establish the 
validity of the measured values of the water solubility.

                      Table 1--Water Solubilities at 25 [deg]C of Some Reference Chemicals
----------------------------------------------------------------------------------------------------------------
                                                                   Water solubility (ppm at 25 [deg]C)
                                                        --------------------------------------------------------
                   Reference chemical                     Wasik (generator                      Other literature
                                                           column method)    Yalkowski\1\ \5\      references
----------------------------------------------------------------------------------------------------------------
2-Heptanone............................................       \2\4080              4300             \5\4330
1-Chlorobutane.........................................        \2\873             872.9              \7\666
Ethylbenzene...........................................        \2\187              208               \7\162
1,2,3-Trimethylbenzene.................................       \2\65.5              75.2             \7\48.2
Biphenyl...............................................     \3\ \10\6.71           7.48             \8\6.62
Phenanthrene...........................................       \4\1.002            1.212                --
2,4,6-Trichlorobiphenyl................................    \3\ \10\0.226          0.225             \8\0.119
2,3,4,5-Tetrachlorobiphenyl............................    \3\ \10\0.0209        0.01396           \8\0.0192
Hexachlorobenzene......................................          --              0.004669          \9\0.00996
2,3,4,5,6-Pentachlorobiphenyl..........................   \3\ \10\0.00548        0.004016          \8\0.0068
----------------------------------------------------------------------------------------------------------------
\1\ Preferred water solubility at 25 [deg]C by Yalkowski et al. (1990) in paragraph (e)(12) of this section
  based on a critical review of all the experimental water solubility data published.
\2\ Tewari et al. (1982) in paragraph (e)(10) of this section.
\3\ Leifer et al. (1983) in paragraph (e)(3) of this section.
\4\ May, Wasik, and Freeman (1978, 1978a) in paragraphs (e)(5) and (6) of this section.
\5\ Yalkowski et al. (1990) in paragraph (e)(12) of this section.
\6\ Hansch et al. (1968) in paragraph (e)(2) of this section.
\7\ Sutton and Calder (1975) in paragraph (e)(9) of this section.
\8\ Mackay et al. (1980) in paragraph (e)(4) of this section.
\9\ The elution chromatographic method from Organization for Economic Cooperation and Development (OECD) (1981)
  in paragraph (e)(8) of this section.
\10\ Miller et al. (1984) in paragraph (e)(7) of this section.


[[Page 314]]

    (5) Applicability and specificity. (i) Procedures are described in 
this section to determine the water solubility for liquid or solid 
compounds. The water solubility can be determined in very pure water, 
buffer solution for compounds that reversibly ionize or protonate, or in 
artificial seawater as a function of temperature (i.e., in the range of 
temperatures of environmental concern). This section is not applicable 
to the water solubility of gases.
    (ii) This section is designed to determine the water solubility of a 
solid or liquid test chemical in the range of 1 ppb to 5,000 ppm. For 
chemicals whose solubility is below 1 ppb, the water solubility should 
be characterized as ``less than 1 ppb'' with no further quantification. 
For solubilities greater than 5,000 ppm, the shake flask method should 
be used, see paragraph (e)(15) of this section.
    (c) Test procedure--(1) Test conditions--(i) Special laboratory 
equipment--(A) Generator column. (1) Either of two different designs 
shall be used depending on whether the eluted aqueous phase is analyzed 
by HPLC in paragraph (c)(3)(ii) of this section or by solvent extraction 
followed by GC (or any other reliable quantitative) analysis of solvent 
extract in paragraph (c)(3)(iv) of this section. The design of the 
generator column is shown in the following figure 1:
                       Figure 1--Generator Column
[GRAPHIC] [TIFF OMITTED] TR15DE00.055

    (2) The column consists of a 6 mm (\1/4\ in) O.D. pyrex tube joined 
to a short enlarged section of 9 mm pyrex tubing which in turn is 
connected to another section of 6 mm (\1/4\ in) O.D. pyrex tubing. 
Connections to the inlet teflon tubing (\1/8\ in O.D.) and to the outlet 
stainless steel tubing (\1/16\ in O.D.) shall be made by means of 
stainless steel fittings with teflon ferrules. The column

[[Page 315]]

is enclosed in a water jacket for temperature control as shown in the 
following figure 2:
Figure 2--Setup Showing Generator Column Enclosed in a Water Jacket and 
       Overall Arrangement of the Apparatus Used in the GC Method
[GRAPHIC] [TIFF OMITTED] TR15DE00.056

    (B) Constant temperature bath with circulation pump-bath and capable 
of controlling temperature to 0.05 [deg]C, see 
paragraph (c)(3) of this section.
    (C) HPLC equipped with a variable wavelenth UV absorption detector 
operating at a suitable wavelength and a recording integrator in 
paragraph (c)(3)(ii) of this section.
    (D) Extractor column--6.6 x 0.6 cm stainless steel tube with end 
fittings containing 5 [micro]m frits filled with a superficially porous 
phase packing (Bondapack C18/Corasil: Waters Associates) in paragraph 
(c)(3)(ii) of this section.
    (E) Two 6-port high-pressure rotary switching valves in paragraph 
(c)(3)(ii) of this section.
    (F) Collection vessel--8 x \3/4\ in section of pyrex tubing with a 
flat bottom connected to a short section of \3/8\ in O.D. borosilicate 
glass tubing in figure 2 in paragraph (c)(1)(i)(A)(2) of this section. 
The collecting vessel is sealed with a \3/8\ in teflon cap fitting in 
paragraph (c)(3)(iii) of this section.
    (G) GC, or any other reliable analytical equipment, which has a 
detector sensitive to the solute of interest in paragraph (c)(3)(iii) of 
this section.
    (ii) Purity of water. Water meeting appropriate American Society for 
Testing and Materials (ASTM) Type II standards, or an equivalent grade, 
are recommended to minimize the effects of dissolved salts and other 
impurities on water solubility. ASTM Type II water is presented in the 
reference listed in paragraph (e)(13) of this section.
    (iii) Purity of solvents. All solvents used in this method must be 
reagent or HPLC grade. Solvents must contain no impurities which could 
interfere with the determination of the test compound.
    (iv) Seawater. When the water solubility in seawater is desired, the 
artificial seawater described in paragraph (c)(2)(ii) of this section 
must be used.
    (v) Effect of pH on solubility. For chemicals that reversibly ionize 
or protonate with a pKa or pKb between 3 and 11, 
experiments must be performed at pH's 5.0, 7.0, and 9.0 using 
appropriate buffers.
    (2) Preparation of reagents and solutions--(i) Buffer solutions. 
Prepare buffer solutions as follows:
    (A) pH 3.0--to 250 mL of 0.10M potassium hydrogen phosphate add 111 
mL of 0.10 M hydrochloric acid; adjust the final volume to 500 mL with 
reagent grade water.
    (B) pH 5.0--to 250 mL of 0.1M potassium hydrogen phthalate add 113 
mL of 0.1M sodium hydroxide; adjust the final volume to 500 mL with 
reagent grade water.
    (C) pH 7.0--to 250 mL of 0.1M potassium dihydrogen phosphate add 145 
mL of 0.1M sodium hydroxide; adjust the final volume to 500 mL with 
reagent grade water.
    (D) pH 9.0--to 250 mL of 0.075M borax add 69 mL of 0.1M HCl; adjust 
the final volume to 500 mL with reagent grade water.
    (E) pH 11.0--to 250 mL of 0.05 M sodium bicarbonate add 3 mL of 0.10 
M

[[Page 316]]

sodium hydroxide; adjust the final volume to 500 mL with reagent grade 
water.
    (ii) Check the pH of each buffer solution with a pH meter at 25 
[deg]C and adjust to pH 5.0, 7.0, or 9.0, if necessary. If the pH of the 
solution has changed by 0.2 pH units or more after 
the addition of the test compound, then a more concentrated buffer is 
required for that pH determination. The sponsor should then choose a 
more suitable buffer.
    (iii) Artificial seawater. Add the reagent-grade chemicals listed in 
table 2 of this section in the specified amounts and order to 890 mL of 
reagent-grade water. Each chemical shall be dissolved before another one 
is added.

             Table 2--Constituents of Artificial Seawater\1\
------------------------------------------------------------------------
                       Chemical                              Amount
------------------------------------------------------------------------
NaF..................................................               3 mg
SrCl2.6H2O...........................................              20 mg
H3BO3................................................              30 mg
KBr..................................................             100 mg
KCl..................................................             700 mg
CaCl2.2H2O...........................................      1.47 gram (g)
Na2SO4...............................................             4.00 g
MgCl2.6H2O...........................................            10.78 g
NaCl.................................................            23.50 g
Na2SiO3.9H2O.........................................              20 mg
NaHCO3...............................................            200 mg
------------------------------------------------------------------------
\1\ If the resulting solution is diluted to 1 L, the salinity should be
  34 0.5 g/kilogram (kg) and the pH 8.0 0.2. The desired test salinity is attained by dilution
  at time of use.

    (3) Performance of the test. Using either the procedures in 
paragraph (c)(3)(ii) or (c)(3)(iii) of this section, determine the water 
solubility of the test compound at 25 [deg]C in reagent-grade water or 
buffer solution, as appropriate. Under certain circumstances, it may be 
necessary to determine the water solubility of a test compound at 25 
[deg]C in artificial seawater. The water solubility can also be 
determined at other temperatures of environmental concern by adjusting 
the temperature of the water bath to the appropriate temperature.
    (i) Prior to the determination of the water solubility of the test 
chemical, two procedures shall be followed.
    (A) The saturated aqueous solution leaving the generator column must 
be tested for the presence of an emulsion, using a Tyndall procedure. If 
colloids are present, they must be eliminated prior to the injection 
into the extractor column. This may be achieved by lowering the flow 
rate of the water.
    (B) The efficiency of the removal of the solute (i.e. test chemical) 
by the solvent extraction from the extraction column must be determined 
and used in the determination of the water solubility of the test 
chemical.
    (ii) Procedure A--HPLC method--(A) Scope. (1) Procedure A covers the 
determination of the aqueous solubility of compounds which absorb in the 
UV.
    (i) The HPLC analytical system is shown schematically in the 
following figure 3:

        Figure 3--Schematic of HPLC--Generator Column Flow System
[GRAPHIC] [TIFF OMITTED] TR15DE00.057


[[Page 317]]


    (ii) Two reciprocating piston pumps deliver the mobile phase (water 
or solvent/water mixture) through two 6-port high-pressure rotary valves 
and a 30 x 0.6 cm C18/Corasil analytical column to a variable wavelength 
UV absorption detector operating at a suitable wavelength; chromatogram 
peaks are recorded and integrated with a recording integrator. One of 
the 6-port valves is the sample injection valve used for injecting 
samples of standard solutions of the solute in an appropriate 
concentration for determining RFs of standard solutions of basic 
chromate for determining the sample-loop volume. The other 6-port valve 
in the system serves as a switching valve for the extractor column which 
is used to remove solute from the aqueous solutions.
    (2) The general procedure for analyzing the aqueous phase is as 
follows (a detailed procedure is given in paragraph (c)(3)(ii)(B)(4) of 
this section).
    (i) Direct the aqueous solution to ``Waste,'' see figure 3 in 
paragraph (c)(3)(ii)(A)(1)(i) of this section, with the switching valve 
in the inject position in order to equilibrate internal surfaces with 
the solution, thus ensuring that the analyzed sample would not be 
depleted by solute adsorption on surfaces upstream from the valve.
    (ii) At the same time, water is pumped from the HPLC pumps in order 
to displace the solvent from the extractor column.
    (iii) The switching valve is next changed to the load position to 
divert a sample of the solution through the extractor column, and the 
liquid leaving this column is collected in a weighing bottle. During 
this extraction step, the mobile phase is changed to a solvent/water 
mixture to condition the analytical column.
    (iv) After the desired volume of sample is extracted, the switching 
valve is returned to the inject position for elution and analysis. 
Assuming that there is no breakthrough of solute from the extractor 
column during the extraction step, the chromatographic peak represents 
all of the solute in the sample, provided that the extraction efficiency 
is 100%. If the extraction efficiency is less than 100%, then the 
extraction efficiency shall be used to determine the actual weight of 
the solute extracted.
    (v) The solute concentration in the aqueous phase is calculated from 
the peak area and the weight of the extracted liquid collected in the 
weighing bottle.
    (B) Determinations--(1) Sample-loop volume. Accurate measurement of 
the sample loop may be accomplished by using the spectrophotometric 
method of Devoe et al. under paragraph (e)(1) of this section. For this 
method measure absorbance, Aloop, at 373 nm of at least three 
solutions, each of which is prepared by collecting from the sample valve 
an appropriate number, n, of loopfuls of an aqueous stock solution of 
K2CrO4 (1.3% by weight) and diluting to 50 mL with 
0.2% KOH. (For a 20 [micro]L loop, use n = 5; for a 50 [micro]L loop, 
use n = 2.) Also measure the absorbance, Astock, of the same 
stock solution after diluting 1:500 with 0.2% KOH. Calculate the loop 
volume to the nearest 0.1 [micro]L using the equation:

                               Equation 1:
[GRAPHIC] [TIFF OMITTED] TR15DE00.059

    (2) RF. (i) For all determinations adjust the mobile phase solvent/
water ratio and flow rate to obtain a reasonable retention time on the 
HPLC column. For example, typical concentrations of solvent in the 
mobile phase range from 50 to 100% while flow rates range from 1 to 3 
mL/min; these conditions give a 3 to 5 min retention time.
    (ii) Prepare standard solutions of known concentrations of the 
solute in a suitable solvent. Concentrations must give a recorder 
response within the maximum response of the detector. Inject samples of 
each standard solution into the HPLC system using the calibrated sample 
loop. Obtain an average peak area from at least three injections of each 
standard sample at a set absorbance unit full scale (AUFS), i.e., at the 
same absorbance scale attenuation setting.
    (iii) Calculate the RF from the following equation:

                               Equation 2:

[[Page 318]]

[GRAPHIC] [TIFF OMITTED] TR15DE00.058

    (3) Loading of the generator column. (i) The design of the generator 
column was described in paragraph (c)(1)(i) of this section and is shown 
in figure 1 in paragraph (c)(1)(i)(A) of this section. To pack the 
column, a plug of silanized glass wool is inserted into one end of the 6 
mm pyrex tubing. Silanized diatomaceous silica support (about 0.5g 100-
120 mesh Chromosorb (W) chromatographic support material) is poured into 
the tube with tapping and retained with a second plug of silanized glass 
wool.
    (ii) If the solute is a liquid, the column is loaded by pulling the 
liquid solute through the dry support with gentle suction. If the solute 
is a solid, a 1% solution of the solid in a volatile solvent is added to 
the dry packing. The solvent is then distilled off the column under 
reduced pressure. After loading the column draw water up through the 
column to remove entrapped air.
    (4) Analysis of the solute. Use the following procedure to collect 
and analyze the solute.
    (i) With the switching valve (figure 3 in paragraph 
(c)(3)(ii)(A)(1)(i) of this section) in the inject position (i.e., water 
to waste), pump water through the generator column at a flow rate of 
approximately 1 mL/min for approximately 5 minutes (min) to bring the 
system into equilibrium. Pump water to the generator column by means of 
a minipump or pressurized water reservoir as shown in the following 
figure 4:
                 Figure 4--Water Reservoir for GC Method
[GRAPHIC] [TIFF OMITTED] TR15DE00.060

    (ii) Flush out the solvent that remains in the system from previous 
runs by changing the mobile phase to 100% H2O and allowing 
the water to reach the HPLC detector, as indicated by a negative 
reading. As soon as this occurs, place a 25 mL weighing bottle (weighed 
to the nearest mg) at the waste position and immediately turn the 
switching valve to the load position.
    (iii) Collect an amount of water (as determined by trial and error) 
in the weighing bottle, corresponding to the amount of solute adsorbed 
by the extractor column that gives a large on-scale detector response. 
During this extraction step, switch back to the original HPLC mobile 
phase composition, i.e., solvent/water mixture, to condition the HPLC 
analytical column.
    (iv) After the desired volume of sample has been extracted, turn the

[[Page 319]]

switching valve back to the inject position (figure 3 in paragraph 
(c)(3)(ii)(A)(1)(i) of this section); at the same time turn on the 
recording integrator. The solvent/water mobile phase will elute the 
solute from the extractor column and transfer the solute to the HPLC 
analytical column.
    (v) Remove the weighing bottle, cap it, and replace it with the 
waste container. Determine the weight of water collected to the nearest 
mg and record the corresponding peak area. Using the same AUFS setting 
repeat the analysis of the solute at least two more times and determine 
the average ratio of peak area to grams of water collected. In this 
equation, s = solubility (M), RF = response factor, Vloop = 
sample-loop volume (L), and R = ratio of area to grams of water. 
Calculate the solute solubility in water using the following equation:

                               Equation 3:
[GRAPHIC] [TIFF OMITTED] TR15DE00.061

    (iii) Procedure B--GC method--(A) Scope. In the GC method, or any 
other analytical method, aqueous solutions from the generator column 
enter a collecting vessel (figure 2 in paragraph (c)(1)(i)(A)(2) of this 
section) containing a known weight of extracting solvent which is 
immiscible in water. The outlet of the generator column is positioned 
such that the aqueous phase always enters below the extracting solvent. 
After the aqueous phase is collected, the collecting vessel is stoppered 
and the quantity of aqueous phase is determined by weighing. The solvent 
and the aqueous phase are equilibrated by slowly rotating the collecting 
vessel. The extraction efficiency of the solvent must be determined at 
this time. A small amount of the extracting solvent is removed and 
injected into a gas chromograph equipped with an appropriate detector. 
The solute concentration in the aqueous phase is determined from a 
calibration curve constructed using known concentrations of the solute.
    (B) Alternative method. If another (approved) analytical method is 
used instead of the GC, that method shall be used to determine 
quantitatively the amount of solute present in the extraction solvent.
    (C) Determinations--(1) Calibration curve. (i) Prepare solute 
standard solutions of concentrations covering the range of the solute 
solubility. Select a column and optimum GC operating conditions for 
resolution between the solute and solvent and the solute and extracting 
solvent. Inject a known volume of each standard solution into the 
injection port of the GC. For each standard solution determine the 
average of the ratio R of peak area to volume (in microliters) for three 
chromatographic peaks from three injections.
    (ii) After running all the standard solutions, determine the 
coefficients, a and b, using a linear regression equation of C vs. R in 
the following form:

                               Equation 4:
[GRAPHIC] [TIFF OMITTED] TR15DE00.062

    (iii) If another analytical method is used, the procedures described 
in paragraph (c)(3)(iii)(C)(1) of this section shall be used to 
determine quantitatively the amount of solute in the extraction solvent.
    (2) Loading of the generator column. The generator column is packed 
and loaded with solute in the same manner as for the HPLC method 
described under paragraph (c)(3)(ii)(B)(3) of this section. As shown in 
figure 2 in paragraph (c)(1)(i)(A)(2) of this section, attach 
approximately 20 cm of straight stainless steel tubing to the bottom of 
the generator column. Connect the top of the generator column to a water 
reservoir (figure 4 in paragraph (c)(3)(ii)(B)(4)(i) of this section) 
using teflon tubing. Use air or nitrogen pressure (5 PSI) from an air or 
nitrogen cylinder to force water from the reservoir through the column. 
Collect water in an Erlenmeyer flask for approximately 15 min while the 
solute concentration in water equilibrates; longer time may be required 
for less soluble compounds.
    (3) Collection and extraction of the solute. During the 
equilibration time, add a known weight of extracting solvent to a 
collection vessel which can be capped. The extracting solvent should

[[Page 320]]

cover the bottom of the collection vessel to a depth sufficient to 
submerge the collecting tube but still maintain 100:1 water/solvent 
ratio. Record the weight (to the nearest mg) of a collection vessel with 
cap and extracting solvent. Place the collection vessel under the 
generator column so that water from the collecting tube enters below the 
level of the extracting solvent (figure 2 in paragraph (c)(1)(i)(A)(2) 
of this section). When the collection vessel is filled, remove it from 
under the generator column, replace cap, and weigh the filled vessel. 
Determine the weight of water collected. Before analyzing for the 
solute, gently shake the collection vessel contents for approximately 30 
min, controlling the rate of shaking so as not to form an emulsion; 
rotating the flask end over end five times per minute is sufficient.
    (4) Analysis of the solute. (i) After shaking, allow the collection 
vessel to stand for approximately 30 min; then remove a known volume of 
the extracting solvent from the vessel using a microliter syringe and 
inject it into the GC. Record the ratio of peak area to volume injected 
and, from the regression equation of the calibration line, determine the 
concentration of solute in the extracting solvent. In this equation, 
Ces is the concentration of solute in extracting solvent (M), 
dH2O and des are the densities of water and 
extracting solvent, respectively, and ges and gH2O 
are the grams of extracting solvent and water, respectively, contained 
in the collection vessel. The concentration of solute in water C(M) is 
determined from the following equation:

                               Equation 5:
[GRAPHIC] [TIFF OMITTED] TR15DE00.063

    (ii) Make replicate injections from each collecting vessel to 
determine the average solute concentration in water for each vessel. To 
make sure the generator column has reached equilibrium, run at least two 
additional (for a total of three) collection vessels and analyze the 
extracted solute as described above. Calculate the water solubility of 
the solute from the average solute concentration in the three vessels.
    (iv) Modification of procedures for potential problems. If the test 
compound decomposes in one or more of the aqueous solvents required 
during the period of the test at a rate such that an accurate value for 
water solubility cannot be obtained, then it will be necessary to carry 
out detailed transformation studies; e.g., hydrolysis in paragraph 
(e)(16) of this section. If decomposition is due to aqueous photolysis, 
then it will be necessary to carry out water solubility studies in the 
dark, under red or yellow lights, or by any other suitable method to 
eliminate this transformation process.
    (d) Data and reporting--(1) Test report. (i) For each set of 
conditions, (e.g., temperature, pure water, buffer solution, artificial 
seawater) required for the study, provide the water solubility value for 
each of three determinations, the mean value, and the standard 
deviation.
    (ii) For compounds that decompose at a rate such that a precise 
value for the water solubility cannot be obtained, provide a statement 
to that effect.
    (iii) For compounds with water solubility below 1 ppb, report the 
value as ``less than 1 ppb.''
    (2) Specific analytical, calibration, and recovery procedures. (i) 
For the HPLC method describe and/or report:
    (A) The method used to determine the sample-loop volume and the 
average and standard deviation of that volume.
    (B) The average and standard deviation of the RF.
    (C) Any changes made or problems encountered in the test procedure.
    (ii) For the GC, or any other analytical, method report:
    (A) The column and GC operating conditions of temperature and flow 
rate, or the operating conditions of any other analytical method used.
    (B) The average and standard deviation of the average area per 
microliter obtained for each of the standard solutions.
    (C) The form of the regression equation obtained in the calibration 
procedure.

[[Page 321]]

    (D) The extracting solvent used, and its extraction efficiency.
    (E) The average and standard deviation of solute concentration in 
each collection vessel.
    (F) Any changes made or problems encountered in the test procedure.
    (G) If applicable, a complete description of the analytical method 
which was used instead of the GC method.
    (e) References. For additional information on this test guideline, 
the following references should be consulted. These references are 
available from the TSCA Nonconfidential Information Center, Rm. NE-B607, 
Environmental Protection Agency, 401 M St., SW., Washington, DC, 12 noon 
to 4 p.m., Monday through Friday, excluding legal holidays.
    (1) DeVoe, H. et al., Generator columns and high pressure liquid 
chromatography for determining aqueous solubilities and octanol-water 
partition coefficients of hydrophobic substances. Journal of Research, 
National Bureau of Standards, 86:361-366 (1981).
    (2) Hansch, C. et al., The linear free-energy relationship between 
partition coefficients, and the aqueous solubility of organic liquids. 
Journal of Organic Chemistry 33:347-350 (1968).
    (3) Leifer, A. et al., Environmental transport and transformation of 
polychlorinated biphenyls. Chapter 1. U.S. Environmental Protection 
Agency Report: EPA-560/5-83-005 (1983).
    (4) Mackay, D. et al., Relationships between aqueous solubility and 
octanol-water partition coefficient. Chemosphere 9:701-711 (1980).
    (5) May, W.E. et al., Determination of the aqueous solubility of 
polynuclear aromatic hydrocarbons by a coupled column liquid 
chromatographic technique. Analytical Chemistry 50:175-179 (1978).
    (6) May, W.E. et al. Determination of the solubility behavior of 
some polycyclic aromatic hydrocarbons in the water. Analytical 
Chemistry, 50:997-1000 (1978a).
    (7) Miller, N.M. et al., Aqueous solubilities, octanol/water 
partition coefficients, and entropy of melting of chlorinated benzenes 
and biphenyls. Journal of Chemical and Engineering Data 29:184-190 
(1984).
    (8) OECD/Organization for Economic Cooperation and Development. Test 
Guideline No. 105. Water solubility column elution-flask method (1981).
    (9) Sutton, C. and Calder, J.A., Solubility of alkylbenzenes in 
distilled water and seawater at 25 [deg]C. Journal of Chemical and 
Engineering Data 20:320-322 (1975).
    (10) Tewari, Y.B. et al., Aqueous solubility and octanol/water 
partition coefficient of organic compounds at 25 [deg]C. Journal of 
Chemical and Engineering Data 27:451-454 (1982).
    (11) Wasik, S.P. et al., Octanol/Water Partition Coefficient and 
Aqueous Solubilities of Organic Compounds. NBS Report NBSIR 81-2406. 
Washington, DC: National Bureau of Standards, U.S. Department of 
Commerce (1981).
    (12) Yalkowski, S.H. et al., ``Aquasol database of aqueous 
solubilities of organic compounds''; Fifth Edition. University of 
Arizona, College of Pharmacy, Tucson, AZ 85721 (1990) (available at 
http://www.pharm.arizona.edu/aquasol/index.html).
    (13) ASTM D 1193-91, Standard Specification for Reagent Water. 
American Society for Testing and Materials (ASTM). 1916 Race St., 
Philadelphia, PA 19103.

Subparts F-G [Reserved]



                Subpart H_Health Effects Test Guidelines

    Source: 62 FR 43824, Aug. 15, 1997, unless otherwise noted.



Sec. 799.9110  TSCA acute oral toxicity.

    (a) Scope. This section is intended to meet the testing requirements 
under section 4 of the Toxic Substances Control Act (TSCA). In the 
assessment and evaluation of the toxic characteristics of a substance, 
determination of acute oral toxicity is usually an initial step. It 
provides information on health hazards likely to arise from short-term 
exposure by the oral route. Data from an acute study may serve as a 
basis for classification and labeling. It is traditionally a step in 
establishing a dosage regimen in subchronic and other studies and may 
provide initial information

[[Page 322]]

on the mode of toxic action of a substance. An evaluation of acute 
toxicity data should include the relationship, if any, between the 
exposure of animals to the test substance and the incidence and severity 
of all abnormalities, including behavioral and clinical abnormalities, 
the reversibility of observed abnormalities, gross lesions, body weight 
changes, effects on mortality, and any other toxic effects.
    (b) Source. The source material used in developing this TSCA test 
guideline is the Office of Prevention, Pesticides, and Toxic Substances 
(OPPTS) harmonized test guideline 870.1100 (August 1998, final 
guideline). This source is available at the address in paragraph (f) of 
this section.
    (c) Definitions. The following definitions apply to this section.
    Acute oral toxicity is the adverse effects occurring within a short 
period of time after oral administration of either a single dose of a 
substance or multiple doses given within a 24-hour period.
    Dosage is a general term comprising the dose, its frequency, and the 
duration of dosing.
    Dose is the amount of test substance administered. Dose is expressed 
as weight of test substance (milligrams, grams) per unit weight of test 
animal (e.g., milligrams per kilogram).
    Dose-effect is the relationship between the dose and the magnitude 
of a defined biological effect either in an individual or in a 
population sample.
    Dose-response is the relationship between the dose and the 
proportion of a population sample showing a defined effect.
    LD50 (median lethal dose) is a statistically derived 
estimate of single dose of a substance that can be expected to cause 
death in 50% of animals when administered by the oral route. The 
LD50 value is expressed in terms of weight of test substance 
per unit weight of test animal (milligrams per kilogram).
    (d) Alternative approaches to the determination of acute toxicity. 
(1) EPA will accept the following procedures to reduce the number of 
animals used to evaluate acute effects of chemical exposure while 
preserving its ability to make reasoned judgments about safety:
    (i) Estimation of acute oral toxicity. When further study is 
warranted, EPA generally supports limiting such tests to those using the 
lowest number of animals feasible. EPA will accept three alternative 
Organization for Economic Cooperation and Development (OECD) test 
methods in place of the ``traditional'' acute oral toxicity test. The 
three OECD alternatives are the following:
    (A) The up and down procedure as described in OECD Guideline 425 
referenced in paragraph (f)(4) of this section.
    (B) The acute toxic class method as described in OECD Guideline 423 
and referenced in paragraph (f)(6) of this section.
    (C) The fixed dose method as described in OECD Guideline 420 and 
referenced in paragraph (f)(5) of this section.
    (ii) Limit test. When data on structurally related chemicals are 
inadequate, a limit test may be considered. If rodents are used, a limit 
dose of at least 2,000 mg per kilogram of body weight may be 
administered to a single group of five males and five females using the 
procedures described in paragraph (e) of this section. If no lethality 
is demonstrated, no further testing for acute oral toxicity is needed. 
(Under current policy and regulations for pesticide products, 
precautionary statements may still be required unless there are data to 
indicate the LD50 is greater than 5,000 mg/kg.) If compound-
related mortality is produced in the limit test, further study may need 
to be considered.
    (2) [Reserved]
    (e) Conventional acute toxicity test--(1) Principle of the test 
method. The test substance is administered orally by gavage in graduated 
doses to several groups of experimental animals, one dose being used per 
group. The doses chosen may be based on the results of a range finding 
test. Subsequently, observations of effects and deaths are made. Animals 
that die during the test are necropsied, and at the conclusion of the 
test the surviving animals are sacrificed and necropsied. This section 
is directed primarily to studies in rodent species but may be adapted 
for studies in nonrodents. Animals showing severe

[[Page 323]]

and enduring signs of distress and pain may need to be humanely 
sacrificed. Dosing test substances in a way known to cause marked pain 
and distress due to corrosive or irritating properties need not be 
carried out.
    (2) Substance to be tested. Test, control, and reference substances 
are described in 40 CFR Part 792--Good Laboratory Practice Standards.
    (3) Test procedures--(i) Preparations. Healthy young adult animals 
are acclimatized to the laboratory conditions for at least 5 days prior 
to the test before the test animals are randomized and assigned to the 
treatment groups.
    (ii) Animal selection--(A) Species and strain. Although several 
mammalian test species may be used, the rat is the preferred species. 
Commonly used laboratory strains must be employed. If another species is 
used, the tester must provide justification and reasoning for its 
selection.
    (B) Age. Young adult rats between 8- and 12-weeks-old at the 
beginning of dosing should be used. Rabbits should be at least 12 weeks 
of age at study initiation. The weight variation of animals used in a 
test must be within 20% of the mean weight for each sex.
    (C) Number and sex of animals. (1) At least five experimentally 
naive rodents are used at each dose level. They should all be of the 
same sex. After completion of the study in one sex, at least one group 
of five animals of the other sex is dosed to establish that animals of 
this sex are not markedly more sensitive to the test substance. The use 
of fewer animals may be justified in individual circumstances. Where 
adequate information is available to demonstrate that animals of the sex 
tested are markedly more sensitive, testing in animals of the other sex 
may be dispensed with. An acceptable option would be to test at least 
one group of five animals per sex at one or more dose levels to 
definitively determine the more sensitive sex prior to conducting the 
main study.
    (2) The females must be nulliparous and nonpregnant.
    (3) In acute toxicity tests with animals of a higher order than 
rodents, the use of smaller numbers should be considered.
    (D) Assignment of animals. Each animal must be assigned a unique 
identification number. A system to assign animals to test groups and 
control groups randomly is required.
    (E) Housing. Animals may be group-caged by sex, but the number of 
animals per cage must not interfere with clear observation of each 
animal. The biological properties of the test substance or toxic effects 
(e.g., morbidity, excitability) may indicate a need for individual 
caging.
    (1) The temperature of the experimental animal rooms should be at 22 
 3 [deg]C for rodents.
    (2) The relative humidity of the experimental animal rooms should be 
30 to 70%.
    (3) Where lighting is artificial, the sequence should be 12-hours 
light/12-hours dark.
    (4) For feeding, conventional laboratory diets may be used with an 
unlimited supply of drinking water.
    (iii) Dose levels and dose selection. (A) Three dose levels must be 
used, spaced appropriately to produce test groups with a range of toxic 
effects and mortality rates. The data collected must be sufficient to 
produce a dose-response curve and permit an acceptable estimation of the 
LD50. Range finding studies using single animals may help to 
estimate the positioning of dose groups so that no more than three dose 
levels will be necessary.
    (B) Limit test. This test has been defined and described in 
paragraph (d)(1)(ii) of this section.
    (C) Vehicle. Where necessary, the test substance is dissolved or 
suspended in a suitable vehicle. If a vehicle or diluent is needed, it 
should not elicit toxic effects itself nor substantially alter the 
chemical or toxicological properties of the test substance. It is 
recommended that wherever possible the use of an aqueous solution be 
considered first, followed by consideration of a solution in oil (e.g., 
corn oil), and then by consideration of possible solution in other 
vehicles. Toxic characteristics of nonaqueous vehicles should be known, 
and, if not known, should be determined before the test.
    (D) Volume. The maximum volume of liquid that can be administered at 
one time depends on the size of the test

[[Page 324]]

animal. In rodents, the volume should not exceed 1 mL/100 g body weight, 
except when an aqueous solution is used in which case 2 mL/100 g may be 
administered. Either constant volume or constant concentration 
administration is acceptable when dosing, provided the following 
guidance is employed. When possible, the liquid test material should be 
dosed neat. Otherwise, it may be diluted, using the highest 
concentration possible, although volumes less than 0.5 mL per animal 
would not be required. Lower dose volumes are acceptable if they can be 
accurately administered. Solid materials should be suspended or 
dissolved in the minimum amount of vehicle and dosed at the highest 
concentration possible.
    (iv) Exposure and exposure duration. (A) Animals must be fasted 
prior to test substance administration. For the rat, feed should be 
withheld overnight; for other rodents with higher metabolic rates a 
shorter period of fasting is appropriate.
    (B) The test substance must be administered in a single dose by 
gavage, using a stomach tube or suitable intubation cannula.
    (C) If a single dose is not possible, the dose may be given in 
smaller fractions over a period not exceeding 24 hours. Where a dose is 
administered in fractions, it may be necessary to provide the animals 
with food and water, depending on the length of the dosing period.
    (D) After the substance has been administered, feed may be withheld 
for an additional 3-4 hours.
    (v) Observation period. Although 14 days is recommended as a minimum 
observation period, the duration of observation should not be fixed 
rigidly. It should be determined by the toxic reactions, rate of onset, 
and length of recovery period, and may thus be extended when considered 
necessary. The time at which signs of toxicity appear, their duration, 
and the time to death are important, especially if there is a tendency 
for deaths to be delayed.
    (vi) Observation of animals. (A) A careful clinical examination must 
be made at least once each day.
    (B) Additional observations must be made daily, especially in the 
early days of the study. Appropriate actions should be taken to minimize 
loss of animals to the study (e.g., necropsy or refrigeration of those 
animals found dead and isolation of weak or moribund animals).
    (C) Observations must be detailed and carefully recorded, preferably 
using explicitly defined scales. Observations should include, but not be 
limited to, evaluation of skin and fur, eyes and mucous membranes, 
respiratory and circulatory effects, autonomic effects such as 
salivation, central nervous system effects, including tremors and 
convulsions, changes in the level of activity, gait and posture, 
reactivity to handling or sensory stimuli, altered strength, and 
stereotypies or bizarre behavior (e.g., self-mutilation, walking 
backwards).
    (D) Individual weights of animals must be determined shortly before 
the test substance is administered, weekly thereafter, and at death. 
Changes in weights should be calculated and recorded when survival 
exceeds 1 day.
    (E) The time of death should be recorded as precisely as possible.
    (vii) Gross pathology. (A) At the end of the test, surviving animals 
must be weighed and sacrificed.
    (B) A gross necropsy must be performed on all animals under test. 
All gross pathology changes should be recorded.
    (C) If necropsy cannot be performed immediately after a dead animal 
is discovered, the animal should be refrigerated (not frozen) at 
temperatures low enough to minimize autolysis. Necropsies should be 
performed as soon as practicable, normally within a day or two.
    (viii) Additional evaluation. Microscopic examination of organs 
showing evidence of gross pathology in animals surviving 24 hours or 
more should also be considered because it may yield useful information.
    (ix) Data and reporting--(A) Treatment of results. Data must be 
summarized in tabular form, showing for each test group the number of 
animals at the start of the test, body weights, time of death of 
individual animals at different dose levels, number of animals 
displaying other signs of toxicity, description of toxic effects, and 
necropsy findings. Any methods used for calculation

[[Page 325]]

of the LD50 or any other parameters should be specified and 
referenced. Methods for parameter estimation are described in the 
references listed in paragraphs (f)(1), (f)(2), and (f)(3) of this 
section.
    (B) Evaluation of results. An evaluation should include the 
relationship, if any, between exposure of the animals to the test 
substance and the incidence and severity of all abnormalities, including 
behavioral and clinical abnormalities, gross lesions, body weight 
changes, effects on mortality, and any other toxic effects. The 
LD50 value should always be considered in conjunction with 
the observed toxic effects and any necropsy findings. The 
LD50 value is a relatively coarse measurement, useful only as 
a reference value for classification and labeling purposes, and for an 
expression of the lethal potential of the test substance by the 
ingestion route. Reference should always be made to the experimental 
animal species in which the LD50 value was obtained.
    (C) Test report. In addition to the reporting requirements specified 
under EPA Good Laboratory Practice Standards at 40 CFR part 792, subpart 
J, the following specific information must be reported. The test report 
shall include:
    (1) Species, strain, sex, and source of test animals.
    (2) Method of randomization in assigning animals to test and control 
groups.
    (3) Rationale for selection of species, if other than that 
recommended.
    (4) Tabulation of individual and test group data by sex and dose 
level (e.g., number of animals exposed, number of animals showing signs 
of toxicity and number of animals that died or were sacrificed during 
the test).
    (i) Description of toxic effects, including their time of onset, 
duration, reversibility, and relationship to dose.
    (ii) Body weights.
    (iii) Time of dosing and time of death after dosing.
    (iv) Dose-response curves for mortality and other toxic effects 
(when permitted by the method of determination).
    (v) Gross pathology findings.
    (vi) Histopathology findings and any additional clinical chemistry 
evaluations, if performed.
    (5) Description of any pretest conditioning, including diet, 
quarantine and treatment for disease.
    (6) Description of caging conditions including: Number (or change in 
number) of animals per cage, bedding material, ambient temperature and 
humidity, photoperiod, and identification of diet of test animals.
    (7) Manufacturer, source, purity, and lot number of test substance.
    (8) Relevant properties of substance tested including physical state 
and pH (if applicable).
    (9) Identification and composition of any vehicles (e.g., diluents, 
suspending agents, and emulsifiers) or other materials used in 
administering the test substance.
    (10) A list of references cited in the body of the report. 
References to any published literature used in developing the test 
protocol, performing the testing, making and interpreting observations, 
and compiling and evaluating the results.
    (f) References. For additional background information on this test 
guideline, the following references should be consulted. These 
references are available for inspection at the TSCA Nonconfidential 
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M 
St., NW., Washington, DC, 12 noon to 4 p.m., Monday through Friday, 
except legal holidays.
    (1) Chanter, D.O. and Heywood, R. The LD50 Test: Some 
Considerations of Precision. Toxicology Letters 10:303-307 (1982).
    (2) Finney, D.J. Chapter 3--Estimation of the median effective dose 
and Chapter 4--Maximum likelihood estimation, Probit Analysis, 3rd ed. 
Cambridge, London (1971).
    (3) Finney, D.J. The Median Lethal Dose and Its Estimation. Archives 
of Toxicology 56:215-218 (1985).
    (4) Organization for Economic Cooperation and Development. OECD 
Guidelines for the Testing of Chemicals. OECD Guideline 425: Acute Oral 
Toxicity: Up-and-Down Procedure, Approved: June 1998.
    (5) Organization for Economic Cooperation and Development. OECD

[[Page 326]]

Guidelines for Testing of Chemicals. Guideline 420: Acute Oral 
Toxicity--Fixed Dose Method, Adopted: July 17, 1992.
    (6) Organization for Economic Cooperation and Development. OECD 
Guidelines for Testing of Chemicals. Guideline 423: Acute Oral 
Toxicity--Acute Toxic Class Method, Adopted: March 22, 1996.
    (7) Organization for Economic Cooperation and Development. OECD 
Guidelines for Testing of Chemicals. Guideline 401: Acute Oral Toxicity, 
Adopted: February 24, 1987.

[65 FR 78771, Dec. 15, 2000]



Sec. 799.9120  TSCA acute dermal toxicity.

    (a) Scope. This section is intended to meet the testing requirements 
under section 4 of the Toxic Substances Control Act (TSCA). In the 
assessment and evaluation of the toxic characteristics of a substance, 
determination of acute dermal toxicity is useful where exposure by the 
dermal route is likely. It provides information on health hazards likely 
to arise from short-term exposure by the dermal route. Data from an 
acute study may serve as a basis for classification and labeling. It is 
an initial step in establishing a dosage regimen in subchronic and other 
studies and may provide information on dermal absorption and the mode of 
toxic action of a substance by this route. An evaluation of acute 
toxicity data should include the relationship, if any, between the 
exposure of animals to the test substance and the incidence and severity 
of all abnormalities, including behavioral and clinical abnormalities, 
the reversibility of observed abnormalities, gross lesions, body weight 
changes, effects on mortality, and any other toxic effects.
    (b) Source. The source material used in developing this TSCA test 
guideline is the Office of Prevention, Pesticides, and Toxic Substances 
(OPPTS) harmonized test guideline 870.1200 (August 1998, final 
guideline). This source is available at the address in paragraph (f) of 
this section.
    (c) Definitions. The following definitions apply to this section.
    Acute dermal toxicity is the adverse effects occurring within a 
short time of dermal application of a single dose of a substance or 
multiple doses given within a 24-hour period.
    Dosage is a general term comprising the dose, its frequency and the 
duration of dosing.
    Dose is the amount of test substance applied. Dose is expressed as 
weight of test substance (grams, milligrams) per unit weight of test 
animal (e.g., milligrams per kilogram).
    Dose-effect is the relationship between the dose and the magnitude 
of a defined biological effect either in an individual or in a 
population sample.
    Dose-response is the relationship between the dose and the 
proportion of a population sample showing a defined effect.
    LD50 (median lethal dose), dermal, is a statistically 
derived estimate of a single dose of a substance that can be expected to 
cause death in 50% of treated animals when applied to the skin. The 
LD50 value is expressed in terms of weight of test substance 
per unit weight of test animal (milligrams per kilogram).
    (d) Approaches to the determination of acute toxicity. (1) EPA 
recommends the following means to reduce the number of animals used to 
evaluate acute effects of chemical exposure while preserving its ability 
to make reasonable judgments about safety:
    (i) Using data from substantially similar mixtures. In order to 
minimize the need for animal testing, the Agency encourages the review 
of existing acute toxicity information on mixtures that are 
substantially similar to the mixture under investigation. In certain 
cases it may be possible to glean enough information to make preliminary 
hazard evaluations that may reduce the need for further animal testing.
    (ii) Limit test. When data on structurally related chemicals are 
inadequate, a limit test may be considered. If rodents are used, a limit 
dose of at least 2,000 mg/kg bodyweight may be administered to a single 
group of five males and five females using the procedures described in 
paragraph (e) of this section. If no lethality is demonstrated, no 
further testing for acute dermal toxicity is needed. If compound-related 
mortality is produced,

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further study may need to be considered.
    (2) [Reserved]
    (e) Conventional acute toxicity test--(1) Principle of the test 
method. The test substance is applied dermally in graduated doses to 
several groups of experimental animals, one dose being used per group. 
The doses chosen may be based on the results of a range finding test. 
Subsequently, observations of effects and deaths are made. Animals that 
die during the test are necropsied, and at the conclusion of the test 
the surviving animals are sacrificed and necropsied. This section is 
directed primarily to studies in either rats, rabbits, or guinea pigs 
but may be adapted for studies in other species. Animals showing severe 
and enduring signs of distress and pain may need to be humanely 
sacrificed. Dosing test substances in a way known to cause marked pain 
and distress due to corrosive or irritating properties need not be 
carried out.
    (2) Substance to be tested. Test, control, and reference substances 
are discussed in 40 CFR Part 792--Good Laboratory Practice Standards.
    (3) Test procedures--(i) Preparations. Healthy young adult animals 
are acclimatized to the laboratory conditions for at least 5 days prior 
to the test before the test animals are randomized and assigned to the 
treatment groups.
    (ii) Animal selection--(A) Species and strain. The rat, rabbit, or 
guinea pig may be used. The albino rabbit is preferred because of its 
size, ease of handling, skin permeability, and extensive data base. 
Commonly used laboratory strains must be employed. If a species other 
than rats, rabbits, or guinea pigs is used, the tester must provide 
justification and reasoning for its selection.
    (B) Age. Young adult animals, rats between 8- and 12-weeks-old, 
rabbits at least 12-weeks-old, and guinea pigs between 5- and 6-weeks-
old at the beginning of dosing should be used. The weight variation of 
animals used in a test must be within 20% of the mean weight for each 
sex.
    (C) Number and sex of animals. (1) At least five experimentally 
naive animals with healthy intact skin are used at each dose level. They 
should all be of the same sex. After completion of the study in one sex, 
at least one group of five animals of the other sex is dosed to 
establish that animals of this sex are not markedly more sensitive to 
the test substance. The use of fewer animals may be justified in 
individual circumstances. Where adequate information is available to 
demonstrate that animals of the sex tested are markedly more sensitive, 
testing in animals of the other sex may be dispensed with. An acceptable 
option would be to test at least one group of five animals per sex at 
one or more dose levels to definitively determine the more sensitive sex 
prior to conducting the main study.
    (2) The females must be nulliparous and nonpregnant.
    (3) In acute toxicity tests with animals of a higher order than 
those mentioned above, the use of smaller numbers should be considered.
    (D) Assignment of animals. Each animal must be assigned a unique 
identification number. A system to randomly assign animals to test 
groups and control groups is required.
    (E) Housing. Animals should be housed in individual cages.
    (1) The temperature of the experimental animal rooms should be at 22 
 3 [deg]C for rodents, 20  3 
[deg]C for rabbits.
    (2) The relative humidity of the experimental animal rooms should be 
30 to 70%.
    (3) Where lighting is artificial, the sequence should be 12-hours 
light/12-hours dark.
    (4) For feeding, conventional laboratory diets may be used with an 
unlimited supply of drinking water.
    (iii) Dose levels and dose selection. (A) Three dose levels must be 
used and spaced appropriately to produce test groups with a range of 
toxic effects and mortality rates. The data must be sufficient to 
produce a dose-response curve and permit an acceptable estimation of the 
median lethal dose. Range finding studies using single animals may help 
to estimate the positioning of the dose groups so that no more than 
three dose levels will be necessary.
    (B) Limit test. This test is described in paragraph (d)(2)(ii) of 
this section.
    (C) Vehicle. Solids should be pulverized when possible. The test 
substance

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should be moistened sufficiently with water or, where necessary, a 
suitable vehicle to ensure good contact with skin. If a vehicle or 
diluent is needed, it should not elicit toxic effects itself nor 
substantially alter the chemical or toxicological properties of the test 
substance. In addition, the influence of the vehicle on penetration of 
skin by the test substance should be taken into account. It is 
recommended that wherever possible the use of an aqueous solution be 
considered first, followed by consideration of a solution in oil (e.g., 
corn oil), and then by consideration of possible solution in other 
vehicles. For nonaqueous vehicles the toxic characteristics of the 
vehicle should be known, and if not known should be determined before 
the test. Acceptable alternative vehicles include gum arabic, ethanol 
and water, carboxymethyl cellulose, glycerol, propylene glycol, PEG 
vegetable oil, and mineral oil as long as the vehicle is not irritating 
and the inability to use water or saline is justified in the report.
    (iv) Exposure and exposure duration. The test substance must be 
administered over a period of 24 hours.
    (v) Preparation of animal skin. Fur must be clipped from the dorsal 
area of the trunk of the test animals. Shaving may be employed, but it 
should be carried out at least 24 hours before dosing. Care must be 
taken to avoid abrading the skin, which would alter its permeability.
    (vi) Application of test substance. (A) The test substance must be 
applied uniformly over a shaved or clipped area which is approximately 
10% of the body surface area. The area starting at the scapulae 
(shoulders) to the wing of the ileum (hip bone) and half way down the 
flank on each side of the animal should be shaved or clipped. Liquid 
test materials should be undiluted if possible. With highly toxic 
substances, the surface area covered may be less, but as much of the 
area as possible should be covered with as thin and uniform a film as 
practical. The test material is not removed until 24 hours after 
application. In the case where less than 10% of the surface area is 
covered an approximation of the exposed areas should be determined.
    (B) The test substance must be held in contact with the skin with a 
porous gauze dressing (<8 ply) and nonirritating tape throughout a 24-
hour exposure period. The test site must be further covered in a 
suitable manner to retain the gauze dressing and test substance and 
ensure that the animals cannot ingest the test substance. Restrainers 
may be used to prevent the ingestion of the test substance, but complete 
immobilization is not a recommended method. Although a semiocclusive 
dressing is preferred, an occlusive dressing will also be acceptable.
    (C) At the end of the exposure period, residual test substance 
should be removed where practicable using water or an appropriate 
solvent.
    (vii) Observation period. Although 14 days is recommended as a 
minimum observation period, the duration of observation should not be 
fixed rigidly. It should be determined by the toxic reactions, rate of 
onset, and length of recovery period, and may thus be extended when 
considered necessary. The time at which signs of toxicity appear, their 
duration, and the time to death are important, especially if there is a 
tendency for deaths to be delayed.
    (viii) Observation of animals. (A) A careful clinical examination 
must be made at least once each day.
    (B) Additional observations must be made daily, especially in the 
early days of the study. Appropriate actions should be taken to minimize 
loss of animals to the study (e.g., necropsy or refrigeration of those 
animals found dead and isolation of weak or moribund animals).
    (C) Observations must be detailed and carefully recorded, preferably 
using explicitly defined scales. Observations should include, but not be 
limited to, evaluation of skin and fur, eyes and mucous membranes, 
respiratory and circulatory effects, autonomic effects such as 
salivation, central nervous system effects, including tremors and 
convulsions, changes in the level of activity, gait and posture, 
reactivity to handling or sensory stimuli, altered strength, and 
stereotypies or bizarre behavior (e.g., self-mutilation, walking 
backwards).

[[Page 329]]

    (D) Individual weights of animals must be determined shortly before 
the test substance is administered, weekly thereafter, and at death. 
Changes in weights should be calculated and recorded when survival 
exceeds one day.
    (E) The time of death should be recorded as precisely as possible.
    (ix) Gross pathology. (A) At the end of the test, surviving animals 
must be weighed and sacrificed.
    (B) A gross necropsy must be performed on all animals under test. 
All gross pathology changes should be recorded.
    (C) If necropsy cannot be performed immediately after a dead animal 
is discovered, the animal should be refrigerated (not frozen) at 
temperatures low enough to minimize autolysis. Necropsies should be 
performed as soon as practicable, normally within a day or two.
    (x) Additional evaluations. Microscopic examination of organs 
showing evidence of gross pathology in animals surviving 24 hours or 
more should also be considered because it may yield useful information.
    (xi) Data and reporting--(A) Treatment of results. Data must be 
summarized in tabular form, showing for each test group the number of 
animals at the start of the test, body weights, time of death of 
individual animals at different dose levels, number of animals 
displaying other signs of toxicity, description of toxic effects and 
necropsy findings. Any methods used for calculation of the 
LD50 or any other parameters should be specified and 
referenced. Methods for parameter estimation are described in the 
references listed in paragraphs (f)(1), (f)(2), and (f)(3) of this 
section.
    (B) Evaluation of results. An evaluation should include the 
relationship, if any, between exposure of the animals to the test 
substance and the incidence and severity of all abnormalities, including 
behavioral and clinical abnormalities, gross lesions, body weight 
changes, effects on mortality, and any other toxic effects. The 
LD50 value should always be considered in conjunction with 
the observed toxic effects and any necropsy findings. The 
LD50 value is a relatively coarse measurement, useful only as 
a reference value for classification and labeling purposes, and for an 
expression of the lethal potential of the test substance by the dermal 
route. Reference should always be made to the experimental animal 
species in which the LD50 value was obtained.
    (C) Test report. In addition to the reporting requirements specified 
under EPA Good Laboratory Practice Standards at 40 CFR part 792, subpart 
J, the following specific information must be reported. The test report 
must include:
    (1) Species, strain, sex, and source of test animals.
    (2) Method of randomization in assigning animals to test and control 
groups.
    (3) Rationale for selection of species, if other than that 
recommended.
    (4) Tabulation of individual and test group data by sex and dose 
level (e.g., number of animals exposed, number of animals showing signs 
of toxicity and number of animals that died or were sacrificed during 
the test).
    (i) Description of toxic effects, including their time of onset, 
duration, reversibility, and relationship to dose.
    (ii) Body weights.
    (iii) Time of dosing and time of death after dosing.
    (iv) Dose-response curves for mortality and other toxic effects 
(when permitted by the method of determination).
    (v) Gross pathology findings.
    (vi) Histopathology findings and any additional clinical chemistry 
evaluations, if performed.
    (5) Description of any pre-test conditioning, including diet, 
quarantine and treatment for disease.
    (6) Description of caging conditions including: Number (or change in 
number) of animals per cage, bedding material, ambient temperature and 
humidity, photoperiod, and identification of diet of test animals.
    (7) Manufacturer, source, purity, and lot number of test substance.
    (8) Relevant properties of substance tested including physical state 
and pH (if applicable).
    (9) Identification and composition of any vehicles (e.g., diluents, 
suspending agents, and emulsifiers) or other materials used in 
administering the test substance.

[[Page 330]]

    (10) A list of references cited in the body of the report. 
References to any published literature used in developing the test 
protocol, performing the testing, making and interpreting observations, 
and compiling and evaluating the results.
    (f) References. For additional background information on this test 
guideline, the following references should be consulted. These 
references are available for inspection at the TSCA Nonconfidential 
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M 
St., NW., Washington, DC, 12 noon to 4 p.m., Monday through Friday, 
except legal holidays.
    (1) Chanter, D.O. and Heywood, R., The LD50 Test: Some 
Considerations of Precision, Toxicology Letters 10:303-307 (1982).
    (2) Finney, D.J. Chapter 3--Estimation of the median effective dose 
and Chapter 4-Maximum likelihood estimation, Probit Analysis, 3rd ed. 
Cambridge, London (1971).
    (3) Finney, D.J. The Median Lethal Dose and Its Estimation. Archives 
of Toxicology 56:215-218 (1985).
    (4) Organization for Economic Cooperation and Development. OECD 
Guideline for the Testing of Chemicals. OECD Guideline 425: Acute Oral 
Toxicity: Up-and-Down Procedure. Adopted: September 21, 1998.
    (5) Organization for Economic Cooperation and Development. OECD 
Guidelines for Testing of Chemicals. Guideline 420: Acute Oral 
Toxicity--Fixed Dose Method. Adopted: July 17, 1992.
    (6) Organization for Economic Cooperation and Development. OECD 
Guidelines for Testing of Chemicals. Guideline 423: Acute Oral 
Toxicity--Acute Toxic Class Method. Adopted: March 22, 1996
    (7) Organization for Economic Cooperation and Development. OECD 
Guidelines for Testing of Chemicals. Guideline 402: Acute Dermal 
Toxicity. Adopted: February 24, 1987.

[65 FR 78774, Dec. 15, 2000]



Sec. 799.9130  TSCA acute inhalation toxicity.

    (a) Scope. This section is intended to meet testing requirements 
under section 4 of the Toxic Substances Control Act (TSCA). 
Determination of acute toxicity is usually an initial step in the 
assessment and evaluation of the toxic characteristics of a substance 
that may be inhaled such as a gas, volatile substance, or aerosol/
particle. It provides information on health hazards likely to arise from 
short-term exposure by the inhalation route. Data from an acute study 
may serve as a basis for classification and labeling. It is 
traditionally a step in establishing a dosage regimen in subchronic and 
other studies and may provide initial information on the mode of toxic 
action of a substance. An evaluation of acute toxicity data should 
include the relationship, if any, between the animals' exposure to the 
test substance and the incidence and severity of all abnormalities, 
including behavioral and clinical abnormalities, the reversibility of 
observed abnormalities, gross lesions, body weight changes, effects on 
mortality, and any other toxic effects.
    (b) Source. The source material used in developing this TSCA test 
guideline is the harmonized Office of Prevention, Pesticides, and Toxic 
Substances (OPPTS) test guideline 870.1300 (August 1998, final 
guideline). These sources are available at the address in paragraph (g) 
of this section.
    (c) Definitions. The definitions in section 3 of TSCA and the 
definitions in 40 CFR Part 792--Good Laboratory Practice Standards apply 
to this section. The following definitions also apply to this section.
    Acute inhalation toxicity is the adverse effect caused by a 
substance following a single uninterrupted exposure by inhalation over a 
short period of time (24 hours or less) to a substance capable of being 
inhaled.
    Aerodynamic equivalent diameter is defined as the diameter of a 
unit-density sphere having the same terminal settling velocity as the 
particle in question, whatever its size, shape, and density. It is used 
to predict where in the respiratory tract such particles may be 
deposited.
    Concentration is expressed as weight of the test substance per unit 
volume of air, e.g., milligrams per liter.
    Geometric standard deviation (GSD) is a dimensionless number equal 
to the

[[Page 331]]

ratio between the mass median aerodynamic diameter (MMAD) and either 84% 
or 16% of the diameter size distribution (e.g., MMAD = 2 m; 84% = 4 m; 
GSD = 4/2 = 2.0.) The MMAD, together with the GSD, describe the particle 
size distribution of an aerosol. Use of the GSD may not be valid for 
non-lognormally distributed aerosols. (If the size distribution deviates 
from the lognormal, it shall be noted).
    Inhalable diameter refers to that aerodynamic diameter of a particle 
which is considered to be inhalable for the organism under study. It is 
used to refer to particles which are capable of being inhaled and 
deposited anywhere within the respiratory tract .
    LC50 (median lethal concentration) is a statistically 
derived estimate of a concentration of a substance that can be expected 
to cause death during exposure or within a fixed time after exposure in 
50% of animals exposed for a specified time. The LC50 value 
is a relatively coarse measurement useful only for classification and 
labeling purposes and an expression of the lethal potential of the test 
substance following inhalation. The LC50 value is expressed 
as weight of test substance per unit volume of air (milligrams per 
liter) or parts per million. For clarity, the exposure duration and test 
animal species should also be specified, e.g., 4 hours LC50 
in F344.
    Mass median aerodynamic diameter (MMAD) is the median aero-dynamic 
diameter and, along with the geometric standard deviation, is used to 
describe the particle size distribution of any aerosol statistically, 
based on the weight and size of the particles. Fifty percent of the 
particles by weight will be smaller than the median diameter and 50% of 
the particles will be larger.
    (d) Approaches to the determination of acute toxicity. (1) EPA 
recommends the following means to reduce the number of animals used to 
evaluate acute effects of chemical exposure while preserving its ability 
to make reasonable judgments about safety:
    (i) Using data from substantially similar mixtures. In order to 
minimize the need for animal testing, the Agency encourages the review 
of existing acute toxicity information on mixtures that are 
substantially similar to mixtures under investigation. In certain cases, 
it may be possible to get enough information to make preliminary hazard 
evaluations that may reduce the need for further animal testing.
    (ii) Limit test. When data on structurally related chemicals are 
inadequate, a limit test may be considered. In the limit test, a single 
group of five males and five females is exposed to 2 mg/L for 4 hours, 
or where this is not possible due to physical or chemical properties of 
the test substance, the maximum attainable concentration where a 
particle size distribution having an MMAD between 1 and 4 [micro]m 
cannot be maintained, using the procedures described under paragraph (e) 
of this section. For fibers, the bivariate distribution of length and 
diameter must ensure inhalability. For gases and vapors, the 
concentrations need not be greater than 50,000 ppm or 50% of the lower 
explosive limit, whichever is lower. If a test at an aerosol or 
particulate exposure of 2 mg/L (actual concentration of respirable 
substance) for 4 hours or, where this is not feasible, the maximum 
attainable concentration, using the procedures described for this study, 
produces no observable toxic effects, then a full study using three 
concentrations will not be necessary. Similarly, if a test at a gas or 
vapor exposure of 50,000 ppm or 50% of the lower explosive limit, 
whichever is lower, produces no observable toxic effects, then a full 
study using three concentrations will not be necessary.
    (2) [Reserved]
    (e) Conventional acute toxicity test--(1) Principle of the test 
method. Several groups of experimental animals are exposed to the test 
substance in graduated concentrations for a defined period, one 
concentration being used per group. When a vehicle other than water is 
used to help generate an appropriate concentration of the substance in 
the atmosphere, a vehicle control group should be used when historical 
data are not available or adequate to determine the acute inhalation 
toxicity of the vehicle. Subsequently, observations of effects and death 
are made. Animals that die during the test are necropsied and at the 
conclusion of the test surviving animals are sacrificed and necropsied. 
This guideline is directed primarily to

[[Page 332]]

studies in rodent species but may be adapted for studies in non-rodents. 
Animals showing severe and enduring signs of distress and pain may need 
to be sacrificed. Dosing test substances in a way known to cause marked 
pain and distress due to corrosive or irritating properties need not be 
carried out.
    (2) Substance to be tested. Test, control, and reference substances 
are discussed under EPA Good Laboratory Practice Standards at 40 CFR 
part 792, subpart f.
    (3) Test procedures--(i) Preparation. Healthy young adult animals 
are acclimatized to the laboratory conditions for at least 5 days prior 
to the test. Before the test, animals are randomized and assigned to the 
required number of groups.
    (ii) Animal selection--(A) Species and strain. (1) Although several 
mammalian test species may be used, the preferred species is the rat. 
Commonly used laboratory strains should be employed. If another 
mammalian species is used, the investigator should provide justification 
and reasoning for the selection.
    (2) Health Status. Body weight and feed consumption are not 
sufficient indicators of the health status of animals prior to 
initiating an inhalation toxicity study. Prior to initiating the study, 
animals must be monitored for known viral and bacterial respiratory 
pathogens determined by conventional microbiological assays (e.g., 
serology). The animals must be free from pathogens at the start of 
exposure.
    (B) Age. Young adult rats between 8-12 weeks old at the beginning of 
dosing, should be used. The weight variation in animals or between 
groups used in a test should not exceed 20% of the 
mean weight of each sex.
    (C) Number of animals and sex. (1) At least five experimentally 
naive animals are used at each concentration and they must be of one 
sex. After completion of the study in one sex, at least one group of 
five animals of the other sex is exposed to establish that animals of 
this sex are not markedly more sensitive to the test substance. The use 
of fewer animals may be justified in individual circumstances. Where 
adequate information is available to demonstrate that animals of the sex 
tested are markedly more sensitive, testing in animals of the other sex 
is not required. An acceptable option would be to test at least one 
group of five animals per sex at one or more dose levels to definitively 
determine the more sensitive sex prior to conducting the main study.
    (2) Females must be nulliparous and nonpregnant.
    (3) In acute toxicity tests with animals of a higher order than 
rodents, the use of fewer animals per concentration group should be 
considered.
    (D) Assignment of animals. (1) Each animal must be assigned a unique 
identification number. A system to assign animals to test groups and 
control groups randomly is required.
    (2) Control groups. A concurrent untreated control group is not 
necessary. Where a vehicle other than water is used to generate an 
appropriate concentration of the test substance in the atmosphere and 
historical data are not available or adequate to determine the acute 
toxicity of the vehicle, a vehicle control group must be used. The 
vehicle control group must be a sham-treated group. Except for treatment 
with the test substance, animals in the vehicle control group must be 
handled in a manner identical to the test-group animals.
    (E) Housing. The animals may be group-caged by sex, but the number 
of animals per cage must not interfere with clear observation of each 
animal. The biological properties of the test substance or toxic effects 
(e.g., morbidity, excitability) may indicate a need for individual 
caging. Animals must be housed individually in inhalation chambers 
during exposure to aerosols.
    (1) Before and after exposure, the temperature of the animal room 
should be 22 3 [deg]C and the relative humidity 
30-70%.
    (2) Where lighting is artificial, the sequence should be 12 hours 
light/12 hours dark.
    (3) For feeding, conventional laboratory diets may be used with an 
unlimited supply of drinking water.
    (F) Inhalation equipment. (1) Animals can be exposed to the 
substance by either a nose-only procedure or in a

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whole-body exposure chamber. Maintenance of slight negative pressure 
inside the chamber will prevent leakage of the test substance into the 
surrounding areas. The nose-only exposure procedure is recommended for 
studies of aerosols to minimize exposures confounding resultant from 
test substance ingestion due to test animal fur licking following 
exposures. Animals must be acclimated to the nose-only exposure chamber 
prior to study and heat stress minimized during testing.
    (2) Inhalation chambers. The animals must be tested in inhalation 
equipment designed to sustain a dynamic airflow for nose-only exposures 
of at least 300 ml/minute/animal or an airflow for whole-body exposures 
of at least 12 to 15 air changes per hour and ensure an adequate oxygen 
content of at least 19% and an evenly distributed exposure atmosphere. 
Where a whole-body chamber is used, its design must minimize crowding by 
providing individual caging. As a general rule, to ensure stability of a 
chamber atmosphere, the total ``volume'' of the test animals should not 
exceed 5% of the volume of the test chamber.
    (3) Environmental conditions. The temperature at which the test is 
performed must be maintained at 22 [deg]C (2 
[deg]C). Ideally, the relative humidity should be maintained between 40% 
and 60%, but in certain instances (e.g., tests using water as a 
vehicle), this may not be practical.
    (G) Physical measurements. Measurements or monitoring must be made 
of the following:
    (1) Chemical purity of the test material must be analyzed. If the 
test substance is present in a mixture, the mass and composition of the 
entire mixture, as well as the principal compound, must be measured. If 
there is some difficulty in measuring chamber analytical concentration 
due to precipitation, nonhomogeneous mixtures, volatile components, or 
other factors, additional analyses of components may be necessary.
    (2) The rate of air flow should be monitored continuously, and must 
be recorded at least every 30 minutes during the exposure period.
    (3) The actual concentrations of the test substance must be measured 
in the breathing zone. During the exposure period, the actual 
concentrations of the test substance must be held as constant as 
practicable, monitored continuously or intermittently depending on the 
method of analysis, and recorded at least three times (i.e., at the 
beginning, at an intermediate time, and at the end) during the exposure 
period. Chamber concentration may be measured using gravimetric or 
analytical methods as appropriate. If trial run measurements are 
reasonably consistent (10% for liquid aerosol, 
gas, or vapor; 20% for dry aerosol), then a 
minimum of two measurements are sufficient. If measurements are not 
consistent, then a minimum of four measurements should be taken.
    (4) During the development of the generating system, particle size 
analysis must be performed to establish the stability of aerosol 
concentrations. During exposure, analysis should be conducted as often 
as necessary to determine the consistency of particle size distribution. 
The MMAD particle size range should be between 1-4 [micro]m. The 
particle size of hygroscopic materials must be small enough when dry to 
assure that the size of the swollen particle will still be within the 1-
4 [micro]m MMAD range. Characterization for fibers must include the 
bivariate distribution of length and diameter; this distribution must 
ensure inhalability. Measurements of aerodynamic particle size in the 
animal's breathing zone must be measured during a trial run. If MMAD 
values for each exposure level are within 10% of each other, then a 
minimum of two measurements during the exposures should be sufficient. 
If pretest measurements are not within 10% of each other, then a minimum 
of four measurements should be taken.
    (5) Temperature and humidity must be monitored continuously, and 
must be recorded at least every 30 minutes.
    (iii) Exposure duration and concentration levels. (A) Exposure 
duration. Shortly before exposure, the animals are weighed and then 
exposed to the test target concentration in the designated apparatus for 
4 hour exposure period after equilibration of the chamber 
concentrations. The target concentration is defined by an average of 5% 
for gases and vapors and 15% for

[[Page 334]]

particles and aerosols. The animals are weighed again at the conclusion 
of the exposure period to determine body weight change. Other durations 
may be needed to meet specific requirements. Food must be withheld 
during exposure. Water may also be withheld in certain circumstances.
    (B) Exposure concentration levels. At least three concentration 
levels and a vehicle control group, if required (see paragraph 
(e)(3)(ii)(D)(2) of this section), must be used. The concentration 
levels should be spaced appropriately to produce a concentration-
response curve and permit an estimation of the median lethal 
concentration (LC50). The concentrations can either be 
linearly or logarithmically spaced depending on the anticipated 
steepness of the concentration-response curve. A rationale for 
concentration selection should be provided to indicate that the selected 
concentrations will maximally support detection of concentration-
response relationship. The high concentration should be clearly toxic or 
a limit concentration, but should not result in an incidence of 
fatalities that would preclude a meaningful evaluation of the data. The 
lowest concentration should define a no-observed-effects level (NOEL). 
Range-finding studies using single animals may help to estimate the 
positioning of the test groups so that no more than three concentration 
levels will be necessary.
    (C) When the physical and chemical properties of the test substance 
show a low flash point or the test substance is otherwise known or 
thought to be explosive, care must be taken to avoid exposure level 
concentrations that could result in an exposure chamber explosion during 
the test.
    (iv) Observation period. The observation period must be at least 14 
days. However, the duration of observation should not be fixed rigidly. 
It should be determined by the toxic reactions, rate of onset, and 
length of recovery period, and thus may be extended when considered 
necessary. The time at which signs of toxicity appear, the duration of 
the signs observed, and the time of death must be recorded and are 
important, especially if there is a tendency for delayed effects.
    (v) Observation of animals. (A) A careful clinical examination must 
be made at least once each day.
    (B) Additional observations should be made daily with appropriate 
actions taken to minimize loss of animals to the study, e.g., necropsy 
or refrigeration of those animals found dead and isolation of weak or 
moribund animals.
    (C) Observations must be detailed and carefully recorded, preferably 
using explicitly defined scales. Observations should include, but not be 
limited to, evaluation of skin and fur, eyes and mucous membranes, 
respiratory and circulatory effects, autonomic effects such as 
salivation, central nervous system effects, including tremors and 
convulsions, changes in the level of activity, gait and posture, 
reactivity to handling or sensory stimuli, altered strength, and 
stereotypies or bizarre behavior (e.g., self mutilation, walking 
backwards).
    (D) Individual weights of animals must be determined pre-exposure 
and post-exposure, weekly after exposure, and at death. Changes in 
weights should be calculated and recorded when survival exceeds 1 day.
    (E) The time of death should be recorded as precisely as possible.
    (vi) Gross pathology. (A) At the end of the test, surviving animals 
must be weighed, sacrificed and a gross necropsy must be performed on 
all animals under test, with particular reference to any changes in the 
respiratory tract. All gross pathology changes must be recorded.
    (1) The gross necropsy must include examination of orifices and the 
cranial, thoracic, and abdominal cavities, and contents.
    (2) At least the lungs, liver, kidneys, adrenals, brain, and gonads 
should be weighed wet, as soon as possible after dissection to avoid 
drying.
    (3) Optionally, the following organs and tissues, or representative 
samples thereof, may be preserved in a suitable medium for possible 
future histopathological examination: All gross lesions; brain-including 
sections of medulla/pons; cerebellar cortex and cerebral cortex; 
pituitary; thyroid/parathyroid; thymus; heart; sternum with bone marrow; 
salivary glands;

[[Page 335]]

liver; spleen; kidneys; adrenals; pancreas; gonads; accessory genital 
organs (epididymis, prostrate, and, if present, seminal vesicles); 
aorta; skin; gall bladder (if present); esophagus; stomach; duodenum; 
jejunum; ileum; cecum; colon; rectum; urinary bladder; representative 
lymph nodes; thigh musculature; peripheral nerve; spinal cord at three 
levels cervical, midthoracic, and lumbar; and eyes. Respiratory tract 
tissues should be perfusion preserved in a suitable medium.
    (B) If necropsy cannot be performed immediately after a dead animal 
is discovered during the observation period, the animal should be 
refrigerated (not frozen) at temperatures low enough to minimize 
autolysis. Necropsies should be performed as soon as possible after 
death (normally within 24 to 48 hours).
    (vii) Additional evaluations. In animals surviving 24 hours or more, 
microscopic examination of organs showing evidence of gross pathology 
should be considered since it may yield useful information on the nature 
of acute toxic effects.
    (f) Data and reporting--(1) Treatment of results. Data must be 
summarized in tabular form showing for each test group the number of 
animals at the start of the test, body weights, time of death of 
individual animals at different exposure levels, number of animals 
displaying other signs of toxicity, description of toxic effects and 
necropsy findings. The method used for calculation of the 
LC50 or any other parameters must be specified and 
referenced. Some acceptable methods for parameter estimation are 
described in the references described in paragraphs (g)(1), (g)(2), and 
(g)(3) of this section.
    (2) Evaluation of results. The LC50 value should be 
considered in conjunction with the observed toxic effects and the 
necropsy findings. The evaluation should include the relationship, if 
any, between exposure of animals to the test substance and the incidence 
and severity of all abnormalities including behavioral and clinical 
abnormalities, gross lesions, body weight changes, mortality, and other 
toxic effects.
    (3) Test report. In addition to the reporting requirements specified 
under EPA Good Laboratory Practice Standards at 40 CFR part 792, subpart 
J, the following specific information must be reported. The test report 
shall include:
    (i) Test conditions. (A) Description of exposure apparatus including 
design, type, dimensions.
    (B) Source of air, system for generating the test article as 
particle, aerosol, gas, or vapor.
    (C) Method for conditioning air, equipment for measuring 
temperature, humidity, particle size or particulate aerosol 
concentration size, and actual concentration.
    (D) Treatment of exhaust air and the method of housing the animals 
in a test chamber when this is used.
    (ii) Exposure data. The exposure data must be tabulated and 
presented with mean values and a measure of variability (e.g., standard 
deviation) and should include:
    (A) Chemical purity of the test material.
    (B) Airflow rates through the inhalation equipment.
    (C) Temperature and humidity of the air.
    (D) Nominal concentration (total amount of test substance fed into 
the inhalation equipment divided by volume of air).
    (E) Actual (analytical or gravimetric) concentration in test 
breathing zone.
    (F) Particle size distribution (calculated MMAD and GSD) and the 
bivariate distribution of fiber length and diameter, where appropriate.
    (G) Explanation as to why the desired chamber concentration and/or 
particle size could not be achieved (if applicable), and the efforts 
taken to comply with these aspects of this section.
    (iii) Species, strain, sex, and source of test animals.
    (iv) Method of randomization in assigning animals to test and 
control groups.
    (v) Rationale for selection of species, if other than that 
recommended.
    (vi) Results. Tabulation of individual and test group data by sex 
and exposure concentration level (e.g., number of animals exposed, 
number of animals showing signs of toxicity and number of animals that 
died or were sacrificed during the test).

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    (A) Description of toxic effects including time of onset, duration, 
reversibility, and relationship to the exposure concentration levels.
    (B) Pre-exposure and post-exposure body weight change in animals, 
and weight change during the observation period.
    (C) Time of dosing and time of death during or following exposure.
    (D) Concentration-response curves for mortality and other toxic 
effects (when permitted by the method of determination).
    (E) Gross pathology necropsy findings in the test animals and 
vehicle control animals, if included. Data must be tabulated to show the 
counts and incidence of gross alterations observed for each group tested 
and the number of animals affected by each type of lesion along with the 
location and frequency of each type of lesion.
    (F) Histopathology findings and any additional evaluations (e.g., 
clinical chemistry), if performed.
    (vii) Description of any pretest conditioning, including diet, 
quarantine and treatment for disease.
    (viii) Description of caging conditions, including: number (or 
change in number) of animals per cage, bedding material, ambient 
temperature and humidity, photoperiod, and identification of diet of 
test animals.
    (ix) Manufacturer (source), lot number, and purity of test 
substance.
    (x) Identification and composition of any vehicles (e.g., diluents, 
suspending agents, and emulsifiers) or other materials , if used in 
administering the test substance.
    (xi) A list of references cited in the body of the report. 
References to any published literature used in developing the test 
protocol, performing the testing, making and interpreting observations, 
and compiling and evaluating the results.
    (g) References. For additional background information on this test 
guideline, the following references should be consulted. These 
references are available for inspection at the TSCA Nonconfidential 
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M 
St., NW., Washington, DC, 12 noon to 4 p.m., Monday through Friday, 
except legal holidays.
    (1) Chanter, D.O. and Heywood, R. The LD50 test: some 
considerations of precision. Toxicology Letters 10:303 307 (1982).
    (2) Finney, D.G. Chapter 3 Estimation of the median effective dose, 
Chapter 4 Maximum likelihood estimation. Probit Analysis. 3rd Ed. 
(Cambridge, London. (1971).
    (3) Finney, D.J. The Median Lethal Dose and Its Estimation, Archives 
of Toxicology 56:215 218 (1985).
    (4) Organization for Economic Cooperation and Development. OECD 
Guidelines for the Testing of Chemicals. Final Draft OECD Guideline 425: 
Acute Oral Toxicity: Up-and-Down Procedure to be adopted in the Tenth 
Addendum to the OECD Guidelines for the Testing of Chemicals.
    (5) Organization for Economic Cooperation and Development. OECD 
Guidelines for Testing of Chemicals. Guideline 403: Acute Inhalation 
Toxicity. Adopted: May 12, 1981.
    (6) Organization for Economic Cooperation and Development. OECD 
Guidelines for Testing of Chemicals. Guideline 420: Acute Oral Toxicity 
Fixed Dose Method. Adopted: July 17, 1992.
    (7) Organization for Economic Cooperation and Development. OECD 
Guidelines for Testing of Chemicals. Guideline 423: Acute Oral Toxicity 
Acute Toxic Class Method. Adopted: March 22, 1996.
    (8) U. S. EPA. Interim Policy for Particle Size and Limit 
Concentration Issues in Inhalation Toxicity Studies. 2/1/94. Health 
Effects Division, Office of Pesticide Programs.

[65 FR 78776, Dec. 15, 2000]



Sec. 799.9135  TSCA acute inhalation toxicity with histopathology.

    (a) Scope. This section is intended to meet the testing requirements 
under section 4 of the Toxic Substances Control Act (TSCA). In the 
assessment and evaluation of the potential human health effects of 
chemical substances, it is appropriate to test for acute inhalation 
toxic effects. The goals of this test are to characterize the exposure-
response relationship for sensitive endpoints following acute exposure 
and to characterize toxicologic response following acute high exposures. 
The

[[Page 337]]

latter is of particular concern in relation to spills and other 
accidental releases. This testing is designed to determine the gross 
pathology and histopathology resulting from acute inhalation exposure to 
a substance. Because toxic effects on the respiratory tract are of 
particular concern following inhalation exposure, several indicators of 
respiratory toxicity consisting of histopathology on fixed tissue and 
evaluation of cellular and biochemical parameters in bronchoalveolar 
lavage fluid should be employed. The respiratory histopathology consists 
of specialized techniques to preserve tissues of the respiratory tract 
in order to allow detailed microscopic examination to identify adverse 
effects of chemical substances on this organ system. The bronchoalveolar 
lavage is designed to be a rapid screening test to provide an early 
indicator of pulmonary toxicity by examining biochemical and cytologic 
endpoints of material from the lungs of animals exposed to potentially 
toxic chemical substances. These acute tests are designed to assess the 
relationship, if any, between the animals' exposure to the test 
substance and to demonstrate relationship between the animals' exposure 
and the incidence and severity of observed abnormalities, including 
gross or histopathologic lesions, body weight changes, effects on 
mortality, and any other toxic effects. These acute tests are not 
intended to provide a complete evaluation of the toxicologic effects of 
a substance, and additional functional and morphological evaluations may 
be necessary to assess completely the potential effects produced by a 
chemical substance. Additional tests may include longer-term exposures, 
or more in-depth evaluation of specific organ systems as indicated by 
signs of toxicity following acute exposure.
    (b) Source. This a new section developed by the United States 
Environmental Protection Agency.
    (c) Definitions. The following definitions apply to this section.
    Aerodynamic diameter (dae) refers to the size of 
particles. It is the diameter of a sphere of unit density that behaves 
aerodynamically (has the same settling velocity in air) as the particle 
of the test substance. It is used to compare particles of different 
size, shape, and density, and to predict where in the respiratory tract 
such particles may be primarily deposited.
    Exposure response is the relationship between the exposure 
concentration and the measured toxic response, whether expressed as a 
group mean standard deviation) in the case of a 
continuous variable or as incidence in the case of a quantal variable. 
This definiton should not preclude the exploration of other dose metrics 
in establishing this relationship.
    Geometric standard deviation (GSD) is a dimensionless number equal 
to the ratio between the mass median aerodynamic diameter (MMAD) and 
either 84% or 16% of the diameter size distribution (e.g., MMAD = 2 
[micro]m; 84% = 4 [micro]m; GSD = 4/2 = 2.0.) The MMAD, together with 
the GSD, describe the particle size distribution of an aerosol. Use of 
the GSD may not be valid for non-lognormally distributed aerosols. (If 
the size distribution deviates from the lognormal, it shall be noted).
    Inhalability is the ratio of the number concentration of particles 
of a certain aerodynamic diameter, dae, that are inspired 
through the nose or mouth to the number concentration of the same 
dae present in the inspired volume of ambient air. In humans, 
inhalability can exceed 15 [micro]m dae, whereas inhalability 
dramatically decreases for particles above 4 [micro]m dae in 
small laboratory animals.
    Lower respiratory tract consists of those structures of the 
respiratory tract below the larynx.
    Mass geometric mean aerodynamic diameter or the mass median 
aerodynamic diameter (MMAD) is the calculated aerodynamic diameter that 
divides the particles of an aerosol (a gaseous suspension of fine liquid 
or solid particles) in half, based on the weight of the particles. By 
weight, 50% of the particles will be larger than the MMAD and 50% of the 
particles will be smaller than the MMAD.
    Particle regional deposition is the fraction of inhaled particles 
that deposits in the specific region of the respiratory tract. The major 
mechanisms of particle deposition in the respiratory tract

[[Page 338]]

include impaction, sedimentation, diffusion, interception, and 
electrostatic precipitation. The deposition mechanism that is dominant 
for a given region depends on the respiratory tract architecture and 
ventilation rate of the species and the aerosol particle size and 
distribution. The respiratory tract in both humans and various 
experimental mammals can be divided into three regions on the basis of 
structure, size, and function:
    (1) The extrathoracic region or upper respiratory tract that 
includes the nose, mouth, nasopharynx, oropharynx, laryngopharynx, and 
larynx.
    (2) The tracheobronchial region that includes the trachea, bronchi, 
and bronchioles (including the terminal bronchioles).
    (3) The alveolar region that includes the respiratory bronchioles 
(if present in the species), alveolar ducts, alveolar sacs, and alveoli.
    Respiratory effects are any adverse effects on the structure or 
functions of the respiratory system related to exposure to a chemical 
substance.
    Target organ is any organ found to be a target of toxicity in the 4-
hour (hr) high concentration group as a result of:
    (1) The initial histopathologic examination (respiratory tract, 
liver, kidney, gross lesions); or
    (2) The retrospective histopathologic examination of archived organs 
triggered by their identification as targets of toxicity in a 90-day 
study.
    Toxic effects are any adverse changes (a change that is 
statistically and biologically significant) in the structure or function 
of an experimental animal as a result of exposure to a chemical 
substance.
    Upper respiratory tract consists of those structures of the 
respiratory tract above and including the larynx.
    (d) Principle of the test method. The test substance shall be 
administered to several groups of experimental animals; one 
concentration level and duration being used per group. Bronchoalveolar 
lavage shall be used to evaluate early effects on the respiratory system 
by examining changes in the content of the lavage fluid of the lung. At 
24 hrs following exposure, the animals shall be sacrificed and 
necropsied, and tissue samples from the respiratory tract and other 
major organs will be prepared for microscopic examination. The exposure 
levels at which significant toxic effects on the respiratory organ 
system are produced are compared to those levels that produce other 
toxic effects. As triggered by the results of the 4-hr test, additional 
exposure periods of 1 hr and 8 hrs will be required to determine the 
effect of exposure time on the toxicity observed. A 1-hr exposure study 
can be elected as an option to provide data suitable for risk assessment 
for very short duration exposures as may occur from chemical releases. 
In the absence of adequate toxicological data for 1-hr exposure, the 
Agency will extrapolate to shorter-term exposures from the 4-hr data on 
the basis of concentration alone. This is a conservative method of 
extrapolation, consistent with general Agency methods for deriving 
criteria for short-term exposure from longer-term studies (a 
concentration x time extrapolation would result in higher concentration 
for a shorter duration).
    (e) Test procedures--(1) Animal selection--(i) Species. In general, 
the laboratory rat and mouse should be used. Under some circumstances, 
other species, such as the hamster or guinea pig, may be more 
appropriate, and if these or other species are used, justification 
should be provided.
    (ii) Strain. If rats and mice are used, the use of the F344 rat and 
the B6C3F1 mouse is preferred to facilitate comparison with existing 
data.
    (iii) Age. Young adults shall be used. The weight variation of 
animals used in a test should not exceed 20% of 
the mean weight for each species.
    (iv) Sex. Equal numbers of animals of each sex shall be used for 
each concentration level. The females shall be nulliparous and 
nonpregnant.
    (v) Health status. Body weight and feed consumption are not 
sufficient indicators of the health status of animals prior to 
initiating an inhalation toxicity study. Prior to initiating the study, 
animals shall be monitored for known viral and bacterial respiratory 
pathogens determined by conventional microbiological assays (e.g., 
serology). The animals shall be free from pathogens at the start of 
exposure.

[[Page 339]]

    (2) Number of animals. At least five males and five females shall be 
used in each concentration/duration and control group. Animals shall be 
randomly assigned to treatment and control groups.
    (3) Control groups. The control group shall be a sham-treated group. 
Except for treatment with the test substance, animals in the control 
group shall be handled in a manner identical to the test-group animals. 
Where a vehicle is used to help generate an appropriate concentration of 
the substance in the atmosphere, a vehicle control group shall be used. 
If the 4- and 8-hr exposure studies are conducted concurrently, a 
concurrent 8-hr sham-exposed control group may serve as the control 
group for both the 4-hr and the 8-hr exposure studies, provided there is 
adequate historical control data showing no changes in histopathology or 
bronchoalveolar lavage of controls exposed for 4 and 8 hrs. Similarly, 
if the optional 1-hr exposure study is conducted concurrently with the 
4- and/or 8-hr study, the concurrent control group for those studies may 
also be used for the 1-hr study, provided adequate historical control 
data show no changes in histopathology or bronchoalveolar lavage between 
controls exposed for these time periods.
    (4) Concentration level and concentration selection. For the 4-hr 
study, at least three concentrations shall be used in addition to the 
control group. Ideally, the data generated from the test should be 
sufficient to produce an exposure-response curve. The concentrations can 
either be linearly or logarithmically spaced depending on the 
anticipated steepness of the concentration-response curve. A rationale 
for concentration selection should be provided to indicate that the 
selected concentrations will maximally support detection of 
concentration-response relationship. The high concentration should be 
clearly toxic or a limit concentration, but should not result in an 
incidence of fatalities that would preclude a meaningful evaluation of 
the data. The lowest concentration should define a no-observed-adverse-
effects level (NOAEL).
    (i) Limit concentration. For aerosols and particles, the high 
concentrations need not be greater than 2 mg/L, or concentrations that 
cannot maintain a particle size distribution having an MMAD between 1 
and 4 [micro]m (i.e., a particle size that permits inhalability and 
deposition throughout the respiratory tract). For fibers, the bivariate 
distribution of length and diameter must ensure inhalability. For gases 
and vapors, the concentrations need not be greater than 50,000 ppm or 
50% of the lower explosive limit, whichever is lower. If a test at an 
aerosol or particulate exposure of 2 mg/L (actual concentration of 
respirable substance) for 4 hrs or, where this is not feasible, the 
maximum attainable concentration, using the procedures described for 
this study, produces no observable toxic effects, then a full study 
using three concentrations will not be necessary. Similarly, if a test 
at a gas or vapor exposure of 50,000 ppm or 50% of the lower explosive 
limit, whichever is lower, produces no observable toxic effects, then a 
full study using three concentrations will not be necessary.
    (ii) 8-hr study and optional 1-hr study. If the 8-hr study is 
triggered, three concentrations shall be tested. These concentrations 
should allow for the determination of an effect level and a NOAEL. If 
the option to perform a 1-hr study is elected, three concentrations 
shall be selected and tested in a similar manner.
    (5) Inhalation exposure. Animals can be exposed to the substance by 
either a nose-only procedure or in a whole-body exposure chamber.
    (i) Inhalation chambers. The animals shall be tested in inhalation 
equipment designed to sustain a dynamic airflow for nose-only exposures 
of at least 300 ml/minute/animal or an airflow for whole-body exposures 
of at least 12 to 15 air changes per hr and ensure an adequate oxygen 
content of at least 19% and an evenly distributed exposure atmosphere. 
Where a whole-body chamber is used, its design shall minimize crowding 
by providing individual caging. As a general rule, to ensure stability 
of a chamber atmosphere, the total ``volume'' of the test animals should 
not exceed 5% of the volume of the test chamber.

[[Page 340]]

    (ii) Environmental conditions. The temperature at which the test is 
performed shall be maintained at 22 [deg]C ( 2 
[deg]C). Ideally, the relative humidity should be maintained between 40% 
and 60%, but in certain instances (e.g., tests using water as a 
vehicle), this may not be practical.
    (iii) Exposure periodicity. For acute testing, the exposure design 
shall enable 4 hrs of exposure to the target concentrations, as defined 
by an average of 5% for gases and vapors and 
15% for particles and aerosols. If triggered by 
the results of the 4-hr exposure, additional testing shall be conducted 
in a comparable manner using an 8-hr exposure period.
    (6) Physical measurements. Measurements or monitoring shall be made 
of the following:
    (i) Chemical purity of the test material shall be analyzed.
    (ii) The rate of airflow shall be monitored continuously, but shall 
be recorded at least every 30 minutes.
    (iii) The actual concentrations of the test substance shall be 
measured in the breathing zone. During the exposure period, the actual 
concentrations of the test substance shall be held as constant as 
practical, monitored continuously or intermittently depending on the 
method of analysis, and recorded at least at the beginning, at an 
intermediate time, and at the end of the exposure period. Well-
established and published monitoring methods should be used where 
available. If no standard methods are available, then accuracy and 
precision information must be supplied.
    (iv) During the development of the generating system, appropriate 
particle size analysis shall be performed to establish the stability of 
the aerosol. During exposure, analysis should be conducted as often as 
necessary to determine the consistency of particle size distribution. 
The particle size distribution shall have an MMAD between 1 and 4 
[micro]m. The particle size of hygroscopic materials shall be small 
enough when dry to assure that the size of the particle at saturation 
will still have an MMAD between 1 and 4 [micro]m. Characterization for 
fibers shall include the bivariate distribution of length and diameter; 
this distribution must ensure inhalability.
    (v) If the test substance is present in a mixture, the mass and 
composition of the entire mixture, as well as the principal compound, 
shall be measured.
    (vi) Temperature and humidity shall be monitored continuously, but 
shall be recorded at least every 30 minutes.
    (7) Food and water during exposure period. Food shall be withheld 
during exposure. Water may also be withheld in certain cases.
    (8) Observation period. The bronchoalveolar lavage and respiratory 
pathology shall be conducted 24 hrs following exposure to allow 
expression of signs of toxicity. There is concern that some latency time 
will be required to allow migration of cells and macromolecules into the 
lungs following exposure, and that some pathology may require 
macromolecular synthesis or degradation before cell damage develops.
    (9) Gross pathology. (i) All animals shall be subjected to a full 
gross necropsy which includes examination of orifices and the cranial, 
thoracic, and abdominal cavities and their contents.
    (ii) At least the lungs, liver, kidneys, adrenals, brain, and gonads 
shall be weighed wet, as soon as possible after dissection to avoid 
drying.
    (iii) The following organs and tissues, or representative samples 
thereof, shall be preserved in a suitable medium for possible future 
histopathological examination: All gross lesions; brain-including 
sections of medulla/pons; cerebellar cortex and cerebral cortex; 
pituitary; thyroid/parathyroid; thymus; heart; sternum with bone marrow; 
salivary glands; liver; spleen; kidneys; adrenals; pancreas; gonads; 
accessory genital organs (epididymis, prostrate, and, if present, 
seminal vesicles); aorta; skin; gall bladder (if present); esophagus; 
stomach; duodenum; jejunum; ileum; cecum; colon; rectum; urinary 
bladder; representative lymph nodes; thigh musculature; peripheral 
nerve; spinal cord at three levels cervical, midthoracic, and lumbar; 
and eyes. Respiratory tract tissues shall also be preserved in a 
suitable medium.
    (10) Histopathology. The following histopathology shall be 
performed:

[[Page 341]]

    (i) Full histopathology shall be performed on the respiratory tract, 
liver and kidney of all animals in the control and high concentration 
groups. The histopathology of the respiratory tract is described under 
paragraph (e)(11) of this section.
    (ii) All gross lesions which differ from controls in frequency, 
distribution, type, or severity in all concentration groups.
    (iii) Target organs in all animals, as indicated by the observations 
in the high concentration group in this study. Histopathologic 
examination of target organs in animals at all concentration levels 
(rather than only to the extent necessary to define the NOAEL) can 
support the application of exposure-response analyses such as the 
benchmark concentration approach.
    (iv) Archived organs identified as targets of toxicity from results 
of the 90-day study (if a 90-day study is required for this substance) 
should be elevated in high concentration animals of the 4-hr acute study 
to determine if they are also targets of acute toxicity.
    (11) Respiratory tract histopathology. (i) Representative sections 
of the respiratory tract shall be examined histologically. These shall 
include the trachea, major conducting airways, alveolar region, terminal 
and respiratory bronchioles (if present), alveolar ducts and sacs, and 
interstitial tissues.
    (ii) Care shall be taken that the method used to kill the animal 
does not result in damage to the tissues of the upper or lower 
respiratory tract. The lungs shall be infused with a fixative while in 
an inflated state of fixed pressure.
    (iii) The upper respiratory tract shall be examined for 
histopathologic lesions. This examination shall use a minimum of four 
sections located as specified under paragraphs (e)(11)(iii)(A) through 
(e)(11)(iii)(D) of this section. An evaluation of the nasal vestibule 
shall be conducted. The method described by the reference under 
paragraph (h)(11) of this section should be given consideration. The use 
of additional sections shall be left to the discretion of the study 
pathologist, but consideration should be given to additional sections as 
recommended in the reference under paragraph (h)(8) of this section to 
ensure adequate evaluation of the entire upper respiratory tract, 
particularly the nasopharyngeal meatus. The following transverse 
sections shall be examined:
    (A) Immediately posterior to the upper incisor teeth.
    (B) At the incisor papilla.
    (C) At the second palatal ridge.
    (D) At the level of the first upper molar teeth.
    (iv) The laryngeal mucosa shall be examined for histopathologic 
changes. Sections of the larynx to be examined include the epithelium 
covering the base of the epiglottis, the ventral pouch, and the medial 
surfaces of the vocal processes of the arytenoid cartilages.
    (12) Bronchoalveolar lavage. (i) Animals can be exposed to the 
substance by either a nose-only procedure or in a whole-body exposure 
chamber.
    (ii) Care should be taken that the method used to kill the animal 
results in minimum changes in the fluid of the lungs of the test 
animals.
    (iii) At the appropriate time, the test animals shall be killed and 
the heart-lung including trachea removed in bloc. Alternatively, lungs 
can be lavaged in situ. If the study will not be compromised, one lobe 
of the lungs may be used for lung lavage while the other is fixed for 
histologic evaluation. The lungs should be lavaged using physiological 
saline. The lavages shall consist of two washes, each of which consists 
of approximately 80% (e.g., 5 ml in rats and 1 ml in mice) of the total 
lung volume. Additional washes merely tend to reduce the concentrations 
of the material collected. The lung lavage fluid shall be stored on ice 
at 5 [deg]C until assayed.
    (iv) The following parameters shall be determined in the lavage 
fluid as indicators of cellular damage in the lungs: total protein, cell 
count, and percent leukocytes. In addition, a phagocytosis assay shall 
be performed to determine macrophage activity. Assay methods described 
in the references under paragraphs (h)(1) and (h)(3) of this section may 
be used.
    (13) Combined protocol. The tests described may be combined with any 
other toxicity study, as long as none of

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the requirements of either are violated by the combination.
    (f) Triggered testing. If no adverse effects are seen in the 4-hr 
study as compared with controls, no further testing is necessary. If the 
4-hr study shows positive effects in histopathology or the 
bronchoalveolar lavage, an 8-hr study shall be conducted. Only those 
tissues showing positive results in the 4-hr study must be pursued in 
the follow-up 8-hr study. Similarly, if the option to perform a 1-hr 
study is exercised, only those tissues showing positive results in the 
4-hr study shall be pursued.
    (g) Data reporting and evaluation. The final test report shall 
include the following information:
    (1) Description of equipment and test methods. A description of the 
general design of the experiment and any equipment used shall be 
provided.
    (i) Description of exposure apparatus, including design, type, 
dimensions, source of air, system for generating particles, aerosols, 
gasses, and vapors, method of conditioning air, treatment of exhaust 
air, and the method of housing animals in a test chamber.
    (ii) Description of the equipment for measuring temperature, 
humidity, and particulate aerosol concentration and size.
    (iii) Exposure data shall be tabulated and presented with mean 
values and measure of variability (e.g., standard deviation) and should 
include:
    (A) Chemical purity of the test material.
    (B) Airflow rates through the inhalation equipment.
    (C) Temperature and humidity of air.
    (D) Nominal concentration (total amount of test substance fed into 
the inhalation equipment divided by the volume of air).
    (E) Actual concentration in test breathing zone.
    (F) Particle size distribution (e.g., MMAD with GSD) and the 
bivariate distribution of fiber length and diameter, where appropriate.
    (2) Results--(i) General group animal data. The following 
information shall be arranged by test group exposure level.
    (A) Number of animals exposed.
    (B) Number of animals dying.
    (C) Number of animals showing overt signs of toxicity.
    (D) Pre- and post-exposure body weight change in animals, and weight 
change during the observation period.
    (ii) Counts and incidence of gross alterations observed at necropsy 
in the test and control groups. Data shall be tabulated to show:
    (A) The number of animals used in each group and the number of 
animals in which any gross lesions were found.
    (B) The number of animals affected by each different type of lesion, 
and the locations and frequency of each type of lesion.
    (iii) Counts and incidence of general histologic alterations in the 
test group. Data shall be tabulated to show:
    (A) The number of animals used in each group and the number of 
animals in which any histopathologic lesions were found.
    (B) The number of animals affected by each different type of lesion, 
and the locations, frequency, and average grade of each type of lesion.
    (iv) Counts and incidence of respiratory histopathologic alterations 
by the test group. Data shall be tabulated to show:
    (A) The number of animals used in each group and the number of 
animals in which any histopathologic lesions were found.
    (B) The number of animals affected by each different type of lesion, 
and the locations, frequency, and average grade of each type of lesion.
    (v) Results of the bronchoalveolar lavage study. Data shall be 
tabulated to show:
    (A) The amount of administered lavage fluid and recovered lavage 
fluid for each test animal.
    (B) The magnitude of change of biochemical and cytologic indices in 
lavage fluids at each test concentration for each animal.
    (C) Results shall be quantified as amount of constituent/mL of 
lavage fluid. This assumes that the amount of lavage fluid recovered is 
a representative sample of the total lavage fluid.
    (3) Evaluation of data. The findings from this acute study should be 
evaluated in the context of preceding and/or concurrent toxicity studies 
and any correlated functional findings. The

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evaluation shall include the relationship between the concentrations of 
the test substance and the presence or absence, incidence, and severity 
of any effects. The evaluation should include appropriate statistical 
analyses, for example, parametric tests for continuous data and non-
parametric tests for the remainder. Choice of analyses should consider 
tests appropriate to the experimental design, including repeated 
measures. The report must include concentration-response curves for the 
bronchoalveolar lavage and tables reporting observations at each 
concentration level for necropsy findings and gross, general, and 
respiratory system histopathology.
    (h) Reference. For additional background information on this test 
guideline, the following references should be consulted. These 
references are available for inspection at the TSCA Nonconfidential 
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M 
St., SW., Washington, DC, 12 noon to 4 p.m., Monday through Friday, 
except legal holidays.
    (1) Burleson, G.R., Fuller, L.B., M[eacute]nache, M.G., and Graham, 
J.A. Poly (I): poly (C)-enhanced alveolar peritoneal macrophage 
phagocytosis: Quantification by a new method utilizing fluorescent 
beads. Proceedings of the Society of Experimental Biology and Medicine. 
184:468-476 (1987).
    (2) Gardner, D.E., Crapo, J.D., and McClellan, R.O. (Eds.) 
Toxicology of the Lung. (Raven Press, New York, 1993) pp. i-xii, 1-30.
    (3) Gilmour, G.I., and Selgrade, M.K. A comparison of the pulmonary 
defenses against streptococcal infection in rats and mice following O3 
exposure: Differences in disease susceptibility and neutrophil 
recruitment. Toxicology and Applied Pharmacology. 123:211-218 (1993).
    (4) Henderson, R.F., Benson, J.M., Hahn, F.F., Hobbs, C.H., Jones, 
R.K., Mauderly, J.L., McClellan, R.O., and Pickrell, J.A. New approaches 
for the evaluation of pulmonary toxicity: Bronchoalveolar lavage fluid 
analysis. Fundamental and Applied Toxicology. 5:451-458 (1985).
    (5) Henderson, R.F. Use of bronchoalveolar lavage to detect lung 
damage. Environmental Health Perspectives. 56:115-129 (1984).
    (6) Henderson, R.F., Rebar, A.H., Pickrell, J.A., and Newton, G.J. 
Early damage indicators in the lung. III. Biochemical and cytological 
response of the lung to inhaled metal salts. Toxicology and Applied 
Pharmacology. 50:123-136 (1979).
    (7) McClellan, R.O. and Henderson, R.F. (Eds.) Second edition. 
Concepts in Inhalation Toxicology. (Taylor and Francis, Washington, DC, 
1995) pp.i-xxiv, 1-24, 441-470.
    (8) Mery, S., Gross, E.A., Joyner, D.R., Godo, M., and Morgan, K.T. 
Nasal Diagrams: A Tool for Recording the Distribution of Nasal Lesions 
in Rats and Mice. Toxicologic Pathology. 22:353-372 (1994).
    (9) Phalen, R.F. (Ed) Methods in Inhalation Toxicology. (CRC Press, 
Boca Raton, FL, 1997) pp. i-xii, 1-12.
    (10) Renne, R.A., Gideon, K.M., Miller, R.A., Mellick, P.W., and 
Grumbein, S.L. Histologic methods and interspecies variations in the 
laryngeal histology of F344/N rats and B6C3F1 mice. Toxicology and 
Pathology. 20:44-51 (1992).
    (11) Young, J.T. Histopathologic examination of the rat nasal 
cavity. Fundamental and Applied Toxicology. 1:309-312 (1981).



Sec. 799.9305  TSCA Repeated dose 28-day oral toxicity study in rodents.

    (a) Scope--(1) Applicability. This section is intended to meet 
testing requirements of the Toxic Substances Control Act (TSCA) (15 
U.S.C. 2601).
    (2) Source. The source material used in developing this TSCA test 
guideline is the Office of Prevention, Pesticides and Toxic Substances 
(OPPTS) harmonized test guideline 870.3050 (July 2000, final 
guidelines). This source is available at the address in paragraph (h) of 
this section.
    (b) Purpose. (1) In the assessment and evaluation of the toxic 
characteristics of a chemical, the determination of oral toxicity using 
repeated doses may be carried out after initial information on toxicity 
has been obtained by acute testing. This study provides information on 
the possible health hazards likely to arise from repeated exposure over 
a relatively limited period of time. The method comprises the basic

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repeated dose toxicity study that may be used for chemicals on which a 
90-day study is not warranted (e.g., when the production volume does not 
exceed certain limits) or as a preliminary to a long term study. The 
duration of exposure should normally be 28 days although a 14-day study 
may be appropriate in certain circumstances; justification for use of a 
14-day exposure period should be provided.
    (2) This section places emphasis on neurological effects as a 
specific endpoint, and the need for careful clinical observations of the 
animals, so as to obtain as much information as possible, is stressed. 
The method should identify chemicals with neurotoxic potential, which 
may warrant further in-depth investigation of this aspect. In addition, 
the method may give an indication of immunological effects and 
reproductive organ toxicity.
    (c) Definitions. The definitions in section 3 of TSCA and in 40 CFR 
Part 792--Good Laboratory Practice Standards apply to this section. The 
following definitions also apply to this section.
    Dosage is a general term comprising of dose, its frequency and the 
duration of dosing.
    Dose is the amount of test substance administered. Dose is expressed 
as weight (g, mg) or as weight of test substance per unit weight of test 
animal (e.g., mg/kg), or as constant dietary concentrations (parts per 
million (ppm)).
    No-observed-effects level (NOEL) is the maximum dose used in a study 
which produces no adverse effects. The NOEL is usually expressed in 
terms of the weight of a test substance given daily per unit weight of 
test animals (milligrams per kilograms per day).
    (d) Principle of the test. The test substance is orally administered 
daily in graduated doses to several groups of experimental animals, one 
dose level per group for a period of 28 days. During the period of 
administration the animals are observed closely, each day for signs of 
toxicity. Animals which die or are sacrificed during the test are 
necropsied and at the conclusion of the test surviving animals are 
sacrificed and necropsied.
    (e) Description of the method--(1) Selection of animal species. The 
preferred rodent species is the rat, although other rodent species may 
be used. Commonly used laboratory strains of young healthy adult animals 
should be employed. The females should be nulliparous and non-pregnant. 
Dosing should begin as soon as possible after weaning and, in any case, 
before the animals are 9 weeks old. At the commencement of the study the 
weight variation of animals used should be minimal and not exceed 20% of the mean weight of each sex. Where a repeated 
dose oral study is conducted as a preliminary to a long term study, 
preferably animals from the same strain and source should be used in 
both studies.
    (2) Housing and feeding conditions. The temperature in the 
experimental animal room should be 22 [deg]C (3 
[deg]C). Although the relative humidity should be at least 30% and 
preferably not to exceed 70% other than during room cleaning, the aim 
should be 50-60%. Lighting should be artificial, the sequence being 12 
hours light, 12 hours dark. For feeding, conventional laboratory diets 
may be used with an unlimited supply of drinking water. The choice of 
diet may be influenced by the need to ensure a suitable admixture of a 
test substance when administered by this method. Animals may be housed 
individually, or be caged in small groups of the same sex; for group 
caging, no more than five animals should be housed per cage.
    (3) Preparation of animals. Healthy young adult animals must be 
randomly assigned to the control and treatment groups. Cages should be 
arranged in such a way that possible effects due to cage placement are 
minimized. The animals are identified uniquely and kept in their cages 
for at least 5 days prior to the start of the study to allow for 
acclimatization to the laboratory conditions.
    (4) Preparation of doses. (i) The test compound must be administered 
by gavage or via the diet or drinking water. The method of oral 
administration is dependent on the purpose of the study, and the 
physical/chemical properties of the test material.
    (ii) Where necessary, the test substance is dissolved or suspended 
in a suitable vehicle. It is recommended that, wherever possible, the 
use of an

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aqueous solution/suspension be considered first, followed by 
consideration of a solution/emulsion in oil (e.g., corn oil) and then by 
possible solution in other vehicles. For vehicles other than water the 
toxic characteristics of the vehicle must be known. The stability of the 
test substance in the vehicle should be determined.
    (f) Procedure--(1)Number and sex of animals. At least 10 animals 
(five female and five male) should be used at each dose level. If 
interim sacrifices are planned, the number should be increased by the 
number of animals scheduled to be sacrificed before the completion of 
the study. Consideration should be given to an additional satellite 
group of 10 animals (five per sex) in the control and in the top dose 
group for observation of reversibility, persistence, or delayed 
occurrence of toxic effects, for at least 14 days post treatment.
    (2) Dosage. (i) Generally, at least three test groups and a control 
group should be used, but if from assessment of other data, no effects 
would be expected at a dose of 1000 mg/kg bodyweight/per day, a limit 
test may be performed. If there are no suitable data available, a range 
finding study may be performed to aid the determination of the doses to 
be used. Except for treatment with the test substance, animals in the 
control group should be handled in an identical manner to the test group 
subjects. If a vehicle is used in administering the test substance, the 
control group should receive the vehicle in the highest volume used.
    (ii) Dose levels should be selected taking into account any existing 
toxicity and (toxico-) kinetic data available for the test compound or 
related materials. The highest dose level should be chosen with the aim 
of inducing toxic effects but not death or severe suffering. Thereafter, 
a descending sequence of dose levels should be selected with a view to 
demonstrating any dosage related response and NOEL at the lowest dose 
level. Two to four fold intervals are frequently optimal for setting the 
descending dose levels and addition of a fourth test group is often 
preferable to using very large intervals (e.g., more than a factor of 
10) between dosages.
    (3) Limit test. If a test at one dose level of at least 1000 mg/kg 
body weight/day or, for dietary or drinking water administration, an 
equivalent percentage in the diet, or drinking water (based upon body 
weight determinations), using the procedures described for this study, 
produces no observable toxic effects and if toxicity would not be 
expected based upon data from structurally related compounds, then a 
full study using three dose levels may not be considered necessary. The 
limit test applies except when human exposure indicates the need for a 
higher dose level to be used.
    (4) Administration of doses. (i) The animals are dosed with the test 
substance daily 7 days each week for a period of 28 days; use of a 5-day 
per week dosing regime or a 14-day exposure period needs to be 
justified. When the test substance is administered by gavage, this 
should be done in a single dose to the animals using a stomach tube or a 
suitable intubation cannula. The maximum volume of liquid that can be 
administered at one time depends on the size of the test animal. The 
volume should not exceed 1ml/100g body weight, except in the case of 
aqueous solutions where 2ml/100g body weight may be used. Except for 
irritating or corrosive substances which will normally reveal 
exacerbated effects with higher concentrations, variability in test 
volume should be minimized by adjusting the concentration to ensure a 
constant volume at all dose levels.
    (ii) For substances administered via the diet or drinking water it 
is important to ensure that the quantities of the test substance 
involved do not interfere with normal nutrition or water balance. When 
the test substance is administered in the diet either a constant dietary 
concentration (parts per million (ppm)) or a constant dose level in 
terms of the animals' body weight may be used; the alternative used must 
be specified. For a substance administered by gavage, the dose should be 
given at similar times each day, and adjusted as necessary to maintain a 
constant dose level in terms of animal body weight. Where a repeated 
dose study is used as a preliminary to a long term study, a similar diet 
should be used in both studies.

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    (5) Observations. (i) The observation period should be 28 days, 
unless the study duration is 14 days (see paragraph (b)(1) of this 
section). Animals in a satellite group scheduled for follow-up 
observations should be kept for at least a further 14 days without 
treatment to detect delayed occurrence, or persistence of, or recovery 
from toxic effects.
    (ii) General clinical observations should be made at least once a 
day, preferably at the same time(s) each day and considering the peak 
period of anticipated effects after dosing. The health condition of the 
animals should be recorded. At least twice daily, all animals are 
observed for morbidity and mortality.
    (iii) Once before the first exposure (to allow for within-subject 
comparisons), and at least once a week thereafter, detailed clinical 
observations should be made in all animals. These observations should be 
made outside the home cage in a standard arena and preferably at the 
same time, each time. They should be carefully recorded, preferably 
using scoring systems, explicitly defined by the testing laboratory. 
Effort should be made to ensure that variations in the test conditions 
are minimal and that observations are preferably conducted by observers 
unaware of the treatment. Signs noted should include, but not be limited 
to, changes in skin, fur, eyes, mucous membranes, occurrence of 
secretions and excretions and autonomic activity (e.g., lacrimation, 
piloerection, pupil size, unusual respiratory pattern). Changes in gait, 
posture and response to handling as well as the presence of clonic or 
tonic movements, stereotypies (e.g., excessive grooming, repetitive 
circling) or bizarre behaviour (e.g., self-mutilation, walking 
backwards) should also be recorded.
    (iv) In the fourth exposure week sensory reactivity to stimuli of 
different types (see paragraph (h)(2) of this section) (e.g., auditory, 
visual and proprioceptive stimuli), assessment of grip strength and 
motor activity assessment should be conducted. Further details of the 
procedures that could be followed are given in the respective 
references. However, alternative procedures than those referenced could 
also be used. Examples of procedures for observation are described in 
the references in paragraphs (h)(1), (h)(2), (h)(3), (h)(4), and (h)(5) 
of this section.
    (v) Functional observations conducted in the fourth exposure week 
may be omitted when the study is conducted as a preliminary study to a 
subsequent subchronic (90-day) study. In that case, the functional 
observations should be included in this follow-up study. On the other 
hand, the availability of data on functional observations from the 
repeated dose study may enhance the ability to select dose levels for a 
subsequent subchronic study.
    (vi) Exceptionally, functional observations may also be omitted for 
groups that otherwise reveal signs of toxicity to an extent that would 
significantly interfere with the functional test performance.
    (6) Body weight and food/water consumption. All animals should be 
weighed at least once a week. Measurements of food consumption should be 
made at least weekly. If the test substance is administered via the 
drinking water, water consumption should also be measured at least 
weekly.
    (7) Hematology. (i) The following hematological examinations should 
be made at the end of the test period: hematocrit, hemoglobin 
concentration, erythrocyte count, total and differential leukocyte 
count, platelet count and a measure of blood clotting time/potential.
    (ii) Blood samples should be taken from a named site just prior to 
or as part of the procedure for sacrificing the animals, and stored 
under appropriate conditions.
    (8) Clinical Biochemistry. (i) Clinical biochemistry determinations 
to investigate major toxic effects in tissues and, specifically, effects 
on kidney and liver, should be performed on blood samples obtained of 
all animals just prior to or as part of the procedure for sacrificing 
the animals (apart from those found moribund and/or intercurrently 
sacrificed). Overnight fasting of the animals prior to blood sampling is

[[Page 347]]

recommended. \1\ Investigations of plasma or serum shall include sodium, 
potassium, glucose, total cholesterol, urea, creatinine, total protein 
and albumin, at least two enzymes indicative of hepatocellular effects 
(such as alanine aminotransferase, aspartate aminotransferase, alkaline 
phosphatase, gamma glutamyl transpeptidase, and sorbitol dehydrogenase). 
Measurements of additional enzymes (of hepatic or other origin) and bile 
acids may provide useful information under certain circumstances.
---------------------------------------------------------------------------

    \1\ For a number of measurements in serum and plasma, most notably 
for glucose, overnight fasting would be preferable. The major reason for 
this preference is that the increased variability which would inevitably 
result from non-fasting, would tend to mask more subtle effects and make 
interpretation difficult. On the other hand, however, overnight fasting 
may interfere with the general metabolism of the animals and, 
particularly in feeding studies, may disturb the daily exposure to the 
test substance. If overnight fasting is adopted, clinical biochemical 
determinations should be performed after the conduct of functional 
observations in week 4 of the study.
---------------------------------------------------------------------------

    (ii) Optionally, the following urinalysis determinations could be 
performed during the last week of the study using timed urine volume 
collection; appearance, volume, osmolality or specific gravity, pH, 
protein, glucose and blood and blood cells.
    (iii) In addition, studies to investigate serum markers of general 
tissue damage should be considered. Other determinations that should be 
carried out if the known properties of the test substance may, or are 
suspected to, affect related metabolic profiles include calcium, 
phosphate, fasting triglycerides, specific hormones, methemoglobin and 
cholinesterase. These must to be identified for chemicals in certain 
classes or on a case-by-case basis.
    (iv) Overall, there is a need for a flexible approach, depending on 
the species and the observed and/or expected effect with a given 
compound.
    (v) If historical baseline data are inadequate, consideration should 
be given to determination of hematological and clinical biochemistry 
variables before dosing commences.
    (9) Pathology--(i)Gross necropsy. (A) All animals in the study must 
be subjected to a full, detailed gross necropsy which includes careful 
examination of the external surface of the body, all orifices, and the 
cranial, thoracic and abdominal cavities and their contents. The liver, 
kidneys, adrenals, testes, epididymides, thymus, spleen, brain and heart 
of all animals (apart from those found moribund and/or intercurrently 
sacrificed) should be trimmed of any adherent tissue, as appropriate, 
and their wet weight taken as soon as possible after dissection to avoid 
drying.
    (B) The following tissues should be preserved in the most 
appropriate fixation medium for both the type of tissue and the intended 
subsequent histopathological examination: all gross lesions, brain 
(representative regions including cerebrum, cerebellum and pons), spinal 
cord, stomach, small and large intestines (including Peyer's patches), 
liver, kidneys, adrenals, spleen, heart, thymus, thyroid, trachea and 
lungs (preserved by inflation with fixative and then immersion), 
ovaries, uterus, testes, epididymides, accessory sex organs (e.g., 
prostate, seminal vesicles), urinary bladder, lymph nodes (preferably 
one lymph node covering the route of administration and another one 
distant from the route of administration to cover systemic effects), 
peripheral nerve (sciatic or tibial) preferably in close proximity to 
the muscle, and a section of bone marrow (or, alternatively, a fresh 
mounted bone marrow aspirate). The clinical and other findings may 
suggest the need to examine additional tissues. Also any organs 
considered likely to be target organs based on the known properties of 
the test substance should be preserved.
    (ii) Histopathology. (A) Full histopathology should be carried out 
on the preserved organs and tissues of all animals in the control and 
high dose groups. These examinations should be extended to animals of 
all other dosage groups, if treatment-related changes are observed in 
the high dose group.
    (B) All gross lesions must be examined.

[[Page 348]]

    (C) When a satellite group is used, histopathology should be 
performed on tissues and organs identified as showing effects in the 
treated groups.
    (g) Data and reporting--(1) Data. (i) Individual data should be 
provided. Additionally, all data should be summarized in tabular form 
showing for each test group the number of animals at the start of the 
test, the number of animals found dead during the test or sacrificed for 
humane reasons and the time of any death or humane sacrifice, the number 
showing signs of toxicity, a description of the signs of toxicity 
observed, including time of onset, duration, and severity of any toxic 
effects, the number of animals showing lesions, the type of lesions and 
the percentage of animals displaying each type of lesion.
    (ii) When possible, numerical results should be evaluated by an 
appropriate and generally acceptable statistical method. The statistical 
methods should be selected during the design of the study.
    (2) Test report. The test report must include the following 
information:
    (i) Test substance:
    (A) Physical nature, purity and physicochemical properties.
    (B) Identification data.
    (ii) Vehicle (if appropriate): Justification for choice of vehicle, 
if other than water.
    (iii) Test animals:
    (A) Species/strain used.
    (B) Number, age and sex of animals.
    (C) Source, housing conditions, diet, etc.
    (D) Individual weights of animals at the start of the test.
    (iv) Test conditions:
    (A) Rationale for dose level selection.
    (B) Details of test substance formulation/diet preparation, achieved 
concentration, stability and homogeneity of the preparation.
    (C) Details of the administration of the test substance.
    (D) Conversion from diet/drinking water test substance concentration 
(parts per million (ppm)) to the actual dose (mg/kg body weight/day), if 
applicable.
    (E) Details of food and water quality.
    (v) Results:
    (A) Body weight/body weight changes.
    (B) Food consumption, and water consumption, if applicable.
    (C) Toxic response data by sex and dose level, including signs of 
toxicity.
    (D) Nature, severity and duration of clinical observations (whether 
reversible or not).
    (E) Sensory activity, grip strength and motor activity assessments.
    (F) Hematological tests with relevant base-line values.
    (G) Clinical biochemistry tests with relevant base-line values.
    (H) Body weight at sacrificing and organ weight data.
    (I) Necropsy findings.
    (J) A detailed description of all histopathological findings.
    (K) Absorption data if available.
    (L) Statistical treatment of results, where appropriate.
    (vi) Discussion of results.
    (vii) Conclusions.
    (h) References. For additional background information on this test 
guideline, the following references should be consulted. These 
references are available for inspection at the TSCA Nonconfidential 
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M 
St., SW., Washington, DC, 12 noon to 4 p.m., Monday through Friday, 
except legal holidays.
    (1) Tupper, D.E., Wallace, R.B. (1980). Utility of the Neurologic 
Examination in Rats. Acta Neurobiological Exposure, 40:999-1003.
    (2) Gad, S.C. (1982). A Neuromuscular Screen for Use in Industrial 
Toxicology. Journal of Toxicology and Environmental Health, 9:691-704.
    (3) Moser, V.C., McDaniel, K.M., Phillips, P.M. (1991). Rat Strain 
and Stock Comparisons Using a Functional Observational Battery: Baseline 
Values and Effects of Amitraz. Toxicology and Applied Pharmacology, 
108:267-283.
    (4) Meyer O.A., Tilson H.A., Byrd W.C., Riley M.T. (1979). A Method 
forthe Routine Assessment of Fore- and Hindlimb Grip Strength of Rats 
and Mice. Neurobehavioral Toxicology, 1:233-236.
    (5) Crofton K.M., Howard J.L., Moser V.C., Gill M.W., Reiter L.W., 
Tilson

[[Page 349]]

H.A., MacPhail R.C. (1991). Interlaboratory Comparison of Motor Activity 
Experiments: Implication for Neurotoxicological Assessments. 
Neurotoxicology and Teratology, 13:599-609.

[65 FR 78780, Dec. 15, 2000]



Sec. 799.9310  TSCA 90-day oral toxicity in rodents.

    (a) Scope. This section is intended to meet the testing requirements 
under section 4 of the Toxic Substances Control Act (TSCA). In the 
assessment and evaluation of the toxic characteristics of a chemical, 
the determination of subchronic oral toxicity may be carried out after 
initial information on toxicity has been obtained by acute testing. The 
subchronic oral study has been designed to permit the determination of 
the no-observed-effects level (NOEL) and toxic effects associated with 
continuous or repeated exposure to a test substance for a period of 90 
days. This study is not capable of determining those effects that have a 
long latency period for development (e.g., carcinogenicity and life 
shortening). Extrapolation from the results of this study to humans is 
valid only to a limited degree. However, it can useful in providing 
information on health hazards likely to arise from repeated exposure by 
the oral route over a limited period of time, such as target organs, the 
possibilities of accumulation, and can be of use in selecting dose 
levels for chronic studies and for establishing safety criteria for 
human exposure.
    (b) Source. The source material used in developing this TSCA test 
guideline is the Office of Prevention, Pesticides, and Toxic Substances 
(OPPTS) harmonized test guideline 870.3100 (August 1998, final 
guideline). This source is available at the address in paragraph (h) of 
this section.
    (c) Definitions. The following definitions apply to this section.
    Cumulative toxicity is the adverse effects of repeated doses 
occurring as a result of prolonged action on, or increased concentration 
of, the administered test substance or its metabolites in susceptible 
tissue.
    Dose in a subchronic oral study is the amount of test substance 
administered daily via the oral route (gavage, drinking water or diet) 
for a period of 90 days. Dose is expressed as weight of the test 
substance (grams, milligrams) per unit body weight of test animal 
(milligram per kilogram) or as weight of the test substance in parts per 
million in food or drinking water per day.
    No-observed-effects level (NOEL) is the maximum dose used in a study 
which produces no adverse effects. The NOEL is usually expressed in 
terms of the weight of a test substance given daily per unit weight of 
test animal (milligrams per kilogram per day).
    Subchronic oral toxicity is the adverse effects occurring as a 
result of the repeated daily exposure of experimental animals to a 
chemical by the oral route for a part (approximately 10%) of the test 
animal's life span.
    Target organ is any organ of a test animal showing evidence of an 
effect induced by a test substance.
    (d) Limit test. If a test at one dose level of at least 1,000 mg/kg 
body weight (expected human exposure may indicate the need for a higher 
dose level), using the procedures described for this study, produces no 
observable toxic effects or if toxic effects would not be expected based 
upon data of structurally related compounds, then a full study using 
three dose levels might not be necessary.
    (e) Test procedures--(1) Animal selection--(i) Species and strain. A 
variety of rodent species may be used, although the rat is the preferred 
species. Commonly used laboratory strains must be employed.
    (ii) Age/weight. (A) Testing should be started with young healthy 
animals as soon as possible after weaning and acclimatization.
    (B) Dosing of rodents should generally begin no later than 8-9 weeks 
of age.
    (C) At the commencement of the study the weight variation of animals 
used must be within 20% of the mean weight for each sex.
    (iii) Sex. Equal numbers of animals of each sex must be used at each 
dose level, and the females shall be nulliparous and nonpregnant.
    (iv) Numbers. (A) At least 20 rodents (10 males and 10 females) at 
each dose level.

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    (B) If interim sacrifices are planned, the number must be increased 
by the number of animals scheduled to be sacrificed before the 
completion of the study.
    (C) To avoid bias, the use of adequate randomization procedures for 
the proper allocation of animals to test and control groups is required.
    (D) Each animal must be assigned a unique identification number. 
Dead animals, their preserved organs and tissues, and microscopic slides 
must be identified by reference to the animal's unique number.
    (v) Husbandry. (A) Animals may be group-caged by sex, but the number 
of animals per cage must not interfere with clear observation of each 
animal. The biological properties of the test substance or toxic effects 
(e.g., morbidity, excitability) may indicate a need for individual 
caging.
    (B) The temperature of the experimental animal rooms should be at 22 
3 [deg]C.
    (C) The relative humidity of the experimental animal rooms should be 
50 20%.
    (D) Where lighting is artificial, the sequence should be 12 hours 
light/12 hours dark.
    (E) Control and test animals must be fed from the same batch and 
lot. The feed should be analyzed to assure adequacy of nutritional 
requirements of the species tested and for impurities that might 
influence the outcome of the test. For feeding, conventional laboratory 
diets may be used with an unlimited supply of drinking water.
    (F) The study should not be initiated until animals have been 
allowed a period of acclimatization/quarantine to environmental 
conditions, nor should animals from outside sources be placed on test 
without an adequate period of quarantine. An acclimation period of at 
least five days is recommended.
    (2) Control and test substances. (i) Where necessary, the test 
substance is dissolved or suspended in a suitable vehicle. If a vehicle 
or diluent is needed, the vehicle should not elicit toxic effects or 
substantially alter the chemical or toxicological properties of the test 
substance. It is recommended that wherever possible the usage of an 
aqueous solution be considered first, followed by consideration of a 
solution in oil and then solution in other vehicles.
    (ii) If possible, one lot of the test substance tested should be 
used throughout the duration of the study and the research sample should 
be stored under conditions that maintain its purity and stability. Prior 
to the initiation of the study, there should be a characterization of 
the test substance, including the purity of the test compound and, if 
technically feasible, the names and quantities of contaminants and 
impurities.
    (iii) If the test or control substance is to be incorporated into 
feed or another vehicle, the period during which the test substance is 
stable in such a mixture should be determined prior to the initiation of 
the study. Its homogeneity and concentration should be determined prior 
to the initiation of the study and periodically during the study. 
Statistically randomized samples of the mixture should be analyzed to 
ensure that proper mixing, formulation, and storage procedures are being 
followed, and that the appropriate concentration of the test or control 
substance is contained in the mixture.
    (3) Control groups. A concurrent control group is required. This 
group must be an untreated or sham-treated control group or, if a 
vehicle is used in administering the test substance, a vehicle control 
group. If the toxic properties of the vehicle are not known or cannot be 
made available, both untreated and vehicle control groups are required.
    (4) Satellite group. A satellite group of 20 animals (10 animals per 
sex) may be treated with the high dose level for 90 days and observed 
for reversibility, persistence, or delayed occurrence of toxic effects 
for a post-treatment period of appropriate length, normally not less 
than 28 days. In addition, a control group of 20 animals (10 animals of 
each sex) should be added to the satellite study.
    (5) Dose levels and dose selection. (i) In subchronic toxicity 
tests, it is desirable to determine a dose-response relationship as well 
as a NOEL. Therefore, at least three dose levels plus a control and, 
where appropriate, a vehicle control (corresponding to the concentration 
of vehicle at the highest dose

[[Page 351]]

level) must be used. Doses should be spaced appropriately to produce 
test groups with a range of toxic effects. The data should be sufficient 
to produce a dose-response curve.
    (ii) The highest dose level should result in toxic effects but not 
produce an incidence of fatalities which would prevent a meaningful 
evaluation.
    (iii) The intermediate dose levels should be spaced to produce a 
gradation of toxic effects.
    (iv) The lowest dose level should produce no evidence of toxicity.
    (6) Administration of the test substance. (i) If the test substance 
is administered by gavage, the animals are dosed with the test substance 
on a 7-day per week basis for a period of at least 90 days. However, 
based primarily on practical considerations, dosing by gavage on a 5-day 
per week basis is acceptable. If the test substance is administered in 
the drinking water, or mixed in the diet, then exposure should be on a 
7-day per week basis.
    (ii) All animals must be dosed by the same method during the entire 
experimental period.
    (iii) For substances of low toxicity, it is important to ensure that 
when administered in the diet the quantities of the test substance 
involved do not interfere with normal nutrition. When the test substance 
is administered in the diet, either a constant dietary concentration 
(parts per million) or a constant dose level in terms of body weight 
should be used; the alternative used should be specified.
    (iv) For a substance administered by gavage, the dose should be 
given at approximately the same time each day, and adjusted at intervals 
(weekly or biweekly) to maintain a constant dose level in terms of body 
weight.
    (7) Observation period. (i) The animals must be observed for a 
period of 90 days.
    (ii) Animals in the satellite group (if used) scheduled for follow-
up observations should be kept for at least 28 days further without 
treatment to detect recovery from, or persistence of, toxic effects.
    (8) Observation of animals. (i) Observations must be made at least 
twice each day for morbidity and mortality. Appropriate actions should 
be taken to minimize loss of animals to the study (e.g., necropsy or 
refrigeration of those animals found dead and isolation or sacrifice of 
weak or moribund animals). General clinical observations should be made 
at least once a day, preferably at the same time each day, taking into 
consideration the peak period of anticipated effects after dosing. The 
clinical condition of the animal should be recorded.
    (ii) A careful clinical examination must be made at least once 
weekly. Observations should be detailed and carefully recorded, 
preferably using explicity defined scales. Observations should include, 
but not be limited to, evaluation of skin and fur, eyes and mucous 
membranes, respiratory and circulatory effects, autonomic effects such 
as salivation, central nervous system effects, including tremors and 
convulsions, changes in the level of activity, gait and posture, 
reactivity to handling or sensory stimuli, altered strength, and 
stereotypes or bizarre behavior (e.g., self-mutilation, walking 
backwards).
    (iii) Signs of toxicity should be recorded as they are observed 
including the time of onset, degree and duration.
    (iv) Measurements of food consumption and water consumption, if 
drinking water is the exposure route, must be made weekly.
    (v) Individual weights of animals must be determined shortly before 
the test substance is administered, weekly thereafter, and at death.
    (vi) Moribund animals should be removed and sacrificed when noticed 
and the time of death should be recorded as precisely as possible.
    (vii) At termination, all survivors in the treatment and control 
groups must be sacrificed.
    (9) Clinical pathology. Hematology and clinical chemistry 
examinations must be made on all animals, including controls, of each 
sex in each group. The hematology and clinical chemistry parameters 
should be examined at terminal sacrifice at the end of the study. 
Overnight fasting of the animals prior to blood sampling is recommended. 
Overall, there is a need for a flexible approach in the measures 
examined, depending on the observed or expected

[[Page 352]]

effects from a chemical, and in the frequency of measures, depending on 
the duration of potential chemical exposures.
    (i) Hematology. The recommended parameters are red blood cell count, 
hemoglobin concentration, hematocrit, mean corpuscular volume, mean 
corpuscular hemoglobin, and mean corpuscular hemoglobin concentration, 
white blood cell count, differential leukocyte count, platelet count, 
and a measure of clotting potential, such as prothrombin time or 
activated partial thromboplastin time.
    (ii) Clinical chemistry. (A) Parameters which are considered 
appropriate to all studies are electrolyte balance, carbohydrate 
metabolism, and liver and kidney function. The selection of specific 
tests will be influenced by observations on the mode of action of the 
substance and signs of clinical toxicity.
    (B) The recommended clinical chemistry determinations are potassium, 
sodium, glucose, total cholesterol, urea nitrogen, creatinine, total 
protein and albumin. More than 2 hepatic enzymes, (such as alanine 
aminotransferase, aspartate aminotransferase, alkaline phosphatase, 
sorbitol dehydrogenase, or gamma glutamyl transpeptidase) should also be 
measured. Measurements of addtional enzymes (of hepatic or other origin) 
and bile acids, may also be useful.
    (C) If a test chemical has an effect on the hematopoietic system, 
reticulocyte counts and bone marrow cytology may be indicated.
    (D) Other determinations that should be carried out if the test 
chemical is known or suspected of affecting related measures include 
calcium, phosphorus, fasting triglycerides, hormones, methemoglobin, and 
cholinesterases.
    (iii) Optionally, the following urinalysis determinations could be 
performed during the last week of the study using timed urine volume 
collection: appearance, volume, osmolality or specific gravity, pH, 
protein, glucose and blood/blood cells.
    (10) Ophthalmological examination. Ophthalmological examinations 
using an ophthalmoscope or an equivalent device must be made on all 
animals prior to the administration of the test substance and on all 
high dose and control groups at termination. If changes in the eyes are 
detected, all animals in the other dose groups must be examined.
    (11) Gross necropsy. (i) All animals must be subjected to a full 
gross necropsy which includes examination of the external surface of the 
body, all orifices, and the cranial, thoracic and abdominal cavities and 
their contents.
    (ii) The liver, kidneys, adrenals, testes, epididymides, ovaries, 
uterus, thymus, spleen, brain, and heart must be trimmed and weighed 
wet, as soon as possible after dissection.
    (iii) The following organs and tissues, or representative samples 
thereof, should be preserved in a suitable medium for possible future 
histopathological examination:
    (A) Digestive system--salivary glands, esophagus, stomach, duodenum, 
jejunum, ileum, cecum, colon, rectum, liver, pancreas, gallbladder (when 
present).
    (B) Nervous system--brain (including sections of medulla/pons, 
cerebellum and cerebrum), pituitary, peripheral nerve (sciatic or 
tibial, preferably in close proximity to the muscle), spinal cord (three 
levels: cervical, mid-thoracic and lumbar), eyes (retina, optic nerve).
    (C) Glandular system--adrenals, parathyroid, thyroid.
    (D) Respiratory system--trachea, lungs, pharynx, larynx, nose.
    (E) Cardiovascular/hemopoietic system--aorta, heart, bone marrow 
(and/or fresh aspirate), lymph nodes (preferably one lymph node covering 
the route of administration and another one distant from the route of 
administration to cover systemic effects), spleen, thymus.
    (F) Urogenital system--kidneys, urinary bladder, prostate, testes, 
epididymides, seminal vesicle(s), uterus, ovaries, female mammary gland.
    (G) Others--all gross lesions and masses, skin.
    (12) Histopathology. (i) The following histopathology must be 
performed:
    (A) Full histopathology on the organs and tissues, listed in 
paragraph (e)(11)(iii) of this section, of all rodents in the control 
and high dose groups, and all rodents that died or were sacrificed 
during the study.

[[Page 353]]

    (B) All gross lesions in all animals.
    (C) Target tissues in all animals.
    (D) When a satellite group is used, histopathology should be 
performed on tissues and organs identified as showing effects in the 
treated groups.
    (ii) If excessive early deaths or other problems occur in the high 
dose group compromising the significance of the data, the next dose 
level should be examined for complete histopathology.
    (iii) An attempt should be made to correlate gross observations with 
microscopic findings.
    (iv) Tissues and organs designated for microscopic examination 
should be fixed in 10% buffered formalin or a recognized suitable 
fixative as soon as necropsy is performed and no less than 48 hours 
prior to trimming.
    (f) Data and reporting--(1) Treatment of results. (i) Data must be 
summarized in tabular form, showing for each test group the number of 
animals at the start of the test, the number of animals showing lesions, 
the types of lesions and the percentage of animals displaying each type 
of lesion.
    (ii) When applicable, all observed results, qualitative and 
quantitative, should be evaluated by an appropriate and generally 
accepted statistical method. Any generally accepted statistical methods 
may be used; the statistical methods, including significance criteria, 
should be selected during the design of the study.
    (2) Evaluation of study results. The findings of a subchronic oral 
toxicity study should be evaluated in conjunction with the findings of 
preceding studies and considered in terms of the toxic effects and the 
necropsy and histopathological findings. The evaluation must include the 
relationship between the dose of the test substance and the presence or 
absence, the incidence and severity, of abnormalities, including 
behavioral and clinical abnormalities, gross lesions, identified target 
organs, body weight changes, effects on mortality and any other general 
or specific toxic effects. A properly conducted subchronic test should 
provide a satisfactory estimation of a NOEL. It also can indicate the 
need for an additional longer-term study and provide information on the 
selection of dose levels.
    (3) Test report. In addition to reporting requirements specified 
under EPA Good Laboratory Practice Standards at 40 CFR part 792, subpart 
J, the following specific information must be reported:
    (i) Test substance characterization should include:
    (A) Chemical identification.
    (B) Lot or batch number.
    (C) Physical properties.
    (D) Purity/impurities.
    (ii) Identification and composition of any vehicle used.
    (iii) Test system should contain data on:
    (A) Species and strain of animals used and rationale for selection 
if other than that recommended.
    (B) Age including body weight data and sex.
    (C) Test environment including cage conditions, ambient temperature, 
humidity, and light/dark periods.
    (D) Identification of animal diet.
    (E) Acclimation period.
    (iv) Test procedure should include the following data:
    (A) Method of randomization used.
    (B) Full description of experimental design and procedure.
    (C) Dose regimen including levels, methods, and volume.
    (v) Test results should include:
    (A) Group animal data. Tabulation of toxic response data by species, 
strain, sex and exposure level for:
    (1) Number of animals exposed.
    (2) Number of animals showing signs of toxicity.
    (3) Number of animals dying.
    (B) Individual animal data. Data should be presented as summary 
(group mean) as well as for individual animals.
    (1) Date of death during the study or whether animals survived to 
termination.
    (2) Date of observation of each abnormal sign and its subsequent 
course.
    (3) Body weight data.
    (4) Feed and water (if collected) consumption data.
    (5) Achieved dose (mg/kg/day) as a time-weighted average if the test 
substance is administered in the diet or drinking water.
    (6) Results of ophthalmological examination.

[[Page 354]]

    (7) Results of hematological tests performed.
    (8) Results of clinical chemistry tests performed.
    (9) Results of urinalysis, if performed.
    (10) Necropsy findings, including absolute and relative (to body 
weight) organ weight data.
    (11) Detailed description of all histopathological findings.
    (12) Statistical treatment of results, where appropriate.
    (g) Quality control. A system must be developed and maintained to 
assure and document adequate performance of laboratory equipment. The 
study must be conducted in compliance with 40 CFR Part 792--Good 
Laboratory Practice Standards.
    (h) References. For additional background information on this test 
guideline, the following references should be consulted. These 
references are available for inspection at the TSCA Nonconfidential 
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M 
St., NW., Washington, DC, 12 noon to 4 p.m., Monday through Friday, 
except legal holidays.
    (1) Boyd, E.M. Chapter 14. Pilot Studies, 15. Uniposal Clinical 
Parameters, 16. Uniposal Autopsy Parameters. Predictive Toxicometrics. 
Williams and Wilkins, Baltimore (1972).
    (2) Fitzhugh, O.G. Subacute Toxicity, Appraisal of the Safety of 
Chemicals in Foods, Drugs and Cosmetics. The Association of Food and 
Drug Officials of the United States (1959, 3rd Printing 1975) pp. 26-35.
    (3) Organization for Economic Cooperation and Development. OECD 
uidelines for Testing of Chemicals. Guideline 408: Subchronic Oral 
Toxicity-Rodent: 90-day Study, Adopted: May 12, 1981.
    (4) Weingand K., Brown G., Hall R. et al. Harmonization of Animal 
Clinical Pathology Testing in Toxicity and Safety Studies. Fundam. & 
Appl. Toxicol. 29:198-201. (1996)

[65 FR 78783, Dec. 15, 2000]



Sec. 799.9325  TSCA 90-day dermal toxicity.

    (a) Scope. This section is intended to meet the testing requirements 
under section 4 of the Toxic Substances Control Act (TSCA). In the 
assessment and evaluation of the toxic characteristics of a chemical, 
the determination of subchronic dermal toxicity may be carried out after 
initial information on toxicity has been obtained by acute testing. The 
subchronic dermal study has been designed to permit the determination of 
the no-observed-effects level (NOEL) and toxic effects associated with 
continuous or repeated exposure to a test substance for a period of 90 
days. This study is not capable of determining those effects that have a 
long latency period for development (e.g., carcinogenicity and life 
shortening). Extrapolation from the results of this study to humans is 
valid only to a limited degree. It can, however, provide useful 
information on the degree of percutaneous absorption, target organs, the 
possibilities of accumulation, and can be of use in selecting dose 
levels for chronic studies and for establishing safety criteria for 
human exposure.
    (b) Source. The source material used in developing this TSCA test 
guideline is the Office of Prevention, Pesticides, and Toxic Substances 
(OPPTS) harmonized test guideline 870.3250 (August 1998, final 
guideline). This source is available at the address in paragraph (h) of 
this section.
    (c) Definitions. The following definitions also apply to this 
section.
    Cumulative toxicity is the adverse effect of repeated doses 
occurring as a result of prolonged action or increased concentration of 
the administered test substance or its metabolites in susceptible 
tissues.
    Dose in a subchronic dermal study is the amount of test substance 
applied daily to the skin for 90 days. Dose is expressed as weight of 
the test substance (grams, milligrams), per unit body weight of test 
animal (milligrams per kilogram), or as weight of the test substance per 
unit of surface area (milligrams per square centimeter) per day.
    No-observed-effects level (NOEL) is the maximum dose used in a study 
which produces no adverse effects. The NOEL is expressed in terms of the 
weight of a test substance given daily per unit weight of test animal 
(milligrams per kilogram per day).

[[Page 355]]

    Subchronic dermal toxicity is the adverse effects occurring as a 
result of the repeated daily exposure of experimental animals to a 
chemical by the dermal route for a part of the test animal's life span.
    Target organ is any organ of a test animal showing evidence of an 
effect induced by a test substance.
    (d) Limit test. If a test at one dose level of at least 1,000 mg/kg 
body weight (expected human exposure may indicate the need for a higher 
dose level), using the procedures described for this section, produces 
no observable toxic effects or if toxic effects would not be expected 
based upon data on structurally related compounds, a full study using 
three dose levels might not be necessary.
    (e) Test procedures--(1) Animal selection--(i) Species and strain. A 
mammalian species must be used for testing. The rat, rabbit, or guinea 
pig may be used. Commonly used laboratory strains must be employed. If 
other mammalian species are used, the tester must provide justification/
reasoning for their selection. When a subchronic dermal study is 
conducted as a preliminary to a chronic dermal study, the same species 
and strain must be used in both studies.
    (ii) Age/weight. (A) Testing should be started with young healthy 
animals as soon as possible after weaning and acclimatization.
    (B) Dosing should generally begin in guinea pigs between 5-6 weeks 
of age, in rats between 8-9 weeks of age, and in rabbits at least 12 
weeks old.
    (C) At the commencement of the study, the weight variation of 
animals used must be within 20% of the mean weight for each sex.
    (iii) Sex. Equal numbers of animals of each sex with healthy skin 
must be used at each dose level. The females shall be nulliparous and 
nonpregnant except for specially designed studies.
    (iv) Numbers. (A) At least 20 animals (10 animals per sex) must be 
used at each dose level.
    (B) If interim sacrifices are planned, the number must be increased 
by the number of animals scheduled to be sacrificed before completion of 
the study.
    (C) To avoid bias, the use of adequate randomization procedures for 
the proper allocation of animals to test and control groups is required.
    (D) Each animal must be assigned a unique identification number. 
Dead animals, their preserved organs and tissues, and microscopic slides 
must be identified by reference to the animal's unique number.
    (v) Husbandry. (A) Animals should be housed in individual cages.
    (B) The temperature of the experimental animal rooms should be at 22 
3 [deg]C
    (C) The relative humidity of the experimental animal rooms should be 
50 20%.
    (D) Where lighting is artificial, the sequence should be 12 hours 
light/12 hours dark.
    (E) Control and test animals must be fed from the same batch and 
lot. The feed should be analyzed to assure adequacy of nutritional 
requirements of the species tested and for impurities that might 
influence the outcome of the test. For feeding, conventional laboratory 
diets may be used with an unlimited supply of drinking water.
    (F) The study should not be initiated until animals have been 
allowed a period of acclimatization/quarantine to environmental 
conditions, nor should animals from outside sources be placed on test 
without an adequate period of quarantine. An acclimation period of at 
least five days is recommended.
    (2) Control and test substances. (i) Where necessary, the test 
substance is dissolved or suspended in a suitable vehicle. If a vehicle 
or diluent is needed, the vehicle should not elicit toxic effects or 
substantially alter the chemical or toxicological properties of the test 
substance. It is recommended that, whenever possible, the usage of an 
aqueous solution be considered first, followed by consideration of a 
solution of oil and then solution of other vehicles.
    (ii) One lot of the test substance should be used, if possible, 
throughout the duration of the study, and the research sample should be 
stored under conditions that maintain its purity and stability. Prior to 
the initiation of the study, there should be a characterization of the 
test substance, including the purity of the test compound and if 
technically feasible, the name and

[[Page 356]]

quantities of unknown contaminants and impurities.
    (iii) If the test substance is dissolved or suspended in a vehicle, 
the period during which the test substance is stable in such a mixture 
should be determined prior to the initiation of the study. Its 
homogeneity and concentration should be determined prior to the 
initiation of the study and periodically during the study. Statistically 
randomized samples of the mixture should be analyzed to ensure that 
proper mixing, formulation, and storage procedures are being followed, 
and that the appropriate concentration of the test or control substance 
is contained in the mixture.
    (3) Control groups. A concurrent control group is required. This 
group must be an untreated or sham-treated control group or, if a 
vehicle is used in the application of the test substance, a vehicle 
control group. If the toxic properties of the vehicle are not known or 
not available, both untreated/sham-treated and vehicle control groups 
are required.
    (4) Satellite group. A satellite group of 20 animals (10 animals per 
sex) may be treated with the high dose level for 90 days and observed 
for reversibility, persistence, or delayed occurrence of toxic effects 
for a post-treatment period of appropriate length, normally not less 
than 28 days. In addition a control group of 20 animals (10 animals per 
sex) should be added to the satellite study.
    (5) Dose levels and dose selection. (i) In subchronic toxicity 
tests, it is desirable to determine a dose-response relationship as well 
as a NOEL. Therefore, at least three dose levels plus a control and, 
where appropriate, a vehicle control (corresponding to the concentration 
of vehicle at the highest dose level) group shall be used. Doses should 
be spaced appropriately to produce test groups with a range of toxic 
effects. The data should be sufficient to produce a dose-response curve.
    (ii) The highest dose level should elicit signs of toxicity but not 
produce severe skin irritation or an incidence of fatality which would 
prevent a meaningful evaluation. If application of the test substance 
produces severe skin irritation, the concentration may be reduced, 
although this may result in a reduction in, or absence of, other toxic 
effects at the high dose level. If the skin has been badly damaged early 
in the study, it may be necessary to terminate the study and undertake a 
new one at lower concentrations.
    (iii) The intermediate dose levels should be spaced to produce a 
gradation of toxic effects.
    (iv) The lowest dose level should not produce any evidence of toxic 
effects.
    (6) Preparation of animal skin. Shortly before testing, fur must be 
clipped from not less than 10% of the body surface area for application 
of the test substance. In order to dose approximately 10% of the body 
surface, the area starting at the scapulae (shoulders) to the wing of 
the ileum (hipbone) and half way down the flank on each side of the 
animal should be shaved. Shaving should be carried out approximately 24 
hours before dosing. Repeated clipping or shaving is usually needed at 
approximately weekly intervals. When clipping or shaving the fur, care 
should be taken to avoid abrading the skin which could alter its 
permeability.
    (7) Preparation of test substance. (i) Liquid test substances are 
generally used undiluted, except as indicated in paragraph (e)(5)(ii) of 
this section.
    (ii) Solids should be pulverized when possible. The substance should 
be moistened sufficiently with water or, when necessary, a suitable 
vehicle to ensure good contact with the skin. When a vehicle is used, 
the influence of the vehicle on toxicity of, and penetration of the skin 
by, the test substance should be taken into account.
    (iii) The volume of application should be kept constant, e.g., less 
than 300 [micro]L for the rat; different concentrations of test solution 
shall be prepared for different dose levels.
    (8) Administration of test substance. (i) The duration of exposure 
should be at least for 90 days.
    (ii) Ideally, the animals should be treated with test substance for 
at least 6 hours per day on a 7-day per week basis. However, based on 
practical considerations, application on a 5-day per week basis is 
acceptable. Dosing should be conducted at approximately the same time 
each day.

[[Page 357]]

    (iii) The test substance must be applied uniformly over the 
treatment site.
    (iv) The surface area covered may be less for highly toxic 
substances. As much of the area should be covered with as thin and 
uniform a film as possible.
    (v) During the exposure period, the test substance must be held in 
contact with the skin with a porous gauze dressing (less than or equal 
to 8 ply). The test site must be further covered with nonirritating tape 
to retain the gauze dressing and the test substance and to ensure that 
the animals cannot ingest the test substance. Restrainers may be used to 
prevent the ingestion of the test substance, but complete immobilization 
is not recommended. The test substance may be wiped from the skin after 
the six-hour exposure period to prevent ingestion.
    (9) Observation of animals. (i) Observations must be made at least 
twice each day for morbidity and mortality. Appropriate actions should 
be taken to minimize loss of animals to the study (e.g., necropsy or 
refrigeration of those animals found dead and isolation or sacrifice of 
weak or moribund animals). General clinical observations must be made at 
least once a day, preferably at the same time each day, taking into 
consideration the peak period of anticipated effects after dosing. The 
clinical condition of the animal should be recorded.
    (ii) A careful clinical examination must be made at least once 
weekly. Observations should be detailed and carefully recorded, 
preferably using explicity defined scales. Observations should include, 
but not be limited to, evaluation of skin and fur, eyes and mucous 
membranes, respiratory and circulatory effects, autonomic effects such 
as salivation, central nervous system effects, including tremors and 
convulsions, changes in the level of activity, gait and posture, 
reactivity to handling or sensory stimuli, altered strength, and 
stereotypes or bizarre behavior (e.g., self-mutilation, walking 
backwards).
    (iii) Signs of toxicity should be recorded as they are observed 
including the time of onset, degree and duration.
    (iv) Individual weights of animals must be determined shortly before 
the test substance is administered, weekly thereafter, and at death.
    (v) Food consumption must also be determined weekly if abnormal body 
weight changes are observed.
    (vi) Moribund animals should be removed and sacrificed when noticed 
and the time of death should be recorded as precisely as possible.
    (vii) At termination, all survivors in the control and treatment 
groups must be sacrificed.
    (10) Clinical pathology. Hematology and clinical chemistry 
examinations must be made on all animals, including controls, of each 
sex in each group. The hematology and clinical chemistry parameters 
should be examined at terminal sacrifice at the end of the study. 
Overnight fasting of the animals prior to blood sampling is recommended. 
Overall, there is a need for a flexible approach in the measures 
examined, depending on the observed or expected effects from a chemical, 
and in the frequency of measures, depending on the duration of potential 
chemical exposures.
    (i) Hematology. The recommended parameters are red blood cell count, 
hemoglobin concentration, hematocrit, mean corpuscular volume, mean 
corpuscular hemoglobin, and mean corpuscular hemoglobin concentration, 
white blood cell count, differential leukocyte count, platelet count, 
and a measure of clotting potential, such as prothrombin time or 
activated partial thromboplastin time.
    (ii) Clinical chemistry. (A) Parameters which are considered 
appropriate to all studies are electrolyte balance, carbohydrate 
metabolism, and liver and kidney function. The selection of specific 
tests will be influenced by observations on the mode of action of the 
substance and signs of clinical toxicity.
    (B) The recommended clinical chemistry determinations are potassium, 
sodium, glucose, total cholesterol, urea nitrogen, creatinine, total 
protein and albumin. More than 2 hepatic enzymes, (such as alanine 
aminotransferase, aspartate aminotransferase, alkaline phosphatase, 
sorbitol dehydrogenase, or gamma glutamyl transpeptidase)

[[Page 358]]

should also be measured. Measurements of additional enzymes (of hepatic 
or other origin) and bile acids, may also be useful.
    (C) If a test chemical has an effect on the hematopoietic system, 
reticulocyte counts and bone marrow cytology may be indicated.
    (D) Other determinations that should be carried out if the test 
chemical is known or suspected of affecting related measures include 
calcium, phosphorus, fasting triglycerides, hormones, methemoglobin, and 
cholinesterases.
    (iii) Optionally, the following urinalysis determinations could be 
performed during the last week of the study using timed urine volume 
collection: appearance, volume, osmolality or specific gravity, pH, 
protein, glucose and blood/blood cells.
    (11) Ophthalmological examination. Using an ophthalmoscope or an 
equivalent device, ophthalmological examinations must be made on all 
animals prior to the administration of the test substance and on all 
high dose and control groups at termination. If changes in the eyes are 
detected, all animals in the other dose groups must be examined.
    (12) Gross necropsy. (i) All animals must be subjected to a full 
gross necropsy which includes examination of the external surface of the 
body, all orifices, and the cranial, thoracic and abdominal cavities and 
their contents.
    (ii) The liver, brain, kidneys, spleen, adrenals, testes, 
epididymides, uterus, ovaries, thymus and heart must be trimmed and 
weighed wet, as soon as possible after dissection.
    (iii) The following organs and tissues, or representative samples 
thereof, must be preserved in a suitable medium for possible future 
histopathological examination:
    (A) Digestive system--salivary glands, esophagus, stomach, duodenum, 
jejunum, ileum, cecum, colon, rectum, liver, pancreas, gallbladder (when 
present).
    (B) Nervous system--brain (multiple sections, including cerebrum, 
cerebellum and medulla/pons), pituitary, peripheral nerve (sciatic or 
tibial, preferably in close proximity to the muscle), spinal cord (three 
levels, cervical, mid-thoracic and lumbar), eyes (retina, optic nerve).
    (C) Glandular system--adrenals, parathyroid, thyroid.
    (D) Respiratory system--trachea, lungs, pharynx, larynx, nose.
    (E) Cardiovascular/Hematopoietic system--aorta, heart, bone marrow 
(and/or fresh aspirate), lymph nodes (preferably one lymph node covering 
the route of administration and another one distant from the route of 
administration to cover systemic effects), spleen, thymus.
    (F) Urogenital system--kidneys, urinary bladder, prostate, testes, 
epididymides, seminal vesicle(s), uterus, ovaries, female mammary gland.
    (G) Other--all gross lesions and masses, skin (both treated and 
adjacent untreated areas).
    (13) Histopathology. (i) The following histopathology must be 
performed:
    (A) Full histopathology on the organs and tissues, listed in 
paragraph (e)(12)(iii) of this section, of all animals in the control 
and high dose groups and all animals that died or were sacrificed during 
the study.
    (B) All gross lesions in all animals.
    (C) Target organs in all animals.
    (D) When a satellite group is used, histopathology must be performed 
on tissues and organs identified as showing toxic effects in the treated 
groups.
    (ii) If excessive early deaths or other problems occur in the high 
dose group compromising the significance of the data, the next dose 
level must be examined for complete histopathology.
    (iii) An attempt should be made to correlate gross observations with 
microscopic findings.
    (iv) Tissues and organs designated for microscopic examination 
should be fixed in 10% buffered formalin or a recognized suitable 
fixative as soon as necropsy is performed and no less than 48 hours 
prior to trimming.
    (f) Data and reporting--(1) Treatment of results. (i) Data must be 
summarized in tabular form, showing for each test group, number of 
animals at the start of the test, the number of animals showing lesions, 
the types of lesions and the percentage of animals displaying each type 
of lesion.
    (ii) When applicable, all observed results, qualitative and 
quantitative,

[[Page 359]]

should be evaluated by an appropriate and generally acceptable 
statistical method. Any generally accepted statistical method should be 
used; the statistical methods including significance criteria should be 
selected during the design of the study.
    (2) Evaluation of study results. The findings of a subchronic dermal 
toxicity study should be evaluated in conjunction with the findings of 
preceding studies and considered in terms of toxic effects and the 
necropsy and histopathological findings. The evaluation should include 
the relationship between the dose of the test substance, the incidence 
and severity of abnormalities including behavioral and clinical 
abnormalities, gross lesions, identified target organs, body weight 
changes, effect on mortality, and any other general or specific toxic 
effects. A properly conducted 90-day subchronic dermal study should 
provide information on the effects of repeated application of a 
substance and a satisfactory estimation of a NOEL. It also can indicate 
the need for an additional longer-term study and provide information on 
the selection of dose levels.
    (3) Test report. In addition to reporting requirements specified 
under EPA Good Laboratory Practice Standards at 40 CFR part 792, subpart 
J, the following specific information must be reported:
    (i) Test substance characterization should include:
    (A) Chemical identification.
    (B) Lot or batch numbers.
    (C) Physical properties.
    (D) Purity/impurities.
    (ii) Identification and composition of any vehicle if used.
    (iii) Test system should contain data on:
    (A) Species and strain of animals used and rationale for selection 
if other than that recommended.
    (B) Age including body weight data and sex.
    (C) Test environment including cage conditions, ambient temperature, 
humidity, and light/dark periods.
    (D) Identification of animal diet.
    (E) Acclimation period.
    (iv) Test procedure should include the following data:
    (A) Method of randomization used.
    (B) Full description of experimental design and procedure.
    (C) Dose regime including levels, method, and volume.
    (v) Test results should include:
    (A) Group animal data. Tabulation of toxic response data by species, 
strain, sex and exposure level for:
    (1) Number of animals exposed.
    (2) Number of animals showing signs of toxicity.
    (3) Number of animals dying.
    (B) Individual animal data. Data should be presented as summary 
(group mean) as well as for individual animals.
    (1) Date of death during the study or whether animals survived to 
termination.
    (2) Date of observation of each abnormal sign and its subsequent 
course.
    (3) Body weight data.
    (4) Feed consumption data, when collected.
    (5) Results of ophthalmological examination.
    (6) Results of hematological tests performed.
    (7) Results of clinical chemistry tests performed.
    (8) Results of urinalysis, when performed.
    (9) Results of observations made.
    (10) Necropsy findings, including absolute and relative (to body 
weight) organ weight data.
    (11) Detailed description of all histopathological findings.
    (12) Statistical treatment of results, where appropriate.
    (g) Quality control. A system must be developed and maintained to 
assure and document adequate performance of laboratory equipment. The 
study must be conducted in compliance with the Good Laboratory Practice 
(GLP) regulations.
    (h) References. For additional background information on this test 
guideline, the following references should be consulted. These 
references are available for inspection at the TSCA Nonconfidential 
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M 
St., NW., Washington, DC, 12 noon to 4 p.m., Monday through Friday, 
except legal holidays.
    (1) Organization for Economic Cooperation and Development. 
Guidelines

[[Page 360]]

for Testing of Chemicals, Section 4-Health Effects, Part 411 Subchronic 
Toxicity Studies, Paris, 1981.
    (2) Weingand K, Brown G, Hall R et al. (1996). Harmonization of 
Animal Clinical Pathology Testing in Toxicity and Safety Studies. 
Fundam. & Appl. Toxicol. 29:198-201.

[65 FR 78786, Dec. 15, 2000]



Sec. 799.9346  TSCA 90-day inhalation toxicity.

    (a) Scope. This section is intended to meet the testing requirements 
under section 4 of TSCA. In the assessment and evaluation of the toxic 
characteristics of a gas, volatile substance, or aerosol/particulate, 
determination of subchronic inhalation toxicity may be carried out after 
initial information on toxicity has been obtained by acute testing. The 
subchronic inhalation study has been designed to permit the 
determination of the no-observed-effect-level (NOEL) and toxic effects 
associated with continuous or repeated exposure to a test substance for 
a period of 90 days. This study is not capable of determining those 
effects that have a long latency period for development (e.g., 
carcinogenicity and life shortening). Extrapolation from the results of 
this study to humans is valid only to a limited degree. It can, however, 
provide useful information on health hazards likely to arise from 
repeated exposures by the inhalation route over a limited period of 
time. It will provide information on target organs and the possibilities 
of accumulation, and can be of use in selecting concentration levels for 
chronic studies and establishing safety criteria for human exposure. 
Hazards of inhaled substances are influenced by the inherent toxicity 
and by physical factors such as volatility and particle size.
    (b) Source. The source material used in developing this TSCA test 
guideline is the OPPTS harmonized test guideline 870.3465 (June 1996 
Public Draft). This source is available at the address in paragraph (h) 
of this section.
    (c) Definitions. The following definitions apply to this section.
    Aerodynamic equivalent diameter is defined as the diameter of a unit 
density sphere having the same terminal settling velocity as the 
particle in question, whatever its size, shape, and density. It is used 
to predict where in the respiratory tract such particles may be 
deposited.
    Concentration in a subchronic inhalation study is the amount of test 
substance administered via inhalation for a period of 90-days. 
Concentration is expressed as weight of the test substance per unit 
volume of air (milligrams per liter or parts per million).
    Cumulative toxicity is the adverse effects of repeated exposures 
occurring as a result of prolonged action on, or increased concentration 
of the administered test substance or its metabolites in susceptible 
tissues.
    Inhalable diameter refers to that aerodynamic diameter of a particle 
which is considered to be inhalable for the organism. It is used to 
refer to particles which are capable of being inhaled and may be 
deposited anywhere within the respiratory tract
    Mass median aerodynamic diameter (MMAD) is the median aerodynamic 
diameter and along with the geometric standard deviation (GSD) is used 
to describe the particle size distribution of any aerosol statistically 
based on the weight and size of the particles. Fifty percent of the 
particles by weight will be smaller than the median diameter and 50% of 
the particles will be larger.
    No-observed-effect-level (NOEL) is the maximum concentration used in 
a study which produces no adverse effects.
    Subchronic inhalation toxicity is the adverse effects occurring as a 
result of the repeated daily exposure of experimental animals to a 
chemical by inhalation for part (approximately 10%) of a life span.
    (d) Limit test. If exposure at a concentration of 1 mg/L (expected 
human exposure may indicate the need for a higher concentration), or 
where this is not possible due to physical or chemical properties of the 
test substance, the maximum attainable concentration produces no 
observable toxic effects, then a full study using three concentrations 
might not be necessary.
    (e) Test procedures--(1) Animal selection--(i) Species and strain. A 
mammalian species shall be used for testing. A variety of rodent species 
may be used,

[[Page 361]]

although the rat is the preferred species. Commonly used laboratory 
strains should be employed. If another mammalian species is used, the 
tester shall provide justification/reasoning for its selection.
    (ii) Age/weight. Testing should be started with young healthy 
animals as soon as possible after weaning and acclimatization.
    (B) Dosing of rodents should generally begin no later than 8 weeks 
of age.
    (C) At the commencement of the study the weight variation of animals 
used shall not exceed  20% of the mean weight for 
each sex.
    (iii) Sex. (A) Equal numbers of animals of each sex shall be used at 
each concentration.
    (B) Females shall be nulliparous and nonpregnant.
    (iv) Numbers. (A) At least 20 animals (10 females and 10 males) 
should be used for each test group.
    (B) If interim sacrifices are planned, the number of animals shall 
be increased by the number of animals scheduled to be sacrificed before 
the completion of the study.
    (C) To avoid bias, the use of adequate randomization procedures for 
the proper allocation of animals to test and control groups is required.
    (D) Each animal shall be assigned a unique identification number. 
Dead animals, their preserved organs and tissues, and microscopic slides 
shall be identified by reference to the animal's unique number.
    (v) Husbandry. (A) Animals may be group-caged by sex, but the number 
of animals per cage must not interfere with clear observation of each 
animal. The biological properties of the test substance or toxic effects 
(e.g., morbidity, excitability) may indicate a need for individual 
caging. Animals must be housed individually in inhalation chambers 
during exposure to aerosols.
    (B) The temperature of the experimental animal rooms should be at 22 
3 [deg]C.
    (C) The relative humidity of the experimental animal rooms should be 
30-70%.
    (D) Where lighting is artificial, the sequence should be 12 h light/
12 h dark.
    (E) Control and test animals should be fed from the same batch and 
lot. The feed should be analyzed to assure adequacy of nutritional 
requirements of the species tested and for impurities that might 
influence the outcome of the rest. For feeding, conventional laboratory 
diets may be used with an unlimited supply of drinking water.
    (F) The study should not be initiated until animals have been 
allowed a period of acclimatization/quarantine to environmental 
conditions, nor should animals from outside sources be placed on test 
without an adequate period of quarantine. An acclimatization period of 
at least 5 days is recommended.
    (2) Control and test substances. (i) Whenever it is necessary to 
formulate the test substance with a vehicle for aerosol generation, the 
vehicle ideally should not elicit toxic effects or substantially alter 
the chemical or toxicological properties of the test substance.
    (ii) One lot of the test substance should be used, if possible 
throughout the duration of the study, and the research sample should be 
stored under conditions that maintain its purity and stability. Prior to 
the initiation of the study, there should be a characterization of the 
test substance, including the purity of the test substance and, if 
technically feasible, the name and quantities of unknown contaminants 
and impurities.
    (3) Control groups. A concurrent control group is required. This 
group shall be an untreated or sham-treated control group. Except for 
treatment with the test substance, animals in the control group shall be 
handled in a manner identical to the test group animals. Where a vehicle 
other than water is used to generate a substance, a vehicle control 
group should be used. If the toxic properties of the vehicle are not 
known or cannot be made available, both untreated and vehicle control 
groups are required.
    (4) Satellite group. A satellite group of 20 animals (10 animals per 
sex) may be treated with the high concentration level for 90 days and 
observed for reversibility, persistence, or delayed occurrence of toxic 
effects for a post-treatment period of appropriate length,

[[Page 362]]

normally not less than 28 days. In addition, a control group of 20 
animals (10 animals of each sex) should be added to the satellite study.
    (5) Concentration levels and concentration selection. (i) In 
subchronic toxicity tests, it is desirable to have a concentration-
response relationship as well as a NOEL. Therefore, at least three 
concentration levels plus a control and, where appropriate, a vehicle 
control (corresponding to the concentration of vehicle at the highest 
exposure level) shall be used. Concentrations should be spaced 
appropriately to produce test groups with a range of toxic effects. The 
data should be sufficient to produce a concentration-response curve.
    (ii) The highest concentration should result in toxic effects but 
not produce an incidence of fatalities which would prevent a meaningful 
evaluation.
    (iii) The intermediate concentrations should be spaced to produce a 
gradation of toxic effects.
    (iv) The lowest concentration should produce no evidence of 
toxicity.
    (v) In the case of potentially explosive test substances, care 
should be taken to avoid generating explosive concentrations.
    (6) Administration of the test substance. Animals should be exposed 
to the test substance for 6 h per day on a 7-day per week basis for a 
period of at least 90 days. Based primarily on practical considerations, 
exposure for 6 h per day on a 5-day per week basis is acceptable.
    (7) Observation period. The animals should be observed for a period 
of 90 days. Animals in the satellite group (if used) scheduled for 
follow-up observations should be kept for at least 28 days further 
without treatment to assess reversibility.
    (8) Exposure specifications. (i) The animals shall be tested in 
dynamic inhalation equipment designed to sustain a minimum airflow of 10 
air changes per hr, an adequate oxygen content of at least 19%, and 
uniform conditions throughout the exposure chamber. Maintenance of 
slight negative pressure inside the chamber will prevent leakage of the 
test substance into the surrounding areas. It is not normally necessary 
to measure chamber oxygen concentration if airflow is adequate.
    (ii) The selection of a dynamic inhalation chamber should be 
appropriate for the test substance and test system. Where a whole body 
chamber is used to expose animals to an aerosol, individual housing must 
be used to minimize crowding of the test animals and maximize their 
exposure to the test substance. To ensure stability of a chamber 
atmosphere, the total volume occupied by the test animals shall not 
exceed 5% of the volume of the test chamber. It is recommended, but not 
required, that nose-only or head-only exposure be used for aerosol 
studies in order to minimize oral exposures due to animals licking 
compound off their fur. Heat stress should be minimized.
    (iii) The temperature at which the test is performed should be 
maintained at 22  2 [deg]C. The relative humidity 
should be maintained between 40 and 60%, but in certain instances (e.g., 
use of water vehicle) this may not be practicable.
    (9) Physical measurements. Measurements or monitoring shall be made 
of the following:
    (i) The rate of airflow shall be monitored continuously but recorded 
at least three times during the exposure.
    (ii) The actual concentrations of the test substance shall be 
measured in the animal's breathing zone. During the exposure period, the 
actual concentrations of the test substance shall be held as constant as 
practicable and monitored continuously or intermittently depending on 
the method of analysis. Chamber concentration may be measured using 
gravimetric or analytical methods as appropriate. If trial run 
measurements are reasonably consistent  10% for 
liquid, aerosol, gas, or vapor;  20% for dry 
aerosol), then two measurements should be sufficient. If measurements 
are not consistent, three to four measurements should be taken. Whenever 
the test substance is a formulation, or it is necessary to formulate the 
test substance with a vehicle for aerosol generation, the analytical 
concentration must be reported for the total formulation, and not just 
for the active ingredient (AI). If, for example, a formulation contains 
10% AI and 90% inerts, a chamber analytical limit concentration of 2 mg/
L would consist

[[Page 363]]

of 0.2 mg/L of the AI. It is not necessary to analyze inert ingredients 
provided the mixture at the animal's breathing zone is analogous to the 
formulation; the grounds for this conclusion must be provided in the 
study report. If there is some difficulty in measuring chamber 
analytical concentration due to precipitation, nonhomogeneous mixtures, 
volatile components, or other factors, additional analyses of inert 
components may be necessary.
    (iii) During the development of the generating system, particle size 
analysis shall be performed to establish the stability of aerosol 
concentrations with respect to particle size. The MMAD particle size 
range should be between 1-3 [micro]m. The particle size of hygroscopic 
materials should be small enough when dry to assure that the size of the 
swollen particle will still be within the 1-3 [micro]m range. 
Measurements of aerodynamic particle size in the animal's breathing zone 
should be measured during a trial run. If MMAD valves for each exposure 
level are within 10% of each other, then two measurements during the 
exposures should be sufficient. If pretest measurements are not within 
10% of each other, three to four measurements should be taken.
    (iv) Temperature and humidity shall be monitored continuously and 
recorded at least three times during an exposure.
    (10) Feed and water during exposure period. Feed shall be withheld 
during exposure. Water may also be withheld during exposure.
    (11) Observation of animals. (i) During and following exposure, 
observations are made and recorded systematically; individual records 
should be maintained for each animal. It is not always possible to 
observe animals during exposure in a whole-body chamber.
    (ii) Observations shall be made at least once each day for morbidity 
and mortality. Appropriate actions should be taken to minimize loss of 
animals to the study (e.g., Necropsy or refrigeration of those animals 
found dead and isolation or sacrifice of weak or moribund animals).
    (iii) A careful clinical examination shall be made at least once 
weekly. Observations should be detailed and carefully recorded, 
preferably using explicitly defined scales. Observations should include, 
but not be limited to, evaluation of skin and fur, eyes and mucous 
membranes, respiratory and circulatory effects, autonomic effects such 
as salivation, central nervous system effects, including tremors and 
convulsions, changes in the level of activity, gait and posture, 
reactivity to handling or sensory stimuli, altered strength, and 
stereotypes or bizarre behavior (e.g., self-mutilation, walking 
backwards).
    (iv) Signs of toxicity should be recorded as they are observed 
including the time of onset, degree and duration.
    (v) Individual weights of animals shall be determined shortly before 
the test substance is administered, and weekly thereafter.
    (vi) Food consumption shall also be determined weekly if abnormal 
body weight changes are observed.
    (vii) Moribund animals should be removed and sacrificed when noticed 
and the time of death should be recorded as precisely as possible.
    (viii) At termination, all survivors in the treatment groups shall 
be sacrificed.
    (12) Clinical pathology. Hematology and clinical chemistry 
examinations shall be made on all animals, including controls, of each 
sex in each group. The hematology and clinical chemistry parameters 
should be examined at terminal sacrifice at the end of the study. 
Overnight fasting of the animals prior to blood sampling is recommended. 
Overall, there is a need for a flexible approach in the measures 
examined, depending on the observed or expected effects from a chemical, 
and in the frequency of measures, depending on the duration of potential 
chemical exposures.
    (i) Hematology. The recommended parameters are red blood cell count, 
hemoglobin concentration, hematocrit, mean corpuscular volume, mean 
corpuscular hemoglobin, and mean corpuscular hemoglobin concentration, 
white blood cell count, differential leukocyte count, platelet count, 
and a measure of clotting potential, such as prothrombin time or 
activated partial thromboplastin time.

[[Page 364]]

    (ii) Clinical chemistry. (A) Parameters which are considered 
appropriate to all studies are electrolyte balance, carbohydrate 
metabolism, and liver and kidney function. The selection of specific 
tests will be influenced by observations on the mode of action of the 
substance and signs of clinical toxicity.
    (B) The recommended clinical chemistry determinations are potassium, 
sodium, glucose, total cholesterol, urea nitrogen, creatinine, total 
protein and albumin. More than 2 hepatic enzymes, (such as alanine 
aminotransferase, aspartate aminotransferase, alkaline phosphatase, 
sorbitol dehydrogenase, or gamma glutamyl transpeptidase) should also be 
measured. Measurements of addtional enzymes (of hepatic or other origin) 
and bile acids, may also be useful.
    (C) If a test chemical has an effect on the hematopoietic system, 
reticulocyte counts and bone marrow cytology may be indicated.
    (D) Other determinations that should be carried out if the test 
chemical is known or suspected of affecting related measures include 
calcium, phosphorus, fasting triglycerides, hormones, methemoglobin, and 
cholinesterases.
    (iii) Optionally, the following urinalysis determinations could be 
performed during the last week of the study using timed urine volume 
collection: appearance, volume, osmolality or specific gravity, pH, 
protein, glucose, and blood/blood cells.
    (13) Ophthalmological examination. Ophthalmological examinations 
shall be made on all animals prior to the administration of the test 
substance and on all high concentration and control groups at 
termination. If changes in the eyes are detected, all animals in the 
other concentration groups shall be examined.
    (14) Gross pathology. (i) All animals shall be subjected to a full 
gross necropsy which includes examination of the external surface of the 
body, all orifices and the cranial, thoracic, and abdominal cavities and 
their contents.
    (ii) At least the liver, kidneys, brain, and gonads shall be trimmed 
and weighed wet, as soon as possible after dissection to avoid drying.
    (iii) The following organs and tissues, or representative samples 
thereof, shall be preserved in a suitable medium for possible future 
histopathological examination:
    (A) Digestive system.
    (1) Salivary glands.
    (2) Esophagus.
    (3) Stomach.
    (4) Duodenum.
    (5) Jejunum.
    (6) Ileum.
    (7) Cecum.
    (8) Colon.
    (9) Rectum.
    (10) Liver.
    (11) Pancreas.
    (12) Gallbladder (dogs).
    (B) Nervous system.
    (1) Brain (multiple sections).
    (2) Pituitary.
    (3) Peripheral nerve(s).
    (4) Spinal cord (three levels).
    (5) Eyes (retina, optic nerve).
    (C) Glandular system.
    (1) Adrenals.
    (2) Parathyroids.
    (3) Thyroids.
    (D) Respiratory system.
    (1) Trachea.
    (2) Lung.
    (3) Pharynx.
    (4) Larynx.
    (5) Nose.
    (E) Cardiovascular/hematopoietic system.
    (1) Aorta (thoracic).
    (2) Heart.
    (3) Bone marrow.
    (4) Lymph nodes.
    (5) Spleen.
    (6) Thymus.
    (F) Urogenital system.
    (1) Kidneys.
    (2) Urinary bladder.
    (3) Prostate.
    (4) Testes.
    (5) Epididymides.
    (6) Seminal vesicle(s).
    (7) Uterus.
    (8) Ovaries.
    (G) Other.
    (1) Lacrimal gland.
    (2) Mammary gland.
    (3) Skin.
    (4) Skeletal muscle.
    (5) All gross lesions and masses.
    (6) Sternum and/or femur.
    (15) Histopathology. (i) The following histopathology shall be 
performed:

[[Page 365]]

    (A) Full histopathology on the respiratory tract and other organs 
and tissues, listed under paragraph (e)(15)(iii) of this section, of all 
animals in the control and high exposure groups and all animals that 
died or were killed during the study.
    (B) All gross lesions in all animals.
    (C) Target organs in all animals.
    (D) Lungs of all animals. Special attention to examination of the 
respiratory tract should be made for evidence of infection as this 
provides a convenient assessment of the state of health of the animals.
    (E) When a satellite group is used, histopathology shall be 
performed on tissues and organs identified as showing effects in the 
treated groups.
    (ii) If excessive early deaths or other problems occur in the high 
exposure group compromising the significance of the data, the next 
concentration should be examined for complete histopathology.
    (iii) An attempt should be made to correlate gross observations with 
microscopic findings.
    (iv) Tissues and organs designated for microscopic examination 
should be fixed in 10% buffered formalin or a recognized suitable 
fixative as soon as necropsy is performed and no less than 48 hrs prior 
to trimming. Tissues should be trimmed to a maximum thickness of 0.4 cm 
for processing.
    (f) Data and reporting--(1) Treatment of results. (i) Data shall be 
summarized in tabular form, showing for each test group the number of 
animals at the start of the test, the number of animals showing lesions, 
the types of lesions, and the percentage of animals displaying each type 
of lesion.
    (ii) All observed results (quantitative and qualitative) should be 
evaluated by an appropriate statistical method. Any generally accepted 
statistical method may be used; the statistical methods including 
significance criteria should be selected during the design of the study.
    (2) Evaluation of study results. The findings of the subchronic 
inhalation toxicity study should be evaluated in conjunction with the 
findings of preceding studies and considered in terms of the observed 
toxic effects and the necropsy and histopathological findings. The 
evaluation will include the relationship between the concentration of 
the test substance and duration of exposure, and the presence or 
absence, the incidence and severity, of abnormalities, including 
behavioral and clinical abnormalities, gross lesions, identified target 
organs, body weight changes, effects on mortality and any other general 
or specific toxic effects. A properly conducted subchronic test should 
provide a satisfactory estimation of a no-effect level. It also can 
indicate the need for an additional longer-term study and provide 
information on the selection of concentrations.
    (3) Test report. In addition to reporting requirements specified 
under 40 CFR part 792, subpart J, the following specific information 
shall be reported. Both individual and summary data should be presented.
    (i) Test substance characterization shall include:
    (A) Chemical identification.
    (B) Lot or batch number.
    (C) Physical properties.
    (D) Purity/impurities.
    (E) Identification and composition of any vehicle used.
    (ii) Test system information shall include:
    (A) Species and strain of animals used and rationale for selection 
if other than that recommended.
    (B) Age, sex, and body weight.
    (C) Test environment including cage conditions, ambient temperature, 
humidity, and light/dark periods.
    (D) Identification of animal diet.
    (E) Acclimation period.
    (iii) Test procedure information shall include:
    (A) Method of randomization used.
    (B) Full description of experimental design and procedure.
    (C) Exposure regimen including concentration levels, methods, and 
volume.
    (D) Description of test conditions; the following exposure 
conditions shall be reported:
    (1) Description of exposure apparatus including design, type, 
volume, source of air, system for generating aerosols, method of 
conditioning air, treatment of exhaust air and the method of housing the 
animals in a test chamber.

[[Page 366]]

    (2) The equipment for measuring temperature, humidity, and 
particulate aerosol concentrations and size should be described.
    (E) Exposure data shall be tabulated and presented with mean values 
and a measure of variability (e.g., standard deviation) and include:
    (1) Airflow rates through the inhalation equipment.
    (2) Temperature and humidity of air.
    (3) Actual (analytical or gravimetric) concentration in the 
breathing zone.
    (4) Nominal concentration (total amount of test substance fed into 
the inhalation equipment divided by volume of air).
    (5) Particle size distribution, calculated mass median aerodynamic 
diameter (MMAD) and geometric standard deviation (GSD).
    (6) Explanation as to why the desired chamber concentration and/or 
particle size could not be achieved (if applicable) and the efforts 
taken to comply with this aspect of the section.
    (iv) Test results information shall include:
    (A) Group animal data. Tabulation of toxic response data by species, 
strain, sex and exposure level for:
    (1) Number of animals exposed.
    (2) Number of animals showing signs of toxicity.
    (3) Number of animals dying.
    (B) Individual animal data. Data should be presented as summary 
(group mean) as well as for individual animals.
    (1) Time of death during the study or whether animals survived to 
termination.
    (2) Time of observation of each abnormal sign and its subsequent 
course.
    (3) Body weight data.
    (4) Feed consumption data, when collected.
    (5) Results of ophthalmological examination, when performed.
    (6) Results of hematological tests performed. .
    (7) Results of clinical chemistry tests performed.
    (8) Results of urinalysis tests performed.
    (9) Necropsy findings, including absolute and relative organ weight 
data.
    (10) Detailed description of all histopathological findings.
    (11) Statistical treatment of results, where appropriate.
    (g) Quality control. A system shall be developed and maintained to 
assure and document adequate performance of laboratory staff and 
equipment. The study shall be conducted in compliance with 40 CFR part 
792--Good Laboratory Practice Standards.
    (h) References. For additional background information on this test 
guideline, the following references should be consulted. These 
references are available for inspection at the TSCA Nonconfidential 
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M 
St., SW., Washington, DC, 12 noon to 4 p.m., Monday through Friday, 
except legal holidays.
    (1) Cage, J.C. Ed. Paget, G.E. Experimental Inhalation Toxicology, 
Methods in Toxicology. (F.A. Davis Co., Philadelphia, PA, 1970) pp. 258-
277.
    (2) Casarett, L.J. and Doull. Chapter 9. Toxicology: The Basic 
Science of Poisons (New York: Macmillan Publishing Co., Inc., 1975).
    (3) U.S. Environmental Protection Agency, Office of Pesticide 
Programs, Health Effects Division. Interim policy for particle size and 
limit concentration issues in inhalation toxicity studies (February 1, 
1994).
    (4) MacFarland, H.N. Ed. Hayes, W.J. Vol. 7. Respiratory Toxicology, 
Essays in Toxicology. (Academic Press, New York, NY, 1976) pp. 121-154.
    (5) Organisation for Economic Co-operation and Development. 
Guidelines for testing of chemicals, section 4-health effects, part 413. 
Subchronic Inhalation Toxicity Studies (Paris, 1981).

[62 FR 43824, Aug. 15, 1997, as amended at 64 FR 35077, June 30, 1999]



Sec. 799.9355  TSCA reproduction/developmental toxicity screening test.

    (a) Scope--(1) Applicability. This section is intended to meet 
testing requirements of the Toxic Substances Control Act (TSCA) (15 
U.S.C. 2601).
    (2) Source. The source material used in developing this TSCA test 
guideline is the Office of Prevention, Pesticides, and Toxic Substances 
(OPPTS) harmonized test guideline 870.3550 (July 2000, final 
guidelines). This source is available at the address in paragraph (h) of 
this section.

[[Page 367]]

    (b) Purpose. (1) This guideline is designed to generate limited 
information concerning the effects of a test substance on male and 
female reproductive performance such as gonadal function, mating 
behavior, conception, development of the conceptus, and parturition. It 
is not an alternative to, nor does it replace, the existing 
comprehensive test standards in Sec. Sec. 799.9370 and 799.9380.
    (2) This screening test guideline can be used to provide initial 
information on possible effects on reproduction and/or development, 
either at an early stage of assessing the toxicological properties of 
chemicals, or on chemicals of high concern. It can also be used as part 
of a set of initial screening tests for existing chemicals for which 
little or no toxicological information is available, as a dose range 
finding study for more extensive reproduction/developmental studies, or 
when otherwise considered relevant.
    (3) This test does not provide complete information on all aspects 
of reproduction and development. In particular, it offers only limited 
means of detecting postnatal manifestations of prenatal exposure, or 
effects that may be induced during postnatal exposure. Due (amongst 
other reasons) to the relatively small numbers of animals in the dose 
groups, the selectivity of the end points, and the short duration of the 
study, this method will not provide evidence for definite claims of no 
effects.
    (c) Definitions. The definitions in section 3 of TSCA and in 40 CFR 
Part 792--Good Laboratory Practice Standards apply to this section. The 
following definitions also apply to this section.
    Dosage is a general term comprising of dose, its frequency and the 
duration of dosing.
    Dose is the amount of test substance administered. Dose is expressed 
as weight (g, mg) as weight of test substance per unit weight of test 
animal (e.g., mg/kg), or as constant dietary concentration parts per 
million (ppm).
    No-observed-effects level (NOEL) is the maximum dose used in a study 
which produces no adverse effects. The NOEL is expressed in terms of the 
weight of a test substance given daily per unit weight of test animal 
(milligrams per kilograms per day).
    (d) Principle of the test. (1) The test substance is administered in 
graduated doses to several groups of males and females. Males should be 
dosed for a minimum of four weeks and up to and including the day before 
scheduled sacrifice (this includes a minimum of two weeks prior to 
mating, during the mating period and, approximately, two weeks post-
mating). In view of the limited pre-mating dosing period in males, 
fertility may not be a particular sensitive indicator of testicular 
toxicity. Therefore, a detailed histological examination of the testes 
is essential. The combination of a pre-mating dosing period of two weeks 
and subsequent mating/fertility observations with an overall dosing 
period of at least four weeks, followed by detailed histopathology of 
the male gonads, is considered sufficient to enable detection of the 
majority of effects on male fertility and spermatogenesis.
    (2) Females should be dosed throughout the study. This includes two 
weeks prior to mating (with the objective of covering at least two 
complete oestrous cycles), the variable time to conception, the duration 
of pregnancy and at least four days after delivery, up to and including 
the day before scheduled sacrifice.
    (3) Duration of study, following acclimatization, is dependent on 
the female performance and is approximately 54 days, (at least 14 days 
premating, (up to) 14 days mating, 22 days gestation, 4 days lactation).
    (4) During the period of administration, the animals are observed 
closely each day for signs of toxicity. Animals which die or are 
sacrificed during the test period are necropsied and, at the conclusion 
of the test, surviving animals are sacrificed and necropsied.
    (e) Description of the method--(1) Selection of animal species. This 
test standard is designed for use with the rat. If other species are 
used, appropriate modifications will be necessary. Strains with low 
fecundity or well-known high incidence of developmental defects should 
not be used. Healthy virgin animals, not subjected to previous 
experimental procedures, should be

[[Page 368]]

used. The test animals should be characterized as to species, strain, 
sex, weight and/or age. At the commencement of the study the weight 
variation of animals used should be minimal and not exceed 20% of the 
mean weight of each sex.
    (2) Housing and feeding conditions. (i) The temperature in the 
experimental animal room should be 22 [deg]C (3[deg]). Although the relative humidity should be at 
least 30% and preferably not exceed 70% other than during room cleaning, 
the aim should be 50-60%. Lighting should be artificial, the sequence 
being 12 hours light, 12 hours dark. For feeding, conventional 
laboratory diets may be used with an unlimited supply of drinking water. 
The choice of diet may be influenced by the need to ensure a suitable 
admixture of a test substance when administered by this method.
    (ii) Animals may be housed individually or be caged in small groups 
of the same sex; for group caging, no more than five animals should be 
housed per cage. Mating procedures should be carried out in cages 
suitable for the purpose. Pregnant females should be caged individually 
and provided with nesting materials.
    (3) Preparation of the animals. Healthy young adult animals must be 
randomly assigned to the control and treatment groups. Cages should be 
arranged in such a way that possible effects due to cage placement are 
minimized. The animals must be uniquely identified and kept in their 
cages for at least five days prior to the start of the study to allow 
for acclimatization to the laboratory conditions.
    (4) Preparation of doses. (i) It is recommended that the test 
substance be administered orally unless other routes of administration 
are considered more appropriate. When the oral route is selected, the 
test compound is usually administered by gavage; however, alternatively, 
test compounds may be administered via the diet or drinking water.
    (ii) Where necessary, the test substance is dissolved or suspended 
in a suitable vehicle. It is recommended that, wherever possible, the 
use of an aqueous solution/suspension be considered first, followed by 
consideration of a solution/emulsion in oil (e.g., corn oil) and then by 
possible solution in other vehicles. For vehicles other than water the 
toxic characteristics of the vehicle must be known. The stability of the 
test substance in the vehicle should be determined.
    (f) Procedure--(1) Number and sex of animals. It is recommended that 
each group be started with at least 10 animals of each sex. Except in 
the case of marked toxic effects, it is expected that this will provide 
at least 8 pregnant females per group which normally is the minimum 
acceptable number of pregnant females per group. The objective is to 
produce enough pregnancies and offspring to assure a meaningful 
evaluation of the potential of the substance to affect fertility, 
pregnancy, maternal and suckling behaviour, and growth and development 
of the F1 offspring from conception to day 4 post-partum.
    (2) Dosage. (i) Generally, at least three test groups and a control 
group should be used. Dose levels may be based on information from acute 
toxicity tests or on results from repeated dose studies. Except for 
treatment with the test substance, animals in the control group should 
be handled in an identical manner to the test group subjects. If a 
vehicle is used in administering the test substance, the control group 
should receive the vehicle in the highest volume used.
    (ii) Dose levels should be selected taking into account any existing 
toxicity and (toxico-) kinetic data available for the test compound or 
related materials. The highest dose level should be chosen with the aim 
of inducing toxic effects but not death or severe suffering. Thereafter, 
a descending sequence of dose levels should be selected in order to 
demonstrate any dose response relationships and no adverse effects at 
the lowest dose level. Two to four fold intervals are frequently optimal 
for setting the descending dose levels and addition of a fourth test 
group is often preferable to using very large intervals (e.g., more than 
a factor of 10) between dosages.
    (3) Limit test. If an oral study at one dose level of at least 1000 
mg/kg body weight/day or, for dietary or drinking water administration, 
an equivalent percentage in the diet, or drinking

[[Page 369]]

water using the procedures described for this study, produces no 
observable toxic effects and if toxicity would not be expected based 
upon data from structurally related compounds, then a full study using 
several dose levels may not be considered necessary. The limit test 
applies except when human exposure indicates the need for a higher oral 
dose level to be used. For other types of administration, such as 
inhalation or dermal application, the physical chemical properties of 
the test substance often may dictate the maximum attainable 
concentration.
    (4) Administration of doses. (i) The animals must be dosed with the 
test substance daily for seven days a week. When the test substance is 
administered by gavage, this should be done in a single dose to the 
animals using a stomach tube or a suitable intubation cannula. The 
maximum volume of liquid that can be administered at one time depends on 
the size of the test animal. The volume should not exceed 1 ml/100 g 
body weight, except in the case of aqueous solutions where 2 ml/100 g 
body weight may be used. Except for irritating substances which will 
normally reveal exacerbated effects with higher concentrations, 
variability in test volume should be minimized by adjusting the 
concentration to ensure a constant volume at all dose levels.
    (ii) For substances administered via the diet or drinking water, it 
is important to ensure that the quantities of the test substance 
involved do not interfere with normal nutrition or water balance. When 
the test substance is administered in the diet either a constant dietary 
concentration (parts per million (ppm)) or a constant dose level in 
terms of the animals' body weight may be used; the alternative used must 
be specified. For a substance administered by gavage, the dose should be 
given at similar times each day, and adjusted at least weekly to 
maintain a constant dose level in terms of animal body weight.
    (5) Experimental schedule. (i) Dosing of both sexes should begin at 
least 2 weeks prior to mating, after they have been acclimatized for at 
least five days. The study should be scheduled in such a way that mating 
begins soon after the animals have attained full sexual maturity. This 
may vary slightly for different strains of rats in different 
laboratories, e.g., Sprague Dawley rats 10 weeks of age, Wistar rats 
about 12 weeks of age. Dams with offspring should be sacrificed on day 4 
post-partum, or shortly thereafter. The day of birth (viz. when 
parturition is complete) is defined as day 0 post-partum. Females 
showing no-evidence of copulation are sacrificed 24-26 days after the 
last day of the mating period. Dosing is continued in both sexes during 
the mating period. Males should further be dosed after the mating period 
at least until the minimum total dosing period of 28 days has been 
completed. They are then sacrificed, or, alternatively, are retained and 
continued to be dosed for the possible conduction of a second mating if 
considered appropriate.
    (ii) Daily dosing of the parental females should continue throughout 
pregnancy and at least up to, and including, day 3 post-partum or the 
day before sacrifice. For studies where the test substance is 
administered by inhalation or by the dermal route, dosing should be 
continued at least up to, and including, day 19 of gestation.
    (iii) The experimental schedule is given in the following figure 1.

[[Page 370]]

[GRAPHIC] [TIFF OMITTED] TR15DE00.064

    (6) Mating procedure. Normally, 1:1 (one male to one female) matings 
should be used in this study. Exceptions can arise in the case of 
occasional deaths of males. The female should be placed with the same 
male until pregnancy occurs or two weeks have elapsed. Each morning the 
females should be examined for the presence of sperm or a vaginal plug. 
Day 0 of pregnancy is defined as the day a vaginal plug or sperm is 
found.
    (7) Observations. (i) Throughout the test period, general clinical 
observations should be made at least once a day, and more frequently 
when signs of toxicity are observed. They should be made preferably at 
the same time(s) each day, considering the peak period of anticipated 
effects after dosing. Pertinent behavioural changes, signs of difficult 
or prolonged parturition and all signs of toxicity, including mortality, 
should be recorded. These records should include time of onset, degree 
and duration of toxicity signs.
    (ii) The duration of gestation should be recorded and is calculated 
from day 0 of pregnancy. Each litter should be examined as soon as 
possible after delivery to establish the number and sex of pups, 
stillbirths, live births, runts (pups that are significantly smaller 
than corresponding control pups) and the presence of gross 
abnormalities.
    (iii) Live pups should be counted and sexed and litters weighed 
within 24 hours of parturition (day 1) and on day 4 post-partum. In 
addition to the observations on parent animals, described by paragraph 
(f)(7) of this section, any abnormal behaviour of the offspring should 
be recorded.
    (8) Body weight and food/water consumption. (i) Males and females 
should be individually weighed on the first day of dosing, at least 
weekly thereafter, and at termination. During pregnancy, females should 
be weighed on days 0, 7, 14 and 20 and within 24 hours of parturition 
(day 1) and day 4 post-partum.
    (ii) During pre-mating, pregnancy and lactation, food consumption 
should be measured at least weekly. The measurement of food consumption 
during mating is optional. Water consumption during these periods should 
also be

[[Page 371]]

measured when the test substance is administered via drinking water.
    (9) Pathology--(i) Gross necropsy. (A) At the time of sacrifice or 
death during the study, the adult animals should be examined 
macroscopically for any abnormalities or pathological changes. Special 
attention should be paid to the organs of the reproductive system. The 
number of implantation sites should be recorded. Corpora lutea should be 
counted.
    (B) The testes and epididymides of all male adult animals should be 
weighed.
    (C) Dead pups and pups sacrificed at day 4 post-partum, or shortly 
thereafter, should, at least, be carefully examined externally for gross 
abnormalities.
    (D) The ovaries, testes, epididymides, accessory sex organs and all 
organs showing macroscopic lesions of all adult animals should be 
preserved. Formalin fixation is not recommended for routine examination 
of testes and epididymides. An acceptable method is the use of Bouin's 
fixative for these tissues.
    (ii) Histopathology. (A) Detailed histological examination should be 
performed on the ovaries, testes and epididymides of the animals of the 
highest dose group and the control group. The other preserved organs may 
be examined when necessary. Examinations should be extended to the 
animals of other dosage groups when changes are seen in the highest dose 
group.
    (B) Detailed testicular histopathological examination (e.g., using 
Bouin's fixative, paraffin embedding and transverse sections of 4-5 
m thickness) should be conducted with special 
emphasis on stages of spermatogenesis and histopathology interstitial 
testicular cell structure. The evaluation should identify treatment-
related effects such as retained spermatids, missing germ cell layers or 
types, multinucleated giant cells or sloughing of spermatogenic cells 
into the lumen (the specifications for the evaluation are discussed in 
paragraph (g)(2) of this section). Examination of the intact epididymis 
should include the caput, corpus, and cauda, which can be accomplished 
by evaluation of a longitudinal section. The epididymis should be 
evaluated for leukocyte infiltration, change in prevalence of cell 
types, aberrant cell types, and phagocytosis of sperm. PAS and 
hematoxylin staining may be used for examination of the male 
reproductive organs. Histopathological examination of the ovary should 
detect qualitative depletion of the primordial follicle population.
    (g) Data and reporting--(1) Data. Individual animal data should be 
provided. Additionally, all data should be summarised in tabular form, 
showing for each test group the number of animals at the start of the 
test, the number of animals found dead during the test or sacrificed for 
humane reasons, the time of any death or humane sacrifice, the number of 
fertile animals, the number of pregnant females, the number of animals 
showing signs of toxicity, a description of the signs of toxicity 
observed, including time of onset, duration, and severity of any toxic 
effects, the types of histopathological changes, and all relevant litter 
data.
    (2) Evaluation of results. (i) The findings of this toxicity study 
should be evaluated in terms of the observed effects, necropsy and 
microscopic findings. This evaluation must include the relationship 
between the dose of the test substance and the presence or absence, 
incidence and severity of abnormalities, including gross lesions, 
identified target organs, infertility, clinical abnormalities, affected 
reproductive and litter performance, body weight changes, effects on 
mortality and any other toxic effects.
    (ii) Because of the short period of treatment of the male, the 
histopathology of the testis and epididymus must be considered along 
with the fertility data, when assessing male reproductive effects.
    (iii) Due to the limited dimensions of the study, statistical 
analysis in the form of tests for ``significance'' are of limited value 
for many endpoints, especially reproductive endpoints. If statistical 
analyses are used then the method chosen should be appropriate for the 
distribution of the variable examined, and be selected prior to the 
start of the study. Because of the small group size, the use of historic 
control data (e.g.,

[[Page 372]]

for litter size), where available, may also be useful as an aid to the 
interpretation of the study.
    (3) Test report. The test report must include the following 
information:
    (i) Test substance:
    (A) Physical nature and, where relevant, physicochemical properties.
    (B) Identification data.
    (ii) Vehicle (if appropriate): Justification for choice of vehicle 
if other than water.
    (iii) Test animals:
    (A) Species/strain used.
    (B) Number, age and sex of animals.
    (C) Source, housing conditions, diet, etc.
    (D) Individual weights of animals at the start of the test.
    (iv) Test conditions:
    (A) Rationale for dose level selection.
    (B) Details of test substance formulation/diet preparation, achieved 
concentrations, stability and homogeneity of the preparation.
    (C) Details of the administration of the test substance.
    (D) Conversion from diet/drinking water test substance concentration 
(parts per million (ppm)) to the actual dose (mg/kg body weight/day), if 
applicable.
    (E) Details of food and water quality.
    (v) Results (toxic response data by sex and dose):
    (A) Time of death during the study or whether animals survived to 
termination.
    (B) Nature, severity and duration of clinical observations (whether 
reversible or not).
    (C) Body weight/body weight change data.
    (D) Food consumption and water consumption, if applicable.
    (E) Effects on reproduction, including information on mating/
precoital interval, fertility, fecundity and gestation duration.
    (F) Effects on offspring, including number of pups born (live and 
dead), sex ratio, postnatal growth (pup weights) and survival (litter 
size), gross abnormalities and clinical observations during lactation.
    (G) Body weight at termination and organ weight data for the 
parental animals.
    (H) Necropsy data, including number of implantations and number of 
corpora lutea.
    (I) Calculations of pre- and postimplantation loss.
    (J) Detailed description of histopathological findings.
    (K) Statistical treatment of results, where appropriate.
    (vi) Discussion of results.
    (vii) Conclusions.
    (4) Interpretation of results. The study will provide evaluations of 
reproduction/developmental toxicity associated with administration of 
repeated doses. It could provide an indication of the need to conduct 
further investigations and provides guidance in the design of subsequent 
studies.
    (h) References. For additional background information on this test 
guideline, the following references should be consulted. These 
references are available for inspection at the TSCA Nonconfidential 
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M 
St., SW., Washington, DC, 12 noon to 4 p.m., Monday through Friday, 
except legal holidays.
    (1) OECD (1995). Reproduction/Developmental Toxicity Screening Test, 
OECD 421, OECD Guidelines for Testing of Chemicals.
    (2) [Reserved]

[65 FR 78789, Dec. 15, 2000]



Sec. 799.9365  TSCA combined repeated dose toxicity study with the 

reproduction/developmental toxicity screening test.

    (a) Scope--(1) Applicability. This section is intended to meet 
testing requirements of the Toxic Substances Control Act (TSCA) (15 
U.S.C. 2601).
    (2) Source. The source material used in developing this TSCA test 
guideline is the Office of Prevention, Pesticides and Toxic Substances 
(OPPTS) harmonized test guideline 870.3650 (July 2000, final 
guidelines). This source is available at the address in paragraph (h) of 
this section.
    (b) Purpose. (1) This screening test provides limited information on 
systemic toxicity, neurotoxicity, and/or immunotoxicity following 
repeated exposure over a limited time period. In addition, it can be 
used to provide initial information on possible effects on

[[Page 373]]

male and female reproductive performance such as gonadal function, 
mating behavior, conception, development of the conceptus, and 
parturition. It is not an alternative to, nor does it replace, the 
existing test guidelines in Sec. Sec. 799.9370, 799.9380, 799.9620, and 
799.9780 of this part.
    (2) This test does not provide complete information on all aspects 
of reproduction and development. In particular, it offers only limited 
means of detecting postnatal manifestations of prenatal exposure, or 
effects that may be induced during postnatal exposure. Due (amongst 
other reasons) to the selectivity of the end points, and the short 
duration of the study, this method will not provide evidence for 
definite claims of no reproduction/developmental effects.
    (3) This test can be used to provide initial information either at 
an early stage of assessing the toxicological properties of chemicals, 
or chemicals of high concern. It can also be used as part of a set of 
initial screening tests for existing chemicals for which little or no 
toxicological information is available or when otherwise considered 
relevant. It also can serve as an alternative to conducting two separate 
screening tests for repeated dose toxicity as described in Sec. 
799.9305 of this part and reproductive/developmental toxicity as 
described in Sec. 799.9355 of this part.
    (c) Definitions. The definitions in section 3 of TSCA and in 40 CFR 
Part 792--Good Laboratory Practice Standards apply to this section. The 
following definitions also apply to this section.
    Dosage is a general term comprising dose, its frequency and the 
duration of dosing.
    Dose is the amount of test substance administered. Dose is expressed 
as weight (g, gm) or as weight of test substance per unit weight of test 
animal (e.g., mg/kg), or as constant dietary concentration (parts per 
million (ppm)).
    No-observed-effects level (NOEL) is the maximum dose used in a study 
which produces no adverse effects. The NOEL is expressed in terms of the 
weight of a test substance given daily per unit weight of test animal 
(milligrams per kilogram per day).
    (d) Principle of the test. (1) The test substance must be 
administered in graduated doses to several groups of males and females. 
Males should be dosed for a minimum of 4 weeks, up to and including the 
day before scheduled sacrifice (this includes a minimum of 2 weeks prior 
to mating, during the mating period and, approximately, 2 weeks post 
mating). In view of the limited pre-mating dosing period in males, 
fertility may not be a particularly sensitive indicator of testicular 
toxicity. Therefore, a detailed histological examination of the testes 
is essential. The combination of a pre-mating dosing period of 2 weeks 
and subsequent mating/fertility observations with an overall dosing 
period of at least 4 weeks, followed by detailed histopathology of the 
male gonads, is considered sufficient to enable detection of the 
majority of effects on male fertility and spermatogenesis.
    (2) Females should be dosed throughout the study. This includes 2 
weeks prior to mating (with the objective of covering at least two 
complete oestrous cycles), the variable time to conception, the duration 
of pregnancy and at least 4 days after delivery, up to and including the 
day before scheduled sacrifice.
    (3) Duration of study, following acclimatization, is dependent on 
the female performance and is approximately 54 days, (at least 14 days 
pre-mating, (up to) 14 days mating, 22 days gestation, 4 days 
lactation).
    (4) During the period of administration, the animals are observed 
closely each day for signs of toxicity. Animals which die or are 
sacrificed during the test are necropsied and, at the conclusion of the 
test, surviving animals are sacrificed and necropsied.
    (e) Description of the method--(1) Selection of animal species. This 
test guideline is designed for use with the rat. If other species are 
used, appropriate modifications will be necessary. Strains with low 
fecundity or well-known high incidence of developmental defects should 
not be used. Healthy virgin animals, not subjected to previous 
experimental procedures, should be used. The test animals should be 
characterised as to species, strain, sex,

[[Page 374]]

weight and/or age. At the commencement of the study the weight variation 
of animals used should be minimal and not exceed 20% of the mean weight of each sex. Where the study is 
conducted as a preliminary study to a long-term or a full-generation 
study, preferably animals from the same strain and source should be used 
in both studies.
    (2) Housing and feeding conditions. (i) The temperature in the 
experimental animal room should be 22 [deg]C (3[deg]). The relative humidity should be at least 30% 
and preferably not exceed 70% other than during room cleaning. Lighting 
should be artificial, the sequence being 12 hours light, 12 hours dark. 
For feeding, conventional laboratory diets may be used with an unlimited 
supply of drinking water. The choice of diet may be influenced by the 
need to ensure a suitable admixture of a test substance when 
administered by this method.
    (ii) Animals may be housed individually or be caged in small groups 
of the same sex; for group caging, no more than five animals should be 
housed per cage. Mating procedures should be carried out in cages 
suitable for the purpose. Pregnant females should be caged individually 
and provided with nesting materials.
    (3) Preparation of the animals. Healthy young adult animals must be 
randomised and assigned to the treatment groups and cages. Cages should 
be arranged in such a way that possible effects due to cage placements 
are minimized. The animals must be uniquely identified and kept in their 
cages for at least 5 days prior to the start of the study to allow for 
acclimatisation to the laboratory conditions.
    (4) Preparation of doses. (i) It is recommended that the test 
substance be administered orally unless other routes of administration 
are considered more appropriate. When the oral route is selected, the 
test compound is usually administered by gavage; however, alternatively, 
test compounds may also be administered via the diet or drinking water.
    (ii) Where necessary, the test substance is dissolved or suspended 
in a suitable vehicle. It is recommended that, wherever possible, the 
use of an aqueous solution/suspension be considered first, followed by 
consideration of a solution/emulsion in oil (e.g., corn oil) and then by 
possible solution in other vehicles. For non-aqueous vehicles the toxic 
characteristics of the vehicle must be known. The stability of the test 
substance in the vehicle should be determined.
    (f) Procedure--(1) Number and sex of animals. It is recommended that 
each group be started with at least 10 animals of each sex. Except in 
the case of marked toxic effects, it is expected that this will provide 
at least eight pregnant females per group which normally is the minimum 
acceptable number of pregnant females per group. The objective is to 
produce enough pregnancies and offspring to assure a meaningful 
evaluation of the potential of the substance to affect fertility, 
pregnancy, maternal and suckling behaviour, and growth and development 
of the F1 offspring from conception to day 4 post-partum. If 
interim sacrifices are planned, the number should be increased by the 
number of animals scheduled to be sacrificed before the completion of 
the study. Consideration should be given to an additional satellite 
group of five animals per sex in the control and the top dose group for 
observation of reversibility, persistence or delayed occurrence of 
systemic toxic effects, for at least 14 days post treatment. Animals of 
the satellite groups must not be mated and, consequently, must not used 
for the assessment of reproduction/developmental toxicity.
    (2) Dosage. (i) Generally, at least three test groups and a control 
group should be used. If there are no suitable general toxicity data 
available, a range finding study may be performed to aid the 
determination of the doses to be used. Except for treatment with the 
test substance, animals in the control group should be handled in an 
identical manner to the test group subjects. If a vehicle is used in 
administering the test substance, the control group should receive the 
vehicle in the highest volume used.
    (ii) Dose levels should be selected taking into account any existing 
toxicity and (toxico-) kinetic data available for the test compound or 
related

[[Page 375]]

materials. It should also be taken into account that there may be 
differences in sensitivity between pregnant and non-pregnant animals. 
The highest dose level should be chosen with the aim of inducing toxic 
effects but not death nor obvious suffering. Thereafter, a descending 
sequence of dose levels should be selected with a view to demonstrating 
any dosage related response and no adverse effects at the lowest dose 
level. Two- to four-fold intervals are frequently optimum and addition 
of a fourth test group is often preferable to using very large intervals 
(e.g., more than a factor of 10) between dosages.
    (3) Limit test. If an oral study at 1-dose level of at least 1000 
mg/kg body weight/day or, for dietary administration, an equivalent 
percentage in the diet, or drinking water (based upon body weight 
determinations), using the procedures described for this study, produces 
no observable toxic effects and if toxicity would not be expected based 
upon data from structurally related compounds, then a full study using 
several dose levels may not be considered necessary. The limit test 
applies except when human exposure indicates the need for a higher dose 
level to be used. For other types of administration, such as inhalation 
or dermal application, the physical chemical properties of the test 
substance often may dictate the maximum attainable exposure.
    (4) Administration of doses. (i) The animals are dosed with the test 
substance daily for 7 days a week. When the test substance is 
administered by gavage, this should be done in a single dose to the 
animals using a stomach tube or a suitable intubation cannula. The 
maximum volume of liquid that can be administered at one time depends on 
the size of the test animal. The volume should not exceed 1 ml/100 g 
body weight, except in the case of aqueous solutions where 2 ml/100 g 
body weight may be used. Except for irritating or corrosive substances 
which will normally reveal exacerbated effects with higher 
concentrations, variability in test volume should be minimized by 
adjusting the concentration to ensure a constant volume at all dose 
levels.
    (ii) For substances administered via the diet or drinking water, it 
is important to ensure that the quantities of the test substance 
involved do not interfere with normal nutrition or water balance. When 
the test substance is administered in the diet either a constant dietary 
concentration (parts per million (ppm)) or a constant dose level in 
terms of the animals' body weight may be used; the alternative used must 
be specified. For a substance administered by gavage, the dose should be 
given at similar times each day, and adjusted at least weekly to 
maintain a constant dose level in terms of animal body weight.
    (5) Experimental schedule. (i) Dosing of both sexes should begin 2 
weeks prior to mating, after they have been acclimatized for at least 5 
days. The study should be scheduled in such a way that mating begins 
soon after the animals have attained full sexual maturity. This may vary 
slightly for different strains of rats in different laboratories, e.g., 
Sprague Dawley rats 10 weeks of age, Wistar rats about 12 weeks of age. 
Dams with offspring should be sacrificed on day 4 post-partum, or 
shortly thereafter. In order to allow for overnight fasting of dams 
prior to blood collection (if this option is preferred), dams and their 
offspring need not necessarily be sacrificed on the same day. The day of 
birth (viz. when parturition is complete) is defined as day 0 post-
partum. Females showing no-evidence of copulation are sacrificed 24-26 
days after the last day of the mating period. Dosing is continued in 
both sexes during the mating period. Males should further be dosed after 
the mating period at least until the minimum total dosing period of 28 
days has been completed. They are then sacrificed, or, alternatively, 
are retained and continued to be dosed for the possible conduction of a 
second mating if considered appropriate.
    (ii) Daily dosing of the parental females should continue throughout 
pregnancy and at least up to, and including, day 3 post-partum or the 
day before sacrifice. For studies where the test substance is 
administered by inhalation or by the dermal route, dosing should be 
continued at least up to, and including, day 19 of gestation.

[[Page 376]]

    (iii) Animals in a satellite group scheduled for follow-up 
observations, if included, must not mated. They should be kept at least 
for a further 14 days after the first scheduled sacrifice of dams, 
without treatment to detect delayed occurrence, or persistence of, or 
recovery from toxic effects.
    (iv) The experimental schedule is given in the following figure 1.
    [GRAPHIC] [TIFF OMITTED] TR15DE00.065
    
    (6) Mating procedure. Normally, 1:1 (one male to one female) matings 
should be used in this study. Exceptions can arise in the case of 
occasional deaths of males. The female should be placed with the same 
male until pregnancy occurs or 2 weeks have elapsed. Each morning the 
females should be examined for the presence of sperm or a vaginal plug. 
Day 0 of pregnancy is defined as the day a vaginal plug or sperm is 
found. In case pairing was unsuccessful, re-mating of females with 
proven males of the same group could be considered.
    (7) Observations. (i) General clinical observations should be made 
at least once a day, preferably at the same time(s) each day and 
considering the peak period of anticipated effects after dosing. The 
health condition of the animals should be recorded. At least twice daily 
all animals must be observed for morbidity and mortality.
    (ii) Once before the first exposure (to allow for within-subject 
comparisons), and at least once a week thereafter, detailed clinical 
observations should be made in all animals. These observations should be 
made outside the home cage in a standard arena and preferably at the 
same time, each day. They should be carefully recorded; preferably using 
scoring systems, explicitly defined by the testing laboratory. Effort 
should be made to ensure that variations in the test conditions are 
minimal and that observations are preferably conducted by observers 
unaware of the treatment. Signs noted should include, but not be limited 
to, changes in skin, fur, eyes, mucous membranes, occurrence of 
secretions and excretions and autonomic activity (e.g., lacrimation, 
piloerection, pupil size, unusual respiratory pattern). Changes in gait, 
posture and response to handling as well as the presence of clonic or 
tonic movements, stereotypies (e.g., excessive grooming, repetitive 
circling), difficult or prolonged parturition or bizarre behaviour 
(e.g., self-mutilation, walking backwards) should also be recorded.

[[Page 377]]

    (iii) At one time during the study, sensory reactivity to stimuli of 
different modalities (e.g., auditory, visual and proprioceptive stimuli) 
assessment of grip strength and motor activity assessment should be 
conducted in five males and five females, randomly selected from each 
group. Further details of the procedures that could be followed are 
given in the respective references. However, alternative procedures than 
those referenced could also be used. In males, these functional 
observations should be made towards the end of their dosing period, 
shortly before scheduled sacrifice but before blood sampling for 
hematology or clinical chemistry. Females should be in a physiologically 
similar state during these functional tests and should preferably be 
tested during lactation, shortly before scheduled sacrifice. In order to 
avoid hypothermia of pups, dams should be removed from the pups for not 
more than 30 to 40 minutes. Examples of procedures for observation are 
described in the references in paragraphs (h)(3), (h)(4), (h)(5), 
(h)(6), and (h)(7) of this section.
    (iv) Functional observations made once towards the end of the study 
may be omitted when the study is conducted as a preliminary study to a 
subsequent subchronic (90-day) or long-term study. In that case, the 
functional observations should be included in this follow-up study. On 
the other hand, the availability of data on functional observations from 
this repeated dose study may enhance the ability to select dose levels 
for a subsequent subchronic or long-term study.
    (v) Functional observations may also be omitted for groups that 
otherwise reveal signs of toxicity to an extent that would significantly 
interfere with the functional test performance.
    (vi) The duration of gestation should be recorded and is calculated 
from day 0 of pregnancy. Each litter should be examined as soon as 
possible after delivery to establish the number and sex of pups, 
stillbirths, live births, runts (pups that are significantly smaller 
than corresponding control pups), and the presence of gross 
abnormalities.
    (vii) Live pups should be counted and sexed and litters weighed 
within 24 hours of parturition (day 0 or 1 post-partum) and on day 4 
post-partum. In addition to the observations on parental animals, 
described by paragraphs (f)(7)(ii) and (f)(7)(iii) of this section, any 
abnormal behaviour of the offspring should be recorded.
    (8) Body weight and food/water consumption. (i) Males and females 
should be weighed on the first day of dosing, at least weekly 
thereafter, and at termination. During pregnancy, females should be 
weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 
0 or 1 post-partum), and day 4 post-partum. These observations should be 
reported individually for each adult animal.
    (ii) During pre-mating, pregnancy and lactation, food consumption 
should be measured at least weekly. The measurement of food consumption 
during mating is optional. Water consumption during these periods should 
also be measured, when the test substance is administered by that 
medium.
    (9) Hematology. (i) Once during the study, the following 
hematological examinations should be made in five males and five females 
randomly selected from each group: hematocrit, hemoglobin concentration, 
erythrocyte count, total and differential leucocyte count, platelet 
count and a measure of blood clotting time/potential.
    (ii) Blood samples should be taken from a named site. Females should 
be in a physiologically similar state during sampling. In order to avoid 
practical difficulties related to the variability in the onset of 
gestation, blood collection in females may be done at the end of the 
pre-mating period as an alternative to sampling just prior to, or as 
part of, the procedure for sacrificing the animals. Blood samples of 
males should preferably be taken just prior to, or as part of, the 
procedure for sacrificing the animals. Alternatively, blood collection 
in males may also be done at the end of the pre-mating period when this 
time point was preferred for females.
    (iii) Blood samples should be stored under appropriate conditions.
    (10) Clinical biochemistry. (i) Clinical biochemistry determinations 
to investigate major toxic effects in tissues and, specifically, effects 
on kidney and liver, should be performed on blood

[[Page 378]]

samples obtained from the selected five males and five females of each 
group. Overnight fasting of the animals prior to blood sampling is 
recommended \1\. Investigations of plasma or serum must include sodium, 
potassium, glucose, total cholesterol, urea, creatinine, total protein 
and albumin, at least two enzymes indicative of hepatocellular effects 
(such as alanine aminotransferase, aspartate aminotransferase and 
sorbitol dehydrogenase) and bile acids. Measurements of additional 
enzymes (of hepatic or other origin) may provide useful information 
under certain circumstances.
---------------------------------------------------------------------------

    \1\ For a number of measurements in serum and plasma, most notably 
for glucose, overnight fasting would be preferable. The major reason for 
this preference is that the increased variability which would inevitably 
result from non-fasting, would tend to mask more subtle effects and make 
interpretation difficult. On the other hand, however, overnight fasting 
may interfere with the general metabolism of the (pregnant) animals, 
disturbs lactation and nursing behaviour, and, particularly in feeding 
studies, may disturb the daily exposure to the test substance. If 
overnight fasting is adopted, clinical biochemical determinations should 
be performed after the conduct of functional observations in week 4 of 
the study.
---------------------------------------------------------------------------

    (ii) Optionally, the following urinalysis determinations could be 
performed in five randomly selected males of each group during the last 
week of the study using timed urine volume collection; appearance, 
volume, osmolality or specific gravity, pH, protein, glucose and blood 
or blood cells.
    (iii) In addition, studies to investigate serum markers of general 
tissue damage should be considered. Other determinations that should be 
carried out if the known properties of the test substance may, or are 
suspected to, affect related metabolic profiles include calcium, 
phosphate, fasting triglycerides and fasting glucose, specific hormones, 
methemoglobin and cholinesterase. These need to be identified on a case-
by-case basis.
    (iv) Overall, there is a need for a flexible approach, depending on 
the observed and/or expected effect with a given compound.
    (v) If historical baseline data are inadequate, consideration should 
be given to determination of hematological and clinical biochemistry 
variables before dosing commences.
    (11) Pathology--(i) Gross necropsy. (A) All adult animals in the 
study must be subjected to a full, detailed gross necropsy which 
includes careful examination of the external surface of the body, all 
orifices, and the cranial, thoracic and abdominal cavities and their 
contents. Special attention should be paid to the organs of the 
reproductive system. The number of implantation sites should be 
recorded. Corpora lutea should be counted.
    (B) The testes and epididymides of all adult males should be weighed 
and the ovaries, testes, epididymides, accessory sex organs, and all 
organs showing macroscopic lesions of all adult animals, should be 
preserved.
    (C) In addition, for five adult males and females, randomly selected 
from each group, the liver, kidneys, adrenals, thymus, spleen, brain and 
heart should be trimmed of any adherent tissue, as appropriate and their 
wet weight taken as soon as possible after dissection to avoid drying. 
Of the selected males and females, the following tissues should also be 
preserved in the most appropriate fixation medium for both the type of 
tissue and the intended subsequent histopathological examination: all 
gross lesions, brain (representative regions including cerebrum, 
cerebellum and pons), spinal cord, stomach, small and large intestines 
(including Peyer's patches), liver, kidneys, adrenals, spleen, heart, 
thymus, thyroid, trachea and lungs (preserved by inflation with fixative 
and then immersion), uterus, urinary bladder, lymph nodes (preferably 1 
lymph node covering the route of administration and another one distant 
from the route of administration to cover systemic effects), peripheral 
nerve (sciatic or tibial) preferably in close proximity to the muscle, 
and a section of bone marrow (or, alternatively, a fresh mounted marrow 
aspirate).
    (D) Formalin fixation is not recommended for routine examination of 
testes and epididymides. An acceptable method is the use of Bouin's 
fixative for these tissues. The clinical and other findings may suggest 
the need to examine additional tissues. Also, any organs

[[Page 379]]

considered likely to be target organs based on the known properties of 
the test substance should be preserved.
    (E) Dead pups and pups sacrificed at day 4 post-partum, or shortly 
thereafter, should, at least, be carefully examined externally for gross 
abnormalities.
    (ii) Histopathology. (A) Full histopathology should be conducted on 
the preserved organs and tissues of the selected animals in the control 
and high dose groups and all gross lesions. These examinations should be 
extended to animals of other dosage groups if treatment-related changes 
are observed in the high dose group.
    (B) Detailed testicular histopathological examination (e.g., using 
Bouin's fixative, paraffin embedding and transverse sections of 4-5 
m thickness) should be conducted with special 
emphasis on stages of spermatogenesis and histopathology interstitial 
testicular cell structure. The evaluation should identify treatment-
related effects such as retained spermatids, missing germ cell layers or 
types, multinucleated giant cells or sloughing of spermatogenic cells 
into the lumen (the specifications for the evaluation are discussed in 
paragraph (g)(2) of this section). Examination of the intact epididymis 
should include the caput, corpus, and cauda, which can be accomplished 
by evaluation of a longitudinal section. The epididymis should be 
evaluated for leukocyte infiltration, change in prevalence of cell 
types, aberrant cell types, and phagocytosis of sperm. Periodic acid-
Schiff (PAS) and hematoxylin staining may be used for examination of the 
male reproductive organs. Histopathological examination of the ovary 
should detect qualitative depletion of the primordial follicle 
population.
    (C) When a satellite group is used, histopathology should be 
performed on tissues and organs identified as showing effects in the 
treated groups.
    (g) Data and reporting--(1) Data. Individual animal data should be 
provided. Additionally, all data should be summarised in tabular form, 
showing for each test group the number of animals at the start of the 
test, the number of animals found dead during the test or sacrificed for 
humane reasons, the time of any death or humane sacrifice, the number of 
fertile animals, the number of pregnant females, the number of animals 
showing signs of toxicity, a description of the signs of toxicity 
observed, including time of onset, duration, and severity of any toxic 
effects, the types of histopathological changes, and all relevant litter 
data.
    (2) Evaluation of results. (i) The findings of this toxicity study 
should be evaluated in terms of the observed effects, necropsy and 
microscopic findings. The evaluation will include the relationship 
between the dose of the test substance and the presence or absence, 
incidence and severity of abnormalities, including gross lesions, 
identified target organs, infertility, clinical abnormalities, affected 
reproductive and litter performance, body weight changes, effects on 
mortality and any other toxic effects.
    (ii) Because of the short period of treatment of the male, the 
histopathology of the testes and epididymides must be considered along 
with the fertility data, when assessing male reproduction effects. The 
use of historic control data on reproduction/development (e.g. for 
litter size) where available may also be useful as an aid to the 
interpretation of the study.
    (iii) When possible, numerical results should be evaluated by an 
appropriate and general acceptable statistical method. The statistical 
methods should be selected during the design of the study. Due to the 
limited dimensions of the study, statistical analysis in the form of 
tests for ``significance'' are of limited value for many endpoints, 
especially reproductive endpoints. Some of the most widely used methods, 
especially parametric tests for measures of central tendency, are 
inappropriate. If statistical analyses are used then the method chosen 
should be appropriate for the distribution of the variable examined and 
be selected prior to the start of the study.
    (3) Test report. The test report must include the following 
information:
    (i) Test substance:
    (A) Physical nature and, where relevant, physicochemical properties.
    (B) Identification data.

[[Page 380]]

    (ii) Vehicle (if appropriate): Justification for choice of vehicle, 
if other than water.
    (iii) Test animals:
    (A) Species/strain used.
    (B) Number, age and sex of animals.
    (C) Source, housing conditions, diet, etc.
    (D) Individual weights of animals at the start of the test.
    (iv) Test conditions:
    (A) Rationale for dose level selection.
    (B) Details of test substance formulation/diet preparation, achieved 
concentration, stability and homogeneity of the preparation.
    (C) Details of the administration of the test substance.
    (D) Conversion from diet/drinking water test substance concentration 
(parts per mission (ppm)) to the actual dose (mg/kg body weight/day), if 
applicable.
    (E) Details of food and water quality.
    (v) Results (toxic response data by sex and dose):
    (A) Time of death during the study or whether animals survived to 
termination.
    (B) Nature, severity and duration of clinical observations (whether 
reversible or not).
    (C) Body weight/body weight change data.
    (D) Food consumption and water consumption, if applicable.
    (E) Sensory activity, grip strength and motor activity assessments.
    (F) Hematological tests with relevant baseline values,
    (G) Clinical biochemistry tests with relevant baseline values.
    (H) Effects on reproduction, including information on mating/
precoital interval, fertility, fecundity and gestation duration.
    (I) Effects on offspring, including number of pups born (live and 
dead), sex ratio, postnatal growth (pup weights) and survival (litter 
size), gross abnormalities and clinical observations during lactation.
    (J) Body weight at termination and organ weight data for the 
parental animals.
    (K) Necropsy data, including number of implantations and number of 
corpora lutea.
    (L) Calculations of pre- and postimplantation loss.
    (M) Detailed description of histopathological findings.
    (N) Statistical treatment of results, where appropriate.
    (vi) Discussion of results.
    (vii) Conclusions.
    (h) References. For additional background information on this test 
guideline, the following references should be consulted. These 
references are available for inspection at the TSCA Nonconfidential 
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M 
St., NW., Washington, DC, 12 noon to 4 p.m., Monday through Friday, 
except legal holidays.
    (1) Mitsumori, K., Kodama, Y., Uchida, O., Takada, K., Saito, M. 
Naito, K., Tanaka, S., Kurokawa, Y., Usami, M., Kawashima, K., Yasuhara, 
K., Toyoda, K., Onodera, H., Furukawa, F., Takahashi, M. and Hayashi, 
Y., (1994). Confirmation Study, Using Nitro-Benzene, of the Combined 
Repeat Dose and Reproductive/ Developmental Toxicity Test Protocol 
Proposed by the Organization for Economic Cooperation and Development 
(OECD). Journal of Toxicology and Science, 19:141-149.
    (2) Tanaka, S., Kawashima, K., Naito, K., Usami, M., Nakadate, M., 
Imaida, K., Takahashi, M., Hayashi, Y., Kurokawa, Y. and Tobe, M. 
(1992). Combined Repeat Dose and Reproductive/Developmental Toxicity 
Screening Test (OECD): Familiarization Using Cyclophosphamide. 
Fundamental and Applied Toxicology, 18:89-95.
    (3) Tupper D.E., Wallace R.B. (1980). Utility of the Neurologic 
Examination in Rats. Acta Neurobiological Exposure, 40:999-1003.
    (4) Gad S.C. (1982). A Neuromuscular Screen for Use in Industrial 
Toxicology. Journal of Toxicology and Environmental Health, 9:691-704.
    (5) Moser V.C., McDaniel K.M., Phillips P.M. (1991). Rat Strain and 
Stock Comparisons Using a Functional Observational Battery: Baseline 
Values and Effects of Amitraz. Toxicology and Applied Pharmacology, 
108:267-283.
    (6) Meyer O.A., Tilson H.A., Byrd W.C., Riley M.T. (1979). A Method 
for the Routine Assessment of Fore- and Hindlimb Grip Strength of Rats 
and

[[Page 381]]

Mice. Neurobehavorial Toxicology, 1:233-236.
    (7) Crofton K.M., Howard J.L., Moser V.C., Gill M.W., Reiter L.W., 
Tilson H.A., MacPhail R.C. (1991). Interlaboratory Comparison of Motor 
Activity Experiments: Implication for Neurotoxicological Assessments. 
Neurotoxicology and Teratology 13:599-609.

[65 FR 78793, Dec. 15, 2000]



Sec. 799.9370  TSCA prenatal developmental toxicity.

    (a) Scope This section is intended to meet the testing requirements 
under section 4 of TSCA. This guideline for developmental toxicity 
testing is designed to provide general information concerning the 
effects of exposure on the pregnant test animal and on the developing 
organism; this may include death, structural abnormalities, or altered 
growth and an assessment of maternal effects. For information on testing 
for functional deficiencies and other postnatal effects, the guidelines 
for the two-generation reproductive toxicity study and the developmental 
neurotoxicity study should be consulted.
    (b) Source. The source material used in developing this TSCA test 
guideline is the OPPTS harmonized test guideline 870.3700 (February 1996 
Public Draft). This source is available at the address in paragraph (h) 
of this section.
    (c) Good laboratory practice standards. The study shall be conducted 
in compliance with 40 CFR Part 792--Good Laboratory Practice Standards.
    (d) Principle of the test method. The test substance is administered 
to pregnant animals at least from implantation to one day prior to the 
expected day of parturition. Shortly before the expected date of 
delivery, the pregnant females are terminated, the uterine contents are 
examined, and the fetuses are processed for visceral and skeletal 
evaluation.
    (e) Test procedures--(1) Animal selection--(i) Species and strain. 
It is recommended that testing be performed in the most relevant 
species, and that laboratory species and strains which are commonly used 
in prenatal developmental toxicity testing be employed. The preferred 
rodent species is the rat and the preferred non-rodent species is the 
rabbit.
    (ii) Age. Young adult animals shall be used.
    (iii) Sex. Nulliparous female animals shall be used at each dose 
level. Animals should be mated with males of the same species and 
strain, avoiding the mating of siblings, if parentage is known. Day 0 in 
the test is the day on which a vaginal plug and/or sperm are observed in 
the rodent or that insemination is performed or observed in the rabbit.
    (iv) Number of animals. Each test and control group shall contain a 
sufficient number of animals to yield approximately 20 animals with 
implantation sites at necropsy.
    (2) Administration of test and control substances--(i) Dose levels 
and dose selection. (A) At least three-dose levels and a concurrent 
control shall be used. Healthy animals shall be randomly assigned to the 
control and treatment groups, in a manner which results in comparable 
mean body weight values among all groups. The dose levels should be 
spaced to produce a gradation of toxic effects. Unless limited by the 
physical/chemical nature or biological properties of the test substance, 
the highest dose shall be chosen with the aim to induce some 
developmental and/or maternal toxicity but not death or severe 
suffering. In the case of maternal mortality, this should not be more 
than approximately 10%. The intermediate dose levels should produce 
minimal observable toxic effects. The lowest dose level should not 
produce any evidence of either maternal or developmental toxicity (i.e., 
the no-observed-adverse-effect level, NOAEL) or should be at or near the 
limit of detection for the most sensitive endpoint. Two- or four-fold 
intervals are frequently optimal for spacing the dose levels, and the 
addition of a fourth test group is often preferable to using very large 
intervals (e.g., more than a factor of 10) between dosages.
    (B) It is desirable that additional information on metabolism and 
pharmacokinetics of the test substance be available to demonstrate the 
adequacy of the dosing regimen. This information should be available 
prior to testing.

[[Page 382]]

    (C) The highest dose tested need not exceed 1,000 mg/kg/day by oral 
or dermal administration, or 2 mg/L (or the maximum attainable 
concentration) by inhalation, unless potential human exposure data 
indicate the need for higher doses. If a test performed at the limit 
dose level, using the procedures described for this study, produces no 
observable toxicity and if an effect would not be expected based upon 
data from structurally related compounds, then a full study using three-
dose levels may not be considered necessary.
    (ii) Control group. (A) A concurrent control group shall be used. 
This group shall be a sham-treated control group or a vehicle-control 
group if a vehicle is used in administering the test substance.
    (B) The vehicle control group should receive the vehicle in the 
highest volume used.
    (C) If a vehicle or other additive is used to facilitate dosing, 
consideration should be given to the following characteristics: Effects 
on the absorption, distribution, metabolism, or retention of the test 
substance; effects on the chemical properties of the test substance 
which may alter its toxic characteristics; and effects on the food or 
water consumption or the nutritional status of the animals.
    (iii) Route of administration. (A) The test substance or vehicle is 
usually administered orally by intubation.
    (B) If another route of administration is used, for example, when 
the route of administration is based upon the principal route of 
potential human exposure, the tester shall provide justification and 
reasoning for its selection, and appropriate modifications may be 
necessary. Care should be taken to minimize stress on the maternal 
animals. For materials administered by inhalation, whole-body exposure 
is preferable to nose-only exposure due to the stress of restraint 
required for nose-only exposure.
    (C) The test substance shall be administered at approximately the 
same time each day.
    (D) When administered by gavage or dermal application, the dose to 
each animal shall be based on the most recent individual body weight 
determination.
    (iv) Dosing schedule. At minimum, the test substance shall be 
administered daily from implantation to the day before cesarean section 
on the day prior to the expected day of parturition. Alternatively, if 
preliminary studies do not indicate a high potential for preimplantation 
loss, treatment may be extended to include the entire period of 
gestation, from fertilization to approximately 1 day prior to the 
expected day of termination.
    (f) Observation of animals--(1) Maternal. (i) Each animal shall be 
observed at least once daily, considering the peak period of anticipated 
effects after dosing. Mortality, moribundity, pertinent behavioral 
changes, and all signs of overt toxicity shall be recorded at this 
cageside observation. In addition, thorough physical examinations shall 
be conducted at the same time maternal body weights are recorded.
    (ii) Animals shall be weighed on day 0, at termination, and at least 
at 3-day intervals during the dosing period.
    (iii) Food consumption shall be recorded on at least 3-day 
intervals, preferably on days when body weights are recorded.
    (iv) (A) Females shall be terminated immediately prior to the 
expected day of delivery.
    (B) Females showing signs of abortion or premature delivery prior to 
scheduled termination shall be killed and subjected to a thorough 
macroscopic examination.
    (v) At the time of termination or death during the study, the dam 
shall be examined macroscopically for any structural abnormalities or 
pathological changes which may have influenced the pregnancy. Evaluation 
of the dams during cesarean section and subsequent fetal analyses should 
be conducted without knowledge of treatment group in order to minimize 
bias.
    (vi) (A) Immediately after termination or as soon as possible after 
death, the uteri shall be removed and the pregnancy status of the 
animals ascertained. Uteri that appear nongravid shall be further 
examined (e.g. by ammonium sulfide staining) to confirm the nonpregnant 
status.
    (B) Each gravid uterus (with cervix) shall be weighed. Gravid 
uterine weights should not be obtained from

[[Page 383]]

dead animals if autolysis or decomposition has occurred.
    (C) The number of corpora lutea shall be determined for pregnant 
animals.
    (D) The uterine contents shall be examined for embryonic or fetal 
deaths and the number of viable fetuses. The degree of resorption shall 
be described in order to help estimate the relative time of death of the 
conceptus.
    (2) Fetal. (i) The sex and body weight of each fetus shall be 
determined.
    (ii) Each fetus shall be examined for external anomalies.
    (iii) Fetuses shall be examined for skeletal and soft tissue 
anomalies (e.g. variations and malformations or other categories of 
anomalies as defined by the performing laboratory).
    (A) For rodents, approximately one-half of each litter shall be 
prepared by standard techniques and examined for skeletal alterations, 
preferably bone and cartilage. The remainder shall be prepared and 
examined for soft tissue anomalies, using appropriate serial sectioning 
or gross dissection techniques. It is also acceptable to examine all 
fetuses by careful dissection for soft tissue anomalies followed by an 
examination for skeletal anomalies.
    (B) For rabbits, all fetuses shall be examined for both soft tissue 
and skeletal alterations. The bodies of these fetuses should be 
evaluated by careful dissection for soft-tissue anomalies, followed by 
preparation and examination for skeletal anomalies. An adequate 
evaluation of the internal structures of the head, including the eyes, 
brain, nasal passages, and tongue, should be conducted for at least half 
of the fetuses.
    (g) Data and reporting--(1) Treatment of results. Data shall be 
reported individually and summarized in tabular form, showing for each 
test group the types of change and the number of dams, fetuses, and 
litters displaying each type of change.
    (2) Evaluation of study results. The following shall be provided:
    (i) Maternal and fetal test results, including an evaluation of the 
relationship, or lack thereof, between the exposure of the animals to 
the test substance and the incidence and severity of all findings.
    (ii) Criteria used for categorizing fetal external, soft tissue, and 
skeletal anomalies.
    (iii) When appropriate, historical control data to enhance 
interpretation of study results. Historical data (on litter incidence 
and fetal incidence within litter), when used, should be compiled, 
presented, and analyzed in an appropriate and relevant manner. In order 
to justify its use as an analytical tool, information such as the dates 
of study conduct, the strain and source of the animals, and the vehicle 
and route of administration should be included.
    (iv) Statistical analysis of the study findings should include 
sufficient information on the method of analysis, so that an independent 
reviewer/statistician can reevaluate and reconstruct the analysis. In 
the evaluation of study data, the litter should be considered the basic 
unit of analysis.
    (v) In any study which demonstrates an absence of toxic effects, 
further investigation to establish absorption and bioavailability of the 
test substance should be considered.
    (3) Test report. In addition to the reporting requirements as 
specified under 40 CFR part 792, subpart J, the following specific 
information shall be reported. Both individual and summary data should 
be presented.
    (i) Species and strain.
    (ii) Maternal toxic response data by dose, including but not limited 
to:
    (A) The number of animals at the start of the test, the number of 
animals surviving, the number pregnant, and the number aborting.
    (B) Day of death during the study or whether animals survived to 
termination.
    (C) Day of observation of each abnormal clinical sign and its 
subsequent course.
    (D) Body weight and body weight change data, including body weight 
change adjusted for gravid uterine weight.
    (E) Food consumption and, if applicable, water consumption data.
    (F) Necropsy findings, including gravid uterine weight.
    (iii) Developmental endpoints by dose for litters with implants, 
including:
    (A) Corpora lutea counts.

[[Page 384]]

    (B) Implantation data, number and percent of live and dead fetuses, 
and resorptions (early and late).
    (C) Pre- and postimplantation loss calculations.
    (iv) Developmental endpoints by dose for litters with live fetuses, 
including:
    (A) Number and percent of live offspring.
    (B) Sex ratio.
    (C) Fetal body weight data, preferably by sex and with sexes 
combined.
    (D) External, soft tissue, and skeletal malformation and variation 
data. The total number and percent of fetuses and litters with any 
external, soft tissue, or skeletal alteration, as well as the types and 
incidences of individual anomalies, should be reported.
    (v) The numbers used in calculating all percentages or indices.
    (vi) Adequate statistical treatment of results.
    (vii) A copy of the study protocol and any amendments should be 
included.
    (h) References. For additional background information on this test 
guideline, the following references should be consulted. These 
references are available for inspection at the TSCA Nonconfidential 
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M 
St., SW., Washington, DC, 12 noon to 4 p.m., Monday through Friday, 
except legal holidays.
    (1) Aliverti, V.L. et al. The extent of fetal ossification as an 
index of delayed development in teratogenicity studies in the rat. 
Teratology. 20:237-242 (1979).
    (2) Barrow, M.V. and W.J. Taylor. A rapid method for detecting 
malformations in rat fetuses. Journal of Morphology 127:291-306 (1969).
    (3) Burdi, A.R. Toluidine blue-alizarin red S staining of cartilage 
and bone in whole-mount skeltons in vitro. Stain Technolology. 40:45-48 
(1965).
    (4) Edwards, J.A. Ed. Woolam,D.H.M. The external development of the 
rabbit and rat embryo. Vol. 3. Advances in Teratology (Academic, NY, 
1968).
    (5) Fritz, H. Prenatal ossification in rabbits as indicative of 
fetal maturity. Teratology. 11:313-320 (1974).
    (6) Fritz, H. and Hess, R. Ossification of the rat and mouse 
skeleton in the perinatal period. Teratology. 3:331-338 (1970).
    (7) Gibson, J.P. et al. Use of the rabbit in teratogenicity studies. 
Toxicology and Applied Pharmacology. 9:398-408 (1966).
    (8) Inouye, M. Differential staining of cartilage and bone in fetal 
mouse skeleton by alcian blue and alizarin red S. Congenital Anomalies. 
16(3):171-173 (1976).
    (9) Igarashi, E. et al. Frequence of spontaneous axial skeletal 
variations detected by the double staining technique for ossified and 
cartilaginous skeleton in rat fetuses. Congenital Anomalies. 32:381-391 
(1992).
    (10) Kimmel, C.A. et al. Skeletal development following heat 
exposure in the rat. Teratology. 47:229-242 (1993).
    (11) Kimmel, C.A. and Francis, E.Z. Proceedings of the workshop on 
the acceptability and interpretation of dermal developmental toxicity 
studies. Fundamental and Applied Toxicology. 14:386-398 (1990).
    (12) Kimmel, C.A. and C. Trammell. A rapid procedure for routine 
double staining of cartilage and bone in fetal and adult animals. Stain 
Technology. 56:271-273 (1981).
    (13) Kimmel, C.A. and Wilson, J.G. Skeletal deviation in rats: 
malformations or variations? Teratology. 8:309-316 (1973).
    (14) Marr, M.C. et al. Comparison of single and double staining for 
evaluation of skeletal development: the effects of ethylene glycol (EG) 
in CD rats. Teratology. 37:476 (1988).
    (15) Marr, M.C. et al. Developmental stages of the CD (Sprague-
Dawley) rat skeleton after maternal exposure to ethylene glycol. 
Teratology. 46:169-181 (1992).
    (16) McLeod, M.J. Differential staining of cartilage and bone in 
whole mouse fetuses by Alcian blue and alizarin red S. Teratology. 
22:299-301 (1980).
    (17) Monie, I.W. et al. Dissection procedures for rat fetuses 
permitting alizarin red staining of skeleton and histological study of 
viscera. Supplement to Teratology Workshop Manual. pp. 163-173 (1965).
    (18) Organisation for Economic Co-operation and Development, No. 
414: Teratogenicity, Guideline for Testing of Chemicals. [C(83)44 
(Final)] (1983).
    (19) Salewski (Koeln), V.E. Faerbermethode zum makroskopischen

[[Page 385]]

nachweis von implantations stellen am uterus der ratte. Naunyn-
Schmeidebergs Archiv f[uuml]r Pharmakologie und Experimentelle 
Pathologie. 247:367 (1964).
    (20) Spark, C. and Dawson,A.B. The order and time of appearance of 
centers of ossification in the fore and hind limbs of the albino rat, 
with special reference to the possible influence of the sex factor. 
American Journal of Anatomy. 41:411-445 (1928).
    (21) Staples, R.E. Detection of visceral alterations in mammalian 
fetuses. Teratology. 9(3):A37-A38 (1974).
    (22) Staples, R.E. and Schnell, V.L. Refinements in rapid clearing 
technique in the KOH--alizarin red S method for fetal bone. Stain 
Technology. 39:61-63 (1964).
    (23) Strong, R.M. The order time and rate of ossification of the 
albino rat (mus norvegicus albinus) skeleton. American Journal of 
Anatomy. 36: 313-355 (1928).
    (24) Stuckhardt, J.L. and Poppe, S.M. Fresh visceral examination of 
rat and rabbit fetuses used in teratogenicity testing. Teratogenesis, 
Carcinogenesis, and Mutagenesis. 4:181-188 (1984).
    (25) Van Julsingha, E.B. and Bennett,C.G. Eds. Neubert, D., Merker, 
H.J., and Kwasigroch, T.E. A dissecting procedure for the detection of 
anomalies in the rabbit foetal head. Methods in Prenatal Toxicology 
(University of Chicago, Chicago, IL, 1977) pp. 126-144.
    (26) Whitaker, J. and Dix, D.M. Double-staining for rat foetus 
skeletons in teratological studies. Laboratory Animals. 13:309-310 
(1979).
    (27) Wilson, J.G. Eds. Wilson, J.G. and Warkany, J. Embryological 
considerations in teratology. Teratology: Principles and Techniques 
(University of Chicago, Chicago, IL, 1965) pp. 251-277.



Sec. 799.9380  TSCA reproduction and fertility effects.

    (a) Scope. This section is intended to meet the testing requirements 
under section 4 of the TSCA. This section is for two-generation 
reproduction testing and is designed to provide general information 
concerning the effects of a test substance on the integrity and 
performance of the male and female reproductive systems, including 
gonadal function, the estrous cycle, mating behavior, conception, 
gestation, parturition, lactation, and weaning, and on the growth and 
development of the offspring. The study may also provide information 
about the effects of the test substance on neonatal morbidity, 
mortality, target organs in the offspring, and preliminary data on 
prenatal and postnatal developmental toxicity and serve as a guide for 
subsequent tests. Additionally, since the study design includes in utero 
as well as postnatal exposure, this study provides the opportunity to 
examine the susceptibility of the immature/neonatal animal.
    (b) Source. The source material used in developing this TSCA test 
guideline is the OPPTS harmonized test guideline 870.3800 (February 1996 
Public Draft). This source is available at the address in paragraph (g) 
of this section.
    (c) Good laboratory practice standards. The study shall be conducted 
in compliance with 40 CFR part 792--Good Laboratory Practice Standards.
    (d) Principle of the test method. The test substance is administered 
to parental (P) animals prior to and during their mating, during the 
resultant pregnancies, and through the weaning of their F1 offspring. 
The substance is then administered to selected F1 offspring during their 
growth into adulthood, mating, and production of an F2 generation, until 
the F2 generation is weaned.
    (e) Test procedures--(1) Animal selection--(i) Species and strain. 
The rat is the most commonly used species for testing. If another 
mammalian species is used, the tester shall provide justification/
reasoning for its selection, and appropriate modifications will be 
necessary. Healthy parental animals, which have been acclimated to 
laboratory conditions for at least 5 days and have not been subjected to 
previous experimental procedures, should be used. Strains of low 
fecundity shall not be used.
    (ii) Age. Parental (P) animals shall be 5 to 9 weeks old at the 
start of dosing. The animals of all test groups should be of uniform 
weight, age, and parity as nearly as practicable, and should be 
representative of the species and strain under study.
    (iii) Sex. (A) For an adequate assessment of fertility, both males 
and females shall be studied.

[[Page 386]]

    (B) The females shall be nulliparous and nonpregnant.
    (iv) Number of animals. Each control group shall contain a 
sufficient number of mating pairs to yield approximately 20 pregnant 
females. Each test group shall contain a similar number of mating pairs.
    (v) Identification of animals. Each animal shall be assigned a 
unique identification number. For the P generation, this should be done 
before dosing starts. For the F1 generation, this should be done for 
animals selected for mating; in addition, records indicating the litter 
of origin shall be maintained for all selected F1 animals.
    (2) Administration of test and control substances--(i) Dose levels 
and dose selection. (A) At least three-dose levels and a concurrent 
control shall be used. Healthy animals should be randomly assigned to 
the control and treatment groups, in a manner which results in 
comparable mean body weight values among all groups. The dose levels 
should be spaced to produce a gradation of toxic effects. Unless limited 
by the physical/chemical nature or biological properties of the test 
substance, the highest dose should be chosen with the aim to induce some 
reproductive and/or systemic toxicity but not death or severe suffering. 
In the case of parental mortality, this should not be more than 
approximately 10%. The intermediate dose levels should produce minimal 
observable toxic effects. The lowest dose level should not produce any 
evidence of either systemic or reproductive toxicity (i.e., the no-
observed-adverse-effect level, NOAEL) or should be at or near the limit 
of detection for the most sensitive endpoint. Two- or four-fold 
intervals are frequently optimal for spacing the dose levels, and the 
addition of a fourth test group is often preferable to using very large 
intervals (e.g., more than a factor of 10) between dosages.
    (B) It is desirable that additional information on metabolism and 
pharmacokinetics of the test substance be available to demonstrate the 
adequacy of the dosing regimen. This information should be available 
prior to testing.
    (C) The highest dose tested should not exceed 1,000 mg/kg/day (or 
20,000 ppm in the diet), unless potential human exposure data indicate 
the need for higher doses. If a test performed at the limit dose level, 
using the procedures described for this study, produces no observable 
toxicity and if an effect would not be expected based upon data from 
structurally related compounds, then a full study using three dose 
levels may not be considered necessary.
    (ii) Control group. (A) A concurrent control group shall be used. 
This group shall be an untreated or sham treated group or a vehicle-
control group if a vehicle is used in administering the test substance.
    (B) If a vehicle is used in administering the test substance, the 
control group shall receive the vehicle in the highest volume used.
    (C) If a vehicle or other additive is used to facilitate dosing, 
consideration should be given to the following characteristics: Effects 
on the absorption, distribution, metabolism, or retention of the test 
substance; effects on the chemical properties of the test substance 
which may alter its toxic characteristics; and effects on the food or 
water consumption or the nutritional status of the animals.
    (D) If a test substance is administered in the diet and causes 
reduced dietary intake or utilization, the use of a pair-fed control 
group may be considered necessary.
    (iii) Route of administration. (A) The test substance is usually 
administered by the oral route (diet, drinking water, or gavage).
    (B) If administered by gavage or dermal application, the dosage 
administered to each animal prior to mating and during gestation and 
lactation shall be based on the individual animal body weight and 
adjusted weekly at a minimum.
    (C) If another route of administration is used, for example, when 
the route of administration is based upon the principal route of 
potential human exposure, the tester should provide justification and 
reasoning for its selection, and appropriate modifications may be 
necessary. Care should be taken to minimize stress on the maternal 
animals and their litters during gestation and lactation.

[[Page 387]]

    (D) All animals should be dosed by the same method during the 
appropriate experimental period.
    (iv) Dosing schedule. (A) The animals should be dosed with the test 
substance on a 7-days-a-week basis.
    (B) Daily dosing of the parental (P) males and females shall begin 
when they are 5 to 9 weeks old. Daily dosing of the F1 males and females 
shall begin at weaning. For both sexes (P and F1), dosing shall be 
continued for at least 10 weeks before the mating period.
    (C) Daily dosing of the P and F1 males and females shall continue 
until termination.
    (3) Mating procedure--(i) Parental. (A) For each mating, each female 
shall be placed with a single randomly selected male from the same dose 
level (1:1 mating) until evidence of copulation is observed or either 3 
estrous periods or 2 weeks has elapsed. Animals should be separated as 
soon as possible after evidence of copulation is observed. If mating has 
not occurred after 2 weeks or 3 estrous periods, the animals should be 
separated without further opportunity for mating. Mating pairs should be 
clearly identified in the data.
    (B) Vaginal smears shall be collected daily and examined for all 
females during mating, until evidence of copulation is observed.
    (C) Each day, the females shall be examined for presence of sperm or 
vaginal plugs. Day 0 of pregnancy is defined as the day a vaginal plug 
or sperm are found.
    (ii) F1 mating. For mating the F1 offspring, at least one male and 
one female should be randomly selected from each litter for mating with 
another pup of the same dose level but different litter, to produce the 
F2 generation.
    (iii) Second mating. In certain instances, such as poor reproductive 
performance in the controls, or in the event of treatment-related 
alterations in litter size, the adults may be remated to produce an F1b 
or F2b litter. If production of a second litter is deemed necessary in 
either generation, the dams should be remated approximately 1-2 weeks 
following weaning of the last F1a or F2a litter.
    (iv) Special housing. After evidence of copulation, animals that are 
presumed to be pregnant shall be caged separately in delivery or 
maternity cages. Pregnant animals shall be provided with nesting 
materials when parturition is near.
    (v) Standardization of litter sizes. (A) Animals should be allowed 
to litter normally and rear their offspring to weaning. Standardization 
of litter sizes is optional.
    (B) If standardization is performed, the following procedure should 
be used. On day 4 after birth, the size of each litter may be adjusted 
by eliminating extra pups by random selection to yield, as nearly as 
possible, four males and four females per litter or five males and five 
females per litter. Selective elimination of pups, i.e. based upon body 
weight, is not appropriate. Whenever the number of male or female pups 
prevents having four (or five) of each sex per litter, partial 
adjustment (for example, five males and three females, or four males and 
six females) is acceptable. Adjustments are not appropriate for litters 
of eight pups or less.
    (4) Observation of animals--(i) Parental. (A) Throughout the test 
period, each animal shall be observed at least once daily, considering 
the peak period of anticipated effects after dosing. Mortality, 
moribundity, pertinent behavioral changes, signs of difficult or 
prolonged parturition, and all signs of overt toxicity shall be recorded 
at this cageside examination. In addition, thorough physical 
examinations should be conducted weekly on each animal.
    (B) Parental animals (P and F1) shall be weighed on the first day of 
dosing and weekly thereafter. Parental females (P and F1) should be 
weighed at a minimum on approximately gestation days 0, 7, 14, and 21, 
and during lactation on the same days as the weighing of litters.
    (C) During the premating and gestation periods, food consumption 
shall be measured weekly at a minimum. Water consumption should be 
measured weekly at a minimum if the test substance is administered in 
the water.
    (D) Estrous cycle length and pattern should be evaluated by vaginal 
smears for all P and F1 females during a minimum of 3 weeks prior to 
mating and throughout cohabitation; care should

[[Page 388]]

be taken to prevent the induction of pseudopregnancy.
    (E) For all P and F1 males at termination, sperm from one testis and 
one epididymis shall be collected for enumeration of homogenization-
resistant spermatids and cauda epididymal sperm reserves, respectively. 
In addition, sperm from the cauda epididymis (or vas deferens) should be 
collected for evaluation of sperm motility and sperm morphology.
    (1) The total number of homogenization-resistant testicular sperm 
and cauda epididymal sperm should be enumerated. The method described in 
the reference under paragraph (g)(8) of this section may be used. Cauda 
sperm reserves can be derived from the concentration and volume of sperm 
in the suspension used to complete the qualitative evaluations, and the 
number of sperm recovered by subsequent mincing and/or homogenizing of 
the remaining cauda tissue. Enumeration in only control and high-dose P 
and F1 males may be performed unless treatment-related effects are 
observed; in that case, the lower dose groups should also be evaluated.
    (2) An evaluation of epididymal (or vas deferens) sperm motility 
should be performed. Sperm should be recovered while minimizing damage 
(the evaluation techniques as described in the reference under paragraph 
(g)(8) of this section may be used), and the percentage of progressively 
motile sperm should be determined either subjectively or objectively. 
For objective evaluations, an acceptable counting chamber of sufficient 
depth can be used to effectively combine the assessment of motility with 
sperm count and sperm morphology. When computer-assisted motion analysis 
is performed, the derivation of progressive motility relies on user-
defined thresholds for average path velocity and straightness or linear 
index. If samples are videotaped, or images otherwise recorded, at the 
time of necropsy, subsequent analysis of only control and high-dose P 
and F1 males may be performed unless treatment-related effects are 
observed; in that case, the lower dose groups should also be evaluated. 
In the absence of a video or digital image, all samples in all treatment 
groups should be analyzed at necropsy.
    (3) A morphological evaluation of an epididymal (or vas deferens) 
sperm sample shall be performed. Sperm (at least 200 per sample) should 
be examined as fixed, wet preparations (the techniques for such 
examinations is described in the references under paragraphs (g)(4) and 
(g)(8) of this section may be used) and classified as either normal 
(both head and midpiece/tail appear normal) or abnormal. Examples of 
morphologic sperm abnormalities would include fusion, isolated heads, 
and misshapen heads and/or tails. Evaluation of only control and high-
dose P and F1 males may be performed unless treatment-related effects 
are observed; in that case, the lower dose groups should also be 
evaluated.
    (ii) Offspring. (A) Each litter should be examined as soon as 
possible after delivery (lactation day 0) to establish the number and 
sex of pups, stillbirths, live births, and the presence of gross 
anomalies. Pups found dead on day 0 should be examined for possible 
defects and cause of death.
    (B) Live pups should be counted, sexed, and weighed individually at 
birth, or soon thereafter, at least on days 4, 7, 14, and 21 of 
lactation, at the time of vaginal patency or balanopreputial separation, 
and at termination.
    (C) The age of vaginal opening and preputial separation should be 
determined for F1 weanlings selected for mating. If there is a 
treatment-related effect in F1 sex ratio or sexual maturation, 
anogenital distance should be measured on day 0 for all F2 pups.
    (5) Termination schedule. (i) All P and F1 adult males and females 
should be terminated when they are no longer needed for assessment of 
reproductive effects.
    (ii) F1 offspring not selected for mating and all F2 offspring 
should be terminated at comparable ages after weaning.
    (6) Gross necropsy. (i) At the time of termination or death during 
the study, all parental animals (P and F1) and when litter size permits 
at least three pups per sex per litter from the unselected F1 weanlings 
and the F2 weanlings shall be examined

[[Page 389]]

macroscopically for any structural abnormalities or pathological 
changes. Special attention shall be paid to the organs of the 
reproductive system.
    (ii) Dead pups or pups that are terminated in a moribund condition 
should be examined for possible defects and/or cause of death.
    (iii) At the time of necropsy, a vaginal smear should be examined to 
determine the stage of the estrous cycle. The uteri of all cohabited 
females should be examined, in a manner which does not compromise 
histopathological evaluation, for the presence and number of 
implantation sites.
    (7) Organ weights. (i) At the time of termination, the following 
organs of all P and F1 parental animals shall be weighed:
    (A) Uterus (with oviducts and cervix), ovaries.
    (B) Testes, epididymides (total weights for both and cauda weight 
for either one or both), seminal vesicles (with coagulating glands and 
their fluids), and prostate.
    (C) Brain, pituitary, liver, kidneys, adrenal glands, spleen, and 
known target organs.
    (ii) For F1 and F2 weanlings that are examined macroscopically, the 
following organs shall be weighed for one randomly selected pup per sex 
per litter.
    (A) Brain.
    (B) Spleen and thymus.
    (8) Tissue preservation. The following organs and tissues, or 
representative samples thereof, shall be fixed and stored in a suitable 
medium for histopathological examination.
    (i) For the parental (P and F1) animals:
    (A) Vagina, uterus with oviducts, cervix, and ovaries.
    (B) One testis (preserved in Bouins fixative or comparable 
preservative), one epididymis, seminal vesicles, prostate, and 
coagulating gland.
    (C) Pituitary and adrenal glands.
    (D) Target organs, when previously identified, from all P and F1 
animals selected for mating.
    (E) Grossly abnormal tissue.
    (ii) For F1 and F2 weanlings selected for macroscopic examination: 
Grossly abnormal tissue and target organs, when known.
    (9) Histopathology--(i) Parental animals. Full histopathology of the 
organs listed in paragraph (e)(8)(i) of this section shall be performed 
for ten randomly chosen high dose and control P and F1 animals per sex, 
for those animals that were selected for mating. Organs demonstrating 
treatment-related changes shall also be examined for the remainder of 
the high-dose and control animals and for all parental animals in the 
low- and mid-dose groups. Additionally, reproductive organs of the low- 
and mid-dose animals suspected of reduced fertility, e.g., those that 
failed to mate, conceive, sire, or deliver healthy offspring, or for 
which estrous cyclicity or sperm number, motility, or morphology were 
affected, shall be subjected to histopathological evaluation. Besides 
gross lesions such as atrophy or tumors, testicular histopathological 
examination should be conducted in order to identify treatment-related 
effects such as retained spermatids, missing germ cell layers or types, 
multinucleated giant cells, or sloughing of spermatogenic cells into the 
lumen. Examination of the intact epididymis should include the caput, 
corpus, and cauda, which can be accomplished by evaluation of a 
longitudinal section, and should be conducted in order to identify such 
lesions as sperm granulomas, leukocytic infiltration (inflammation), 
aberrant cell types within the lumen, or the absence of clear cells in 
the cauda epididymal epithelium. The postlactational ovary should 
contain primordial and growing follicles as well as the large corpora 
lutea of lactation. Histopathological examination should detect 
qualitative depletion of the primordial follicle population. A 
quantitative evaluation of primordial follicles should be conducted for 
all F1 females; the number of animals, ovarian section selection, and 
section sample size should be statistically appropriate for the 
evaluation procedure used. Examination should include enumeration of the 
number of primordial follicles, which can be combined with small growing 
follicles (see paragraphs (g)(1) and (g)(2) of this section), for 
comparison of treated and control ovaries.

[[Page 390]]

    (ii) Weanling. For F1 and F2 weanlings, histopathological 
examination of treatment-related abnormalities noted in macroscopic 
examination should be considered, if such evaluation were deemed 
appropriate and would contribute to the interpretation of the study 
data.
    (f) Data and reporting--(1) Treatment of results. Data shall be 
reported individually and summarized in tabular form, showing for each 
test group the types of change and the number of animals displaying each 
type of change.
    (2) Evaluation of study results. (i) An evaluation of test results, 
including the statistical analysis, shall be provided. This should 
include an evaluation of the relationship, or lack thereof, between the 
exposure of the animals to the test substance and the incidence and 
severity of all abnormalities.
    (ii) When appropriate, historical control data should be used to 
enhance interpretation of study results. Historical data, when used, 
should be compiled, presented, and analyzed in an appropriate and 
relevant manner. In order to justify its use as an analytical tool, 
information such as the dates of study conduct, the strain and source of 
the animals, and the vehicle and route of administration should be 
included.
    (iii) Statistical analysis of the study findings should include 
sufficient information on the method of analysis, so that an independent 
reviewer/statistician can reevaluate and reconstruct the analysis.
    (iv) In any study which demonstrates an absence of toxic effects, 
further investigation to establish absorption and bioavailability of the 
test substance should be considered.
    (3) Test report. In addition to the reporting requirements as 
specified under 40 CFR part 792, subpart J, the following specific 
information shall be reported. Both individual and summary data should 
be presented.
    (i) Species and strain.
    (ii) Toxic response data by sex and dose, including indices of 
mating, fertility, gestation, birth, viability, and lactation; offspring 
sex ratio; precoital interval, including the number of days until mating 
and the number of estrous periods until mating; and duration of 
gestation calculated from day 0 of pregnancy. The report should provide 
the numbers used in calculating all indices.
    (iii) Day (week) of death during the study or whether animals 
survived to termination; date (age) of litter termination.
    (iv) Toxic or other effects on reproduction, offspring, or postnatal 
growth.
    (v) Developmental milestone data (mean age of vaginal opening and 
preputial separation, and mean anogenital distance, when measured).
    (vi) Number of P and F1 females cycling pattern and mean estrous 
cycle length.
    (vii) Day (week) of observation of each abnormal sign and its 
subsequent course.
    (viii) Body weight and body weight change data by sex for P, F1, and 
F2 animals.
    (ix) Food (and water, if applicable) consumption, food efficiency 
(body weight gain per gram of food consumed), and test material 
consumption for P and F1 animals, except for the period of cohabitation.
    (x) Total cauda epididymal sperm number, homogenization-resistant 
testis spermatid number, number and percent of progressively motile 
sperm, number and percent of morphologically normal sperm, and number 
and percent of sperm with each identified anomaly.
    (xi) Stage of the estrous cycle at the time of termination for P and 
F1 parental females.
    (xii) Necropsy findings.
    (xiii) Implantation data and postimplantation loss calculations for 
P and F1 parental females.
    (xiv) Absolute and adjusted organ weight data.
    (xv) Detailed description of all histopathological findings.
    (xvi) Adequate statistical treatment of results.
    (xvii) A copy of the study protocol and any amendments should be 
included.
    (g) References. For additional backgound information on this test 
guideline, the following references should be consulted. These 
references are available for inspection at the TSCA Nonconfidential 
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M 
St., SW.,

[[Page 391]]

Washington, DC, 12 noon to 4 p.m., Monday through Friday, except legal 
holidays.
    (1) Gray, L.E. et al. A dose-response analysis of methoxychlor-
induced alterations of reproductive development and function in the rat. 
Fundamental and Applied Toxicology. 12:92-108 (1989).
    (2) Heindel, J.J. et al. Ed. Hirshfield, A.N. Histological 
assessment of ovarian follicle number in mice as a screen of ovarian 
toxicity. Growth Factors and the Ovary (Plenum, NY, 1989) pp. 421-426.
    (3) Korenbrot, C.C. et al. Preputial separation as an external sign 
of pubertal development in the male rat. Biology of Reproduction. 
17:298-303 (1977).
    (4) Linder, R.E. et al. Endpoints of spermatoxicity in the rat after 
short duration exposures to fourteen reproductive toxicants. 
Reproductive Toxicology. 6:491-505 (1992).
    (5) Manson, J.M. and Kang, Y.J. Ed. Hayes, A.W. Test methods for 
assessing female reproductive and developmental toxicology. Principles 
and Methods of Toxicology (Raven, NY, 1989).
    (6) Organisation for Economic Co-operation and Development, No. 416: 
Two Generation Reproduction Toxicity Study, Guidelines for Testing of 
Chemicals. [C(83)44 (Final)] (1983).
    (7) Pederson, T. and Peters, H. Proposal for classification of 
oocytes and follicles in the mouse ovary. Journal of Reproduction and 
Fertility. 17:555-557 (1988).
    (8) Seed, J., Chapin, R.E. E.D. Clegg, L.A. Dostal, R.H. Foote, M.E. 
Hurtt, G.R. Klinefelter, S.L. Makris, S.D. Perreault, S. Schrader, D. 
Seyler, R. Sprando, K.A. Treinen, D.N.R. Veeramachaneni, and Wise, L.D. 
Methods for assessing sperm motility, morphology, and counts in the rat, 
rabbit, and dog: a consensus report. Reproductive Toxicology. 10(3):237-
244 (1996).
    (9) Smith, B.J. et al. Comparison of random and serial sections in 
assessment of ovarian toxicity. Reproductive Toxicology. 5:379-383 
(1991).
    (10) Thomas, J.A. Eds. M.O. Amdur, J. Doull, and C.D. Klaassen. 
Toxic responses of the reproductive system. Casarett and Doull's 
Toxicology (Pergamon, NY, 1991).
    (11) Working, P.K. and Hurtt, M. Computerized videomicrographic 
analysis of rat sperm motility. Journal of Andrology. 8:330-337 (1987).
    (12) Zenick, H. et al. Ed. Hayes, A.W. Assessment of male 
reproductive toxicity: a risk assessment approach. Principles and 
Methods of Toxicology (Raven, NY, 1994).

[62 FR 43824, Aug. 15, 1997, as amended at 64 FR 35078, June 30, 1999]



Sec. 799.9410  TSCA chronic toxicity.

    (a) Scope--(1) Applicability. This section is intended to meet the 
testing requirement of the Toxic Substances Control Act (TSCA) (15 
U.S.C. 2601).
    (2) Source. The source material used in developing this TSCA test 
guideline is the Office of Prevention, Pesticides and Toxic Substances 
(OPPTS) harmonized test guideline 870.4100 (August 1998, final 
guidelines). This source is available at the address in paragraph (h) of 
this section
    (b) Purpose. The objective of a chronic toxicity study is to 
determine the effects of a substance in a mammalian species following 
prolonged and repeated exposure. A chronic toxicity study should 
generate data from which to identify the majority of chronic effects and 
to define long-term dose-response relationships. The design and conduct 
of chronic toxicity tests should allow for the detection of general 
toxic effects, including neurological, physiological, biochemical, and 
hematological effects and exposure-related morphological (pathological) 
effects.
    (c) Definitions. The definitions in section 3 of TSCA and in 40 CFR 
Part 792--Good Laboratory Practice Standards apply to this section. The 
following definitions also apply to this section.
    Chronic toxicity is the adverse effects occurring as a result of the 
repeated daily exposure of experimental animals to a chemical by the 
oral, dermal, or inhalation routes of exposure.
    Cumulative toxicity is the adverse effects of repeated doses 
occurring as a result of prolonged action on, or increased concentration 
of, the administered test substance or its metabolites in susceptible 
tissue.
    Dose in a chronic toxicity study is the amount of test substance 
administered daily via the oral, dermal or inhalation routes for a 
period of at least 12

[[Page 392]]

months. Dose is expressed as weight of the test substance (grams, 
milligrams) per unit body weight of test animal (milligram per 
kilogram), or as weight of the test substance in parts per million (ppm) 
in food or drinking water per day. For inhalation exposure, dose is 
expressed as weight of the test substance per unit volume of air 
(milligrams per liter) or as parts per million per day. For dermal 
exposure, dose is expressed as weight of the test substance (grams, 
milligrams) per unit body weight of the test animal (milligrams per 
kilogram) or as weight of the substance per unit of surface area 
(milligrams per square centimeter) per day.
    No-observed-effects level (NOEL) is the maximum dose used in a study 
which produces no adverse effects. The NOEL is usually expressed in 
terms of the weight of a test substance given daily per unit weight of 
test animal (milligrams per kilogram per day).
    Target organ is any organ of a test animal showing evidence of an 
effect induced by a test substance.
    (d) Limit test. If a test at one dose level of at least 1,000 mg/kg 
body weight (expected human exposure may indicate the need for a higher 
dose level), using the procedures described for this study, produces no 
observable toxic effects and if toxicity would not be expected based 
upon data of structurally related compounds, a full study using three 
dose levels might not be necessary.
    (e) Test procedures--(1) Animal selection--(i) Species and strain. 
Testing should be performed with two mammalian species, one a rodent and 
the other a nonrodent. The rat is the preferred rodent species. Commonly 
used laboratory strains must be employed.
    (ii) Age/weight. (A) Testing must be started with young healthy 
animals as soon as possible after weaning and acclimatization.
    (B) Dosing of rodents should generally begin no later than 8 weeks 
of age.
    (C) Dosing of non-rodents should begin between 4 and 6 months of age 
and in no case later than 9 months of age.
    (D) At commencement of the study, the weight variation of animals 
used should be within 20% of the mean weight for each sex.
    (E) Studies using prenatal or neonatal animals may be recommended 
under special conditions.
    (iii) Sex. (A) Equal numbers of animals of each sex should be used 
at each dose level.
    (B) Females should be nulliparous and nonpregnant.
    (iv) Numbers. (A) For rodents, at least 40 animals (20 males and 20 
females) and for nonrodents at least 8 animals (4 females and 4 males) 
should be used at each dose level and concurrent control group.
    (B) If interim sacrifices are planned, the number should be 
increased by the number of animals scheduled to be sacrificed during the 
course of the study.
    (C) The number of animals at the termination of the study must be 
adequate for a meaningful and valid statistical evaluation of chronic 
effects. The Agency must be notified if excessive early deaths or other 
problems are encountered that might compromise the integrity of the 
study.
    (D) To avoid bias, the use of adequate randomization procedures for 
the proper allocation of animals to test and control groups is required.
    (E) Each animal should be assigned a unique identification number. 
Dead animals, their preserved organs and tissues, and microscopic slides 
should be identified by reference to the unique numbers assigned.
    (v) Husbandry. (A) Rodents may be group-caged by sex, but the number 
of animals per cage must not interfere with clear observation of each 
animal. The biological properties of the test substance or toxic effects 
(e.g., morbidity, excitability) may indicate a need for individual 
caging. Rodents should be housed individually in dermal studies and 
during exposure in inhalation studies. Caging should be appropriate to 
the nonrodent species.
    (B) The temperature of the experimental animal rooms should be at 22 
3 [deg]C.
    (C) The relative humidity of the experimental animal rooms should be 
50 20%.
    (D) Where lighting is artificial, the sequence should be 12 hours 
light/12 hours dark.

[[Page 393]]

    (E) Control and test animals should be fed from the same batch and 
lot. The feed should be analyzed to assure adequacy of nutritional 
requirements of the species tested and for impurities that might 
influence the outcome of the test. Animals should be fed and watered ad 
libitum with food replaced at least weekly.
    (F) The study should not be initiated until animals have been 
allowed a period of acclimatization/quarantine to environmental 
conditions, nor should animals from outside sources be placed on test 
without an adequate period of quarantine. An acclimation period of at 
least 5 days is recommended.
    (2) Control and test substances. (i) Where necessary, the test 
substance is dissolved or suspended in a suitable vehicle. If a vehicle 
or diluent is needed it should not elicit toxic effects itself nor 
substantially alter the chemical or toxicological properties of the test 
substance. It is recommended that wherever possible the use of an 
aqueous solution be the first choice, followed by consideration of 
solution in oil, and finally, solution in other vehicles.
    (ii) One lot of the test substance should be used, if possible, 
throughout the duration of the study, and the research sample should be 
stored under conditions that maintain its purity and stability. Prior to 
the initiation of the study, there should be a characterization of the 
test substance, including the purity of the test compound, and, if 
technically feasible, the names and quantities of contaminants and 
impurities.
    (iii) If the test or control substance is to be incorporated into 
feed or another vehicle, the period during which the test substance is 
stable in such a mixture should be determined prior to the initiation of 
the study. Its homogeneity and concentration should be determined prior 
to the initiation of the study and periodically during the study. 
Statistically randomized samples of the mixture should be analyzed to 
ensure that proper mixing, formulation, and storage procedures are being 
followed, and that the appropriate concentration of the test or control 
substance is contained in the mixture.
    (3) Control groups. A concurrent control group is required. This 
group should be an untreated or sham-treated control group or, if a 
vehicle is used in administering the test substance, a vehicle control 
group. If the toxic properties of the vehicle are not known or cannot be 
made available, both untreated and vehicle control groups are required.
    (4) Satellite group. A satellite group of 40 animals (20 animals per 
sex) for rodents and 8 animals (4 animals per sex) for nonrodents may be 
treated with the high-dose level for 12 months and observed for 
reversibility, persistence, or delayed occurrence of toxic effects for a 
post-treatment of appropriate length, normally not less than 28 days. In 
addition, a control group of 40 animals (20 animals per sex) for rodents 
and 8 animals (4 animals per sex) for nonrodents should be added to the 
satellite study.
    (5) Dose levels and dose selections. (i) In chronic toxicity tests, 
it is desirable to determine a dose-response relationship as well as a 
NOEL. Therefore, at least three dose levels with a control group and, 
where appropriate, a vehicle control (corresponding to the concentration 
of the vehicle at the highest exposure level) should be used. Dose 
levels should be spaced to produce a gradation of effects. A rationale 
must be provided for the doses selected.
    (ii) The highest-dose level should elicit signs of toxicity without 
substantially altering the normal life span of the animal. The highest 
dose should be determined based on the findings from a 90-day study to 
ensure that the dose used is adequate to assess the chronic toxicity of 
the test substance. Thus, the selection of the highest dose to be tested 
is dependent upon changes observed in several toxicological parameters 
in subchronic studies. The highest dose tested need not exceed 1,000 mg/
kg/day. If dermal application of the test substance produces severe skin 
irritation, then it may be necessary either to terminate the study and 
choose a lower high-dose level or to reduce the dose level. Gross 
criteria for defining severe irritation would include ulcers, fissures, 
exudate/crust(eschar), dead tissue, or anything leading to destruction 
of the functional integrity of the epidermis (e.g. caking,

[[Page 394]]

open sores, fissuring, eschar). Histological criteria for defining 
severe irritation would include follicular and interfollicular crust, 
microulcer, mild/moderate degeneration/necrosis, moderate/marked 
epidermal edema, marked dermal edema, and marked inflammation.
    (iii) The intermediate dose levels should be spaced to produce a 
gradation of toxic effects.
    (iv) The lowest-dose level should produce no evidence of toxicity.
    (6) Administration of the test substance. The three main routes of 
administration are oral, dermal, and inhalation. The choice of the route 
of administration depends upon the physical and chemical characteristics 
of the test substance and the form typifying exposure in humans.
    (i) Oral studies. Ideally, the animals should be dosed by gavage or 
with capsules on a 7-day per week basis for a period of at least 12 
months. However, based primarily on practical considerations, dosing by 
gavage or capsules on a 5-day per week schedule is acceptable. If the 
test substance is administered via in the drinking water or mixed in the 
diet, exposure should be on a 7-day per week basis.
    (ii) Dermal studies. (A) Preparation of animal skin. Shortly before 
testing, fur should be clipped from not less than 10% of the body 
surface area for application of the test substance. In order to dose 
approximately 10% of the body surface, the area starting at the scapulae 
(shoulders) to the wing of the ileum (hipbone) and half way down the 
flank on each side of the animal should be shaved. Shaving should be 
carried out approximately 24 hours before dosing. Repeated clipping or 
shaving is usually needed at approximately weekly intervals. When 
clipping or shaving the fur, care should be taken to avoid abrading the 
skin which could alter its permeability.
    (B) Preparation of test substance. Liquid test substances are 
generally used undiluted, except as indicated in paragraph (e)(5)(ii) of 
this section. Solids should be pulverized when possible. The substance 
should be moistened sufficiently with water or, when necessary, with a 
suitable vehicle to ensure good contact with the skin. When a vehicle is 
used, the influence of the vehicle on toxicity of, and penetration of 
the skin by, the test substance should be taken into account. The volume 
of application should be kept constant, e.g., less than 100 [micro]L for 
the mouse and less than 300 [micro]L for the rat. Different 
concentrations of test solution should be prepared for different dose 
levels.
    (C) Administration of test substance. The duration of exposure 
should be at least for 12 months. Ideally, the animals should be treated 
with test substance for at least 6 hours per day on a 7-day per week 
basis. However, based on practical considerations, application on a 5-
day per week basis is acceptable. Dosing should be conducted at 
approximately the same time each day. The test substance should be 
applied uniformly over the treatment site. The surface area covered may 
be less for highly toxic substances. As much of the area should be 
covered with as thin and uniform a film as possible. For rats, the test 
substance may be held in contact with the skin with a porous gauze 
dressing and nonirritating tape if necessary. The test site should be 
further covered in a suitable manner to retain the gauze dressing plus 
test substance and to ensure that the animals cannot ingest the test 
substance. The application site should not be covered when the mouse is 
the species of choice. The test substance may be wiped from the skin 
after the six-hour exposure period to prevent ingestion.
    (iii) Inhalation studies. (A) The animals should be exposed to the 
test substance for 6 hours per day on a 7-day per week basis, for a 
period of at least 12 months. However, based primarily on practical 
considerations, exposure for 6 hours per day on a 5-day per week basis 
is acceptable.
    (B) The animals should be tested in dynamic inhalation equipment 
designed to sustain a minimum air flow of 10 air changes per hour, an 
adequate oxygen content of at least 19%, and uniform conditions 
throughout the exposure chamber. Maintenance of slight negative pressure 
inside the chamber will prevent leakage of the test substance into 
surrounding areas. It is not

[[Page 395]]

normally necessary to measure chamber oxygen concentration if airflow is 
adequate.
    (C) The selection of a dynamic inhalation chamber should be 
appropriate for the test substance and test system. When a whole body 
chamber is used, individual housing must be used to minimize crowding of 
the test animals and maximize their exposure to the test substance. To 
ensure stability of a chamber atmosphere, the total volume occupied by 
the test animals should not exceed 5% of the volume of the test chamber. 
It is recommended, but not required, that nose-only or head-only 
exposure be used for aerosol studies in order to minimize oral exposures 
due to animals licking compound off their fur. The animals should be 
acclimated and heat stress minimized.
    (D) The temperature at which the test is performed should be 
maintained at 22 2 [deg]C. The relative humidity 
should be maintained between 40-60%, but in certain instances (e.g., use 
of water vehicle) this may not be practicable.
    (E) The rate of air flow should be monitored continuously but 
recorded at least three times during the exposure.
    (F) Temperature and humidity should be monitored continuously but 
should be recorded at least every 30 min.
    (G) The actual concentrations of the test substance should be 
measured in the breathing zone. During the exposure period, the actual 
concentrations of the test substance should be held as constant as 
practicable, monitored continuously or intermittently depending on the 
method of analysis. Chamber concentration may be measured using 
gravimetric or analytical methods, as appropriate. If trial run 
measurements are reasonably consistent (10% for 
liquid aerosol, gas, or vapor; 20% for dry 
aerosol), then two measurements should be sufficient. If measurements 
are not consistent, three to four measurements should be taken. If there 
is some difficulty measuring chamber analytical concentration due to 
precipitation, nonhomogeneous mixtures, volatile components, or other 
factors, additional analysis of inert components may be necessary.
    (H) During the development of the generating system, particle size 
analysis should be performed to establish the stability of aerosol 
concentrations with respect to particle size. The mass median 
aerodynamic diameter (MMAD) particle size range should be between 1-3 
[micro]m. The particle size of hygroscopic materials should be small 
enough when dry to assure that the size of the swollen particle will 
still be within the 1-3 [micro]m range. Measurements of aerodynamic 
particle size in the animal's breathing zone should be measured during a 
trial run. If MMAD values for each exposure level are within 10% of each 
other, then two measurements during the exposures should be sufficient. 
If pretest measurements are not within 10% of each other, three to four 
measurements should be taken.
    (I) Feed should be withheld during exposure. Water may also be 
withheld during exposure.
    (7) Observation period. (i) Animals should be observed for a period 
of at least 12 months.
    (ii) Animals in a satellite group (if used) scheduled for follow-up 
observations should be kept for at least 28 days further without 
treatment to detect recovery from, or persistence of, toxic effects.
    (8) Observation of animals. (i) Observations should be made at least 
twice each day for morbidity and mortality. Appropriate actions should 
be taken to minimize loss of animals to the study (e.g., necropsy or 
refrigeration of those animals found dead and isolation or sacrifice of 
weak or moribund animals). General clinical observations should be made 
at least once a day, preferably at the same time each day, taking into 
consideration the peak period of anticipated effects after dosing. The 
clinical condition of the animal should be recorded.
    (ii) A careful clinical examination should be made at least once 
prior to the initiation of treatment (to allow for within subject 
comparisons) and once weekly during treatment in all animals. These 
observations should be made outside the home cage, preferably in a 
standard arena, and at similar times on each occasion. Effort should be 
made to ensure that variations in the observation conditions

[[Page 396]]

are minimal. Observations should be detailed and carefully recorded, 
preferably using scoring systems, explicitly defined by the testing 
laboratory. Signs noted should include, but not be limited to, changes 
in skin, fur, eyes, mucous membranes, occurrence of secretions and 
excretions and autonomic activity (e.g., lacrimation, piloerection, 
pupil size, unusual respiratory pattern). Changes in gait, posture and 
response to handling as well as the presence of clonic or tonic 
movements, stereotypies (e.g., excessive grooming, repetitive circling) 
or bizarre behavior (e.g., self-mutilation, walking backwards) should be 
recorded.
    (iii) Once, near the end of the first year of the exposure period 
and in any case not earlier than in month 11, assessment of motor 
activity, grip strength, and sensory reactivity to stimuli of different 
types (e.g., visual, auditory, and proprioceptive stimuli) should be 
conducted in rodents. Further details of the procedures that could be 
followed are described in the references listed under paragraphs (h)(2), 
(h)(7), (h)(8), and (h)(11) of this section.
    (iv) Functional observations conducted towards the end of the study 
may be omitted when data on functional observations are available from 
other studies and the daily clinical observations did not reveal any 
functional deficits.
    (v) Exceptionally, functional observations may be omitted for groups 
that otherwise reveal signs of toxicity to an extent that would 
significantly interfere with functional test performance.
    (vi) Body weights should be recorded individually for all animals 
once prior to the administration of the test substance, once a week 
during the first 13 weeks of study and at least once every 4 weeks 
thereafter, unless signs of clinical toxicity suggest more frequent 
weighing to facilitate monitoring of health status.
    (vii) Measurements of feed consumption should be determined weekly 
during the first 13 weeks of the study and at approximately monthly 
intervals thereafter unless health status or body weight changes dictate 
otherwise. Measurements of water consumption should be determined at the 
same intervals if the test substance is administered in the drinking 
water.
    (viii) Moribund animals should be removed and sacrificed when 
noticed and the time of death should be recorded as precisely as 
possible. All survivors should be sacrificed at the end of the study 
period.
    (9) Clinical pathology. Hematology, clinical chemistry, and 
urinalysis should be performed on 10 rats per sex per group, and on all 
nonrodents. In rodents, the parameters should be examined at 
approximately 6 month intervals during the conduct of the study and at 
termination. If possible, these collections should be from the same 
animals at each interval. In nonrodents, the parameters should be 
examined once or twice prior to initiation of treatment, at 6-month 
intervals during the conduct of the study, and at termination. If 
hematological and biochemical effects were seen in the subchronic study, 
testing should also be performed at 3 months. Overnight fasting of 
animals prior to blood sampling is recommended.
    (i) Hematology. The recommended parameters are red blood cell count, 
hemoglobin concentration, hematocrit, mean corpuscular volume, mean 
corpuscular hemoglobin, and mean corpuscular hemoglobin concentration, 
white blood cell count, differential leukocyte count, platelet count, 
and a measure of clotting potential, such as prothrombin time or 
activated partial thromboplastin time.
    (ii) Clinical chemistry. (A) Parameters which are considered 
appropriate to all studies are electrolyte balance, carbohydrate 
metabolism, and liver and kidney function. The selection of specific 
tests will be influenced by observations on the mode of action of the 
substance and signs of clinical toxicity.
    (B) The recommended clinical chemistry determinations are potassium, 
sodium, calcium (nonrodent), phosphorus (nonrodent), chloride 
(nonrodent), glucose, total cholesterol, urea nitrogen, creatinine, 
total protein, total bilirubin (nonrodent), and albumin. More than two 
hepatic enzymes, (such as alanine aminotransferase, aspartate 
aminotransferase, alkaline phosphatase, sorbitol dehydrogenase, or

[[Page 397]]

gamma glutamyl transpeptidase) should also be measured. Measurements of 
additional enzymes (of hepatic or other origin) and bile acids, may also 
be useful.
    (C) If a test chemical has an effect on the hematopoietic system, 
reticulocyte counts and bone marrow cytology may be indicated.
    (D) Other determinations that should be carried out if the test 
chemical is known or suspected of affecting related measures include 
calcium, phosphorus, fasting triglycerides, hormones, methemoglobin, and 
cholinesterases.
    (iii) Urinalysis. Urinalysis for rodents should be performed at the 
end of the study using timed urine collection. Urinalysis for nonrodents 
should be performed prior to treatment, midway through treatment and at 
the end of the study using timed urine collection. Urinalysis 
determinations include: appearance, volume, osmolality or specific 
gravity, pH, protein, glucose, and blood/blood cells.
    (10) Ophthalmological examination. Examinations should be made of 
all animals using an ophthalmoscope or equivalent device prior to the 
administration of the test substance and at termination of the study on 
10 rats of each sex in the high-dose and control groups and preferably 
in all nonrodents, but at least the control and high-dose groups should 
be examined. If changes in eyes are detected, all animals should be 
examined.
    (11) Gross necropsy. (i) All animals should be subjected to a full 
gross necropsy which includes examination of the external surface of the 
body, all orifices, and the cranial, thoracic and abdominal cavities and 
their contents.
    (ii) At least the liver, kidneys, adrenals, testes, epididymides, 
ovaries, uterus, nonrodent thyroid (with parathyroid), spleen, brain, 
and heart should be weighed wet as soon as possible after dissection to 
avoid drying. The lungs should be weighed if the test substance is 
administered by the inhalation route.
    (iii) The following organs and tissues, or representative samples 
thereof, should be preserved in a suitable medium for possible future 
histopathological examination:
    (A) Digestive system--salivary glands, esophagus, stomach, duodenum, 
jejunum, ileum, cecum, colon, rectum, liver, pancreas, gallbladder (when 
present).
    (B) Nervous system--brain (multiple sections, including cerebrum, 
cerebellum and medulla/pons), pituitary, peripheral nerve (sciatic or 
tibial, preferably in close proximity to the muscle), spinal cord (three 
levels, cervical, mid-thoracic and lumbar), eyes (retina, optic nerve).
    (C) Glandular system--adrenals, parathyroid, thyroid.
    (D) Respiratory system--trachea, lungs, pharynx, larynx, nose.
    (E) Cardiovascular/hematopoietic system--aorta, heart, bone marrow 
(and/or fresh aspirate), lymph nodes (preferably one lymph node covering 
the route of administration and another one distant from the route of 
administration to cover systemic effects), spleen.
    (F) Urogenital system--kidneys, urinary bladder, prostate, testes, 
epididymides, seminal vesicle(s), uterus, ovaries, female mammary gland.
    (G) Other--all gross lesions and masses, skin.
    (iv) In inhalation studies, the entire respiratory tract, including 
nose, pharynx, larynx, and paranasal sinuses should be examined and 
preserved. In dermal studies, skin from treated and adjacent control 
skin sites should be examined and preserved.
    (v) Inflation of lungs and urinary bladder with a fixative is the 
optimal method for preservation of these tissues. The proper inflation 
and fixation of the lungs in inhalation studies is considered essential 
for appropriate and valid histopathological examination.
    (vi) Information from clinical pathology and other in-life data 
should be considered before microscopic examination, since they may 
provide significant guidance to the pathologist.
    (12) Histopathology. (i) The following histopathology should be 
performed:
    (A) Full histopathology on the organs and tissues (listed under 
paragraph (e)(11)(iii) of this section) of all rodents and nonrodents in 
the control and high-dose groups, and all rodents

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and nonrodents that died or were sacrificed during the study. The 
examination should be extended to all animals in all dosage groups if 
treatment-related changes are observed in the high-dose group.
    (B) All gross lesions in all animals.
    (C) Target tissues in all animals.
    (ii) If the results show substantial alteration of the animal's 
normal life span, or other effects that might compromise the 
significance of the data, the next lower levels should be examined fully 
as described in paragraph (e)(12)(i) of this section.
    (iii) An attempt should be made to correlate gross observations with 
microscopic findings.
    (iv) Tissues and organs designated for microscopic examination 
should be fixed in 10% buffered formalin or a recognized suitable 
fixative as soon as necropsy is performed and no less than 48 hours 
prior to trimming.
    (f) Data and reporting--(1) Treatment of results. (i) Data should be 
summarized in tabular form, showing for each test group the number of 
animals at the start of the test, the number of animals showing lesions, 
the types of lesions and the percentage of animals displaying each type 
of lesion.
    (ii) When applicable, all observed results (quantitative and 
qualitative) should be evaluated by an appropriate statistical method. 
Any generally accepted statistical methods may be used; the statistical 
methods including significance criteria should be selected during the 
design of the study.
    (2) Evaluation of study results. The findings of a chronic toxicity 
study should be evaluated in conjunction with the findings of preceding 
studies and considered in terms of the toxic effects as well as the 
necropsy and histopathological findings. The evaluation will include the 
relationship between the dose of the test substance and the presence, 
incidence, and severity of abnormalities (including behavioral and 
clinical abnormalities), gross lesions, identified target organs, body 
weight changes, effects on mortality and any other general or specific 
toxic effects.
    (3) Test report. In addition to the reporting requirements specified 
under EPA Good Laboratory Practice Standards at 40 CFR part 792, subpart 
J, the following specific information must be reported:
    (i) Test substance characterization should include:
    (A) Chemical identification.
    (B) Lot or batch number.
    (C) Physical properties.
    (D) Purity/impurities.
    (ii) Identification and composition of any vehicle used.
    (iii) Test system should contain data on:
    (A) Species and strain of animals used and rationale for selection 
if other than that recommended.
    (B) Age including body weight data and sex.
    (C) Test environment including cage conditions, ambient temperature, 
humidity, and light/dark periods.
    (D) Identification of animal diet.
    (E) Acclimation period.
    (iv) Test procedure should include the following data:
    (A) Method of randomization used.
    (B) Full description of experimental design and procedure.
    (C) Dose regimen including levels, methods, and volume.
    (v) Test results.
    (A) Group animal data. Tabulation of toxic response data by species, 
strain, sex and exposure level for:
    (1) Number of animals exposed.
    (2) Number of animals showing signs of toxicity.
    (3) Number of animals dying.
    (B) Individual animal data. Data should be presented as summary 
(group mean) as well as for individual animals.
    (1) Time of death during the study or whether animals survived to 
termination.
    (2) Time of observation of each abnormal sign and its subsequent 
course.
    (3) Body weight data.
    (4) Feed and water (if collected) consumption data.
    (5) Achieved dose (mg/kg/day) as a time-weighted average if the test 
substance is administered in the diet or drinking water.
    (6) Results of ophthalmological examinations.
    (7) Results of hematological tests performed.

[[Page 399]]

    (8) Results of clinical chemistry tests performed.
    (9) Urinalysis tests performed and results.
    (10) Results of observations made.
    (11) Necropsy findings, including absolute and relative (to body 
weight) organ weight data.
    (12) Detailed description of all histopathological findings.
    (13) Statistical treatment of results, where appropriate.
    (vi) In addition, for inhalation studies the following should be 
reported:
    (A) Test conditions. The following exposure conditions must be 
reported:
    (1) Description of exposure apparatus including design, type, 
dimensions, source of air, system for generating particulate and 
aerosols, method of conditioning air, treatment of exhaust air and the 
method of housing the animals in a test chamber.
    (2) The equipment for measuring temperature, humidity, and 
particulate aerosol concentrations and size should be described.
    (B) Exposure data. These data should be tabulated and presented with 
mean values and a measure of variability (e.g., standard deviation) and 
should include:
    (1) Airflow rates through the inhalation equipment.
    (2) Temperature and humidity of air.
    (3) Actual (analytical or gravimetric) concentration in the 
breathing zone.
    (4) Nominal concentration (total amount of test substance fed into 
the inhalation equipment divided by volume of air).
    (5) Particle size distribution, calculated MMAD, and geometric 
standard deviation.
    (6) Explanation as to why the desired chamber concentration and/or 
particle size could not be achieved (if applicable) and the efforts 
taken to comply with this aspect of the guidelines.
    (g) Quality control. A system should be developed and maintained to 
assure and document adequate performance of laboratory staff and 
equipment. The study must be conducted in compliance with 40 CFR Part 
792--Good Laboratory Practice Standards.
    (h) References. For additional background information on this test 
guideline, the following references should be consulted. These 
references are available for inspection at the TSCA Nonconfidential 
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M 
St., SW., Washington, DC, 12 noon to 4 p.m., Monday through Friday, 
except legal holidays.
    (1) Benitz, K.F. Measurement of Chronic Toxicity. Methods of 
Toxicology. Ed. G.E. Paget. Blackwell, Oxford. pp. 82-131 (1970).
    (2) Crofton K.M., Howard J.L., Moser V.C., Gill M.W., Leiter L.W., 
Tilson H.A., MacPhail, R.C. Interlaboratory Comparison of Motor Activity 
Experiments: Implication for Neurotoxicological Assessments. 
Neurotoxicol. Teratol. 13, 599-609. (1991)
    (3) D'Aguanno, W. Drug Safety Evaluation-Pre-Clinical 
Considerations. Industrial Pharmacology: Neuroleptic. Vol. I, Ed. S. 
Fielding and H. Lal. Futura, Mt. Kisco, NY. pp. 317-332 (1974).
    (4) Fitzhugh, O.G. Chronic Oral Toxicity, Appraisal of the Safety of 
Chemicals in Foods, Drugs and Cosmetics. The Association of Food and 
Drug Officials of the United States. pp. 36-45 (1959, 3rd Printing 
1975).
    (5) Gad S.C. A Neuromuscular Screen for Use in Industrial 
Toxicology. Journal of Toxicology and Environmental Health. 9, 691-704. 
(1982)
    (6) Goldenthal, E.I. and D'Aguanno, W. Evaluation of Drugs, 
Appraisal of the Safety of Chemicals in Foods, Drugs, and Cosmetics. The 
Association of Food and Drug Officials of the United States. pp. 60-67 
(1959, 3rd Printing 1975).
    (7) Meyer O.A., Tilson H.A., Byrd W.C., Riley M.T. A Method for the 
Routine Assessment of Fore- and Hind-Limb Grip Strength of Rats and 
Mice. Neurobehav. Toxicol. 1, 233-236. (1979)
    (8) Moser V.C., McDaniel K.M., Phillips P.M. Rat Strain and Stock 
Comparisons using a Functional Observational Battery: Baseline Values 
and Effects of Amitraz. Toxicol. Appl. Pharmacol. 108, 267-283 (1991)
    (9) Organization for Economic Cooperation and Development. 
Guidelines for Testing of Chemicals, Section 4-Health Effects, Part 452 
Chronic Toxicity Studies, Paris (1981).
    (10) Page, N.P. Chronic Toxicity and Carcinogenicity Guidelines. 
Journal of

[[Page 400]]

Environmental Pathology and Toxicology. 11:161-182 (1977).
    (11) Tupper, D.E., Wallace R.B. Utility of the Neurologic 
Examination in Rats. Acta. Neurobiol. Exp. 40, 999-1003 (1980).
    (12) Weingand K., Brown G., Hall R. et al. (1996). Harmonization of 
Animal Clinical Pathology Testing in Toxicity and Safety Studies. 
Fundam. and Appl. Toxicol. 29:198-201.

[65 FR 78797, Dec. 15, 2000]



Sec. 799.9420  TSCA carcinogenicity.

    (a) Scope. This section is intended to meet the testing requirements 
under section 4 of TSCA. The objective of a long-term carcinogenicity 
study is to observe test animals for a major portion of their life span 
for development of neoplastic lesions during or after exposure to 
various doses of a test substance by an appropriate route of 
administration.
    (b) Source. The source material used in developing this TSCA test 
guideline is the OPPTS harmonized test guideline 870.4200 (June 1996 
Public Draft). This source is available at the address in paragraph (g) 
of this section.
    (c) Definitions. The following definitions apply to this section.
    Carcinogenicity is the development of neoplastic lesions as a result 
of the repeated daily exposure of experimental animals to a chemical by 
the oral, dermal, or inhalation routes of exposure.
    Cumulative toxicity is the adverse effects of repeated dose 
occurring as a result of prolonged action on, or increased concentration 
of, the administered test substance or its metabolites in susceptible 
tissues.
    Dose in a carcinogenicity study is the amount of test substance 
administered via the oral, dermal or inhalation routes for a period of 
up to 24 months. Dose is expressed as weight of the test substance 
(grams, milligrams) per unit body weight of test animal (milligram per 
kilogram), or as weight of the test substance in parts per million (ppm) 
in food or drinking water. When exposed via inhalation, dose is 
expressed as weight of the test substance per unit volume of air 
(milligrams per liter) or as parts per million.
    Target organ is any organ of a test animal showing evidence of an 
effect induced by a test substance.
    (d) Test procedures--(1) Animal selection--(i) Species and strain. 
Testing shall be performed on two mammalian species. Rats and mice are 
the species of choice because of their relatively short life spans, 
limited cost of maintenance, widespread use in pharmacological and 
toxicological studies, susceptibility to tumor induction, and the 
availability of inbred or sufficiently characterized strains. Commonly 
used laboratory strains shall be used. If other mammalian species are 
used, the tester shall provide justification/reasoning for their 
selection.
    (ii) Age/weight. (A) Testing shall be started with young healthy 
animals as soon as possible after weaning and acclimatization.
    (B) Dosing should generally begin no later than 8 weeks of age.
    (C) At commencement of the study, the weight variation of animals 
used shall not exceed  20% of the mean weight for 
each sex.
    (D) Studies using prenatal or neonatal animals may be recommended 
under special conditions.
    (iii) Sex. (A) Equal numbers of animals of each sex shall be used at 
each dose level.
    (B) Females shall be nulliparous and nonpregnant.
    (iv) Numbers. (A) At least 100 rodents (50 males and 50 females) 
shall be used at each dose level and concurrent control group.
    (B) If interim sacrifices are planned, the number shall be increased 
by the number of animals scheduled to be sacrificed during the course of 
the study.
    (C) For a meaningful and valid statistical evaluation of long term 
exposure and for a valid interpretation of negative results, the number 
of animals in any group should not fall below 50% at 15 months in mice 
and 18 months in rats. Survival in any group should not fall below 25% 
at 18 months in mice and 24 months in rats.
    (D) The use of adequate randomization procedures for the proper 
allocation of animals to test and control groups is required to avoid 
bias.
    (E) Each animal shall be assigned a unique identification number. 
Dead

[[Page 401]]

animals, their preserved organs and tissues, and microscopic slides 
shall be identified by reference to the unique numbers assigned.
    (v) Husbandry. (A) Animals may be group-caged by sex, but the number 
of animals per cage must not interfere with clear observation of each 
animal. The biological properties of the test substance or toxic effects 
(e.g., morbidity, excitability) may indicate a need for individual 
caging. Animals should be housed individually in dermal studies and 
during exposure in inhalation studies.
    (B) The temperature of the experimental animal rooms should be at 22 
 3 [deg]C.
    (C) The relative humidity of the experimental animal rooms should be 
30 to 70%.
    (D) Where lighting is artificial, the sequence should be 12 h light/
12 h dark.
    (E) Control and test animals should be fed from the same batch and 
lot. The feed should be analyzed to assure uniform distribution and 
adequacy of nutritional requirements of the species tested and for 
impurities that might influence the outcome of the test. Animals should 
be fed and watered ad libitum with food replaced at least weekly.
    (F) The study should not be initiated until animals have been 
allowed a period of acclimatization/quarantine to environmental 
conditions, nor should animals from outside sources be placed on test 
without an adequate period of quarantine.
    (2) Control and test substances. (i) Where necessary, the test 
substance is dissolved or suspended in a suitable vehicle. If a vehicle 
or diluent is needed, it should not elicit toxic effects itself. It is 
recommended that wherever possible the use of an aqueous solution be 
considered first, followed by consideration of solution in oil, and 
finally solution in other vehicles.
    (ii) One lot of the test substance should be used, if possible, 
throughout the duration of the study, and the research sample should be 
stored under conditions that maintain its purity and stability. Prior to 
the initiation of the study, there should be a characterization of the 
test substance, including the purity of the test compound, and, if 
possible, the name and quantities of contaminants and impurities.
    (iii) If the test or control substance is to be incorporated into 
feed or another vehicle, the period during which the test substance is 
stable in such a mixture should be determined prior to the initiation of 
the study. Its homogeneity and concentration should be determined prior 
to the initiation of the study and periodically during the study. 
Statistically randomized samples of the mixture should be analyzed to 
ensure that proper mixing, formulation, and storage procedures are being 
followed, and that the appropriate concentration of the test or control 
substance is contained in the mixture.
    (3) Control groups. A concurrent control group (50 males and 50 
females) is required. This group shall be untreated or if a vehicle is 
used in administering the test substance, a vehicle control group. If 
the toxic properties of the vehicle are not known, both untreated and 
vehicle control groups are required.
    (4) Dose levels and dose selection. (i) For risk assessment 
purposes, at least three dose levels shall be used, in addition to the 
concurrent control group. Dose levels should be spaced to produce a 
gradation of effects. A rationale for the doses selected must be 
provided.
    (ii) The highest dose level should elicit signs of toxicity without 
substantially altering the normal life span due to effects other than 
tumors. The highest dose should be determined based on the findings from 
a 90-day study to ensure that the dose used is adequate to asses the 
carcinogenic potential of the test substance. Thus, the selection of the 
highest dose to be tested is dependent upon changes observed in several 
toxicological parameters in subchronic studies. The highest dose tested 
need not exceed 1,000 mg/kg/day.
    (iii) The intermediate-dose level should be spaced to produce a 
gradation of toxic effects.
    (iv) The lowest dose level should produce no evidence of toxicity.

[[Page 402]]

    (v) For skin carcinogenicity studies, when toxicity to the skin is a 
determining factor, the highest dose selected should not destroy the 
functional integrity of the skin, the intermediate dose should be a 
minimally irritating dose, and the low dose should be the highest 
nonirritating dose.
    (vi) The criteria for selecting the dose levels for skin 
carcinogenicity studies, based on gross and histopathologic dermal 
lesions, are as follows:
    (A) Gross criteria for reaching the high dose:
    (1) Erythema (moderate).
    (2) Scaling.
    (3) Edema (mild).
    (4) Alopecia.
    (5) Thickening.
    (B) Histologic criteria for reaching the high dose:
    (1) Epidermal hyperplasia.
    (2) Epidermal hyperkeratosis.
    (3) Epidermal parakeratosis.
    (4) Adnexal atrophy/hyperplasia.
    (5) Fibrosis.
    (6) Spongiosis (minimal-mild).
    (7) Epidermal edema (minimal-mild).
    (8) Dermal edema (minimal-moderate).
    (9) Inflammation (moderate).
    (C) Gross criteria for exceeding the high dose:
    (1) Ulcers, fissures.
    (2) Exudate/crust (eschar).
    (3) nonviable (dead) tissues.
    (4) Anything leading to destruction of the functional integrity of 
the epidermis (e.g., caking, fissuring, open sores, eschar).
    (D) Histologic criteria for exceeding the high dose:
    (1) Crust (interfollicular and follicular).
    (2) Microulcer.
    (3) Degeneration/necrosis (mild to moderate).
    (4) Epidermal edema (moderate to marked).
    (5) Dermal edema (marked).
    (6) Inflammation (marked).
    (5) Administration of the test substance. The three main routes of 
administration are oral, dermal, and inhalation. The choice of the route 
of administration depends upon the physical and chemical characteristics 
of the test substance and the form typifying exposure in humans.
    (i) Oral studies. If the test substance is administered by gavage, 
the animals are dosed with the test substance on a 7-day per week basis 
for a period of at least 18 months for mice and hamsters and 24 months 
for rats. However, based primarily on practical considerations, dosing 
by gavage or via a capsule on a 5-day per week basis is acceptable. If 
the test substance is administered in the drinking water or mixed in the 
diet, then exposure should be on a 7-day per week basis.
    (ii) Dermal studies. (A) The animals should be treated with the test 
substance for at least 6 h/day on a 7-day per week basis for a period of 
at least 18 months for mice and hamsters and 24 months for rats. 
However, based primarily on practical considerations, application on a 
5-day per week basis is acceptable. Dosing should be conducted at 
approximately the same time each day.
    (B) Fur should be clipped weekly from the dorsal area of the trunk 
of the test animals. Care should be taken to avoid abrading the skin 
which could alter its permeability. A minimum of 24 hrs should be 
allowed for the skin to recover before the next dosing of the animal.
    (C) Preparation of test substance. Liquid test substances are 
generally used undiluted, except as indicated in paragraph (e)(4)(vi) of 
this section. Solids should be pulverized when possible. The substance 
should be moistened sufficiently with water or, when necessary, with a 
suitable vehicle to ensure good contact with the skin. When a vehicle is 
used, the influence of the vehicle on toxicity of, and penetration of 
the skin by, the test substance should be taken into account. The volume 
of application should be kept constant, e.g. less than 100 uL for the 
mouse and less than 300 uL for the rat. Different concentrations of test 
solution should be prepared for different dose levels.
    (D) The test substance shall be applied uniformly over a shaved area 
which is approximately 10 percent of the total body surface area. In 
order to dose approximately 10 percent of the body surface, the area 
starting at the

[[Page 403]]

scapulae (shoulders) to the wing of the ileum (hipbone) and half way 
down the flank on each side of the animal should be shaved. With highly 
toxic substances, the surface area covered may be less, but as much of 
the area as possible should be covered with as thin and uniform a film 
as practical.
    (iii) Inhalation studies. (A) The animals should be exposed to the 
test substance for 6 h/day on a 7-day per week basis, for a period of at 
least 18 months in mice and 24 months in rats. However, based primarily 
on practical considerations, exposure for 6 h/day on a 5-day per week 
basis is acceptable.
    (B) The animals shall be tested in dynamic inhalation equipment 
designed to sustain a minimum air flow of 10 air changes per hr, an 
adequate oxygen content of at least 19%, and uniform conditions 
throughout the exposure chamber. Maintenance of slight negative pressure 
inside the chamber will prevent leakage of the test substance into 
surrounding areas.
    (C) The selection of a dynamic inhalation chamber should be 
appropriate for the test substance and test system. Where a whole body 
chamber is used to expose animals to an aerosol, individual housing must 
be used to minimize crowding of the test animals and maximize their 
exposure to the test substance. To ensure stability of a chamber 
atmosphere, the total volume occupied by the test animals shall not 
exceed 5% of the volume of the test chamber. It is recommended, but not 
required, that nose-only or head-only exposure be used for aerosol 
studies in order to minimize oral exposures due to animals licking 
compound off their fur. Heat stress to the animals should be minimized.
    (D) The temperature at which the test is performed should be 
maintained at 22  2 [deg]C. The relative humidity 
should be maintained between 40 to 60%, but in certain instances (e.g., 
tests of aerosols, use of water vehicle) this may not be practicable.
    (E) The rate of air flow shall be monitored continuously but 
recorded at least three times during exposure.
    (F) Temperature and humidity shall be monitored continuously but 
should be recorded at least every 30 minutes.
    (G) The actual concentration of the test substance shall be measured 
in the breathing zone. During the exposure period, the actual 
concentrations of the test substance should be held as constant as 
practicable, monitored continuously or intermittently depending on the 
method of analysis. Chamber concentrations may be measured using 
gravimetric or analytical methods as appropriate. If trial run 
measurements are reasonably consistent (plus or minus 10 percent for 
liquid aerosol, gas, or vapor; plus or minus 20 percent for dry 
aerosol), the two measurements should be sufficient. If measurements are 
not consistent, then three to four measurements should be taken.
    (H) During the development of the generating system, particle size 
analysis shall be performed to establish the stability of aerosol 
concentrations with respect to particle size. Measurement of aerodynamic 
particle size in the animals's breathing zone should be measured during 
a trial run. If median aerodynamic diameter (MMAD) values for each 
exposure level are within 10% of each other, then two measurements 
during the exposures should be sufficient. If pretest measurements are 
not within 10% of each other, three to four measurements should be 
taken. The MMAD particle size range should be between 1-3 [micro]m. The 
particle size of hygroscopic materials should be small enough to allow 
pulmonary deposition once the particles swell in the moist environment 
of the respiratory tract.
    (I) Feed shall be withheld during exposure. Water may also be 
withheld during exposure.
    (6) Observation period. It is necessary that the duration of the 
carcinogenicity study comprise the majority of the normal life span of 
the strain of animals used. This time period shall not be less than 24 
months for rats and 18 months for mice, and ordinarily not longer than 
30 months for rats and 24 months for mice. For longer time periods, and 
where any other species are used, consultation with the Agency in regard 
to the duration of the study is advised.
    (7) Observation of animals. (i) Observations shall be made at least 
once each

[[Page 404]]

day for morbidity and mortality. Appropriate actions should be taken to 
minimize loss of animals from the study (e.g., necropsy or refrigeration 
of those animals found dead and isolation or sacrifice of weak or 
moribund animals).
    (ii) A careful clinical examination shall be made at least once 
weekly. Observations should be detailed and carefully recorded, 
preferably using explicitly defined scales. Observations should include, 
but not be limited to, evaluation of skin and fur, eyes and mucous 
membranes, respiratory and circulatory effects, autonomic effects such 
as salivation, central nervous system effects, including tremors and 
convulsions, changes in the level of activity, gait and posture, 
reactivity to handling or sensory stimuli, altered strength and 
stereotypes or bizarre behavior (e.g., self-mutilation, walking 
backwards).
    (iii) Body weights shall be recorded individually for all animals; 
once a week during the first 13 weeks of the study and at least once 
every 4 weeks, thereafter, unless signs of clinical toxicity suggest 
more frequent weighing to facilitate monitoring of health status.
    (iv) Measurements of feed consumption should be determined weekly 
during the first 13 weeks of the study and at approximately monthly 
intervals thereafter unless health status or body weight changes dictate 
otherwise. Measurement of water consumption should be determined at the 
same intervals if the test substance is administered in the drinking 
water.
    (v) Moribund animals shall be removed and sacrificed when noticed 
and the time of death should be recorded as precisely as possible. At 
the end of the study period, all survivors shall be sacrificed.
    (8) Clinical pathology. At 12 months, 18 months, and at terminal 
sacrifice, a blood smear shall be obtained from all animals. A 
differential blood count should be performed on blood smears from those 
animals in the highest dosage group and the controls from the terminal 
sacrifice. If these data, or data from the pathological examination 
indicate a need, then the 12- and 18-month blood smears should also be 
examined. Differential blood counts should be performed for the next 
lower groups if there is a major discrepancy between the highest group 
and the controls. If clinical observations suggest a deterioration in 
health of the animals during the study, a differential blood count of 
the affected animals shall be performed.
    (9) Gross necropsy. (i) A complete gross examination shall be 
performed on all animals, including those that died during the 
experiment or were killed in a moribund condition.
    (ii) At least the liver, kidneys, adrenals, testes, epididymides, 
ovaries, uterus, spleen, brain, and heart should be weighed wet as soon 
as possible after dissection to avoid drying. The lungs should be 
weighed if the test substance is administered by the inhalation route. 
The organs should be weighed from interim sacrifice animals as well as 
from at least 10 animals per sex per group at terminal sacrifice.
    (iii) The following organs and tissues, or representative samples 
thereof, shall be preserved in a suitable medium for possible future 
histopathological examination.
    (A) Digestive system.
    (1) Salivary glands.
    (2) Esophagus.
    (3) Stomach.
    (4) Duodenum.
    (5) Jejunum.
    (6) Ileum.
    (7) Cecum.
    (8) Colon.
    (9) Rectum.
    (10) Liver.
    (11) Pancreas.
    (12) Gallbladder (mice).
    (B) Nervous system.
    (1) Brain (multiple sections).
    (2) Pituitary.
    (3) Peripheral nerves.
    (4) Spinal cord (three levels).
    (5) Eyes (retina, optic nerve).
    (C) Glandular system.
    (1) Adrenals.
    (2) Parathyroids.
    (3) Thyroids.
    (D) Respiratory system.
    (1) Trachea.
    (2) Lung.
    (3) Pharynx.
    (4) Larynx.
    (5) Nose.

[[Page 405]]

    (E) Cardiovascular/hematopoietic system.
    (1) Aorta (thoracic).
    (2) Heart.
    (3) Bone marrow.
    (4) Lymph nodes.
    (5) Spleen.
    (F) Urogenital system.
    (1) Kidneys.
    (2) Urinary bladder.
    (3) Prostate.
    (4) Testes/epididymides.
    (5) Seminal vesicles.
    (6) Uterus.
    (7) Ovaries.
    (8) Female mammary gland.
    (G) Other.
    (1) Skin.
    (2) All gross lesions and masses.
    (iv) In inhalation studies, the entire respiratory tract, including 
nose, pharynx, larynx, and paranasal sinuses should be examined and 
preserved. In dermal studies, skin from treated and adjacent control 
skin sites should be examined and preserved.
    (v) Inflation of lungs and urinary bladder with a fixative is the 
optimal method for preservation of these tissues. The proper inflation 
and fixation of the lungs in inhalation studies is essential for 
appropriate and valid histopathological examination.
    (vi) Information from clinical pathology, and other in-life data 
should be considered before microscopic examination, since they may 
provide significant guidance to the pathologist.
    (10) Histopathology. (i) The following histopathology shall be 
performed:
    (A) Full histopathology on the organs and tissues under paragraph 
(d)(9) (iii) of this section of all animals in the control and high dose 
groups and all animals that died or were killed during the study.
    (B) All gross lesions in all animals.
    (C) Target organs in all animals.
    (ii) If the results show substantial alteration of the animal's 
normal life span, the induction of effects that might affect a 
neoplastic response, or other effects that might compromise the 
significance of the data, the next lower dose levels shall be examined 
as described in paragraph (d)(10)(i) of this section.
    (iii) An attempt should be made to correlate gross observations with 
microscopic findings.
    (iv) Tissues and organs designated for microscopic examination 
should be fixed in 10 percent buffered formalin or a recognized suitable 
fixative as soon as necropsy is performed and no less than 48 hours 
prior to trimming.
    (e) Data and reporting--(1) Treatment of results. (i) Data shall be 
summarized in tabular form, showing for each test group the number of 
animals at the start of the test, the number of animals showing lesions, 
the types of lesions, and the percentage of animals displaying each type 
of lesion.
    (ii) All observed results (quantitative and qualitative) shall be 
evaluated by an appropriate statistical method. Any generally accepted 
statistical methods may be used; the statistical methods including 
significance criteria shall be selected during the design of the study.
    (2) Evaluation of study results. (i) The findings of a 
carcinogenicity study should be evaluated in conjunction with the 
findings of previous studies and considered in terms of the toxic 
effects, the necropsy and histopathological findings. The evaluation 
shall include the relationship between the dose of the test substance 
and the presence, incidence, and severity of abnormalities (including 
behavioral and clinical abnormalities), gross lesions, identified target 
organs, body weight changes, effects on mortality, and any other general 
or specific toxic effects.
    (ii) In any study which demonstrates an absence of toxic effects, 
further investigation to establish absorption and bioavailablity of the 
test substance should be considered.
    (iii) In order for a negative test to be acceptable, it must meet 
the following criteria: No more than 10% of any group is lost due to 
autolysis, cannibalism, or management problems; and survival in each 
group is no less than 50% at 15 months for mice and 18 months for rats. 
Survival should not fall below 25% at 18 months for mice and 24 months 
for rats.
    (iv) The use of historical control data from an appropriate time 
period from the same testing laboratory (i.e., the incidence of tumors 
and other suspect

[[Page 406]]

lesions normally occurring under the same laboratory conditions and in 
the same strain of animals employed in the test) is helpful for 
assessing the significance of changes observed in the current study.
    (3) Test report. (i) In addition to the reporting requirements as 
specified under 40 CFR part 792, subpart J, the following specific 
information shall be reported. Both individual and summary data should 
be presented.
    (A) Test substance characterization should include:
    (1) Chemical identification.
    (2) Lot or batch number.
    (3) Physical properties.
    (4) Purity/impurities.
    (5) Identification and composition of any vehicle used.
    (B) Test system should contain data on:
    (1) Species and strain of animals used and rationale for selection 
if other than that recommended.
    (2) Age including body weight data and sex.
    (3) Test environment including cage conditions, ambient temperature, 
humidity, and light/dark periods.
    (4) Identification of animal diet.
    (5) Acclimation period.
    (C) Test procedure should include the following data:
    (1) Method of randomization used.
    (2) Full description of experimental design and procedure.
    (3) Dose regimen including levels, methods, and volume.
    (4) Test results--(i) Group animal data. Tabulation of toxic 
response data by species, strain, sex, and exposure level for:
    (A) Number of animals exposed.
    (B) Number of animals showing signs of toxicity.
    (C) Number of animals dying.
    (ii) Individual animal data. Data should be presented as summary 
(group mean) as well as for individual animals.
    (A) Time of death during the study or whether animals survived to 
termination.
    (B) Time of observation of each abnormal sign and its subsequent 
course.
    (C) Body weight data.
    (D) Feed and water consumption data, when collected.
    (E) Results of clinical pathology and immunotoxicity screen when 
performed.
    (F) Necropsy findings including absolute/relative organ weight data.
    (G) Detailed description of all histopathological findings.
    (H) Statistical treatment of results where appropriate.
    (I) Historical control data.
    (J) Achieved dose (mg/kg/day) as a time-weighted average if the test 
substance is administered in the diet or drinking water.
    (iii) Inhalation studies. In addition, for inhalation studies the 
following shall be reported:
    (A) Test conditions. The following exposure conditions shall be 
reported.
    (1) Description of exposure apparatus including design, type, 
dimensions, source of air, system for generating particulate and 
aerosols, method of conditioning air, treatment of exhaust air and the 
method of housing the animals in a test chamber.
    (2) The equipment for measuring temperature, humidity, and 
particulate aerosol concentrations and size should be described.
    (B) Exposure data. These shall be tabulated and presented with mean 
values and a measure of variability (e.g. standard deviation) and should 
include:
    (1) Airflow rates through the inhalation equipment.
    (2) Temperature and humidity of air.
    (3) Actual (analytical or gravimetric) concentration in the 
breathing zone.
    (4) Nominal concentration (total amount of test substance fed into 
the inhalation equipment divided by volume of air).
    (5) Particle size distribution, calculated MMAD and geometric 
standard deviation (GSD).
    (6) Explanation as to why the desired chamber concentration and/or 
particle size could not be achieved (if applicable) and the efforts 
taken to comply with this aspect of the sections.
    (f) Quality assurance. A system shall be developed and maintained to 
assure and document adequate performance of laboratory staff and 
equipment. The study shall be conducted in compliance with 40 CFR part 
792--Good Laboratory Practice Standards.

[[Page 407]]

    (g) References. For additional background information on this test 
guideline, the following references should be consulted. These 
references are available for inspection at the TSCA Nonconfidential 
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M 
St., SW., Washington, DC, 12 noon to 4 p.m., Monday through Friday, 
except legal holidays.
    (1) Benitz, K.F. Ed. Paget, G.E. Measurement of Chronic Toxicity. 
Methods of Toxicology (Blackwell, Oxford, 1970) pp. 82-131.
    (2) Fitzhugh, O.G. Chronic Oral Toxicity, Appraisal of the Safety of 
Chemicals in Foods, Drugs and Cosmetics. The Association of Food and 
Drug Officials of the United States. pp. 36-45 (1959, 3rd Printing 
1975).
    (3) Goldenthal, E.I. and D'Aguanno, W. Evaluation of Drugs, 
Appraisal of the Safety of Chemicals in Foods, Drugs, and Cosmetics. The 
Association of Food and Drug Officials of the United States. pp. 60-67 
(1959, 3rd Printing 1975).
    (4) Organisation for Economic Co-operation and Development. 
Guidelines for Testing of Chemicals, Section 4-Health Effects, Part 451 
Carcinogenicity Studies (Paris, 1981).
    (5) Page, N.P. Chronic Toxicity and Carcinogenicity Guidelines. 
Journal of Environmental Pathology and Toxicology. 11:161-182 (1977).
    (6) Page, N.P. Eds. Kraybill and Mehlman. Concepts of a Bioassay 
Program in Environmental Carcinogenesis. Vol.3. Advances in Modern 
Toxicology (Hemisphere, Washington, DC., 1977) pp. 87-171.
    (7) Sontag, J.M. et al. Guidelines for Carcinogen Bioassay in Small 
Rodents. NCI-CS-TR-1 United States Cancer Institute, Division of Cancer 
Control and Prevention, Carcinogenesis Bioassay Program (Bethesda, MD).

[62 FR 43824, Aug. 15, 1997, as amended at 64 FR 35078, June 30, 1999]



Sec. 799.9430  TSCA combined chronic toxicity/carcinogenicity.

    (a) Scope. This section is intended to meet the testing requirements 
under section 4 of the Toxic Substances Control Act (TSCA). The 
objective of a combined chronic toxicity/carcinogenicity study is to 
determine the effects of a substance in a mammalian species following 
prolonged and repeated exposure. The application of this section should 
generate data which identify the majority of chronic and carcinogenicity 
effects and determine dose-response relationships. The design and 
conduct should allow for the detection of neoplastic effects and a 
determination of the carcinogenic potential as well as general toxicity, 
including neurological, physiological, biochemical, and hematological 
effects and exposure-related morphological (pathology) effects.
    (b) Source. The source material used in developing this TSCA test 
guideline is the Office of Prevention, Pesticides, and Toxic Substances 
(OPPTS) harmonized test guideline 870.4300 (August 1998, final 
guideline). This source is available at the address in paragraph (h) of 
this section.
    (c) Definitions. The following definitions apply to this section.
    Carcinogenicity is the development of neoplastic lesions as a result 
of the repeated daily exposure of experimental animals to a chemical by 
the oral, dermal, or inhalation routes of exposure.
    Chronic toxicity is the adverse effects occurring as a result of the 
repeated daily exposure of experimental animals to a chemical by the 
oral, dermal, or inhalation routes of exposure.
    Cumulative toxicity is the adverse effects of repeated dose 
occurring as a result of prolonged action on, or increased concentration 
of, the administered test substance or its metabolites in susceptible 
tissues.
    Dose in a combined chronic toxicity/carcinogenicity study is the 
amount of test substance administered via the oral, dermal, or 
inhalation routes for a period of up to 24 months. Dose is expressed as 
weight of the test substance per unit body weight of test animal 
(milligrams per kilogram), or as weight of the test substance in parts 
per million (ppm) in food or drinking water. When exposed via 
inhalation, dose is expressed as weight of the test substance per unit 
volume of air (milligrams per liter) or as parts per million per day. 
For dermal application, dose is expressed as weight of the test 
substance (grams, milligrams) per unit

[[Page 408]]

body weight of the test animal (milligrams per kilogram) or as weight of 
the substance per unit surface area (milligrams per square centimeter) 
per day.
    No-observed-effects level (NOEL) is the maximum dose used in a study 
which produces no observed adverse effects. The NOEL is usually 
expressed in terms of the weight of a test substance given daily per 
unit weight of test animal (milligrams per kilogram per day).
    Target organ is any organ of a test animal showing evidence of an 
effect induced by a test substance.
    (d) Limit test. If a test at one dose level of at least 1,000 mg/kg 
body weight (expected human exposure may indicate the need for a higher 
dose level), using the procedures described for this study, produces no 
observable toxic effects or if toxic effects would not be expected based 
upon data of structurally related compounds, then a full study using 
three dose levels might not be necessary.
    (e) Test procedures--(1) Animal selection--(i) Species and strain. 
Preliminary studies providing data on acute, subchronic, and metabolic 
responses should have been carried out to permit an appropriate choice 
of animals (species and strain). As discussed in other guidelines, the 
mouse and rat have been most widely used for assessment of carcinogenic 
potential, while the rat and dog have been most often studied for 
chronic toxicity. For the combined chronic toxicity/carcinogenicity 
study via the oral and inhalation routes, the rat is the species of 
choice and for the dermal route, the mouse is species of choice. If 
other species are used, the tester must provide justification/reasoning 
for their selection. The strain selected should be susceptible to the 
carcinogenic or toxic effect of the class of substances being tested, if 
known, and provided it does not have a spontaneous background incidence 
too high for meaningful assessment. Commonly used laboratory strains 
must be employed.
    (ii) Age/weight. (A) Testing must be started with young healthy 
animals as soon as possible after weaning and acclimatization.
    (B) Dosing should generally begin no later than 8 weeks of age.
    (C) At commencement of the study, the weight variation of animals 
used must be within 20% of the mean weight for each sex.
    (D) Studies using prenatal or neonatal animals may be recommended 
under special conditions.
    (iii) Sex. (A) Equal numbers of animals of each sex must be used at 
each dose level.
    (B) Females must be nulliparous and nonpregnant.
    (iv) Numbers. (A) At least 100 rodents (50 males and 50 females) 
must be used at each dose level and concurrent control group. At least 
20 additional rodents (10 males and 10 females) should be used for 
satellite dose groups and the satellite control group. The purpose of 
the satellite group is to allow for the evaluation of chronic toxicity 
after 12 months of exposure to the test substance.
    (B) For a meaningful and valid statistical evaluation of long term 
exposure and for a valid interpretation of negative results, the number 
of animals in any group should not fall below 50% at 15 months in mice 
and 18 months in rats. Survival in any group should not fall below 25% 
at 18 months in mice and 24 months in rats.
    (C) To avoid bias, the use of adequate randomization procedures for 
the proper allocation of animals to test and control groups is required.
    (D) Each animal must be assigned a unique identification number. 
Dead animals (and their preserved organs) and tissues, and microscopic 
slides shall be identified by reference to the unique numbers assigned.
    (v) Husbandry. (A) Animals may be group-caged by sex, but the number 
of animals per cage must not interfere with clear observation of each 
animal. The biological properties of the test substance or toxic effects 
(e.g., morbidity, excitability) may indicate a need for individual 
caging. Rodents should be housed individually in dermal studies and 
during exposure in inhalation studies.
    (B) The temperature of the experimental animal rooms should be at 22 
3 [deg]C.
    (C) The relative humidity of the experimental animal rooms should be 
50 20%.

[[Page 409]]

    (D) Where lighting is artificial, the sequence should be 12 hours 
light/12 hours dark.
    (E) Control and test animals should be fed from the same batch and 
lot. The feed should be analyzed to assure uniform distribution and 
adequacy of nutritional requirements of the species tested and for 
impurities that might influence the outcome of the test. Animals should 
be fed and watered ad libitum with food replaced at least weekly.
    (F) The study should not be initiated until animals have been 
allowed a period of acclimatization/quarantine to environmental 
conditions, nor should animals from outside sources be placed on test 
without an adequate period of quarantine. An acclimation period of at 
least five days is recommended.
    (2) Control and test substances. (i) Where necessary, the test 
substance is dissolved or suspended in a suitable vehicle. If a vehicle 
or diluent is needed, it should not elicit toxic effects itself nor 
substantially alter the chemical or toxicological properties of the test 
substance. It is recommended that wherever possible the usage of an 
aqueous solution be considered first, followed by consideration of a 
solution in oil, and finally solution in other vehicles.
    (ii) One lot of the test substance should be used throughout the 
duration of the study if possible, and the research sample should be 
stored under conditions that maintain its purity and stability. Prior to 
the initiation of the study, there should be a characterization of the 
test substance, including the purity of the test compound, and, if 
possible, the name and quantities of contaminants and impurities.
    (iii) If the test or control substance is to be incorporated into 
feed or another vehicle, the period during which the test substance is 
stable in such a mixture should be determined prior to the initiation of 
the study. Its homogeneity and concentration should be determined prior 
to the initiation of the study and periodically during the study. 
Statistically randomized samples of the mixture should be analyzed to 
ensure that proper mixing, formulation, and storage procedures are being 
followed, and that the appropriate concentration of the test or control 
substance is contained in the mixture.
    (3) Control groups. A concurrent control group is required. This 
group should be an untreated or sham-treated control group or, if a 
vehicle is used in administering the test substance, a vehicle control 
group. If the toxic properties of the vehicle are not known or cannot be 
made available, both untreated and vehicle control groups are required.
    (4) Dose levels and dose selection. (i) For risk assessment 
purposes, at least three dose levels must be used, in addition to the 
concurrent control group. Dose levels should be spaced to produce a 
gradation of effects. A rationale for the doses selected must be 
provided.
    (ii) The highest dose level in rodents should elicit signs of 
toxicity without substantially altering the normal life span due to 
effects other than tumors. The highest dose should be determined based 
on the findings from a 90-day study to ensure that the dose used is 
adequate to assess the chronic toxicity and the carcinogenic potential 
of the test substance. Thus, the selection of the highest dose to be 
tested is dependent upon changes observed in several toxicological 
parameters in subchronic studies. The highest dose tested need not 
exceed 1,000 mg/kg/day.
    (iii) The intermediate-dose levels should be spaced to produce a 
gradation of toxic effects.
    (iv) The lowest-dose level should produce no evidence of toxicity.
    (v) For skin carcinogenicity studies, when toxicity to the skin is a 
determining factor, the highest dose selected should not destroy the 
functional integrity of the skin, the intermediate doses should be a 
minimally irritating dose and the low dose should be the highest 
nonirritating dose.
    (vi) The criteria for selecting the dose levels for skin 
carcinogenicity studies, based on gross and histopathologic dermal 
lesions, are as follows:
    (A) Gross criteria for reaching the high dose:
    (1) Erythema (moderate).
    (2) Scaling.
    (3) Edema (mild).
    (4) Alopecia.
    (5) Thickening.

[[Page 410]]

    (B) Histologic criteria for reaching the high dose:
    (1) Epidermal hyperplasia.
    (2) Epidermal hyperkeratosis.
    (3) Epidermal parakeratosis.
    (4) Adnexal atrophy/hyperplasia.
    (5) Fibrosis.
    (6) Spongiosis (minimal-mild).
    (7) Epidermal edema (minimal-mild).
    (8) Dermal edema (minimal-moderate).
    (9) Inflammation (moderate).
    (C) Gross criteria for exceeding the high dose:
    (1) Ulcers-fissures, exudate/crust (eschar), nonviable (dead) 
tissues.
    (2) Anything leading to destruction of the functional integrity of 
the epidermis (e.g., caking, fissuring, open sores, eschar).
    (D) Histologic criteria for exceeding the high-dose:
    (1) Crust (interfollicular and follicular).
    (2) Microulcer.
    (3) Degeneration/necrosis (mild to moderate).
    (4) Epidermal edema (moderate to marked).
    (5) Dermal edema (marked).
    (6) Inflammation (marked).
    (5) Administration of the test substance. The three main routes of 
administration are oral, dermal, and inhalation. The choice of the route 
of administration depends upon the physical and chemical characteristics 
of the test substance and the form typifying exposure in humans.
    (i) Oral studies. If the test substance is administered by gavage, 
the animals are dosed with the test substance on a 7-day per week basis 
for a period of at least 18 months for mice and hamsters and 24 months 
for rats. However, based primarily on practical considerations, dosing 
by gavage on a 5-day per week basis is acceptable. If the test substance 
is administered in the drinking water or mixed in the diet, then 
exposure should be on a 7-day per week basis.
    (ii) Dermal studies. (A) Preparation of animal skin. Shortly before 
testing, fur should be clipped from not less than 10% of the body 
surface area for application of the test substance. In order to dose 
approximately 10% of the body surface, the area starting at the scapulae 
(shoulders) to the wing of the ileum (hipbone) and half way down the 
flank on each side of the animal should be shaved. Shaving should be 
carried out approximately 24 hours before dosing. Repeated clipping or 
shaving is usually needed at approximately weekly intervals. When 
clipping or shaving the fur, care should be taken to avoid abrading the 
skin which could alter its permeability.
    (B) Preparation of test substance. Liquid test substances are 
generally used undiluted, except as indicated in paragraph (e)(4)(vi) of 
this section. Solids should be pulverized when possible. The substance 
should be moistened sufficiently with water or, when necessary, with a 
suitable vehicle to ensure good contact with the skin. When a vehicle is 
used, the influence of the vehicle on toxicity of, and penetration of 
the skin by, the test substance should be taken into account. The volume 
of application should be kept constant, e.g., less than 100 [micro]L for 
the mouse and less than 300 [micro]L for the rat. Different 
concentrations of test solution should be prepared for different dose 
levels.
    (C) Administration of test substance. The duration of exposure 
should be at least 18 months for mice and hamsters and 24 months for 
rats. Ideally, the animals should be treated with test substance for at 
least 6 hours per day on a 7-day per week basis. However, based on 
practical considerations, application on a 5-day per week basis is 
acceptable. Dosing should be conducted at approximately the same time 
each day. The test substance must be applied uniformly over the 
treatment site. The surface area covered may be less for highly toxic 
substances. As much of the area should be covered with as thin and 
uniform a film as possible. For rats, the test substance may be held in 
contact with the skin with a porous gauze dressing and nonirritating 
tape if necessary. The test site should be further covered in a suitable 
manner to retain the gauze dressing plus test substance and to ensure 
that the animals cannot ingest the test substance. The application site 
should not be covered when the mouse is the species of choice. The test 
substance may

[[Page 411]]

be wiped from the skin after the 6-hour exposure period to prevent 
ingestion.
    (iii) Inhalation studies. (A) The animals should be exposed to the 
test substance, for 6 hours per day on a 7-day per week basis, for a 
period of at least 18 months in mice and 24 months in rats. However, 
based primarily on practical considerations, exposure for 6 hours per 
day on a 5-day per week basis is acceptable.
    (B) The animals must be tested in dynamic inhalation equipment 
designed to sustain a minimum air flow of 10 air changes per hour, an 
adequate oxygen content of at least 19%, and uniform conditions 
throughout the exposure chamber. Maintenance of slight negative pressure 
inside the chamber will prevent leakage of the test substance into 
surrounding areas. It is not normally necessary to measure chamber 
oxygen concentration if airflow is adequate.
    (C) The selection of a dynamic inhalation chamber should be 
appropriate for the test substance and test system. Where a whole body 
chamber is used, individual housing must be used to minimize crowding of 
the test animals and maximize their exposure to the test substance. To 
ensure stability of a chamber atmosphere, the total volume occupied by 
the test animals shall not exceed 5% of the volume of the test chamber. 
It is recommended, but not required, that nose-only or head-only 
exposure be used for aerosol studies in order to minimize oral exposures 
due to animals licking compound off their fur. The animals should be 
acclimated and heat stress minimized.
    (D) The temperature at which the test is performed should be 
maintained at 22 2 [deg]C. The relative humidity 
should be maintained between 40 to 60%, but in certain instances (e.g., 
tests of aerosols, use of water vehicle) this may not be practicable.
    (E) The rate of air flow must be monitored continuously but recorded 
at least three times during the exposure.
    (F) Temperature and humidity must be monitored continuously but 
should be recorded at least every 30 minutes.
    (G) The actual concentrations of the test substance must be measured 
in the animal's breathing zone. During the exposure period, the actual 
concentrations of the test substance must be held as constant as 
practicable and monitored continuously or intermittently depending on 
the method of analysis. Chamber concentration may be measured using 
gravimetric or analytical methods as appropriate. If trial run 
measurements are reasonably consistent (10% for 
liquid aerosol, gas, or vapor; 20% for dry 
aerosol), then two measurements should be sufficient. If measurements 
are not consistent, three to four measurements should be taken. If there 
is some difficulty in measuring chamber analytical concentration due to 
precipitation, nonhomogeneous mixtures, volatile components, or other 
factors, additional analyses of inert components may be necessary.
    (H) During the development of the generating system, particle size 
analysis must be performed to establish the stability of aerosol 
concentrations with respect to particle size. The mass median 
aerodynamic diameter (MMAD) particle size range should be between 1-3 
[micro]m. The particle size of hygroscopic materials should be small 
enough when dry to assure that the size of the swollen particle will 
still be within the 1-3 [micro]m range. Measurements of aerodynamic 
particle size in the animal's breathing zone should be measured during a 
trial run. If MMAD values for each exposure level are within 10% of each 
other, then two measurements during the exposures should be sufficient. 
If pretest measurements are not within 10% of each other, three to four 
measurements should be taken.
    (I) Feed must be withheld during exposure. Water may also be 
withheld during exposure.
    (J) When the physical and chemical properties of the test substance 
show a low flash point or the test substance is otherwise known or 
thought to be explosive, care must be taken to avoid exposure level 
concentrations that could result in an exposure chamber explosion during 
the test.
    (6) Observation period. (i) This time period must not be less than 
24 months for rats and 18 months for mice, and ordinarily not longer 
than 30 months for rats and 24 months for mice. For longer time periods, 
and where any other species are used, consultation with the

[[Page 412]]

Agency in regard to the duration of the study is advised.
    (ii) Animals in a satellite group to assess chronic toxicity should 
be observed for 12 months.
    (7) Observation of animals. (i) Observations must be made at least 
twice each day for morbidity and mortality. Appropriate actions should 
be taken to minimize loss of animals to the study (e.g., necropsy or 
refrigeration of those animals found dead and isolation or sacrifice of 
weak or moribund animals). General clinical observations shall be made 
at least once a day, preferably at the same time each day, taking into 
consideration the peak period of anticipated effects after dosing. The 
clinical condition of the animal should be recorded.
    (ii) A careful clinical examination must be made at least once 
weekly. Observations should be detailed and carefully recorded, 
preferably using explicity defined scales. Observations should include, 
but not be limited to, evaluation of skin and fur, eyes and mucous 
membranes, respiratory and circulatory effects, autonomic effects such 
as salivation, central nervous system effects, including tremors and 
convulsions, changes in the level of activity, gait and posture, 
reactivity to handling or sensory stimuli, altered strength, and 
stereotypes or bizarre behavior (e.g., self-mutilation, walking 
backwards).
    (iii) Signs of toxicity should be recorded as they are observed 
including the time of onset, degree and duration.
    (iv) Body weights must be recorded individually for all animals once 
prior to administration of the test substance, once a week during the 
first 13 weeks of the study and at least once every 4 weeks thereafter 
unless signs of clinical toxicity suggest more frequent weighing to 
facilitate monitoring of health status.
    (v) Measurements of feed consumption should be determined weekly 
during the first 13 weeks of the study and then at approximately monthly 
intervals unless health status or body weight changes dictate otherwise. 
Measurements of water consumption should be determined at the same 
intervals if the test material is administered in drinking water.
    (vi) Moribund animals must be removed and sacrificed when noticed 
and the time of death should be recorded as precisely as possible. At 
the end of the study period, all survivors must be sacrificed. Animals 
in the satellite group must be sacrificed after 12 months of exposure to 
the test substance (interim sacrifice).
    (8) Clinical pathology. Hematology, clinical chemistry and 
urinalyses must be performed from 10 animals per sex per group. The 
parameters should be examined at approximately 6 month intervals during 
the first 12 months of the study. If possible, these collections should 
be from the same animals at each interval. If hematological and 
biochemical effects are seen in the subchronic study, testing shall also 
be performed at 3 months. Overnight fasting of animals prior to blood 
sampling is recommended.
    (i) Hematology. The recommended parameters are red blood cell count, 
hemoglobin concentration, hematocrit, mean corpuscular volume, mean 
corpuscular hemoglobin, and mean corpuscular hemoglobin concentration, 
white blood cell count, differential leukocyte count, platelet count, 
and a measure of clotting potential, such as prothrombin time or 
activated partial thromboplastin time.
    (ii) Clinical chemistry. (A) Parameters which are considered 
appropriate to all studies are electrolyte balance, carbohydrate 
metabolism, and liver and kidney function. The selection of specific 
tests will be influenced by observations on the mode of action of the 
substance and signs of clinical toxicity.
    (B) The recommended clinical chemistry determinations are potassium, 
sodium, glucose, total cholesterol, urea nitrogen, creatinine, total 
protein, and albumin. More than two hepatic enzymes, (such as alanine 
aminotransferase, aspartate aminotransferase, alkaline phosphatase, 
sorbitol dehydrogenase, or gamma glutamyl transpeptidase) should also be 
measured. Measurements of addtional enzymes (of hepatic or other origin) 
and bile acids, may also be useful.
    (iii) If a test chemical has an effect on the hematopoietic system,

[[Page 413]]

reticulocyte counts and bone marrow cytology may be indicated.
    (iv) Other determinations that should be carried out if the test 
chemical is known or suspected of affecting related measures include 
calcium, phosphorus, fasting triglycerides, hormones, methemoglobin, and 
cholinesterases.
    (v) Urinalyses. Urinalysis for rodents must be performed at the end 
of the first year of the study using timed urine collection. Urinalysis 
determinations include: appearance, volume, osmolality or specific 
gravity, pH, protein, glucose, and blood/blood cells.
    (9) Ophthalmological examination. Examinations must be made on all 
animals using an ophthalmoscope or an equivalent device prior to the 
administration of the test substance and at termination of the study on 
10 animals per sex in the high-dose and control groups. If changes in 
eyes are detected, all animals must be examined.
    (10) Gross necropsy. (i) A complete gross examination must be 
performed on all animals, including those which died during the 
experiment or were sacrificed in a moribund condition.
    (ii) At least, the liver, kidneys, adrenals, testes, epididymides, 
ovaries, uterus, spleen, brain, and heart should be trimmed and weighed 
wet, as soon as possible after dissection to avoid drying. The lungs 
should be weighed if the test substance is administered by the 
inhalation route. The organs should be weighed from interim sacrifice 
animals as well as from at least 10 animals per sex per group at 
terminal sacrifice.
    (iii) The following organs and tissues, or representative samples 
thereof, must be preserved in a suitable medium for possible future 
histopathological examination:
    (A) Digestive system--salivary glands, esophagus, stomach, duodenum, 
jejunum, ileum, cecum, colon, rectum, liver, pancreas, gallbladder (when 
present) .
    (B) Nervous system--brain (multiple sections, including cerebrum, 
cerebellum and medulla/pons), pituitary, peripheral nerve (sciatic or 
tibial, preferably in close proximity to the muscle), spinal cord (three 
levels, cervical, mid-thoracic, and lumbar), eyes (retina, optic nerve).
    (C) Glandular system--adrenals, parathyroid, thyroid.
    (D) Respiratory system--trachea, lungs, pharynx, larynx, nose.
    (E) Cardiovascular/Hematopoietic system--aorta, heart, bone marrow 
(and/or fresh aspirate), lymph nodes (preferably one lymph node covering 
the route of administration and another one distant from the route of 
administration to cover systemic effects), spleen.
    (F) Urogenital system--kidneys, urinary bladder, prostate, testes, 
epididymides, seminal vesicle(s), uterus, ovaries, female mammary gland.
    (G) Other--all gross lesions and masses, skin.
    (iv) In inhalation studies, the entire respiratory tract, including 
nose, pharynx, larynx, and paranasal sinuses should be examined and 
preserved. In dermal studies, skin from treated and adjacent control 
skin sites should be examined and preserved.
    (v) Inflation of lungs and urinary bladder with a fixative is the 
optimal method for preservation of these tissues. The proper inflation 
and fixation of the lungs in inhalation studies is essential for 
appropriate and valid histopathological examination.
    (vi) Information from clinical pathology and other in-life data 
should be considered before microscopic examination, since these data 
may provide significant guidance to the pathologist.
    (11) [Reserved]
    (12) Histopathology. (i) The following histopathology must be 
performed:
    (A) Full histopathology on the organs and tissues, listed in 
paragraph (e)(10)(iii) of this section of all animals in the control and 
high dose groups and of all animals that died or were sacrificed during 
the study.
    (B) All gross lesions in all animals.
    (C) Target organs in all animals.
    (ii) If the results show substantial alteration of the animal's 
normal life span, the induction of effects that might affect a 
neoplastic response, or other effects that might compromise the 
significance of the data, the next lower levels should be examined fully 
as described in paragraph (e)(12)(i) of this section.

[[Page 414]]

    (iii) An attempt should be made to correlate gross observations with 
microscopic findings.
    (iv) Tissues and organs designated for microscopic examination 
should be fixed in 10% buffered formalin or a recognized suitable 
fixative as soon as necropsy is performed and no less than 48 hours 
prior to trimming.
    (f) Data and reporting--(1) Treatment of results. (i) Data must be 
summarized in tabular form, showing for each test group the number of 
animals at the start of the test, the number of animals showing lesions, 
the types of lesions and the percentage of animals displaying each type 
of lesion.
    (ii) When applicable, all observed results, quantitative and 
qualitative, must be evaluated by an appropriate statistical method. Any 
generally accepted statistical methods may be used; the statistical 
methods including significance criteria should be selected during the 
design of the study.
    (2) Evaluation of study results. (i) The findings of a combined 
chronic toxicity/carcinogenicity study should be evaluated in 
conjunction with the findings of previous studies and considered in 
terms of the toxic effects, the necropsy and histopathological findings. 
The evaluation must include the relationship between the dose of the 
test substance and the presence, incidence and severity of abnormalities 
(including behavioral and clinical abnormalities), gross lesions, 
identified target organs, body weight changes, effects on mortality and 
any other general or specific toxic effects.
    (ii) In any study which demonstrates an absence of toxic effects, 
further investigation to establish absorption and bioavailablity of the 
test substance should be considered.
    (iii) In order for a negative test to be acceptable, it should meet 
the following criteria--no more than 10% of any group is lost due to 
autolysis, cannibalism, or management problems, and survival in each 
group is no less than 50% at 15 months for mice and 18 months for rats. 
Survival should not fall below 25% at 18 months for mice and 24 months 
for rats.
    (iv) The use of historical control data from an appropriate time 
period from the same testing laboratory (i.e, the incidence of tumors 
and other suspect lesions normally occurring under the same laboratory 
conditions and in the same strain of animals employed in the test) is 
helpful for assessing the significance of changes observed in the 
current study.
    (3) Test report. (i) In addition to the reporting requirements 
specified under EPA Good Laboratory Practice Standards at 40 CFR part 
792, subpart J, the following specific information must be reported:
    (A) Test substance characterization should include:
    (1) Chemical identification.
    (2) Lot or batch number.
    (3) Physical properties.
    (4) Purity/impurities.
    (5) Identification and composition of any vehicle used.
    (B) Test system should contain data on:
    (1) Species and strain of animals used and rationale for selection 
if other than that recommended.
    (2) Age including body weight data and sex.
    (3) Test environment including cage conditions, ambient temperature, 
humidity, and light/dark periods.
    (4) Identification of animal diet.
    (5) Acclimation period.
    (C) Test procedure should include the following data:
    (1) Method of randomization used.
    (2) Full description of experimental design and procedure.
    (3) Dose regimen including levels, methods, and volume.
    (4) Test results. (i) Group animal data. Tabulation of toxic 
response data by species, strain, sex, and exposure level for:
    (A) Number of animals exposed.
    (B) Number of animals showing signs of toxicity.
    (C) Number of animals dying.
    (ii) Individual animal data. Data should be presented as summary 
(group mean) as well as for individual animals.
    (A) Time of death during the study or whether animals survived to 
termination.
    (B) Time of observation of each abnormal sign and its subsequent 
course.
    (C) Body weight data.

[[Page 415]]

    (D) Feed and water consumption data, when collected.
    (E) Achieved dose (milligrams/kilogram body weight) as a time-
weighed average is the test substance is administered in the diet or 
drinking water.
    (F) Results of ophthalmological examination, when performed.
    (G) Results of hematological tests performed.
    (H) Results of clinical chemistry tests performed.
    (I) Results of urinalysis tests performed.
    (J) Results of observations made.
    (K) Necropsy findings including absolute/relative organ weight data.
    (L) Detailed description of all histopathological findings.
    (M) Statistical treatment of results where appropriate.
    (N) Historical control data.
    (iii) In addition, for inhalation studies the following should be 
reported:
    (A) Test conditions. The following exposure conditions must be 
reported.
    (1) Description of exposure apparatus including design, type, 
dimensions, source of air, system for generating particulates and 
aerosols, method of conditioning air, treatment of exhaust air and the 
method of housing the animals in a test chamber.
    (2) The equipment for measuring temperature, humidity, and 
particulate aerosol concentrations and size should be described.
    (B) Exposure data. These must be tabulated and presented with mean 
values and a measure of variability (e.g., standard deviation) and 
should include:
    (1) Airflow rates through the inhalation equipment.
    (2) Temperature and humidity of air.
    (3) Actual (analytical or gravimetric) concentration in the 
breathing zone.
    (4) Nominal concentration (total amount of test substance fed into 
the inhalation equipment divided by volume of air).
    (5) Particle size distribution, and calculated MMAD and geometric 
standard deviation.
    (6) Explanation as to why the desired chamber concentration and/or 
particle size could not be achieved (if applicable) and the efforts 
taken to comply with this aspect of the guidelines.
    (g) Quality control. A system must be developed and maintained to 
assure and document adequate performance of laboratory equipment. The 
study must be conducted in compliance with 40 CFR Part 792--Good 
Laborary Practice Standards.
    (h) References. For additional background information on this test 
guideline, the following references should be consulted. These 
references are available for inspection at the TSCA Nonconfidential 
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M 
St., NW., Washington, DC, 12 noon to 4 p.m., Monday through Friday, 
except legal holidays.
    (1) Benitz, K.F. Measurement of Chronic Toxicity. Methods of 
Toxicology. Ed. G.E. Paget. Blackwell, Oxford. pp. 82-131 (1970).
    (2) Crofton K.M., Howard J.L., Moser V.C., Gill M.W., Leiter L.W., 
Tilson H.A., MacPhail, R.C. Interlaboratory Comparison of Motor Activity 
Experiments: Implication for Neurotoxicological Assessments. 
Neurotoxicol. Teratol. 13, 599-609. (1991)
    (3) D'Aguanno, W. Drug Safety Evaluation--Pre-Clinical 
Considerations. Industrial Pharmacology: Neuroleptic. Vol. I, Ed. S. 
Fielding and H. Lal. Futura, Mt. Kisco, NY. pp. 317-332 (1974).
    (4) Fitzhugh, O.G. Chronic Oral Toxicity, Appraisal of the Safety of 
Chemicals in Foods, Drugs and Cosmetics. The Association of Food and 
Drug Officials of the United States. pp. 36-45 (1959, 3rd Printing 
1975).
    (5) Goldenthal, E.I. and D'Aguanno, W. Evaluation of Drugs, 
Appraisal of the Safety of Chemicals in Foods, Drugs, and Cosmetics. The 
Association of Food and Drug Officials of the United States. pp. 60-67 
(1959, 3rd Printing 1975).
    (6) Organization for Economic Cooperation and Development. 
Guidelines for Testing of Chemicals, Section 4-Health Effects, Part 453 
Combined Chronic Toxicity/Carcinogenicity Studies, Paris. (1981).
    (7) Page, N.P. Chronic Toxicity and Carcinogenicity Guidelines. 
Journal of Environmental Pathology and Toxicology 11:161-182 (1977).
    (8) Page, N.P. Concepts of a Bioassay Program in Environmental 
Carcinogenesis, Advances in Modern Toxicology. Vol.3, Ed. Kraybill and

[[Page 416]]

Mehlman. Hemisphere, Washington, DC pp. 87-171 (1977)
    (9) Sontag, J.M. et al. Guidelines for Carcinogen Bioassay in Small 
Rodents. NCI-CS-TR-1 (Bethesda: United States Cancer Institute, Division 
of Cancer Control and Prevention, Carcinogenesis Bioassay Program.
    (10) Summary of the EPA Workshop on Carcinogenesis Bioassay via the 
Dermal Route. EPA Report 50/6-89-002; 50/6-89-003. Washington, DC.
    (11) The Atlas Of Dermal Lesions, EPA Report 20T-004, U.S 
Environmental Protection Agency, Washington, DC.

[65 FR 78802, Dec. 15, 2000]



Sec. 799.9510  TSCA bacterial reverse mutation test.

    (a) Scope. This section is intended to meet the testing requirements 
under section 4 of TSCA.
    (1) The bacterial reverse mutation test uses amino-acid requiring 
strains of Salmonella typhimurium and Escherichia coli to detect point 
mutations, which involve substitution, addition or deletion of one or a 
few DNA base pairs. The principle of this bacterial reverse mutation 
test is that it detects mutations which revert mutations present in the 
test strains and restore the functional capability of the bacteria to 
synthesize an essential amino acid. The revertant bacteria are detected 
by their ability to grow in the absence of the amino acid required by 
the parent test strain.
    (2) Point mutations are the cause of many human genetic diseases and 
there is substantial evidence that point mutations in oncogenes and 
tumor suppressor genes of somatic cells are involved in tumor formation 
in humans and experimental animals. The bacterial reverse mutation test 
is rapid, inexpensive and relatively easy to perform. Many of the test 
strains have several features that make them more sensitive for the 
detection of mutations, including responsive DNA sequences at the 
reversion sites, increased cell permeability to large molecules and 
elimination of DNA repair systems or enhancement of error-prone DNA 
repair processes. The specificity of the test strains can provide some 
useful information on the types of mutations that are induced by 
genotoxic agents. A very large data base of results for a wide variety 
of structures is available for bacterial reverse mutation tests and 
well-established methodologies have been developed for testing chemicals 
with different physico-chemical properties, including volatile 
compounds.
    (b) Source. The source material used in developing this TSCA test 
guideline are the OECD replacement guidelines for 471 and 472 (February 
1997). This source is available at the address in paragraph (g) of this 
section.
    (c) Definitions. The following definitions apply to this section:
    A reverse mutation test in either Salmonella typhimurium or 
Escherichia coli detects mutation in an amino-acid requiring strain 
(histidine or tryptophan, respectively) to produce a strain independent 
of an outside supply of amino-acid.
    Base pair substitution mutagens are agents that cause a base change 
in DNA. In a reversion test this change may occur at the site of the 
original mutation, or at a second site in the bacterial genome.
    Frameshift mutagens are agents that cause the addition or deletion 
of one or more base pairs in the DNA, thus changing the reading frame in 
the RNA
    (d) Initial considerations. (1) The bacterial reverse mutation test 
utilizes prokaryotic cells, which differ from mammalian cells in such 
factors as uptake, metabolism, chromosome structure and DNA repair 
processes. Tests conducted in vitro generally require the use of an 
exogenous source of metabolic activation. In vitro metabolic activation 
systems cannot mimic entirely the mammalian in vivo conditions. The test 
therefore does not provide direct information on the mutagenic and 
carcinogenic potency of a substance in mammals.
    (2) The bacterial reverse mutation test is commonly employed as an 
initial screen for genotoxic activity and, in particular, for point 
mutation-inducing activity. An extensive data base has demonstrated that 
many chemicals that are positive in this test also exhibit mutagenic 
activity in other tests. There are examples of mutagenic agents which 
are not detected by this

[[Page 417]]

test; reasons for these shortcomings can be ascribed to the specific 
nature of the endpoint detected, differences in metabolic activation, or 
differences in bioavailability. On the other hand, factors which enhance 
the sensitivity of the bacterial reverse mutation test can lead to an 
overestimation of mutagenic activity.
    (3) The bacterial reverse mutation test may not be appropriate for 
the evaluation of certain classes of chemicals, for example highly 
bactericidal compounds (e.g. certain antibiotics) and those which are 
thought (or known) to interfere specifically with the mammalian cell 
replication system (e.g. some topoisomerase inhibitors and some 
nucleoside analogues). In such cases, mammalian mutation tests may be 
more appropriate.
    (4) Although many compounds that are positive in this test are 
mammalian carcinogens, the correlation is not absolute. It is dependent 
on chemical class and there are carcinogens that are not detected by 
this test because they act through other, non-genotoxic mechanisms or 
mechanisms absent in bacterial cells.
    (e) Test method--(1) Principle. (i) Suspensions of bacterial cells 
are exposed to the test substance in the presence and in the absence of 
an exogenous metabolic activation system. In the plate incorporation 
method, these suspensions are mixed with an overlay agar and plated 
immediately onto minimal medium. In the preincubation method, the 
treatment mixture is incubated and then mixed with an overlay agar 
before plating onto minimal medium. For both techniques, after 2 or 3 
days of incubation, revertant colonies are counted and compared to the 
number of spontaneous revertant colonies on solvent control plates.
    (ii) Several procedures for performing the bacterial reverse 
mutation test have been described. Among those commonly used are the 
plate incorporation method, the preincubation method, the fluctuation 
method, and the suspension method. Suggestions for modifications for the 
testing of gases or vapors are described in the reference in paragraph 
(g)(12) of this section.
    (iii) The procedures described in this section pertain primarily to 
the plate incorporation and preincubation methods. Either of them is 
acceptable for conducting experiments both with and without metabolic 
activation. Some compounds may be detected more efficiently using the 
preincubation method. These compounds belong to chemical classes that 
include short chain aliphatic nitrosamines, divalent metals, aldehydes, 
azo-dyes and diazo compounds, pyrollizidine alkaloids, allyl compounds 
and nitro compounds. It is also recognized that certain classes of 
mutagens are not always detected using standard procedures such as the 
plate incorporation method or preincubation method. These should be 
regarded as ``special cases'' and it is strongly recommended that 
alternative procedures should be used for their detection. The following 
``special cases'' could be identified (together with examples of 
procedures that could be used for their detection): azo-dyes and diazo 
compounds (alterative procedures are described in the references in 
paragraphs (g)(3), (g)(5), (g)(6), and (g)(13) of this section), gases 
and volatile chemicals (alterative procedures are described in the 
references in paragraphs (g)(12), (g)(14), (g)(15), and (g)(16) of this 
section), and glycosides (alterative procedures are described in the 
references in paragraphs (g)(17) and (g)(18) of this section). A 
deviation from the standard procedure needs to be scientifically 
justified.
    (2) Description--(i) Preparations--(A) Bacteria. (1) Fresh cultures 
of bacteria should be grown up to the late exponential or early 
stationary phase of growth (approximately 10\9\ cells per ml). Cultures 
in late stationary phase should not be used. The cultures used in the 
experiment shall contain a high titre of viable bacteria. The titre may 
be demonstrated either from historical control data on growth curves, or 
in each assay through the determination of viable cell numbers by a 
plating experiment.
    (2) The culture temperature shall be 37 [deg]C.
    (3) At least five strains of bacteria should be used. These should 
include four strains of S. typhimurium (TA1535; TA1537 or TA97a or TA97; 
TA98; and

[[Page 418]]

TA100) that have been shown to be reliable and reproducibly responsive 
between laboratories. These four S. typhimurium strains have GC base 
pairs at the primary reversion site and it is known that they may not 
detect certain oxidizing mutagens, cross-linking agents, and hydrazines. 
Such substances may be detected by E. coli WP2 strains or S. typhimurium 
TA102 (see reference in paragraph (g)(19) of this section) which have an 
AT base pair at the primary reversion site. Therefore the recommended 
combination of strains is:
    (i) S. typhimurium TA1535.
    (ii) S. typhimurium TA1537 or TA97 or TA97a.
    (iii) S. typhimurium TA98.
    (iv) S. typhimurium TA100.
    (v) E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. 
typhimurium TA102. In order to detect cross-linking mutagens it may be 
preferable to include TA102 or to add a DNA repair-proficient strain of 
E.coli [e.g. E.coli WP2 or E.coli WP2 (pKM101).]
    (4) Established procedures for stock culture preparation, marker 
verification and storage should be used. The amino-acid requirement for 
growth should be demonstrated for each frozen stock culture preparation 
(histidine for S. typhimurium strains, and tryptophan for E. coli 
strains). Other phenotypic characteristics should be similarly checked, 
namely: the presence or absence of R-factor plasmids where appropriate 
[i.e. ampicillin resistance in strains TA98, TA100 and TA97a or TA97, 
WP2 uvrA and WP2 uvrA (pKM101), and ampicillin = tetracycline resistance 
in strain TA102]; the presence of characteristic mutations (i.e. rfa 
mutation in S. typhimurium through sensitivity to crystal violet, and 
uvrA mutation in E. coli or uvrB mutation in S. typhimurium, through 
sensitivity to ultra-violet light). The strains should also yield 
spontaneous revertant colony plate counts within the frequency ranges 
expected from the laboratory's historical control data and preferably 
within the range reported in the literature.
    (B) Medium. An appropriate minimal agar (e.g. containing Vogel-
Bonner minimal medium E and glucose) and an overlay agar containing 
histidine and biotin or tryptophan, to allow for a few cell divisions, 
shall be used. The procedures described in the references under 
paragraphs (g)(1), (g)(2), and (g)(9) of this section may be used for 
this analysis.
    (C) Metabolic activation. Bacteria shall be exposed to the test 
substance both in the presence and absence of an appropriate metabolic 
activation system. The most commonly used system is a cofactor-
supplemented post-mitochondrial fraction (S9) prepared from the livers 
of rodents treated with enzyme-inducing agents such as Aroclor 1254 (the 
system described in the references under paragraphs (g)(1) and (g)(2) of 
this section may be used) or a combination of phenobarbitone and [beta]-
naphthoflavone (the system described in the references under paragraphs 
(g)(18), (g)(20), and (g)(21) of this section may be used). The post-
mitochondrial fraction is usually used at concentrations in the range 
from 5 to 30% v/v in the S9-mix. The choice and condition of a metabolic 
activation system may depend upon the class of chemical being tested. In 
some cases it may be appropriate to utilize more than one concentration 
of post-mitochondrial fraction. For azo-dyes and diazo-compounds, using 
a reductive metabolic activation system may be more appropriate (the 
system described in the references under paragraphs (g)(6) and (g)(13) 
of this section may be used).
    (D) Test substance/preparation. Solid test substances should be 
dissolved or suspended in appropriate solvents or vehicles and diluted 
if appropriate prior to treatment of the bacteria. Liquid test 
substances may be added directly to the test systems and/or diluted 
prior to treatment. Fresh preparations should be employed unless 
stability data demonstrate the acceptability of storage.
    (ii) Test conditions--(A) Solvent/vehicle. The solvent/vehicle 
should not be suspected of chemical reaction with the test substance and 
shall be compatible with the survival of the bacteria and the S9 
activity (for further information see the reference in paragraph (g)(22) 
of this section). If other than well-known

[[Page 419]]

solvent/vehicles are used, their inclusion should be supported by data 
indicating their compatibility. It is recommended that wherever 
possible, the use of an aqueous solvent/vehicle be considered first. 
When testing water-unstable substances, the organic solvents used be 
free of water.
    (B) Exposure concentrations. (1) Amongst the criteria to be taken 
into consideration when determining the highest amount of test substance 
to be used are cytotoxicity and solubility in the final treatment 
mixture. It may be useful to determine toxicity and insolubility in a 
preliminary experiment. Cytotoxicity may be detected by a reduction in 
the number of revertant colonies, a clearing or diminution of the 
background lawn, or the degree of survival of treated cultures. The 
cytotoxicity of a substance may be altered in the presence of metabolic 
activation systems. Insolubility should be assessed as precipitation in 
the final mixture under the actual test conditions and evident to the 
unaided eye. The recommended maximum test concentration for soluble non-
cytotoxic substances is 5 mg/plate or 5 [micro]l/plate. For non-
cytotoxic substances that are not soluble at 5 mg/plate or 5 [micro]l/
plate, one or more concentrations tested should be insoluble in the 
final treatment mixture. Test substances that are cytotoxic already 
below 5mg/plate or 5 [micro]l/plate should be tested up to a cytotoxic 
concentration. The precipitate should not interfere with the scoring.
    (2) At least five different analyzable concentrations of the test 
substance shall be used with approximately half log (i.e. [radic]10) 
intervals between test points for an initial experiment. Smaller 
intervals may be appropriate when a concentration-response is being 
investigated.
    (3) Testing above the concentration of 5 mg/plate or 5 [micro]l/
plate may be considered when evaluating substances containing 
substantial amounts of potentially mutagenic impurities.
    (C) Controls. (1) Concurrent strain-specific positive and negative 
(solvent or vehicle) controls, both with and without metabolic 
activation, shall be included in each assay. Positive control 
concentrations that demonstrate the effective performance of each assay 
should be selected.
    (2)(i) For assays employing a metabolic activation system, the 
positive control reference substance(s) should be selected on the basis 
of the type of bacteria strains used. The following chemicals are 
examples of suitable positive controls for assays with metabolic 
activation:

------------------------------------------------------------------------
                 Chemical                              CAS No.
------------------------------------------------------------------------
9,10-Dimethylanthracene...................  [CAS no. 781-43-1]
7,12-Dimethylbenzanthracene...............  [CAS no. 57-97-6]
Congo Red (for the reductive metabolic      [CAS no. 573-58-0]
 activation method).
Benzo(a)pyrene............................  [CAS no. 50-32-8]
Cyclophosphamide (monohydrate)............  [CAS no. 50-18-0]
                                            [CAS no. 6055-19-2]
2-Aminoanthracene.........................  [CAS no. 613-13-8]
------------------------------------------------------------------------

    (ii) 2-Aminoanthracene should not be used as the sole indicator of 
the efficacy of the S9-mix. If 2-aminoanthracene is used, each batch of 
S9 should also be characterized with a mutagen that requires metabolic 
activation by microsomal enzymes, e.g., benzo(a)pyrene, 
dimethylbenzanthracene.
    (3) For assays performed without metabolic activation system, 
examples of strain-specific positive controls are:

------------------------------------------------------------------------
            Chemical                    CAS No.             Strain
------------------------------------------------------------------------
(a) Sodium azide................  [CAS no. 26628-22-  TA1535 and TA100
                                   8].
(b) 2-Nitrofluorene.............  [CAS no. 607-57-8]  TA 98
(c) 9-Aminoacridine or ICR 191..  [CAS no. 90-45-9]   TA1537, TA97 and
                                   or.                 TA97a
                                  [CAS no. 17070-45-
                                   0].
(d) Cumene hydroperoxide........  [CAS no. 80-15-9].  TA102
(e) Mitomycin C.................  [CAS no. 50-07-7].  WP2 uvrA and TA102
(f) N-Ethyl-N-nitro-N-            [CAS no. 70-25-7]   WP2, WP2 uvrA and
 nitrosoguanidine or               or.                 WP2 uvrA (pKM101)
4-nitroquinoline 1-oxide........  [CAS no. 56-57-5].
(g) Furylfuramide (AF-2)........  [CAS no. 3688-53-   Plasmid-containing
                                   7].                 strains
------------------------------------------------------------------------


[[Page 420]]

    (4) Other appropriate positive control reference substances may be 
used. The use of chemical class-related positive control chemicals may 
be considered, when available.
    (5) Negative controls, consisting of solvent or vehicle alone, 
without test substance, and otherwise treated in the same way as the 
treatment groups, shall be included. In addition, untreated controls 
should also be used unless there are historical control data 
demonstrating that no deleterious or mutagenic effects are induced by 
the chosen solvent.
    (3) Procedure--(i) Treatment with test substance. (A) For the plate 
incorporation method, without metabolic activation, usually 0.05 ml or 
0.1 ml of the test solutions, 0.1 ml of fresh bacterial culture 
(containing approximately 10\8\ viable cells) and 0.5 ml of sterile 
buffer are mixed with 2.0 ml of overlay agar. For the assay with 
metabolic activation, usually 0.5 ml of metabolic activation mixture 
containing an adequate amount of post-mitochondrial fraction (in the 
range from 5 to 30% v/v in the metabolic activation mixture) are mixed 
with the overlay agar (2.0 ml), together with the bacteria and test 
substance/test solution. The contents of each tube are mixed and poured 
over the surface of a minimal agar plate. The overlay agar is allowed to 
solidify before incubation.
    (B) For the preincubation method the test substance/test solution is 
preincubated with the test strain (containing approximately 10\8\ viable 
cells) and sterile buffer or the metabolic activation system (0.5 ml) 
usually for 20 min. or more at 30-37 [deg]C prior to mixing with the 
overlay agar and pouring onto the surface of a minimal agar plate. 
Usually, 0.05 or 0.1 ml of test substance/test solution, 0.1 ml of 
bacteria, and 0.5 ml of S9-mix or sterile buffer, are mixed with 2.0 ml 
of overlay agar. Tubes should be aerated during pre-incubation by using 
a shaker.
    (C) For an adequate estimate of variation, triplicate plating should 
be used at each dose level. The use of duplicate plating is acceptable 
when scientifically justified. The occasional loss of a plate does not 
necessarily invalidate the assay.
    (D) Gaseous or volatile substances should be tested by appropriate 
methods, such as in sealed vessels (methods described in the references 
under paragraphs (g)(12), (g)(14), (g)(15), and (g)(16) of this section 
may be used).
    (ii) Incubation. All plates in a given assay shall be incubated at 
37 [deg]C for 48-72 hrs. After the incubation period, the number of 
revertant colonies per plate is counted.
    (f) Data and reporting--(1) Treatment of results. (i) Data shall be 
presented as the number of revertant colonies per plate. The number of 
revertant colonies on both negative (solvent control, and untreated 
control if used) and positive control plates shall also be given.
    (ii) Individual plate counts, the mean number of revertant colonies 
per plate and the standard deviation shall be presented for the test 
substance and positive and negative (untreated and/or solvent) controls.
    (iii) There is no requirement for verification of a clear positive 
response. Equivocal results shall be clarified by further testing 
preferably using a modification of experimental conditions. Negative 
results need to be confirmed on a case-by-case basis. In those cases 
where confirmation of negative results is not considered necessary, 
justification should be provided. Modification of study parameters to 
extend the range of conditions assessed should be considered in follow-
up experiments. Study parameters that might be modified include the 
concentration spacing, the method of treatment (plate incorporation or 
liquid preincubation), and metabolic activation conditions.
    (2) Evaluation and interpretation of results. (i) There are several 
criteria for determining a positive result, such as a concentration-
related increase over the range tested and/or a reproducible increase at 
one or more concentrations in the number of revertant colonies per plate 
in at least one strain with or without metabolic activation system. 
Biological relevance of the results should be considered first. 
Statistical methods may be used as an aid in evaluating the test 
results. However, statistical significance should not be the only 
determining factor for a positive response.

[[Page 421]]

    (ii) A test substance for which the results do not meet the criteria 
described under paragraph (f)(2)(i) of this section is considered non-
mutagenic in this test
    (iii) Although most experiments will give clearly positive or 
negative results, in rare cases the data set will preclude making a 
definite judgement about the activity of the test substance. Results may 
remain equivocal or questionable regardless of the number of times the 
experiment is repeated.
    (iv) Positive results from the bacterial reverse mutation test 
indicate that a substance induces point mutations by base substitutions 
or frameshifts in the genome of either Salmonella typhimurium and/or 
Escherichia coli. Negative results indicate that under the test 
conditions, the test substance is not mutagenic in the tested species.
    (3) Test report. The test report shall include the following 
information:
    (i) Test substance:
    (A) Identification data and CAS no., if known.
    (B) Physical nature and purity.
    (C) Physicochemical properties relevant to the conduct of the study.
    (D) Stability of the test substance, if known.
    (ii) Solvent/vehicle:
    (A) Justification for choice of solvent/vehicle.
    (B) Solubility and stability of the test substance in solvent/
vehicle, if known.
    (iii) Strains:
    (A) Strains used.
    (B) Number of cells per culture.
    (C) Strain characteristics.
    (iv) Test conditions:
    (A) Amount of test substance per plate (mg/plate or ml/plate) with 
rationale for selection of dose and number of plates per concentration.
    (B) Media used.
    (C) Type and composition of metabolic activation system, including 
acceptability criteria.
    (D) Treatment procedures.
    (v) Results:
    (A) Signs of toxicity.
    (B) Signs of precipitation.
    (C) Individual plate counts.
    (D) The mean number of revertant colonies per plate and standard 
deviation.
    (E) Dose-response relationship, where possible.
    (F) Statistical analyses, if any.
    (G) Concurrent negative (solvent/vehicle) and positive control data, 
with ranges, means and standard deviations.
    (H) Historical negative (solvent/vehicle) and positive control data, 
with e.g. ranges, means and standard deviations.
    (vi) Discussion of the results.
    (vii) Conclusion.
    (g) References. For additional background information on this test 
guideline, the following references should be consulted. These 
references are available for inspection at the TSCA Nonconfidential 
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M 
St., SW., Washington, DC, 12 noon to 4 p.m., Monday through Friday, 
except legal holidays.
    (1) Ames, B.N., McCann, J., and Yamasaki, E. Methods for Detecting 
Carcinogens and Mutagens With the Salmonella/Mammalian-Microsome 
Mutagenicity Test. Mutation Research. 31, 347-364 (1975).
    (2) Maron, D.M. and Ames, B.N. Revised Methods for the Salmonella 
Mutagenicity Test. Mutation Research. 113, 173-215 (1983).
    (3) Gatehouse, D., Haworth, S., Cebula, T., Gocke, E., Kier, L., 
Matsushima, T., Melcion, C., Nohmi, T., Venitt, S., and Zeiger, E. 
Recommendations for the Performance of Bacterial Mutation Assays. 
Mutation Research. 312, 217-233 (1994).
    (4) Kier, L.D., Brusick, D.J., Auletta, A.E., Von Halle, E.S., 
Brown, M.M., Simmon, V.F., Dunkel, V., McCann, J., Mortelmans, K., 
Prival, M., Rao, T.K., and Ray V. The Salmonella Typhimurium/Mammalian 
Microsomal Assay: A Report of the U.S. Environmental Protection Agency 
Gene-Tox Program. Mutation Research. 168, 69-240 (1986).
    (5) Yahagi, T., Degawa, M., Seino, Y.Y., Matsushima, T., Nagao, M., 
Sugimura, T., and Hashimoto, Y. Mutagenicity of Carcinogen Azo Dyes and 
Their Derivatives. Cancer Letters, 1. 91-96 (1975).
    (6) Matsushima, M., Sugimura, T., Nagao, M., Yahagi, T., Shirai, A., 
and

[[Page 422]]

Sawamura, M. Factors Modulating Mutagenicity Microbial Tests. Eds. 
Norpoth, K.H. and Garner, R.C. Short-Term Test Systems for Detecting 
Carcinogens (Springer, Berlin-Heidelberg-New York, 1980) pp. 273-285.
    (7) Gatehouse, D.G., Rowland, I.R., Wilcox, P., Callender, R.D., and 
Foster, R. Bacterial Mutation Assays. Ed. Kirkland, D.J. Basic 
Mutagenicity Tests. UKEMS Part 1 Revised (Cambridge University Press, 
1990) pp. 13-61.
    (8) Aeschbacher, H.U., Wolleb, U., and Porchet, L.J. Liquid 
Preincubation Mutagenicity Test for Foods. Food Safety. 8, 167-177 
(1987).
    (9) Green, M.H.L., Muriel, W.J., and Bridges, B.A. Use of a 
Simplified Fluctuation Test to Detect Low Levels of Mutagens. Mutation 
Research. 38, 33-42 (1976).
    (10) Hubbard, S.A., Green, M.H.L., Gatehouse, D., and J.W. Bridges. 
The Fluctuation Test in Bacteria. 2nd Edition. Ed. Kilbey, B.J., 
Legator, M., Nichols, W., and Ramel C. Handbook of Mutagenicity Test 
Procedures (Elsevier, Amsterdam-New York-Oxford, 1984) pp. 141-161.
    (11) Thompson, E.D. and Melampy, P.J. An Examination of the 
Quantitative Suspension Assay for Mutagenesis With Strains of Salmonella 
Typhimurium. Environmental Mutagenesis. 3, 453-465 (1981).
    (12) Araki, A., Noguchi, T., Kato, F., and T. Matsushima. Improved 
Method for Mutagenicity Testing of Gaseous Compounds by Using a Gas 
Sampling Bag. Mutation Research. 307, 335-344 (1994).
    (13) Prival, M.J., Bell, S.J., Mitchell, V.D., Reipert, M.D., and 
Vaughn, V.L. Mutagenicity of Benzidine and Benzidine-Congener Dyes and 
Selected Monoazo Dyes in a Modified Salmonella Assay. Mutation Research. 
136, 33-47 (1984).
    (14) Zeiger, E., Anderson, B. E., Haworth, S, Lawlor, T., and 
Mortelmans, K. Salmonella Mutagenicity Tests. V. Results from the 
Testing of 311 Chemicals. Environ. Mol. Mutagen. 19, 2-141 (1992).
    (15) Simmon, V., Kauhanen, K., and Tardiff, R.G. Mutagenic Activity 
of Chemicals Identified in Drinking Water. Ed. Scott, D., Bridges, B., 
and Sobels, F. Progress in Genetic Toxicology (Elsevier, Amsterdam, 
1977) pp. 249-258.
    (16) Hughes, T.J., Simmons, D.M., Monteith, I.G., and Claxton, L.D. 
Vaporization Technique to Measure Mutagenic Activity of Volatile Organic 
Chemicals in the Ames/Salmonella Assay. Environmental Mutagenesis. 9, 
421-441 (1987).
    (17) Matsushima, T., Matsumoto, A., Shirai, M., Sawamura, M., and 
Sugimura, T. Mutagenicity of the Naturally Occurring Carcinogen Cycasin 
and Synthetic Methylazoxy Methane Conjugates in Salmonella Typhimurium. 
Cancer Research. 39, 3780-3782 (1979).
    (18) Tamura, G., Gold, C., Ferro-Luzzi, A., and Ames. B.N. Fecalase: 
A Model for Activation of Dietary Glycosides to Mutagens by Intestinal 
Flora. Proc. National Academy of Science. (USA, 1980) 77, 4961-4965.
    (19) Wilcox, P., Naidoo, A., Wedd, D. J., and Gatehouse, D. G. 
Comparison of Salmonella Typhimurium TA 102 With Escherichia Coli WP2 
Tester Strains. Mutagenesis. 5, 285-291 (1990).
    (20) Matsushima, T., Sawamura, M., Hara, K., and Sugimura, T. A Safe 
Substitute for Polychlorinated Biphenyls as an Inducer of Metabolic 
Activation Systems. Ed. F.J. de Serres et al. In Vitro Metabolic 
Activation in Mutagenesis Testing. (Elsevier, North Holland, 1976) pp. 
85-88.
    (21) Elliott, B.M., Combes, R.D., Elcombe, C.R., Gatehouse, D.G., 
Gibson, G.G., Mackay, J.M., and Wolf, R.C. Alternatives to Aroclor 1254-
Induced S9 in In Vitro Genotoxicity Assays. Mutagenesis. 7, 175-177 
(1992).
    (22) Maron, D., Katzenellenbogen, J., and Ames, B.N. Compatibility 
of Organic Solvents With the Salmonella/Microsome Test. Mutation 
Research. 88, 343-350 (1981).
    (23) Claxton, L.D., Allen, J., Auletta, A., Mortelmans, K., 
Nestmann, E., and Zeiger, E. Guide for the Salmonella Typhimurium/
Mammalian Microsome Tests for Bacterial Mutagenicity. Mutation Research. 
189, 83-91 (1987).
    (24) Mahon, G.A.T., Green, M.H.L., Middleton, B., Mitchell, I., 
Robinson, W.D., and Tweats, D.J. Analysis of Data from Microbial Colony 
Assays. UKEMS Sub-Committee on Guidelines for Mutagenicity Testing Part 
II. Ed.

[[Page 423]]

Kirkland, D.J. Statistical Evaluation of Mutagenicity Test Data 
(Cambridge University Press, 1989) pp. 28-65.

[62 FR 43824, Aug. 15, 1997, as amended at 64 FR 35079, June 30, 1999]



Sec. 799.9530  TSCA in vitro mammalian cell gene mutation test.

    (a) Scope. This section is intended to meet the testing requirements 
under section 4 of TSCA. The in vitro mammalian cell gene mutation test 
can be used to detect gene mutations induced by chemical substances. 
Suitable cell lines include L5178Y mouse lymphoma cells, the CHO, AS52 
and V79 lines of Chinese hamster cells, and TK6 human lymphoblastoid 
cells under paragraph (g)(1) of this section. In these cell lines the 
most commonly-used genetic endpoints measure mutation at thymidine 
kinase (TK) and hypoxanthine-guanine phosphoribosyl transferase (HPRT), 
and a transgene of xanthine-guanine phosphoribosyl transferase (XPRT). 
The TK, HPRT and XPRT mutation tests detect different spectra of genetic 
events. The autosomal location of TK and XPRT may allow the detection of 
genetic events (e.g. large deletions) not detected at the HPRT locus on 
X-chromosomes (For a discussion see the references in paragraphs (g)(2), 
(g)(3), (g)(4),(g)(5), and (g)(6) of this section).
    (b) Source. The source material used in developing this TSCA test 
guideline is the OECD guideline 476 (February 1997). This source is 
available at the address in paragraph (g) of this section.
    (c) Definitions. The following definitions apply to this section:
    Base pair substitution mutagens are substances which cause 
substitution of one or several base pairs in the DNA.
    Forward mutation is a gene mutation from the parental type to the 
mutant form which gives rise to an alteration or a loss of the enzymatic 
activity or the function of the encoded protein.
    Frameshift mutagens are substances which cause the addition or 
deletion of single or multiple base pairs in the DNA molecule.
    Mutant frequency is the number of mutant cells observed divided by 
the number of viable cells.
    Phenotypic expression time is a period during which unaltered gene 
products are depleted from newly mutated cells.
    Relative suspension growth is an increase in cell number over the 
expression period relative to the negative control.
    Relative total growth is an increase in cell number over time 
compared to a control population of cells; calculated as the product of 
suspension growth relative to the negative control times cloning 
efficiency relative to negative control.
    Survival is the cloning efficiency of the treated cells when plated 
at the end of the treatment period; survival is usually expressed in 
relation to the survival of the control cell population.
    Viability is the cloning efficiency of the treated cells at the time 
of plating in selective conditions after the expression period.
    (d) Initial considerations. (1) In the in vitro mammalian cell gene 
mutation test, cultures of established cell lines or cell strains can be 
used. The cells used are selected on the basis of growth ability in 
culture and stability of the spontaneous mutation frequency. Tests 
conducted in vitro generally require the use of an exogenous source of 
metabolic activation. This metabolic activation system cannot mimic 
entirely the mammalian in vivo conditions. Care should be taken to avoid 
conditions which would lead to results not reflecting intrinsic 
mutagenicity. Positive results which do not reflect intrinsic 
mutagenicity may arise from changes in pH, osmolality or high levels of 
cytotoxicity.
    (2) This test is used to screen for possible mammalian mutagens and 
carcinogens. Many compounds that are positive in this test are mammalian 
carcinogens; however, there is not a perfect correlation between this 
test and carcinogenicity. Correlation is dependent on chemical class and 
there is increasing evidence that there are carcinogens that are not 
detected by this test because they appear to act through other, non-
genotoxic mechanisms or mechanisms absent in bacterial cells.
    (e) Test method--(1) Principle. (i) Cells deficient in thymidine 
kinase (TK) due

[[Page 424]]

to the mutation TK=/- -<= TK-/
- are resistant to the cytotoxic effects of the pyrimidine 
analogue trifluorothymidine (TFT). Thymidine kinase proficient cells are 
sensitive to TFT, which causes the inhibition of cellular metabolism and 
halts further cell division. Thus mutant cells are able to proliferate 
in the presence of TFT, whereas normal cells, which contain thymidine 
kinase, are not. Similarly, cells deficient in HPRT or XPRT are selected 
by resistance to 6-thioguanine (TG) or 8-azaguanine (AG). The properties 
of the test substance should be considered carefully if a base analogue 
or a compound related to the selective agent is tested in any of the 
mammalian cell gene mutation tests. For example, any suspected selective 
toxicity by the test substance for mutant and non-mutant cells should be 
investigated. Thus, performance of the selection system/agent shall be 
confirmed when testing chemicals structurally related to the selective 
agent.
    (ii) Cells in suspension or monolayer culture shall be exposed to 
the test substance, both with and without metabolic activation, for a 
suitable period of time and subcultured to determine cytotoxicity and to 
allow phenotypic expression prior to mutant selection. Cytotoxicity is 
usually determined by measuring the relative cloning efficiency 
(survival) or relative total growth of the cultures after the treatment 
period. The treated cultures shall be maintained in growth medium for a 
sufficient period of time, characteristic of each selected locus and 
cell type, to allow near-optimal phenotypic expression of induced 
mutations. Mutant frequency is determined by seeding known numbers of 
cells in medium containing the selective agent to detect mutant cells, 
and in medium without selective agent to determine the cloning 
efficiency (viability). After a suitable incubation time, colonies shall 
be counted. The mutant frequency is derived from the number of mutant 
colonies in selective medium and the number of colonies in non-selective 
medium.
    (2) Description--(i) Preparations--(A) Cells. (1) A variety of cell 
types are available for use in this test including subclones of L5178Y, 
CHO, CHO-AS52, V79, or TK6 cells. Cell types used in this test should 
have a demonstrated sensitivity to chemical mutagens, a high cloning 
efficiency and a stable spontaneous mutant frequency. Cells should be 
checked for mycoplasma contamination and should not be used if 
contaminated.
    (2) The test should be designed to have a predetermined sensitivity 
and power. The number of cells, cultures, and concentrations of test 
substance used should reflect these defined parameters. The parameters 
discussed in the reference under paragraph (g)(13) of this section may 
be used. The minimal number of viable cells surviving treatment and used 
at each stage in the test should be based on the spontaneous mutation 
frequency. A general guide is to use a cell number which is at least ten 
times the inverse of the spontaneous mutation frequency. However, it is 
recommended to utilize at least 10\6\ cells. Adequate historical data on 
the cell system used should be available to indicate consistent 
performance of the test.
    (B) Media and culture conditions. Appropriate culture media and 
incubation conditions (culture vessels, temperature, CO2 
concentration and humidity) shall be used. Media should be chosen 
according to the selective systems and cell type used in the test. It is 
particularly important that culture conditions should be chosen that 
ensure optimal growth of cells during the expression period and colony 
forming ability of both mutant and non-mutant cells.
    (C) Preparation of cultures. Cells are propagated from stock 
cultures, seeded in culture medium and incubated at 37 [deg]C. Prior to 
use in this test, cultures may need to be cleansed of pre-existing 
mutant cells.
    (D) Metabolic activation. Cells shall be exposed to the test 
substance both in the presence and absence of an appropriate metabolic 
activation system. The most commonly used system is a co-factor-
supplemented post-mitochondrial fraction (S9) prepared from the livers 
of rodents treated with enzyme-inducing agents such as Aroclor 1254 or a 
combination of phenobarbitone and [beta]-naphthoflavone. The post-
mitochondrial fraction is usually used at concentrations in the range 
from 1-10% v/v in the final test

[[Page 425]]

medium. The choice and condition of a metabolic activation system may 
depend upon the class of chemical being tested. In some cases it may be 
appropriate to utilize more than one concentration of post-mitochondrial 
fraction. A number of developments, including the construction of 
genetically engineered cell lines expressing specific activating 
enzymes, may provide the potential for endogenous activation. The choice 
of the cell lines used should be scientifically justified (e.g. by the 
relevance of the cytochrome P450 isoenzyme to the metabolism of the test 
substance).
    (E) Test substance/preparations. Solid test substances should be 
dissolved or suspended in appropriate solvents or vehicles and diluted 
if appropriate prior to treatment of the cells. Liquid test substances 
may be added directly to the test systems and/or diluted prior to 
treatment. Fresh preparations should be employed unless stability data 
demonstrate the acceptability of storage.
    (ii) Test conditions--(A) Solvent/vehicle. The solvent/vehicle shall 
not be suspected of chemical reaction with the test substance and shall 
be compatible with the survival of the cells and the S9 activity. If 
other than well-known solvent/vehicles are used, their inclusion should 
be supported by data indicating their compatibility. It is recommended 
that wherever possible, the use of an aqueous solvent/vehicle be 
considered first. When testing water-unstable substances, the organic 
solvents used should be free of water. Water can be removed by adding a 
molecular sieve.
    (B) Exposure concentrations. (1) Among the criteria to be considered 
when determining the highest concentration are cytotoxicity and 
solubility in the test system and changes in pH or osmolality.
    (2) Cytotoxicity should be determined with and without metabolic 
activation in the main experiment using an appropriate indicator of cell 
integrity and growth, such as relative cloning efficiency (survival) or 
relative total growth. It may be useful to determine cytotoxicity and 
solubility in a preliminary experiment.
    (3) At least four analyzable concentrations shall be used. Where 
there is cytotoxicity, these concentrations shall cover a range from the 
maximum to little or no toxicity; this will usually mean that the 
concentration levels should be separated by no more than a factor 
between 2 and [radic]10. If the maximum concentration is based on 
cytotoxicity then it shall result in approximately 10-20% but not less 
than 10% relative survival (relative cloning efficiency) or relative 
total growth. For relatively non-cytotoxic compounds the maximum 
concentration should be 5 mg/ml, 5 [micro]l/ml, or 0.01 M, whichever is 
the lowest.
    (4) Relatively insoluble substances should be tested up to or beyond 
their limit of solubility under culture conditions. Evidence of 
insolubility should be determined in the final treatment medium to which 
cells are exposed. It may be useful to assess solubility at the 
beginning and end of the treatment, as solubility can change during the 
course of exposure in the test system due to presence of cells, S9, 
serum etc. Insolubility can be detected by using the unaided eye. The 
precipitate should not interfere with the scoring.
    (C) Controls. (1) Concurrent positive and negative (solvent or 
vehicle) controls both with and without metabolic activation shall be 
included in each experiment. When metabolic activation is used the 
positive control chemical shall be one that requires activation to give 
a mutagenic response.
    (2) Examples of positive control substances include:

----------------------------------------------------------------------------------------------------------------
    Metabolic Activation condition              Locus                   Chemical                 CAS No.
----------------------------------------------------------------------------------------------------------------
Absence of exogenous metabolic         HPRT...................  Ethylmethanesulfonate..  [CAS no. 62-50-0]
 activation
                                       .......................  Ethylnitrosourea.......  [CAS no. 759-73-9]
                                       TK (small and large      Methylmethanesulfonate.  [CAS no. 66-27-3]
                                        colonies).
                                       XPRT...................  Ethylmethanesulfonate..  [CAS no. 62-50-0]
                                                                Ethylnitrosourea.......  [CAS no. 759-73-9]

[[Page 426]]

 
Presence of exogenous metabolic        HPRT...................  3-Methylcholanthrene...  [CAS no. 56-49-5]
 activation.
                                                                N-Nitrosodimethylamine.  [CAS no. 62-75-9]
                                                                7,12-                    [CAS no. 57-97-6]
                                                                 Dimethylbenzanthracene.
                                       TK (small and large      Cyclophosphamide         [CAS no. 50-18-0]
                                        colonies).               (monohydrate).          [CAS no. 6055-19-2]
                                                                Benzo(a)pyrene.........  [CAS no. 50-32-8]
                                                                3-Methylcholanthrene...  [CAS no. 56-49-5]
                                       XPRT...................  N-Nitrosodimethylamine   [CAS no. 62-75-9]
                                                                 (for high levels of S-
                                                                 9).
                                                                Benzo(a)pyrene.........  [CAS no. 50-32-8]
----------------------------------------------------------------------------------------------------------------

    (3) Other appropriate positive control reference substances may be 
used, e.g., if a laboratory has a historical data base on 5-Bromo 2'-
deoxyuridine [CAS No. 59-14-3], this reference substance could be used 
as well. The use of chemical class-related positive control chemicals 
may be considered, when available.
    (4) Negative controls, consisting of solvent or vehicle alone in the 
treatment medium, and treated in the same way as the treatment groups 
shall be included. In addition, untreated controls should also be used 
unless there are historical control data demonstrating that no 
deleterious or mutagenic effects are induced by the chosen solvent.
    (3) Procedure--(i) Treatment with test substance. (A) Proliferating 
cells shall be exposed to the test substance both with and without 
metabolic activation. Exposure shall be for a suitable period of time 
(usually 3 to 6 hrs is effective). Exposure time may be extended over 
one or more cell cycles.
    (B) Either duplicate or single treated cultures may be used at each 
concentration tested. When single cultures are used, the number of 
concentrations should be increased to ensure an adequate number of 
cultures for analysis (e.g. at least eight analyzsable concentrations). 
Duplicate negative (solvent) control cultures should be used.
    (C) Gaseous or volatile substances should be tested by appropriate 
methods, such as in sealed culture vessels. Methods described in the 
references under paragraphs (g)(20) and (g)(21) of this section may be 
used.
    (ii) Measurement of survival, viability, and mutant frequency. (A) 
At the end of the exposure period, cells shall be washed and cultured to 
determine survival and to allow for expression of the mutant phenotype. 
Measurement of cytotoxicity by determining the relative cloning 
efficiency (survival) or relative total growth of the cultures is 
usually initiated after the treatment period.
    (B) Each locus has a defined minimum time requirement to allow near 
optimal phenotypic expression of newly induced mutants (HPRT and XPRT 
require at least 6-8 days, and TK at least 2 days). Cells are grown in 
medium with and without selective agent(s) for determination of numbers 
of mutants and cloning efficiency, respectively. The measurement of 
viability (used to calculate mutant frequency) is initiated at the end 
of the expression time by plating in non-selective medium.
    (C) If the test substance is positive in the L5178Y TK=/
- test, colony sizing should be performed on at least one of 
the test cultures (the highest positive concentration) and on the 
negative and positive controls. If the test substance is negative in the 
L5178Y TK=/- test, colony sizing should be 
performed on the negative and positive controls. In studies using 
TK6TK=/-, colony sizing may also be performed.
    (f) Data and reporting--(1) Treatment of results. (i) Data shall 
include cytotoxicity and viability determination, colony counts and 
mutant frequencies for the treated and control cultures. In the case of 
a positive response in the L5178Y TK=/- test, 
colonies are scored using the criteria of small and large colonies on at 
least one concentration of the test substance (highest positive 
concentration) and on the negative and positive control. The molecular 
and cytogenetic nature of both large and small colony mutants

[[Page 427]]

has been explored in detail and is discussed in the references under 
paragraphs (g)(22) and (g)(23) of this section. In the TK=/
- test, colonies are scored using the criteria of normal 
growth (large) and slow growth (small) colonies (a scoring system 
similar to the one described in the reference under paragraph (g)(24) of 
this section may be used). Mutant cells that have suffered the most 
extensive genetic damage have prolonged doubling times and thus form 
small colonies. This damage typically ranges in scale from the losses of 
the entire gene to karyotypically visible chromosome aberrations. The 
induction of small colony mutants has been associated with chemicals 
that induce gross chromosome aberrations. Less seriously affected mutant 
cells grow at rates similar to the parental cells and form large 
colonies.
    (ii) Survival (relative cloning efficiencies) or relative total 
growth shall be given. Mutant frequency shall be expressed as number of 
mutant cells per number of surviving cells.
    (iii) Individual culture data shall be provided. Additionally, all 
data shall be summarized in tabular form.
    (iv) There is no requirement for verification of a clear positive 
response. Equivocal results shall be clarified by further testing 
preferably using a modification of experimental conditions. Negative 
results need to be confirmed on a case-by-case basis. In those cases 
where confirmation of negative results is not considered necessary, 
justification should be provided. Modification of study parameters to 
extend the range of conditions assessed should be considered in follow-
up experiments for either equivocal or negative results. Study 
parameters that might be modified include the concentration spacing, and 
the metabolic activation conditions.
    (2) Evaluation and interpretation of results. (i) There are several 
criteria for determining a positive result, such as a concentration-
related, or a reproducible increase in mutant frequency. Biological 
relevance of the results should be considered first. Statistical methods 
may be used as an aid in evaluating the test results. Statistical 
significance should not be the only determining factor for a positive 
response.
    (ii) A test substance, for which the results do not meet the 
criteria described in paragraph (f)(2)(i) of this section is considered 
non-mutagenic in this system.
    (iii) Although most studies will give clearly positive or negative 
results, in rare cases the data set will preclude making a definite 
judgement about the activity of the test substance. Results may remain 
equivocal or questionable regardless of the number of times the 
experiment is repeated.
    (iv) Positive results for an in vitro mammalian cell gene mutation 
test indicate that the test substance induces gene mutations in the 
cultured mammalian cells used. A positive concentration-response that is 
reproducible is most meaningful. Negative results indicate that, under 
the test conditions, the test substance does not induce gene mutations 
in the cultured mammalian cells used.
    (3) Test report. The test report shall include the following 
information:
    (i) Test substance:
    (A) Identification data and CAS no., if known.
    (B) Physical nature and purity.
    (C) Physicochemical properties relevant to the conduct of the study.
    (D) Stability of the test substance.
    (ii) Solvent/vehicle:
    (A) Justification for choice of vehicle/solvent.
    (B) Solubility and stability of the test substance in solvent/
vehicle, if known.
    (iii) Cells:
    (A) Type and source of cells.
    (B) Number of cell cultures.
    (C) Number of cell passages, if applicable.
    (D) Methods for maintenance of cell cultures, if applicable.
    (E) Absence of mycoplasma.
    (iv) Test conditions:
    (A) Rationale for selection of concentrations and number of cell 
cultures including e.g., cytotoxicity data and solubility limitations, 
if available.
    (B) Composition of media, CO2 concentration.
    (C) Concentration of test substance.
    (D) Volume of vehicle and test substance added.

[[Page 428]]

    (E) Incubation temperature.
    (F) Incubation time.
    (G) Duration of treatment.
    (H) Cell density during treatment.
    (I) Type and composition of metabolic activation system including 
acceptability criteria.
    (J) Positive and negative controls.
    (K) Length of expression period (including number of cells seeded, 
and subcultures and feeding schedules, if appropriate).
    (L) Selective agent(s).
    (M) Criteria for considering tests as positive, negative or 
equivocal.
    (N) Methods used to enumerate numbers of viable and mutant cells.
    (O) Definition of colonies of which size and type are considered 
(including criteria for ``small'' and ``large'' colonies, as 
appropriate).
    (v) Results:
    (A) Signs of toxicity.
    (B) Signs of precipitation.
    (C) Data on pH and osmolality during the exposure to the test 
substance, if determined.
    (D) Colony size if scored for at least negative and positive 
controls.
    (E) Laboratory's adequacy to detect small colony mutants with the 
L5178Y TK=/- system, where appropriate.
    (F) Dose-response relationship, where possible.
    (G) Statistical analyses, if any.
    (H) Concurrent negative (solvent/vehicle) and positive control data.
    (I) Historical negative (solvent/vehicle) and positive control data 
with ranges, means, and standard deviations.
    (J) Mutant frequency.
    (vi) Discussion of the results.
    (vii) Conclusion.
    (g) References. For additional background information on this test 
guideline, the following references should be consulted. These 
references are available for inspection at the TSCA Nonconfidential 
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M 
St., SW., Washington, DC, 12 noon to 4 p.m., Monday through Friday, 
except legal holidays.
    (1) Chu, E.H.Y. and Malling, H.V. Mammalian Cell Genetics. II. 
Chemical Induction of Specific Locus Mutations in Chinese Hamster Cells 
In Vitro, Proc. National Academy Science (USA, 1968) 61, 1306-1312.
    (2) Liber, H.L. and Thilly, W.G. Mutation Assay at the Thymidine 
Kinase Locus in Diploid Human Lymphoblasts. Mutation Research. 94, 467-
485 (1982).
    (3) Moore, M.M., Harrington-Brock, K., Doerr, C.L., and Dearfield, 
K.L. Differential Mutant Quantitation at the Mouse Lymphoma TK and CHO 
HGPRT Loci. Mutagenesis. 4, 394-403 (1989).
    (4) Aaron, C.S. and Stankowski, Jr., L.F. Comparison of the AS52/
XPRT and the CHO/HPRT Assays: Evaluation of Six Drug Candidates. 
Mutation Research. 223, 121-128 (1989).
    (5) Aaron, C.S., Bolcsfoldi, G., Glatt, H.R., Moore, M., Nishi, Y., 
Stankowski, L., Theiss, J., and Thompson, E. Mammalian Cell Gene 
Mutation Assays Working Group Report. Report of the International 
Workshop on Standardization of Genotoxicity Test Procedures. Mutation 
Research. 312, 235-239 (1994).
    (6) Scott, D., Galloway, S.M., Marshall, R.R., Ishidate, M., 
Brusick, D., Ashby, J., and Myhr, B.C. Genotoxicity Under Extreme 
Culture Conditions. A report from ICPEMC Task Group 9. Mutation 
Research. 257, 147-204 (1991).
    (7) Clive, D., McCuen, R., Spector, J.F.S., Piper, C., and 
Mavournin, K.H. Specific Gene Mutations in L5178Y Cells in Culture. A 
Report of the U.S. Environmental Protection Agency Gene-Tox Program. 
Mutation Research. 115, 225-251 (1983).
    (8) Li, A.P., Gupta, R.S., Heflich, R.H., and Wasson, J. S. A Review 
and Analysis of the Chinese Hamster Ovary/Hypoxanthine Guanine 
Phosphoribosyl Transferase System to Determine the Mutagenicity of 
Chemical Agents: A Report of Phase III of the U.S. Environmental 
Protection Agency Gene-Tox Program. Mutation Research. 196, 17-36 
(1988).
    (9) Li, A.P., Carver, J.H., Choy, W.N., Hsie, A.W., Gupta, R.S., 
Loveday, K.S., O'Neill, J.P., Riddle, J.C., Stankowski, Jr., L.F., and 
Yang, L.L. A Guide for the Performance of the Chinese Hamster Ovary 
Cell/Hypoxanthine-Guanine Phosphoribosyl Transferase Gene Mutation 
Assay. Mutation Research. 189, 135-141 (1987).
    (10) Liber, H.L., Yandell, D.W., and Little, J.B. A Comparison of 
Mutation

[[Page 429]]

Induction at the tk and hprt Loci in Human Lymphoblastoid Cells; 
Quantitative Differences are Due to an Additional Class of Mutations at 
the Autosomal TK Locus. Mutation Research. 216, 9-17 (1989).
    (11) Stankowski, L.F. Jr., Tindall, K.R., and Hsie, A.W. 
Quantitative and Molecular Analyses of Ethyl Methanesulfonate- and ICR 
191-Induced Molecular Analyses of Ethyl Methanesulfonate- and ICR 191-
Induced Mutation in AS52 Cells. Mutation Reseach. 160, 133-147 (1986).
    (12) Turner, N.T., Batson, A.G., and Clive, D. Eds. Kilbey, B.J. et 
al. Procedures for the L5178Y/TK=/-  
TK=/- Mouse Lymphoma Cell Mutagenicity Assay. 
Handbook of Mutagenicity Test Procedures (Elsevier Science Publishers, 
New York, 1984) pp. 239-268.
    (13) Arlett, C.F., Smith, D.M., Clarke, G.M., Green, M.H.L., Cole, 
J., McGregor, D.B., and Asquith, J.C. Ed. Kirkland, D.J. Mammalian Cell 
Gene Mutation Assays Based Upon Colony Formation. Statistical Evaluation 
of Mutagenicity Test Data (Cambridge University Press, 1989) pp. 66-101.
    (14) Abbondandolo, A., Bonatti, S., Corti, G., Fiorio, R., Loprieno, 
N., and Mazzaccaro, A. Induction of 6-Thioguanine-Resistant Mutants in 
V79 Chinese Hamster Cells by Mouse-Liver Microsome-Activated 
Dimethylnitrosamine. Mutation Research. 46, 365-373 (1977).
    (15) Ames, B.N., McCann, J., and Yamasaki, E. Methods for Detecting 
Carcinogens and Mutagens with the Salmonella/Mammalian-Microsome 
Mutagenicity Test. Mutation Reseach. 31, 347-364 (1975).
    (16) Clive, D., Johnson, K.O., Spector, J.F.S., Batson, A.G., and 
Brown M.M.M. Validation and Characterization of the L5178Y/
TK=/- Mouse Lymphoma Mutagen Assay System. 
Mutation Reseach. 59, 61-108 (1979).
    (17) Maron, D.M. and Ames, B.N. Revised Methods for the Salmonella 
Mutagenicity Test. Mutation Reseach. 113, 173, 215 (1983).
    (18) Elliott, B.M., Combes, R.D., Elcombe, C.R., Gatehouse, D.G., 
Gibson, G.G., Mackay, J.M., and Wolf, R.C. Alternatives to Aroclor 1254-
Induced S9 in In Vitro Genotoxicity Assays. Mutagenesis. 7, 175-177 
(1992).
    (19) Matsushima, T., Sawamura, M., Hara, K., and Sugimura, T. A Safe 
Substitute for Polychlorinated Biphenyls as an Inducer of Metabolic 
Activation Systems. (Eds.) de Serres, F.J., Fouts, J.R., Bend, J.R., and 
Philpot, R.M. In Vitro Metabolic Activation in Mutagenesis Testing 
(Elsevier, North-Holland, 1976) pp. 85-88.
    (20) Krahn, D.F., Barsky, F.C., and McCooey, K.T. Eds. Tice, R.R., 
Costa, D.L., and Schaich, K.M. CHO/HGPRT Mutation Assay: Evaluation of 
Gases and Volatile Liquids. Genotoxic Effects of Airborne Agents (New 
York, Plenum, 1982) pp. 91-103.
    (21) Zamora, P.O., Benson, J.M., Li, A.P., and Brooks, A.L. 
Evaluation of an Exposure System Using Cells Grown on Collagen Gels for 
Detecting Highly Volatile Mutagens in the CHO/HGPRT Mutation Assay. 
Environmental Mutagenesis. 5, 795-801 (1983).
    (22) Applegate, M.L., Moore, M.M., Broder, C.B., Burrell, A., and 
Hozier, J.C. Molecular Dissection of Mutations at the Heterozygous 
Thymidine Kinase Locus in Mouse Lymphoma Cells. Proc. National Academy 
Science (USA, 1990) 87, 51-55.
    (23) Moore, M.M., Clive, D., Hozier, J.C., Howard, B.E., Batson, 
A.G., Turner, N.T., and Sawyer, J. Analysis of Trifluorothymidine-
Resistant (TFT\r\) Mutants of L5178Y/TK=/- Mouse 
Lymphoma Cells. Mutation Research. 151, 161-174 (1985).
    (24) Yandell, D.W., Dryja, T.P., and Little J.B. Molecular Genetic 
Analysis of Recessive Mutations at a Heterozygous Autosomal Locus in 
Human Cells. Mutation Research. 229, 89-102 (1990).
    (25) Moore, M.M. and Doerr, C.L. Comparison of Chromosome Aberration 
Frequency and Small-Colony TK-Deficient Mutant Frequency in L5178Y/
TK=/- 3.7.2C Mouse Lymphoma Cells. Mutagenesis. 5, 
609-614 (1990).



Sec. 799.9537  TSCA in vitro mammalian chromosome aberration test.

    (a) Scope--(1) Applicability. This section is intended to meet 
testing requirements under section 4 of the Toxic Substances Control Act 
(TSCA) (15 U.S.C. 2601).
    (2) Background. The source material used in developing this TSCA 
test

[[Page 430]]

guideline is the Office of Prevention, Pesticides, and Toxic Substances 
(OPPTS) harmonized test guideline 870.5375 (August 1998, final 
guidelines). The source is available at the address in paragraph (i) of 
this section.
    (b) Purpose. (1) The purpose of the in vitro chromosome aberration 
test is to identify agents that cause structural chromosome aberrations 
in cultured mammalian cells (see paragraphs (i)(1), (i)(2), and (i)(3) 
of this section). Structural aberrations may be of two types, chromosome 
or chromatid. With the majority of chemical mutagens, induced 
aberrations are of the chromatid type, but chromosome-type aberrations 
also occur. An increase in polyploidy may indicate that a chemical has 
the potential to induce numerical aberrations. However, this guideline 
is not designed to measure numerical aberrations and is not routinely 
used for that purpose. Chromosome mutations and related events are the 
cause of many human genetic diseases and there is substantial evidence 
that chromosome mutations and related events causing alterations in 
oncogenes and tumour-suppressor genes of somatic cells are involved in 
cancer induction in humans and experimental animals.
    (2) The in vitro chromosome aberration test may employ cultures of 
established cell lines, cell strains or primary cell cultures. The cells 
used are selected on the basis of growth ability in culture, stability 
of the karyotype, chromosome number, chromosome diversity, and 
spontaneous frequency of chromosome aberrations.
    (c) Definitions. The definitions in section 3 of TSCA and in 40 CFR 
Part 792--Good Laboratory Practice Standards apply to this test 
guideline. The following definitions also apply to this test guideline.
    Chromatid-type aberration is structural chromosome damage expressed 
as breakage of single chromatids or breakage and reunion between 
chromatids.
    Chromosome-type aberration is structural chromosome damage expressed 
as breakage, or breakage and reunion, of both chromatids at an identical 
site.
    Endoreduplication is a process in which after an S period of DNA 
replication, the nucleus does not go into mitosis but starts another S 
period. The result is chromosomes with 4, 8, 16,...chromatids.
    Gap is an achromatic lesion smaller than the width of one chromatid, 
and with minimum misalignment of the chromatid(s).
    Mitotic index is the ratio of cells in metaphase divided by the 
total number of cells observed in a population of cells; an indication 
of the degree of proliferation of that population.
    Numerical aberration is a change in the number of chromosomes from 
the normal number characteristic of the cells utilized.
    Polyploidy is a multiple of the haploid chromosome number (n) other 
than the diploid number (i.e., 3n, 4n, and so on).
    Structural aberration is a change in chromosome structure detectable 
by microscopic examination of the metaphase stage of cell division, 
observed as deletions and fragments, intrachanges, and interchanges.
    (d) Initial considerations. (1) Tests conducted in vitro generally 
require the use of an exogenous source of metabolic activation. This 
metabolic activation system cannot mimic entirely the mammalian in vivo 
conditions. Care should be taken to avoid conditions which would lead to 
positive results which do not reflect intrinsic mutagenicity and may 
arise from changes in pH, osmolality, or high levels of cytotoxicity 
(the test techniques described in the references under paragraphs (i)(4) 
and (i)(5) of this section may be used).
    (2) This test is used to screen for possible mammalian mutagens and 
carcinogens. Many compounds that are positive in this test are mammalian 
carcinogens; however, there is not a perfect correlation between this 
test and carcinogenicity. Correlation is dependent on chemical class and 
there is increasing evidence that there are carcinogens that are not 
detected by this test because they appear to act through mechanisms 
other than direct DNA damage.
    (e) Principle of the test method. Cell cultures are exposed to the 
test substance both with and without metabolic activation. At 
predetermined intervals after exposure of cell cultures

[[Page 431]]

to the test substance, they are treated with a metaphase-arresting 
substance (e.g., Colcemid [reg] or colchicine), harvested, 
stained, and metaphase cells are analysed microscopically for the 
presence of chromosome aberrations.
    (f) Description of the method--(1) Preparations--(i) Cells. A 
variety of cell lines, strains, or primary cell cultures, including 
human cells, may be used (e.g., Chinese hamster fibroblasts, human, or 
other mammalian peripheral blood lymphocytes).
    (ii) Media and culture conditions. Appropriate culture media, and 
incubation conditions (culture vessels, CO2 concentration, temperature 
and humidity) must be used in maintaining cultures. Established cell 
lines and strains must be checked routinely for stability in the modal 
chromosome number and the absence of Mycoplasma contamination and should 
not be used if contaminated. The normal cell-cycle time for the cells 
and culture conditions used should be known.
    (iii) Preparation of cultures--(A) Established cell lines and 
strains. Cells are propagated from stock cultures, seeded in culture 
medium at a density such that the cultures will not reach confluency 
before the time of harvest, and incubated at 37 [deg]C.
    (B) Lymphocytes. Whole blood treated with an anti-coagulant (e.g., 
heparin) or separated lymphocytes obtained from healthy subjects are 
added to culture medium containing a mitogen (e.g., phytohemagglutinin) 
and incubated at 37 [deg]C.
    (iv) Metabolic activation. Cells must be exposed to the test 
substance both in the presence and absence of an appropriate metabolic 
activation system. The most commonly used system is a co-factor-
supplemented post-mitochondrial fraction (S9) prepared from the livers 
of rodents treated with enzyme-inducing agents such as Aroclor 1254 (the 
test techniques described in the references under paragraphs (i)(6), 
(i)(7), (8)(i), and (i)(9) of this section may be used), or a mixture of 
phenobarbitone and [beta]-naphthoflavone (the test techniques described 
in the references under paragraphs (i)(10), (i)(11), and (i)(12) of this 
section may be used). The post-mitochondrial fraction is usually used at 
concentrations in the range from 1-10% v/v in the final test medium. The 
condition of a metabolic activation system may depend upon the class of 
chemical being tested. In some cases, it may be appropriate to utilize 
more than one concentration of post-mitochondrial fraction. A number of 
developments, including the construction of genetically engineered cell 
lines expressing specific activating enzymes, may provide the potential 
for endogenous activation. The choice of the cell lines used should be 
scientifically justified (e.g., by the relevance of the cytochrome P450 
isoenzyme for the metabolism of the test substance).
    (v) Test substance/preparation. Solid test substances should be 
dissolved or suspended in appropriate solvents or vehicles and diluted, 
if appropriate, prior to treatment of the cells. Liquid test substances 
may be added directly to the test systems and/or diluted prior to 
treatment. Fresh preparations of the test substance should be employed 
unless stability data demonstrate the acceptability of storage.
    (2) Test conditions--(i) Solvent/vehicle. The solvent/vehicle should 
not be suspected of chemical reaction with the test substance and must 
be compatible with the survival of the cells and the S9 activity. If 
other than well-known solvent/vehicles are used, their inclusion should 
be supported by data indicating their compatibility. It is recommended 
that wherever possible, the use of an aqueous solvent/vehicle be 
considered first. When testing water-unstable substances, the organic 
solvents used should be free of water. Water can be removed by adding a 
molecular sieve.
    (ii) Exposure concentrations. (A) Among the criteria to be 
considered when determining the highest concentration are cytotoxicity, 
solubility in the test system, and changes in pH or osmolality.
    (B) Cytotoxicity should be determined with and without metabolic 
activation in the main experiment using an appropriate indication of 
cell integrity and growth, such as degree of confluency, viable cell 
counts, or mitotic index. It may be useful to determine cytotoxicity and 
solubility in a preliminary experiment.

[[Page 432]]

    (C) At least three analyzable concentrations should be used. Where 
cytotoxicity occurs, these concentrations should cover a range from the 
maximum to little or no toxicity; this will usually mean that the 
concentrations should be separated by no more than a factor between 2 
and [radic]10. At the time of harvesting, the highest concentration 
should show a significant reduction in degree of confluency, cell count 
or mitotic index, (all greater than 50%). The mitotic index is only an 
indirect measure of cytotoxic/cytostatic effects and depends on the time 
after treatment. However, the mitotic index is acceptable for suspension 
cultures in which other toxicity measurements may be cumbersome and 
impractical. Information on cell-cycle kinetics, such as average 
generation time (AGT), could be used as supplementary information. AGT, 
however, is an overall average that does not always reveal the existence 
of delayed subpopulations, and even slight increases in average 
generation time can be associated with very substantial delay in the 
time of optimal yield of aberrations. For relatively non-cytotoxic 
compounds the maximum concentration should be 5 [micro]g/ml, 5mg/ml, or 
0.01M, whichever is the lowest.
    (D) For relatively insoluble substances that are not toxic at 
concentrations lower than the insoluble concentration, the highest dose 
used should be a concentration above the limit of solubility in the 
final culture medium at the end of the treatment period. In some cases 
(e.g., when toxicity occurs only at higher than the lowest insoluble 
concentration) it is advisable to test at more than one concentration 
with visible precipitation. It may be useful to assess solubility at the 
beginning and the end of the treatment, as solubility can change during 
the course of exposure in the test system due to presence of cells, S9, 
serum etc. Insolubility can be detected by using the unaided eye. The 
precipitate should not interfere with the scoring.
    (iii) Controls. (A) Concurrent positive and negative (solvent or 
vehicle) controls both with and without metabolic activation must be 
included in each experiment. When metabolic activation is used, the 
positive control chemical must be the one that requires activation to 
give a mutagenic response.
    (B) Positive controls must employ a known clastogen at exposure 
levels expected to give a reproducible and detectable increase over 
background which demonstrates the sensitivity of the test system. 
Positive control concentrations should be chosen so that the effects are 
clear but do not immediately reveal the identity of the coded slides to 
the reader. Examples of positive-control substances include:

------------------------------------------------------------------------
 Metabolic activation condition        Chemical           CAS number
------------------------------------------------------------------------
Absence of exogenous metabolic    Methyl              [66-27-3]
 activation.                       methanesulfonate.
                                  Ethyl               [62-50-0]
                                   methanesulfonate.
                                  Ethylnitrosourea..  [759-73-9]
                                  Mitomycin C.......  [50-07-7]
                                  4-Nitroquinoline-N- [56-57-5]
                                   Oxide.
Presence of exogenous metabolic   Benzo(a)pyrene....  [50-32-8]
 activation.
                                  Cyclophosphamide..  [50-18-0]
                                  (monohydrate).....  ([6055-19-2])
------------------------------------------------------------------------

    (C) Other appropriate positive control substances may be used. The 
use of chemical class-related positive-control chemicals may be 
considered, when available.
    (D) Negative controls, consisting of solvent or vehicle alone in the 
treatment medium, and treated in the same way as the treatment cultures, 
must be included for every harvest time. In addition, untreated controls 
should also be used unless there are historical-control data 
demonstrating that no deleterious or mutagenic effects are induced by 
the chosen solvent.
    (g) Procedure--(1) Treatment with test substance. (i) Proliferating 
cells are treated with the test substance in the presence and absence of 
a metabolic-activation system. Treatment of lymphocytes should commence 
at about 48 hours after mitogenic stimulation.
    (ii) Duplicate cultures must be used at each concentration, and are 
strongly recommended for negative/solvent control cultures. Where 
minimal variation between duplicate cultures can be demonstrated (the 
test techniques described in the references under paragraphs (i)(13) and 
(i)(14) of this section

[[Page 433]]

may be used), from historical data, it may be acceptable for single 
cultures to be used at each concentration.
    (iii) Gaseous or volatile substances should be tested by appropriate 
methods, such as in sealed culture vessels (the test techniques 
described in the references under paragraphs (i)(15) and (i)(16) of this 
section may be used).
    (2) Culture harvest time. In the first experiment, cells should be 
exposed to the test substance both with and without metabolic activation 
for 3-6 hours, and sampled at a time equivalent to about 1.5 normal 
cell-cycle length after the beginning of treatment (the test techniques 
described in the references under paragraph (i)(12) of this section may 
be used). If this protocol gives negative results both with and without 
activation, an additional experiment without activation should be done, 
with continuous treatment until sampling at a time equivalent to about 
1.5 normal cell-cycle lengths. Certain chemicals may be more readily 
detected by treatment/sampling times longer than 1.5 cycle lengths. 
Negative results with metabolic activation need to be confirmed on a 
case-by-case basis. In those cases where confirmation of negative 
results is not considered necessary, justification should be provided.
    (3) Chromosome preparation. Cell cultures must be treated with 
Colcemid [reg] or colchicine usually for 1 to 3 hours prior 
to harvesting. Each cell culture must be harvested and processed 
separately for the preparation of chromosomes. Chromosome preparation 
involves hypotonic treatment of the cells, fixation and staining.
    (4) Analysis. (i) All slides, including those of positive and 
negative controls, must be independently coded before microscopic 
analysis. Since fixation procedures often result in the breakage of a 
proportion of metaphase cells with loss of chromosomes, the cells scored 
must therefore contain a number of centromeres equal to the modal number 
2 for all cell types. At least 200 well-spread 
metaphases should be scored per concentration and control equally 
divided amongst the duplicates, if applicable. This number can be 
reduced when high numbers of aberrations are observed.
    (ii) Though the purpose of the test is to detect structural 
chromosome aberrations, it is important to record polyploidy and 
endoreduplication when these events are seen.
    (h) Data and reporting--(1) Treatment of results. (i) The 
experimental unit is the cell, and therefore the percentage of cells 
with structural chromosome aberration(s) should be evaluated. Different 
types of structural chromosome aberrations must be listed with their 
numbers and frequencies for experimental and control cultures. Gaps are 
recorded separately and reported but generally not included in the total 
aberration frequency.
    (ii) Concurrent measures of cytotoxicity for all treated and 
negative control cultures in the main aberration experiment(s) should 
also be recorded.
    (iii) Individual culture data should be provided. Additionally, all 
data should be summarized in tabular form.
    (iv) There is no requirement for verification of a clear positive 
response. Equivocal results should be clarified by further testing 
preferably using modification of experimental conditions. The need to 
confirm negative results has been discussed in paragraph (g)(2) of this 
section. Modification of study parameters to extend the range of 
conditions assessed should be considered in follow-up experiments. Study 
parameters that might be modified include the concentration spacing and 
the metabolic activation conditions.
    (2) Evaluation and interpretation of results. (i) There are several 
criteria for determining a positive result, such as a concentration-
related increase or a reproducible increase in the number of cells with 
chromosome aberrations. Biological relevance of the results should be 
considered first. Statistical methods may be used as an aid in 
evaluating the test results (see paragraphs (i)(3) and (i)(13) of this 
section). Statistical significance should not be the only determining 
factor for a positive response.
    (ii) An increase in the number of polyploid cells may indicate that 
the test substance has the potential to inhibit mitotic processes and to 
induce numerical chromosome aberrations. An increase in the number of 
cells with

[[Page 434]]

endoreduplicated chromosomes may indicate that the test substance has 
the potential to inhibit cell-cycle progression (the test techniques 
described in the references under paragraphs (i)(17) and (i)(18) of this 
section may be used).
    (iii) A test substance for which the results do not meet the 
criteria in paragraphs (h)(2)(i) and (h)(2)(ii) of this section is 
considered nonmutagenic in this system.
    (iv) Although most experiments will give clearly positive or 
negative results, in rare cases the data set will preclude making a 
definite judgement about the activity of the test substance. Results may 
remain equivocal or questionable regardless of the number of times the 
experiment is repeated.
    (v) Positive results from the in vitro chromosome aberration test 
indicate that the test substance induces structural chromosome 
aberrations in cultured mammalian somatic cells. Negative results 
indicate that, under the test conditions, the test substance does not 
induce chromosome aberrations in cultured mammalian somatic cells.
    (3) Test report. The test report must include the following 
information.
    (i) Test substance.
    (A) Identification data and CAS no., if known.
    (B) Physical nature and purity.
    (C) Physicochemical properties relevant to the conduct of the study.
    (D) Stability of the test substance, if known.
    (ii) Solvent/vehicle.
    (A) Justification for choice of solvent/vehicle.
    (B) Solubility and stability of the test substance in solvent/
vehicle, if known.
    (iii) Cells.
    (A) Type and source of cells.
    (B) Karyotype features and suitability of the cell type used.
    (C) Absence of Mycoplasma, if applicable.
    (D) Information on cell-cycle length.
    (E) Sex of blood donors, whole blood or separated lymphocytes, 
mitogen used.
    (F) Number of passages, if applicable.
    (G) Methods for maintenance of cell cultures if applicable.
    (H) Modal number of chromosomes.
    (iv) Test conditions.
    (A) Identity of metaphase arresting substance, its concentration and 
duration of cell exposure.
    (B) Rationale for selection of concentrations and number of cultures 
including, e.g., cytotoxicity data and solubility limitations, if 
available.
    (C) Composition of media, CO2 concentration if applicable.
    (D) Concentration of test substance.
    (E) Volume of vehicle and test substance added.
    (F) Incubation temperature.
    (G) Incubation time.
    (H) Duration of treatment.
    (I) Cell density at seeding, if appropriate.
    (J) Type and composition of metabolic activation system, including 
acceptability criteria.
    (K) Positive and negative controls.
    (L) Methods of slide preparation.
    (M) Criteria for scoring aberrations.
    (N) Number of metaphases analyzed.
    (O) Methods for the measurements of toxicity.
    (P) Criteria for considering studies as positive, negative or 
equivocal.
    (v) Results.
    (A) Signs of toxicity, e.g., degree of confluency, cell-cycle data, 
cell counts, mitotic index.
    (B) Signs of precipitation.
    (C) Data on pH and osmolality of the treatment medium, if 
determined.
    (D) Definition for aberrations, including gaps.
    (E) Number of cells with chromosome aberrations and type of 
chromosome aberrations given separately for each treated and control 
culture.
    (F) Changes in ploidy if seen.
    (G) Dose-response relationship, where possible.
    (H) Statistical analyses, if any.
    (I) Concurrent negative (solvent/vehicle) and positive control data.
    (J) Historical negative (solvent/vehicle) and positive control data, 
with ranges, means and standard deviations.
    (vi) Discussion of the results.
    (vii) Conclusion.
    (i) References. For additional background information on this test 
guideline, the following references should be consulte. These references 
are available for inspection at the TSCA Nonconfidential Information 
Center, Rm.

[[Page 435]]

NE-B607, Environmental Protection Agency, 401 M St., SW., Washington, 
DC, 12 noon to 4 p.m., Monday through Friday, except legal holidays.
    (1) Evans, H.J. Cytological Methods for Detecting Chemical Mutagens. 
Chemical Mutagens, Principles and Methods for their Detection, Vol. 4, 
Hollaender, A. Ed. Plenum Press, New York and London, pp. 1-29 (1976).
    (2) Ishidate, M. Jr. and Sofuni, T. The In Vitro Chromosomal 
Aberration Test Using Chinese Hamster Lung (CHL) Fibroblast Cells in 
Culture. Progress in Mutation Research, Vol. 5, Ashby, J. et al., Eds. 
Elsevier Science Publishers, Amsterdam-New York-Oxford, pp. 427-432 
(1985).
    (3) Galloway, S.M. et al. Chromosome aberration and sister chromatid 
exchanges in Chinese hamster ovary cells: Evaluation of 108 chemicals. 
Environmental and Molecular Mutagenesis 10 (suppl. 10), 1-175 (1987).
    (4) Scott, D. et al. Genotoxicity under Extreme Culture Conditions. 
A report from ICPEMC Task Group 9. Mutation Research 257, 147-204 
(1991).
    (5) Morita, T. et al. Clastogenicity of Low pH toVarious Cultured 
Mammalian Cells. Mutation Research 268, 297-305 (1992).
    (6) Ames, B.N., McCann, J. and Yamasaki, E. Methods for Detecting 
Carcinogens and Mutagens with the Salmonella/Mammalian Microsome 
Mutagenicity Test. Mutation Research 31, 347-364 (1975).
    (7) Maron, D.M. and Ames, B.N. Revised Methods for the Salmonella 
Mutagenicity Test. Mutation Research 113, 173-215 (1983).
    (8) Natarajan, A.T. et al. Cytogenetic Effects of Mutagens/
Carcinogens after Activation in a Microsomal System In Vitro, I. 
Induction of Chromosome Aberrations and Sister Chromatid Exchanges by 
Diethylnitrosamine (DEN) and Dimethylnitrosamine (DMN) in CHO Cells in 
the Presence of Rat-Liver Microsomes. Mutation Research 37, 83-90 
(1976).
    (9) Matsuoka, A., Hayashi, M. and Ishidate, M., Jr. Chromosomal 
Aberration Tests on 29 Chemicals Combined with S9 Mix In Vitro. Mutation 
Research 66, 277-290 (1979).
    (10) Elliot, B.M. et al. Report of UK Environmental Mutagen Society 
Working Party. Alternatives to Aroclor 1254-induced S9 in In Vitro 
Genotoxicity Assays. Mutagenesis 7, 175-177 (1992).
    (11) Matsushima, T. et al. A Safe Substitute for Polychlorinated 
Biphenyls as an Inducer of Metabolic Activation Systems. de Serres, 
F.J., Fouts, J.R., Bend, J.R. and Philpot, R.M. Eds. In Vitro Metabolic 
Activation in Mutagenesis Testing, Elsevier, North-Holland, pp. 85-88 
(1976).
    (12) Galloway, S.M. et al. Report from Working Group on In Vitro 
Tests for Chromosomal Aberrations. Mutation Research 312, 241-261 
(1994).
    (13) Richardson, C. et al. Analysis of Data from In Vitro 
Cytogenetic Assays. Statistical Evaluation of Mutagenicity Test Data. 
Kirkland, D.J., Ed. Cambridge University Press, Cambridge, pp. 141-154 
(1989).
    (14) Soper, K.A. and Galloway S.M. Replicate Flasks are not 
Necessary for In Vitro Chromosome Aberration Assays in CHO Cells. 
Mutation Research 312, 139-149 (1994).
    (15) Krahn, D.F., Barsky, F.C. and McCooey, K.T. CHO/HGPRT Mutation 
Assay: Evaluation of Gases and Volatile Liquids. Tice, R.R., Costa, 
D.L., Schaich, K.M. Eds. Genotoxic Effects of Airborne Agents. New York, 
Plenum, pp. 91-103 (1982).
    (16) Zamora, P.O. et al. Evaluation of an Exposure System Using 
Cells Grown on Collagen Gels for Detecting Highly Volatile Mutagens in 
the CHO/HGPRT Mutation Assay. Environmental Mutagenesis 5, 795-801 
(1983).
    (17) Locke-Huhle, C. Endoreduplication in Chinese hamster cells 
during alpha-radiation induced G2 arrest. Mutation Research 119, 403-413 
(1983).
    (18) Huang, Y., Change, C. and Trosko, J.E. Aphidicolin--induced 
endoreduplication in Chinese hamster cells. Cancer Research 43, 1362-
1364 (1983).

[65 FR 78807, Dec. 15, 2000]



Sec. 799.9538  TSCA mammalian bone marrow chromosomal aberration test.

    (a) Scope. This section is intended to meet the testing requirements 
under section 4 of TSCA. The mammalian bone marrow chromosomal 
aberration test is used for the detection of structural chromosome 
aberrations induced

[[Page 436]]

by test compounds in bone marrow cells of animals, usually rodents. 
Structural chromosome aberrations may be of two types, chromosome or 
chromatid. An increase in polyploidy may indicate that a chemical has 
the potential to induce numerical aberrations. With the majority of 
chemical mutagens, induced aberrations are of the chromatid-type, but 
chromosome-type aberrations also occur. Chromosome mutations and related 
events are the cause of many human genetic diseases and there is 
substantial evidence that chromosome mutations and related events 
causing alterations in oncogenes and tumor suppressor genes are involved 
in cancer in humans and experimental systems.
    (b) Source. The source material used in developing this TSCA test 
guideline is the OECD guideline 475 (February 1997). This source is 
available at the address in paragraph (g) of this section.
    (c) Definitions. The following definitions apply to this section:
    Chromatid-type aberration is structural chromosome damage expressed 
as breakage of single chromatids or breakage and reunion between 
chromatids.
    Chromosome-type aberration is structural chromosome damage expressed 
as breakage, or breakage and reunion, of both chromatids at an identical 
site.
    Endoreduplication is a process in which after an S period of DNA 
replication, the nucleus does not go into mitosis but starts another S 
period. The result is chromosomes with 2,4,8,...chromatids.
    Gap is an achromatic lesion smaller than the width of one chromatid, 
and with minimum misalignment of the chromatids.
    Numerical aberration is a change in the number of chromosomes from 
the normal number characteristic of the animals utilized.
    Polyploidy is a multiple of the haploid chromosome number (n) other 
than the diploid number (i.e., 3n, 4n and so on).
    Structural aberration is a change in chromosome structure detectable 
by microscopic examination of the metaphase stage of cell division, 
observed as deletions and fragments, intrachanges or interchanges.
    (d) Initial considerations. (1) Rodents are routinely used in this 
test. Bone marrow is the target tissue in this test, since it is a 
highly vascularised tissue, and it contains a population of rapidly 
cycling cells that can be readily isolated and processed. Other species 
and target tissues are not the subject of this section.
    (2) This chromosome aberration test is especially relevant to 
assessing mutagenic hazard in that it allows consideration of factors of 
in vivo metabolism, pharmacokinetics and DNA-repair processes although 
these may vary among species and among tissues. An in vivo test is also 
useful for further investigation of a mutagenic effect detected by an in 
vitro test.
    (3) If there is evidence that the test substance, or a reactive 
metabolite, will not reach the target tissue, it is not appropriate to 
use this test.
    (e) Test method--(1) Principle. Animals are exposed to the test 
substance by an appropriate route of exposure and are sacrificed at 
appropriate times after treatment. Prior to sacrifice, animals are 
treated with a metaphase-arresting agent (e.g., colchicine or Colcemid 
[reg]). Chromosome preparations are then made from the bone 
marrow cells and stained, and metaphase cells are analyzed for 
chromosome aberrations.
    (2) Description--(i) Preparations--(A) Selection of animal species. 
Rats, mice and Chinese hamsters are commonly used, although any 
appropriate mammalian species may be used. Commonly used laboratory 
strains of young healthy adult animals should be employed. At the 
commencement of the study, the weight variation of animals should be 
minimal and not exceed 20% of the mean weight of 
each sex.
    (B) Housing and feeding conditions. The temperature in the 
experimental animal room should be 22 [deg]C 3 
[deg]C). Although the relative humidity should be at least 30% and 
preferably not exceed 70% other than during room cleaning, the aim 
should be 50-60%. Lighting should be artificial, the sequence being 12 
hrs light, 12 hrs dark. For feeding, conventional laboratory diets may 
be used with an unlimited supply of drinking water. The choice of diet 
may be influenced by the need to ensure a suitable admixture of a test 
substance

[[Page 437]]

when administered by this method. Animals may be housed individually, or 
be caged in small groups of the same sex.
    (C) Preparation of the animals. Healthy young adult animals shall be 
randomly assigned to the control and treatment groups. Cages should be 
arranged in such a way that possible effects due to cage placement are 
minimized. The animals are identified uniquely. The animals are 
acclimated to the laboratory conditions for at least 5 days.
    (D) Preparation of doses. Solid test substances shall be dissolved 
or suspended in appropriate solvents or vehicles and diluted, as 
appropriate, prior to dosing of the animals. Liquid test substances may 
be dosed directly or diluted prior to dosing. Fresh preparations of the 
test substance should be employed unless stability data demonstrate the 
acceptability of storage.
    (ii) Test conditions--(A) Solvent/vehicle. The solvent/vehicle shall 
not produce toxic effects at the dose levels used, and shall not be 
suspected of chemical reaction with the test substance. If other than 
well-known solvents/vehicles are used, their inclusion should be 
supported with data indicating their compatibility. It is recommended 
that wherever possible, the use of an aqueous solvent/vehicle should be 
considered first.
    (B) Controls. (1) Concurrent positive and negative (solvent/vehicle) 
controls shall be included for each sex in each test. Except for 
treatment with the test substance, animals in the control groups should 
be handled in an identical manner to the animals in the treated groups.
    (2) Positive controls shall produce structural chromosome 
aberrations in vivo at exposure levels expected to give a detectable 
increase over background. Positive control doses should be chosen so 
that the effects are clear but do not immediately reveal the identity of 
the coded slides to the reader. It is acceptable that the positive 
control be administered by a route different from the test substance and 
sampled at only a single time. The use of chemical class related 
positive control chemicals may be considered, when available. Examples 
of positive control substances include:

------------------------------------------------------------------------
                 Chemical                              CAS No.
------------------------------------------------------------------------
Triethylenemelamine.......................  [CAS no. 51-18-3]
Ethyl methanesulphonate...................  [CAS no. 62-50-0]
Ethyl nitrosourea.........................  [CAS no. 759-73-9]
Mitomycin C...............................  [CAS no. 50-07-7]
Cyclophosphamide (monohydrate)............  [CAS no. 50-18-0]
                                            [CAS no. 6055-19-2]
------------------------------------------------------------------------

    (3) Negative controls, treated with solvent or vehicle alone, and 
otherwise treated in the same way as the treatment groups, shall be 
included for every sampling time, unless acceptable inter-animal 
variability and frequencies of cells with chromosome aberrations are 
available from historical control data. If single sampling is applied 
for negative controls, the most appropriate time is the first sampling 
time. In the absence of historical or published control data 
demonstrating that no deleterious or mutagenic effects are induced by 
the chosen solvent/vehicle, untreated animals should be used.
    (3) Procedure--(i) Number and sex of animals. Each treated and 
control group shall include at least 5 analyzable animals per sex. If at 
the time of the study there are data available from studies in the same 
species and using the same route of exposure that demonstrate that there 
are no substantial differences in toxicity between sexes, then testing 
in a single sex will be sufficient. Where human exposure to chemicals 
may be sex-specific, as for example with some pharmaceutical agents, the 
test should be performed with animals of the appropriate sex.
    (ii) Treatment schedule. (A) Test substances are preferably 
administered as a single treatment. Test substances may also be 
administered as a split dose, i.e. two treatments on the same day 
separated by no more than a few hrs, to facilitate administering a large 
volume of material. Other dose regimens should be scientifically 
justified.
    (B) Samples shall be taken at two separate times following treatment 
on one day. For rodents, the first sampling interval is 1.5 normal cell 
cycle length (the latter being normally 12-18 hr) following treatment. 
Since the time required for uptake and metabolism of the test substance 
as well as its

[[Page 438]]

effect on cell cycle kinetics can affect the optimum time for chromosome 
aberration detection, a later sample collection 24 hr after the first 
sample time is recommended. If dose regimens of more than one day are 
used, one sampling time at 1.5 normal cell cycle lengths after the final 
treatment should be used.
    (C) Prior to sacrifice, animals shall be injected intraperitoneally 
with an appropriate dose of a metaphase arresting agent (e.g. Colcemid 
[reg] or colchicine). Animals are sampled at an appropriate 
interval thereafter. For mice this interval is approximately 3-5 hrs; 
for Chinese hamsters this interval is approximately 4-5 hrs. Cells shall 
be harvested from the bone marrow and analyzed from chromosome 
aberrations.
    (iii) Dose levels. If a range finding study is performed because 
there are no suitable data available, it shall be performed in the same 
laboratory, using the same species, strain, sex, and treatment regimen 
to be used in the main study (an approach to dose selection is presented 
in the reference under paragraph (g)(5) of this section). If there is 
toxicity, three dose levels shall be used for the first sampling time. 
These dose levels shall cover a range from the maximum to little or no 
toxicity. At the later sampling time only the highest dose needs to be 
used. The highest dose is defined as the dose producing signs of 
toxicity such that higher dose levels, based on the same dosing regimen, 
would be expected to produce lethality. Substances with specific 
biological activities at low non-toxic doses (such as hormones and 
mitogens) may be exceptions to the dose-setting criteria and should be 
evaluated on a case-by-case basis. The highest dose may also be defined 
as a dose that produces some indication of toxicity in the bone marrow 
(e.g. greater than 50% reduction in mitotic index).
    (iv) Limit test. If a test at one dose level of at least 2,000 mg/kg 
body weight using a single treatment, or as two treatments on the same 
day, produces no observable toxic effects, and if genotoxicity would not 
be expected based on data from structurally related compounds, then a 
full study using three dose levels may not be considered necessary. For 
studies of a longer duration, the limit dose is 2,000 mg/kg/body weight/
day for treatment up to 14 days, and 1,000 mg/kg/body weight/day for 
treatment longer than 14 days. Expected human exposure may indicate the 
need for a higher dose level to be used in the limit test.
    (v) Administration of doses. The test substance is usually 
administered by gavage using a stomach tube or a suitable intubation 
cannula, or by intraperitoneal injection. Other routes of exposure may 
be acceptable where they can be justified. The maximum volume of liquid 
that can be administered by gavage or injection at one time depends on 
the size of the test animal. The volume should not exceed 2 ml/100g body 
weight. The use of volumes higher than these must be justified. Except 
for irritating or corrosive substances which will normally reveal 
exacerbated effects with higher concentrations, variability in test 
volume should be minimized by adjusting the concentration to ensure a 
constant volume at all dose levels.
    (vi) Chromosome preparation. Immediately after sacrifice, bone 
marrow shall be obtained, exposed to hypotonic solution and fixed. The 
cells shall be then spread on slides and stained.
    (vii) Analysis. (A) The mitotic index should be determined as a 
measure of cytotoxicity in at least 1,000 cells per animal for all 
treated animals (including positive controls) and untreated negative 
control animals.
    (B) At least 100 cells should be analyzed for each animal. This 
number could be reduced when high numbers of aberrations are observed. 
All slides, including those of positive and negative controls, shall be 
independently coded before microscopic analysis. Since slide preparation 
procedures often result in the breakage of a proportion of metaphases 
with loss of chromosomes, the cells scored should therefore contain a 
number of centromeres equal to the number 2n 2.
    (f) Data and reporting--(1) Treatment of results. Individual animal 
data shall be presented in tabular form. The experimental unit is the 
animal. For each animal the number of cells scored, the number of 
aberrations per cell and the

[[Page 439]]

percentage of cells with structural chromosome aberration(s) shall be 
evaluated. Different types of structural chromosome aberrations shall be 
listed with their numbers and frequencies for treated and control 
groups. Gaps shall be recorded separately and reported but generally not 
included in the total aberration frequency. If there is no evidence for 
a difference in response between the sexes, the data may be combined for 
statistical analysis.
    (2) Evaluation and interpretation of results. (i) There are several 
criteria for determining a positive result, such as a dose-related 
increase in the relative number of cells with chromosome aberrations or 
a clear increase in the number of cells with aberrations in a single 
dose group at a single sampling time. Biological relevance of the 
results should be considered first. Statistical methods may be used as 
an aid in evaluating the test results (some statistical methods are 
described in the reference under paragraph (g)(6) of this section). 
Statistical significance should not be the only determining factor for a 
positive response. Equivocal results should be clarified by further 
testing preferably using a modification of experimental conditions.
    (ii) An increase in polyploidy may indicate that the test substance 
has the potential to induce numerical chromosome aberrations. An 
increase in endoreduplication may indicate that the test substance has 
the potential to inhibit cell cycle progression. This phenomenon is 
described in the references under paragraphs (g)(7) and (g)(8) of this 
section.
    (iii) A test substance for which the results do not meet the 
criteria described in paragraph (f)(2)(i) of this section is considered 
non-mutagenic in this test.
    (iv) Although most experiments will give clearly positive or 
negative results, in rare cases the data set will preclude making a 
definite judgment about the activity of the test substance. Results may 
remain equivocal or questionable regardless of the number of experiments 
performed.
    (v) Positive results from the in vivo chromosome aberration test 
indicate that a substance induces chromosome aberrations in the bone 
marrow of the species tested. Negative results indicate that, under the 
test conditions, the test substance does not induce chromosome 
aberrations in the bone marrow of the species tested.
    (vi) The likelihood that the test substance or its metabolites reach 
the general circulation or specifically the target tissue (e.g., 
systemic toxicity) should be discussed.
    (3) Test report. The test report shall include the following 
information:
    (i) Test substance:
    (A) Identification data and CAS No., if known.
    (B) Physical nature and purity.
    (C) Physicochemical properties relevant to the conduct of the study.
    (D) Stability of the test substance, if known.
    (ii) Solvent/vehicle:
    (A) Justification for choice of vehicle.
    (B) Solubility and stability of the test substance in solvent/
vehicle, if known.
    (iii) Test animals:
    (A) Species/strain used.
    (B) Number, age and sex of animals.
    (C) Source, housing conditions, diet, etc.
    (D) Individual weight of the animals at the start of the test, 
including body weight range, mean and standard deviation for each group.
    (iv) Test conditions:
    (A) Positive and negative (vehicle/solvent) controls.
    (B) Data from range-finding study, if conducted.
    (C) Rationale for dose level selection.
    (D) Details of test substance preparation.
    (E) Details of the administration of the test substance.
    (F) Rationale for route of administration.
    (G) Methods for verifying that the test substance reached the 
general circulation or target tissue, if applicable.
    (H) Conversion from diet/drinking water test substance concentration 
parts per million (ppm) to the actual dose (mg/kg body weight/day), if 
applicable.
    (I) Details of food and water quality.
    (J) Detailed description of treatment and sampling schedules.

[[Page 440]]

    (K) Methods for measurement of toxicity.
    (L) Identity of metaphase arresting substance, its concentration and 
duration of treatment.
    (M) Methods of slide preparation.
    (N) Criteria for scoring aberrations.
    (O) Number of cells analyzed per animal.
    (P) Criteria for considering studies as positive, negative or 
equivocal.
    (v) Results:
    (A) Signs of toxicity.
    (B) Mitotic index.
    (C) Type and number of aberrations, given separately for each 
animal.
    (D) Total number of aberrations per group with means and standard 
deviations.
    (E) Number of cells with aberrations per group with means and 
standard deviations.
    (F) Changes in ploidy, if seen.
    (G) Dose-response relationship, where possible.
    (H) Statistical analyses, if any.
    (I) Concurrent negative control data.
    (J) Historical negative control data with ranges, means and standard 
deviations.
    (K) Concurrent positive control data.
    (vi) Discussion of the results.
    (vii) Conclusion.
    (g) References. For additional background information on this test 
guideline, the following references should be consulted. These 
references are available for inspection at the TSCA Nonconfidential 
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M 
St., SW., Washington, DC, 12 noon to 4 p.m., Monday through Friday, 
except legal holidays.
    (1) Adler, I.D. Eds. S. Venitt and J.M. Parry. Cytogenetic Tests in 
Mammals. Mutagenicity Testing: A Practical Approach. (IRL Press, Oxford, 
Washington DC, 1984) pp. 275-306.
    (2) Preston, R.J., Dean, B.J., Galloway, S., Holden, H., McFee, 
A.F., and Shelby, M. Mammalian In Vivo Cytogenetic Assays: Analysis of 
Chromosome Aberrations in Bone Marrow Cells. Mutation Research. 189, 
157-165 (1987).
    (3) Richold, M., Chandley, A., Ashby, J., Gatehouse, D.G., Bootman, 
J., and Henderson, L. Ed. D.J. Kirkland. In Vivo Cytogenetic Assays. 
Basic Mutagenicity Tests, UKEMS Recommended Procedures. UKEMS 
Subcommittee on Guidelines for Mutagenicity Testing. Report. Part I 
revised. (Cambridge University Press, Cambridge, NY, Port Chester, 
Melbourne, Sydney, 1990) pp. 115-141.
    (4) Tice, R.R., Hayashi, M., MacGregor, J.T., Anderson, D., Blakey, 
D.H., Holden, H.E., Kirsch-Volders, M., Oleson Jr., F.B., Pacchierotti, 
F., Preston, R.J., Romagna, F., Shimada, H., Sutou, S., and Vannier, B. 
Report from the Working Group on the In Vivo Mammalian Bone Marrow 
Chromosomal Aberration Test. Mutation Research. 312, 305-312 (1994).
    (5) Fielder, R.J., Allen, J.A., Boobis, A.R., Botham, P.A., Doe, J., 
Esdaile, D.J., Gatehouse, D.G., Hodson-Walker, G., Morton, D.B., 
Kirkland, D. J., and Richold, M. Report of British Toxicology Society/UK 
Environmental Mutagen Society Working Group: Dose Setting in In Vivo 
Mutagenicity Assays. Mutagenesis. 7, 313-319 (1992).
    (6) Lovell, D.P., Anderson, D., Albanese, R., Amphlett, G.E., Clare, 
G., Ferguson, R., Richold, M., Papworth, D.G., and Savage, J.R.K. Ed. 
Kirkland,D. J. Statistical Analysis of In Vivo Cytogenetic Assays. UKEMS 
Sub-Committee on Guidelines for Mutagenicity Testing. Report Part III. 
Statistical Evaluation of Mutagenicity Test Data (Cambridge University 
Press, Cambridge, 1989) pp. 184-232.
    (7) Locke-Huhle, C. Endoreduplication in Chinese Hamster Cells 
During Alpha-Radiation Induced G2 Arrest. Mutation Research. 119, 403-
413 (1983).
    (8) Huang, Y., Change, C., and Trosko, J. E. Aphidicolin-Induced 
Endoreduplication in Chinese Hamster Cells. Cancer Research. 43, 1362-
1364 (1983).

[62 FR 43824, Aug. 15, 1997, as amended at 64 FR 35079, June 30, 1999]



Sec. 799.9539  TSCA mammalian erythrocyte micronucleus test.

    (a) Scope. This section is intended to meet the testing requirements 
under section 4 of TSCA.

[[Page 441]]

    (1) The mammalian erythrocyte micronucleus test is used for the 
detection of damage induced by the test substance to the chromosomes or 
the mitotic apparatus of erythroblasts by analysis of erythrocytes as 
sampled in bone marrow and/or peripheral blood cells of animals, usually 
rodents.
    (2) The purpose of the micronucleus test is to identify substances 
that cause cytogenetic damage which results in the formation of 
micronuclei containing lagging chromosome fragments or whole 
chromosomes.
    (3) When a bone marrow erythroblast develops into a polychromatic 
erythrocyte, the main nucleus is extruded; any micronucleus that has 
been formed may remain behind in the otherwise anucleated cytoplasm. 
Visualization of micronuclei is facilitated in these cells because they 
lack a main nucleus. An increase in the frequency of micronucleated 
polychromatic erythrocytes in treated animals is an indication of 
induced chromosome damage.
    (b) Source. The source material used in developing this TSCA test 
guideline is the OECD guideline 474 (February 1997). This source is 
available at the address in paragraph (g) of this section.
    (c) Definitions. The following definitions apply to this section:
    Centromere (kinetochore) is a region of a chromosome with which 
spindle fibers are associated during cell division, allowing orderly 
movement of daughter chromosomes to the poles of the daughter cells.
    Micronuclei are small nuclei, separate from and additional to the 
main nuclei of cells, produced during telophase of mitosis (meiosis) by 
lagging chromosome fragments or whole chromosomes.
    Normochromatic erythrocyte is a mature erythrocyte that lacks 
ribosomes and can be distinguished from immature, polychromatic 
erythrocytes by stains selective for ribosomes.
    Polychromatic erythrocyte is an immature erythrocyte, in an 
intermediate stage of development, that still contains ribosomes and 
therefore can be distinguished from mature, normochromatic erythrocytes 
by stains selective for ribosomes.
    (d) Initial considerations. (1) The bone marrow of rodents is 
routinely used in this test since polychromatic erythrocytes are 
produced in that tissue. The measurement of micronucleated immature 
(polychromatic) erythrocytes in peripheral blood is equally acceptable 
in any species in which the inability of the spleen to remove 
micronucleated erythrocytes has been demonstrated, or which has shown an 
adequate sensitivity to detect agents that cause structural or numerical 
chromosome aberrations. Micronuclei can be distinguished by a number of 
criteria. These include identification of the presence or absence of a 
kinetochore or centromeric DNA in the micronuclei. The frequency of 
micronucleated immature (polychromatic) erythrocytes is the principal 
endpoint. The number of mature (normochromatic) erythrocytes in the 
peripheral blood that contain micronuclei among a given number of mature 
erythrocytes can also be used as the endpoint of the assay when animals 
are treated continuously for 4 weeks or more. This mammalian in vivo 
micronucleus test is especially relevant to assessing mutagenic hazard 
in that it allows consideration of factors of in vivo metabolism, 
pharmacokinetics and DNA-repair processes although these may vary among 
species, among tissues and among genetic endpoints. An in vivo assay is 
also useful for further investigation of a mutagenic effect detected by 
an in vitro system.
    (2) If there is evidence that the test substance, or a reactive 
metabolite, will not reach the target tissue, it is not appropriate to 
use this test.
    (e) Test method--(1) Principle. Animals are exposed to the test 
substance by an appropriate route. If bone marrow is used, the animals 
are sacrificed at appropriate times after treatment, the bone marrow 
extracted, and preparations made and stained (test techniques described 
in the references under paragraphs (g)(1), (g)(2), and (g)(3) of this 
section may be used). When peripheral blood is used, the blood is 
collected at appropriate times after treatment and smear preparations 
are made and stained (the test techniques described in the references

[[Page 442]]

under paragraphs (g)(3), (g)(4), (g)(5), and (g)(6) of this section may 
be used). For studies with peripheral blood, as little time as possible 
should elapse between the last exposure and cell harvest. Preparations 
are analyzed for the presence of micronuclei.
    (2) Description--(i) Preparations--(A) Selection of animal species. 
Mice or rats are recommended if bone marrow is used, although any 
appropriate mammalian species may be used. When peripheral blood is 
used, mice are recommended. However, any appropriate mammalian species 
may be used provided it is a species in which the spleen does not remove 
micronucleated erythrocytes or a species which has shown an adequate 
sensitivity to detect agents that cause structural or numerical 
chromosome aberrations. Commonly used laboratory strains of young 
healthy animals should be employed. At the commencement of the study, 
the weight variation of animals should be minimal and not exceed 20% of the mean weight of each sex.
    (B) Housing and feeding conditions. The temperature in the 
experimental animal room should be 22 [deg]C 3 
[deg]C). Although the relative humidity should be at least 30% and 
preferably not exceed 70% other than during room cleaning, the aim 
should be 50-60%. Lighting should be artificial, the sequence being 12 
hrs light, 12 hrs dark. For feeding, conventional laboratory diets may 
be used with an unlimited supply of drinking water. The choice of diet 
may be influenced by the need to ensure a suitable admixture of a test 
substance when administered by this route. Animals may be housed 
individually, or caged in small groups of the same sex.
    (C) Preparation of the animals. Healthy young adult animals shall be 
randomly assigned to the control and treatment groups. The animals are 
identified uniquely. The animals are acclimated to the laboratory 
conditions for at least 5 days. Cages should be arranged in such a way 
that possible effects due to cage placement are minimized.
    (D) Preparation of doses. Solid test substances shall be dissolved 
or suspended in appropriate solvents or vehicles and diluted, if 
appropriate, prior to dosing of the animals. Liquid test substances may 
be dosed directly or diluted prior to dosing. Fresh preparations of the 
test substance should be employed unless stability data demonstrate the 
acceptability of storage.
    (ii) Test conditions--(A) Solvent/vehicle. The solvent/vehicle shall 
not produce toxic effects at the dose levels used, and shall not be 
suspected of chemical reaction with the test substance. If other than 
well-known solvents/vehicles are used, their inclusion should be 
supported with reference data indicating their compatibility. It is 
recommended that wherever possible, the use of an aqueous solvent/
vehicle should be considered first.
    (B) Controls. (1) Concurrent positive and negative (solvent/vehicle) 
controls shall be included for each sex in each test. Except for 
treatment with the test substance, animals in the control groups should 
be handled in an identical manner to animals of the treatment groups.
    (2) Positive controls shall produce micronuclei in vivo at exposure 
levels expected to give a detectable increase over background. Positive 
control doses should be chosen so that the effects are clear but do not 
immediately reveal the identity of the coded slides to the reader. It is 
acceptable that the positive control be administered by a route 
different from the test substance and sampled at only a single time. In 
addition, the use of chemical class-related positive control chemicals 
may be considered, when available. Examples of positive control 
substances include:

------------------------------------------------------------------------
                 Chemical                              CAS No.
------------------------------------------------------------------------
Ethyl methanesulphonate...................  [CAS no. 62-50-0]
Ethyl nitrosourea.........................  [CAS no. 759-73-9]
Mitomycin C...............................  [CAS no. 50-07-7]
Cyclophosphamide (monohydrate)............  [CAS no. 50-18-0]
                                            [CAS no. 6055-19-2]
Triethylenemelamine.......................  [CAS no. 51-18-3]
------------------------------------------------------------------------

    (3) Negative controls, treated with solvent or vehicle alone, and 
otherwise treated in the same way as the treatment groups shall be 
included for every sampling time, unless acceptable inter-animal 
variability and frequencies of cells with micronuclei are demonstrated 
by historical control data. If single sampling is applied for negative 
controls, the most appropriate time is

[[Page 443]]

the first sampling time. In addition, untreated controls should also be 
used unless there are historical or published control data demonstrating 
that no deleterious or mutagenic effects are induced by the chosen 
solvent/vehicle.
    (4) If peripheral blood is used, a pre-treatment sample may also be 
acceptable as a concurrent negative control, but only in the short 
peripheral blood studies (e.g., one to three treatment(s)) when the 
resulting data are in the expected range for the historical control.
    (3) Procedure--(i) Number and sex of animals. Each treated and 
control group shall include at least 5 analyzable animals per sex 
(techniques described in the reference under paragraph (g)(7) of this 
section may be used). If at the time of the study there are data 
available from studies in the same species and using the same route of 
exposure that demonstrate that there are no substantial differences 
between sexes in toxicity, then testing in a single sex will be 
sufficient. Where human exposure to chemicals may be sex-specific, as 
for example with some pharmaceutical agents, the test should be 
performed with animals of the appropriate sex.
    (ii) Treatment schedule. (A) No standard treatment schedule (i.e. 
one, two, or more treatments at 24 h intervals) can be recommended. The 
samples from extended dose regimens are acceptable as long as a positive 
effect has been demonstrated for this study or, for a negative study, as 
long as toxicity has been demonstrated or the limit dose has been used, 
and dosing continued until the time of sampling. Test substances may 
also be administered as a split dose, i.e., two treatments on the same 
day separated by no more than a few hrs, to facilitate administering a 
large volume of material.
    (B) The test may be performed in two ways:
    (1) Animals shall be treated with the test substance once. Samples 
of bone marrow shall be taken at least twice, starting not earlier than 
24 hrs after treatment, but not extending beyond 48 hrs after treatment 
with appropriate interval(s) between samples. The use of sampling times 
earlier than 24 hrs after treatment should be justified. Samples of 
peripheral blood shall be taken at least twice, starting not earlier 
than 36 hrs after treatment, with appropriate intervals following the 
first sample, but not extending beyond 72 hrs. When a positive response 
is recognized at one sampling time, additional sampling is not required.
    (2) If two or more daily treatments are used (e.g. two or more 
treatments at 24 hr intervals), samples shall be collected once between 
18 and 24 hrs following the final treatment for the bone marrow and once 
between 36 and 48 hrs following the final treatment for the peripheral 
blood (techniques described in the reference under paragraph (g)(8) of 
this section may be used).
    (C) Other sampling times may be used in addition, when relevant.
    (iii) Dose levels. If a range finding study is performed because 
there are no suitable data available, it shall be performed in the same 
laboratory, using the same species, strain, sex, and treatment regimen 
to be used in the main study (guidance on dose setting is provided in 
the reference in paragraph (g)(9) of this section). If there is 
toxicity, three dose levels shall be used for the first sampling time. 
These dose levels shall cover a range from the maximum to little or no 
toxicity. At the later sampling time only the highest dose needs to be 
used. The highest dose is defined as the dose producing signs of 
toxicity such that higher dose levels, based on the same dosing regimen, 
would be expected to produce lethality. Substances with specific 
biological activities at low non-toxic doses (such as hormones and 
mitogens) may be exceptions to the dose-setting criteria and should be 
evaluated on a case-by-case basis. The highest dose may also be defined 
as a dose that produces some indication of toxicity in the bone marrow 
(e.g. a reduction in the proportion of immature erythrocytes among total 
erythrocytes in the bone marrow or peripheral blood).
    (iv) Limit test. If a test at one dose level of at least 2,000 mg/kg 
body weight using a single treatment, or as two treatments on the same 
day, produces no observable toxic effects, and if genotoxicity would not 
be expected based upon data from structurally related substances, then a 
full study

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using three dose levels may not be considered necessary. For studies of 
a longer duration, the limit dose is 2,000 mg/kg/body weight/day for 
treatment up to 14 days, and 1,000 mg/kg/body weight/day for treatment 
longer than 14 days. Expected human exposure may indicate the need for a 
higher dose level to be used in the limit test.
    (v) Administration of doses. The test substance is usually 
administered by gavage using a stomach tube or a suitable intubation 
cannula, or by intraperitoneal injection. Other routes of exposure may 
be acceptable where they can be justified. The maximum volume of liquid 
that can be administered by gavage or injection at one time depends on 
the size of the test animal. The volume should not exceed 2 ml/100g body 
weight. The use of volumes higher than these must be justified. Except 
for irritating or corrosive substances which will normally reveal 
exacerbated effects with higher concentrations, variability in test 
volume should be minimized by adjusting the concentration to ensure a 
constant volume at all dose levels.
    (vi) Bone marrow/blood preparation. Bone marrow cells shall be 
obtained from the femurs or tibias immediately following sacrifice. 
Cells shall be removed from femurs or tibias, prepared and stained using 
established methods. Peripheral blood is obtained from the tail vein or 
other appropriate blood vessel. Blood cells are immediately stained 
supravitally (the test techniques described in the references under 
paragraphs (g)(4), (g)(5), and (g)(6) of this section may be used) or 
smear preparations are made and then stained. The use of a DNA specific 
stain (e.g. acridine orange (techniques described in the reference under 
paragraph (g)(10) of this section may be used) or Hoechst 33258 plus 
pyronin-Y) can eliminate some of the artifacts associated with using a 
non-DNA specific stain. This advantage does not preclude the use of 
conventional stains (e.g., Giemsa). Additional systems (e.g. cellulose 
columns to remove nucleated cells (the test techniques described in the 
references under paragraph (g)(12) of this section may be used)) can 
also be used provided that these systems have been shown to adequately 
work for micronucleus preparation in the laboratory.
    (vii) Analysis. The proportion of immature among total (immature = 
mature) erythrocytes is determined for each animal by counting a total 
of at least 200 erythrocytes for bone marrow and 1,000 erythrocytes for 
peripheral blood (techniques described in the reference under paragraph 
(g)(13) of this section maybe used). All slides, including those of 
positive and negative controls, shall be independently coded before 
microscopic analysis. At least 2,000 immature erythrocytes per animal 
shall be scored for the incidence of micronucleated immature 
erythrocytes. Additional information may be obtained by scoring mature 
erythrocytes for micronuclei. When analyzing slides, the proportion of 
immature erythrocytes among total erythrocytes should not be less than 
20% of the control value. When animals are treated continuously for 4 
weeks or more, at least 2,000 mature erythrocytes per animal can also be 
scored for the incidence of micronuclei. Systems for automated analysis 
(image analysis) and cell suspensions (flow cytometry) are acceptable 
alternatives to manual evaluation if appropriately justified and 
validated.
    (f) Data and reporting--(1) Treatment of results. Individual animal 
data shall be presented in tabular form. The experimental unit is the 
animal. The number of immature erythrocytes scored, the number of 
micronucleated immature erythrocytes, and the number of immature among 
total erythrocytes shall be listed separately for each animal analyzed. 
When animals are treated continuously for 4 weeks or more, the data on 
mature erythrocytes should also be given if it is collected. The 
proportion of immature among total erythrocytes and, if considered 
applicable, the percentage of micronucleated erythrocytes shall be given 
for each animal. If there is no evidence for a difference in response 
between the sexes, the data from both sexes may be combined for 
statistical analysis.
    (2) Evaluation and interpretation of results. (i) There are several 
criteria for determining a positive result, such as a dose-related 
increase in the number of

[[Page 445]]

micronucleated cells or a clear increase in the number of micronucleated 
cells in a single dose group at a single sampling time. Biological 
relevance of the results should be considered first. Statistical methods 
may be used as an aid in evaluating the test results (the test 
techniques described in the references paragraphs (g)(14) and (g)(15) of 
this section may be used). Statistical significance should not be the 
only determining factor for a positive response. Equivocal results 
should be clarified by further testing preferably using a modification 
of experimental conditions.
    (ii) A test substance for which the results do not meet the criteria 
in paragraph (f)(2)(i) of this section is considered non-mutagenic in 
this test.
    (iii) Although most experiments will give clearly positive or 
negative results, in rare cases the data set will preclude making a 
definite judgement about the activity of the test substance. Results, 
may remain equivocal or questionable regardless of the number of times 
the experiment is repeated. Positive results in the micronucleus test 
indicate that a substance induces micronuclei which are the result of 
chromosomal damage or damage to the mitotic apparatus in the 
erythroblasts of the test species. Negative results indicate that, under 
the test conditions, the test substance does not produce micronuclei in 
the immature erythrocytes of the test species.
    (iv) The likelihood that the test substance or its metabolites reach 
the general circulation or specifically the target tissue (e.g. systemic 
toxicity) should be discussed.
    (3) Test report. The test report shall include the following 
information:
    (i) Test substance:
    (A) Identification data and CAS no., if known.
    (B) Physical nature and purity.
    (C) Physiochemical properties relevant to the conduct of the study.
    (D) Stability of the test substance, if known.
    (ii) Solvent/vehicle:
    (A) Justification for choice of vehicle.
    (B) Solubility and stability of the test substance in the solvent/
vehicle, if known.
    (iii) Test animals:
    (A) Species/strain used.
    (B) Number, age, and sex of animals.
    (C) Source, housing conditions, diet, etc.
    (D) Individual weight of the animals at the start of the test, 
including body weight range, mean and standard deviation for each group.
    (iv) Test conditions:
    (A) Positive and negative (vehicle/solvent) control data.
    (B) Data from range-finding study, if conducted.
    (C) Rationale for dose level selection.
    (D) Details of test substance preparation.
    (E) Details of the administration of the test substance.
    (F) Rationale for route of administration.
    (G) Methods for verifying that the test substance reached the 
general circulation or target tissue, if applicable.
    (H) Conversion from diet/drinking water test substance concentration 
parts per million (ppm) to the actual dose (mg/kg body weight/day), if 
applicable.
    (I) Details of food and water quality.
    (J) Detailed description of treatment and sampling schedules.
    (K) Methods of slide preparation.
    (L) Methods for measurement of toxicity.
    (M) Criteria for scoring micronucleated immature erythrocytes.
    (N) Number of cells analyzed per animal.
    (O) Criteria for considering studies as positive, negative or 
equivocal.
    (v) Results:
    (A) Signs of toxicity.
    (B) Proportion of immature erythrocytes among total erythrocytes.
    (C) Number of micronucleated immature erythrocytes, given separately 
for each animal.
    (D) Mean = standard deviation of 
micronucleated immature erythrocytes per group.
    (E) Dose-response relationship, where possible.
    (F) Statistical analyses and method applied.
    (G) Concurrent and historical negative control data.

[[Page 446]]

    (H) Concurrent positive control data.
    (vi) Discussion of the results.
    (vii) Conclusion.
    (g) References. For additional background information on this test 
guideline, the following references should be consulted. These 
references are available for inspection at the TSCA Nonconfidential 
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M 
St., SW., Washington, DC, 12 noon to 4 p.m., Monday through Friday, 
except legal holidays.
    (1) Heddle, J.A. A Rapid In Vivo Test for Chromosomal Damage. 
Mutation Research. 18, 187-190 (1973).
    (2) Schmid, W. The Micronucleus Test. Mutation Research. 31, 9-15 
(1975).
    (3) Mavournin, K.H., Blakey, D.H., Cimino, M.C., Salamone, M.F., and 
Heddle, J.A. The In Vivo Micronucleus Assay in Mammalian Bone Marrow and 
Peripheral Blood. A report of the U.S. Environmental Protection Agency 
Gene-Tox Program. Mutation Research. 239, 29-80 (1990).
    (4) Hayashi, M., Morita, T., Kodama, Y., Sofuni, T., and Ishidate, 
Jr., M. The Micronucleus Assay with Mouse Peripheral Blood Reticulocytes 
Using Acridine Orange-Coated Slides. Mutation Research. 245, 245-249 
(1990).
    (5) The Collaborative Study Group for the Micronucleus Test (1992). 
Micronucleus Test with Mouse Peripheral Blood Erythrocytes by Acridine 
Orange Supravital Staining: The Summary Report of the 5th Collaborative 
Study by CSGMT/JEMS. MMS. Mutation Research. 278, 83-98.
    (6) The Collaborative Study Group for the Micronucleus Test (CSGMT/
JEMMS.MMS, The Mammalian Mutagenesis Study Group of the Environmental 
Mutagen Society of Japan) Protocol recommended for the short-term mouse 
peripheral blood micronucleus test. Mutagenesis. 10, 153-159 (1995).
    (7) Hayashi, M., Tice, R.R., MacGregor, J.T., Anderson, D., Blakey, 
D.H., Kirsch-Volders, M., Oleson, Jr. F.B., Pacchierotti, F., Romagna, 
F., Shimada, H., Sutou, S., and Vannier, B. In Vivo Rodent Erythrocyte 
Micronucleus Assay. Mutation Research. 312, 293-304 (1994).
    (8) Higashikuni, N. and Sutou, S. An optimal, generalized sampling 
time of 30 =/- 6 h after double dosing in the mouse 
peripheral blood micronucleus test. Mutagenesis. 10, 313-319 (1995).
    (9) Fielder, R.J., Allen, J.A., Boobis, A.R., Botham, P.A., Doe, J., 
Esdaile, D.J., Gatehouse, D.G., Hodson-Walker, G., Morton, D.B., 
Kirkland, D. J., and Richold, M. Report of British Toxicology Society/UK 
Environmental Mutagen Society Working Group: Dose Setting in In Vivo 
Mutagenicity Assays. Mutagenesis. 7, 313-319 (1992).
    (10) Hayashi, M., Sofuni, T., and Ishidate, Jr., M. An Application 
of Acridine Orange Fluorescent Staining to the Micronucleus Test. 
Mutation Research. 120, 241-247 (1983).
    (11) MacGregor, J.T., Wehr, C.M., and Langlois, R.G. A Simple 
Fluorescent Staining Procedure for Micronuclei and RNA in Erythrocytes 
Using Hoechst 33258 and Pyronin Y. Mutation Research. 120, 269-275 
(1983).
    (12) Romagna, F. and Staniforth, C.D. The automated bone marrow 
micronucleus test. Mutation Research. 213, 91-104 (1989).
    (13) Gollapudi, B. and McFadden, L.G. Sample size for the estimation 
of polychromatic to normochromatic eruthrocyte ratio in the bone marrow 
micronucleus test. Mutation Research. 347, 97-99 (1995).
    (14) Richold, M., Ashby, J., Bootman, J., Chandley, A., Gatehouse, 
D.G., and Henderson, L. Ed. Kirkland, D.J. In Vivo Cytogenetics Assays. 
Basic Mutagenicity Tests, UKEMS Recommended Procedures. UKEMS 
Subcommittee on Guidelines for Mutagenicity Testing. Report. Part I 
revised (Cambridge University Press, Cambridge, New York, Port Chester, 
Melbourne, Sydney, 1990) pp. 115-141.
    (15) Lovell, D.P., Anderson, D., Albanese, R., Amphlett, G.E., 
Clare, G., Ferguson, R., Richold, M., Papworth, D.G., and Savage, J.R.K. 
Ed. D.J. Kirkland. Statistical Analysis of In Vivo Cytogenetic Assays. 
Statistical Evaluation of Mutagenicity Test Data. UKEMS Sub-Committee on 
Guidelines for Mutagenicity Testing, Report, Part III. (Cambridge 
University Press, Cambridge, New York, Port Chester, Melbourne, Sydney, 
1989) pp. 184-232.
    (16) Heddle, J.A., Salamone, M.F., Hite, M., Kirkhart, B., 
Mavournin, K., MacGregor, J.G., and Newell, G.W. The

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Induction of Micronuclei as a Measure of Genotoxicity. Mutation 
Research. 123: 61-118 (1983).
    (17) MacGregor, J.T., Heddle, J.A., Hite, M., Margolin, G.H., Ramel 
C., Salamone, M.F., Tice, R.R., and Wild, D. Guidelines for the Conduct 
of Micronucleus Assays in Mammalian Bone Marrow Erythrocytes. Mutation 
Research. 189: 103-112 (1987).
    (18) MacGregor, J.T., Wehr, C.M., Henika, P.R., and Shelby, M.E. 
(1990). The In Vivo Erythrocyte Micronucleus Test: Measurement at Steady 
State Increases Assay Efficiency and Permits Integration with Toxicity 
Studies. Fundamental Applied Toxicology. 14: 513-522.
    (19) MacGregor, J.T., Schlegel, R. Choy, W.N., and Wehr, C.M. Eds. 
Hayes, A.W., Schnell, R.C., and Miya, T.S. Micronuclei in Circulating 
Erythrocytes: A Rapid Screen for Chromosomal Damage During Routine 
Toxicity Testing in Mice. Developments in Science and Practice of 
Toxicology (Elsevier, Amsterdam, 1983) pp. 555-558.

[62 FR 43824, Aug. 15, 1997, as amended at 64 FR 35079, June 30, 1999]



Sec. 799.9620  TSCA neurotoxicity screening battery.

    (a) Scope. This section is intended to meet the testing requirements 
under section 4 of TSCA. This neurotoxicity screening battery consists 
of a functional observational battery, motor activity, and 
neuropathology. The functional observational battery consists of 
noninvasive procedures designed to detect gross functional deficits in 
animals and to better quantify behavioral or neurological effects 
detected in other studies. The motor activity test uses an automated 
device that measures the level of activity of an individual animal. The 
neuropathological techniques are designed to provide data to detect and 
characterize histopathological changes in the central and peripheral 
nervous system. This battery is designed to be used in conjunction with 
general toxicity studies and changes should be evaluated in the context 
of both the concordance between functional neurological and 
neuropatholgical effects, and with respect to any other toxicological 
effects seen. This test battery is not intended to provide a complete 
evaluation of neurotoxicity, and additional functional and morphological 
evaluation may be necessary to assess completely the neurotoxic 
potential of a chemical.
    (b) Source. The source material used in developing this TSCA test 
guideline is the OPPTS harmonized test guideline 870.6200 (June 1996 
Public Draft). This source is available at the address in paragraph (g) 
of this section.
    (c) Definitions. The following definitions apply to this section.
    ED is effective dose.
    Motor activity is any movement of the experimental animal.
    Neurotoxicity is any adverse effect on the structure or function of 
the nervous system related to exposure to a chemical substance.
    Toxic effect is an adverse change in the structure or function of an 
experimental animal as a result of exposure to a chemical substance.
    (d) Principle of the test method. The test substance is administered 
to several groups of experimental animals, one dose being used per 
group. The animals are observed under carefully standardized conditions 
with sufficient frequency to ensure the detection and quantification of 
behavioral and/or neurologic abnormalities, if present. Various 
functions that could be affected by neurotoxicants are assessed during 
each observation period. Measurements of motor activity of individual 
animals are made in an automated device. The animals are perfused and 
tissue samples from the nervous system are prepared for microscopic 
examination. The exposure levels at which significant neurotoxic effects 
are produced are compared to one another and to those levels that 
produce other toxic effects.
    (e) Test procedures--(1) Animal selection--(i) Species. In general, 
the laboratory rat should be used. Under some circumstances, other 
species, such as the mouse or the dog, may be more appropriate, although 
not all of the battery may be adaptable to other species.
    (ii) Age. Young adults (at least 42 days old for rats) shall be 
used.
    (iii) Sex. Both males and females shall be used. Females shall be 
nulliparous and nonpregnant.

[[Page 448]]

    (2) Number of animals. At least 10 males and 10 females should be 
used in each dose and control group for behavioral testing. At least 
five males and five females should be used in each dose and control 
group for terminal neuropathology. If interim neuropathological 
evaluations are planned, the number should be increased by the number of 
animals scheduled to be perfused before the end of the study. Animals 
shall be randomly assigned to treatment and control groups.
    (3) Control groups. (i) A concurrent (vehicle) control group is 
required. Subjects shall be treated in the same way as for an exposure 
group except that administration of the test substance is omitted. If 
the vehicle used has known or potential toxic properties, both untreated 
or saline treated and vehicle control groups are required.
    (ii) Positive control data from the laboratory performing the 
testing shall provide evidence of the ability of the observational 
methods used to detect major neurotoxic endpoints including limb 
weakness or paralysis, tremor, and autonomic signs. Positive control 
data are also required to demonstrate the sensitivity and reliability of 
the activity-measuring device and testing procedures. These data should 
demonstrate the ability to detect chemically induced increases and 
decreases in activity. Positive control groups exhibiting central 
nervous system pathology and peripheral nervous system pathology are 
also required. Separate groups for peripheral and central neuropathology 
are acceptable (e.g. acrylamide and trimethyl tin). Permanently 
injurious substances need not be used for the behavioral tests. 
Historical data may be used if the essential aspects of the experimental 
procedure remain the same. Periodic updating of positive control data is 
recommended. New positive control data should also be collected when 
personnel or some other critical element in the testing laboratory has 
changed.
    (4) Dose level and dose selection. At least three doses shall be 
used in addition to the vehicle control group. The data should be 
sufficient to produce a dose-effect curve. The Agency strongly encourage 
the use of equally spaced doses and a rationale for dose selection that 
will maximally support detection of dose-effect relations. For acute 
studies, dose selection may be made relative to the establishment of a 
benchmark dose (BD). That is, doses may be specified as successive 
fractions, e.g. 0.5, 0.25, ...n of the BD. The BD itself may be 
estimated as the highest nonlethal dose as determined in a preliminary 
range-finding lethality study. A variety of test methodologies may be 
used for this purpose, and the method chosen may influence subsequent 
dose selection. The goal is to use a dose level that is sufficient to be 
judged a limit dose, or clearly toxic.
    (i) Acute studies. The high dose need not be greater than 2 g/kg. 
Otherwise, the high dose should result in significant neurotoxic effects 
or other clearly toxic effects, but not result in an incidence of 
fatalities that would preclude a meaningful evaluation of the data. This 
dose may be estimated by a BD procedure as described under paragraph 
(e)(4) of this section, with the middle and low dose levels chosen as 
fractions of the BD dose. The lowest dose should produce minimal effect, 
e.g. an ED10, or alternatively, no effects.
    (ii) Subchronic and chronic studies. The high dose need not be 
greater than 1 g/kg. Otherwise, the high dose level should result in 
significant neurotoxic effects or other clearly toxic effects, but not 
produce an incidence of fatalities that would prevent a meaningful 
evaluation of the data. The middle and low doses should be fractions of 
the high dose. The lowest dose should produce minimal effects, e.g. an 
ED10, or alternatively, no effects.
    (5) Route of exposure. Selection of route may be based on several 
criteria including, the most likely route of human exposure, 
bioavailability, the likelihood of observing effects, practical 
difficulties, and the likelihood of producing nonspecific effects. For 
many materials, it should be recognized that more than one route of 
exposure may be important and that these criteria may conflict with one 
another. Initially only one route is required for screening for 
neurotoxicity. The route that best meets these criteria should

[[Page 449]]

be selected. Dietary feeding will generally be acceptable for repeated 
exposures studies.
    (6) Combined protocol. The tests described in this screening battery 
may be combined with any other toxicity study, as long as none of the 
requirements of either are violated by the combination.
    (7) Study conduct--(i) Time of testing. All animals shall be weighed 
on each test day and at least weekly during the exposure period.
    (A) Acute studies. At a minimum, for acute studies observations and 
activity testing shall be made before the initiation of exposure, at the 
estimated time of peak effect within 8 hrs of dosing, and at 7 and 14 
days after dosing. Estimation of times of peak effect may be made by 
dosing pairs of rats across a range of doses and making regular 
observations of gait and arousal.
    (B) Subchronic and chronic studies. In a subchronic study, at a 
minimum, observations and activity measurements shall be made before the 
initiation of exposure and before the daily exposure, or for feeding 
studies at the same time of day, during the 4th, 8th, and 13th weeks of 
exposure. In chronic studies, at a minimum, observations and activity 
measurements shall be made before the initiation of exposure and before 
the daily exposure, or for feeding studies at the same time of day, 
every 3 months.
    (ii) Functional observational battery--(A) General conduct. All 
animals in a given study shall be observed carefully by trained 
observers who are unaware of the animals' treatment, using standardized 
procedures to minimize observer variability. Where possible, it is 
advisable that the same observer be used to evaluate the animals in a 
given study. If this is not possible, some demonstration of 
interobserver reliability is required. The animals shall be removed from 
the home cage to a standard arena for observation. Effort should be made 
to ensure that variations in the test conditions are minimal and are not 
systematically related to treatment. Among the variables that can affect 
behavior are sound level, temperature, humidity, lighting, odors, time 
of day, and environmental distractions. Explicit, operationally defined 
scales for each measure of the battery are to be used. The development 
of objective quantitative measures of the observational end-points 
specified is encouraged. Examples of observational procedures using 
defined protocols may be found in the references under paragraphs 
(g)(5), (g)(6), and (g)(9) of this section. The functional observational 
battery shall include a thorough description of the subject's 
appearance, behavior, and functional integrity. This shall be assessed 
through observations in the home cage and while the rat is moving freely 
in an open field, and through manipulative tests. Testing should proceed 
from the least to the most interactive with the subject. Scoring 
criteria, or explicitly defined scales, should be developed for those 
measures which involve subjective ranking.
    (B) List of measures. The functional observational battery shall 
include the following list of measures:
    (1) Assessment of signs of autonomic function, including but not 
limited to:
    (i) Ranking of the degree of lacrimation and salivation, with a 
range of severity scores from none to severe.
    (ii) Presence or absence of piloerection and exophthalmus.
    (iii) Ranking or count of urination and defecation, including 
polyuria and diarrhea. This is most easily conducted during the open 
field assessment.
    (iv) Pupillary function such as constriction of the pupil in 
response to light or a measure of pupil size.
    (v) Degree of palpebral closure, e.g., ptosis.
    (2) Description, incidence, and severity of any convulsions, 
tremors, or abnormal motor movements, both in the home cage and the open 
field.
    (3) Ranking of the subject's reactivity to general stimuli such as 
removal from the cage or handling, with a range of severity scores from 
no reaction to hyperreactivity.
    (4) Ranking of the subject's general level of activity during 
observations of the unperturbed subject in the open field, with a range 
of severity scores from unresponsive to hyperactive.
    (5) Descriptions and incidence of posture and gait abnormalities 
observed in the home cage and open field.

[[Page 450]]

    (6) Ranking of any gait abnormalities, with a range of severity 
scores from none to severe.
    (7) Forelimb and hindlimb grip strength measured using an objective 
procedure (the procedure described in the reference under paragraph 
(g)(8) of this section may be used).
    (8) Quantitative measure of landing foot splay (the procedure 
described in the reference under paragraph (g)(3) of this section may be 
used).
    (9) Sensorimotor responses to stimuli of different modalities will 
be used to detect gross sensory deficits. Pain perception may be 
assessed by a ranking or measure of the reaction to a tail-pinch, tail-
flick, or hot-plate. The response to a sudden sound, e.g., click or 
snap, may be used to assess audition.
    (10) Body weight.
    (11) Description and incidence of any unusual or abnormal behaviors, 
excessive or repetitive actions (stereotypies), emaciation, dehydration, 
hypotonia or hypertonia, altered fur appearance, red or crusty deposits 
around the eyes, nose, or mouth, and any other observations that may 
facilitate interpretation of the data.
    (C) Additional measures. Other measures may also be included and the 
development and validation of new tests is encouraged. Further 
information on the neurobehavioral integrity of the subject may be 
provided by:
    (1) Count of rearing activity on the open field.
    (2) Ranking of righting ability.
    (3) Body temperature.
    (4) Excessive or spontaneous vocalizations.
    (5) Alterations in rate and ease of respiration, e.g., rales or 
dyspnea.
    (6) Sensorimotor responses to visual or proprioceptive stimuli.
    (iii) Motor activity. Motor activity shall be monitored by an 
automated activity recording apparatus. The device used must be capable 
of detecting both increases and decreases in activity, i.e., baseline 
activity as measured by the device must not be so low as to preclude 
detection of decreases nor so high as to preclude detection of increases 
in activity. Each device shall be tested by standard procedures to 
ensure, to the extent possible, reliability of operation across devices 
and across days for any one device. In addition, treatment groups must 
be balanced across devices. Each animal shall be tested individually. 
The test session shall be long enough for motor activity to approach 
asymptotic levels by the last 20% of the session for nontreated control 
animals. All sessions shall have the same duration. Treatment groups 
shall be counterbalanced across test times. Effort should be made to 
ensure that variations in the test conditions are minimal and are not 
systematically related to treatment. Among the variables which can 
affect motor activity are sound level, size and shape of the test cage, 
temperature, relative humidity, lighting conditions, odors, use of the 
home cage or a novel test cage, and environmental distractions.
    (iv) Neuropathology: Collection, processing and examination of 
tissue samples. To provide for adequate sampling as well as optimal 
preservation of cellular integrity for the detection of 
neuropathological alterations, tissue shall be prepared for histological 
analysis using in situ perfusion and paraffin and/or plastic embedding 
procedures. Paraffin embedding is acceptable for tissue samples from the 
central nervous system. Plastic embedding of tissue samples from the 
central nervous system is encouraged, when feasible. Plastic embedding 
is required for tissue samples from the peripheral nervous system. 
Subject to professional judgment and the type of neuropathological 
alterations observed, it is recommended that additional methods, such as 
glial fibrillary acidic protein (GFAP) immunohistochemistry and/or 
methods known as Bodian's or Bielchowsky's silver methods be used in 
conjunction with more standard stains to determine the lowest dose level 
at which neuropathological alterations are observed. When new or 
existing data provide evidence of structural alterations it is 
recommended that the GFAP immunoassay also be considered. A description 
of this technique can be found in the reference under paragraph (g)(10) 
of this section.
    (A) Fixation and processing of tissue. The nervous system shall be 
fixed by in situ perfusion with an appropriate

[[Page 451]]

aldehyde fixative. Any gross abnormalities should be noted. Tissue 
samples taken should adequately represent all major regions of the 
nervous system. The tissue samples should be postfixed and processed 
according to standardized published histological protocols (protocols 
described in the references under paragraphs (g)(1), (g)(2), or (g)(11) 
of this section may be used). Tissue blocks and slides should be 
appropriately identified when stored. Histological sections should be 
stained for hematoxylin and eosin (H&E), or a comparable stain according 
to standard published protocols (some of these protocols are described 
in the references under paragraphs (g)(1) and (g)(11) of this section).
    (B) Qualitative examination. Representative histological sections 
from the tissue samples should be examined microscopically by an 
appropriately trained pathologist for evidence of neuropathological 
alterations. The nervous system shall be thoroughly examined for 
evidence of any treatment-related neuropathological alterations. 
Particular attention should be paid to regions known to be sensitive to 
neurotoxic insult or those regions likely to be affected based on the 
results of functional tests. Such treatment-related neuropathological 
alterations should be clearly distinguished from artifacts resulting 
from influences other than exposure to the test substance. A stepwise 
examination of tissue samples is recommended. In such a stepwise 
examination, sections from the high dose group are first compared with 
those of the control group. If no neuropathological alterations are 
observed in samples from the high dose group, subsequent analysis is not 
required. If neuropathological alterations are observed in samples from 
the high dose group, samples from the intermediate and low dose groups 
are then examined sequentially.
    (C) Subjective diagnosis. If any evidence of neuropathological 
alterations is found in the qualitative examination, then a subjective 
diagnosis shall be performed for the purpose of evaluating dose-response 
relationships. All regions of the nervous system exhibiting any evidence 
of neuropathological changes should be included in this analysis. 
Sections from all dose groups from each region will be coded and 
examined in randomized order without knowledge of the code. The 
frequency of each type and severity of each lesion will be recorded. 
After all samples from all dose groups including all regions have been 
rated, the code will be broken and statistical analysis performed to 
evaluate dose-response relationships. For each type of dose-related 
lesion observed, examples of different degrees of severity should be 
described. Photomicrographs of typical examples of treatment-related 
regions are recommended to augment these descriptions. These examples 
will also serve to illustrate a rating scale, such as 1=, 2=, and 3= for 
the degree of severity ranging from very slight to very extensive.
    (f) Data reporting and evaluation. The final test report shall 
include the following information:
    (1) Description of equipment and test methods. A description of the 
general design of the experiment and any equipment used shall be 
provided. This shall include a short justification explaining any 
decisions involving professional judgment.
    (i) A detailed description of the procedures used to standardize 
observations, including the arena and scoring criteria.
    (ii) Positive control data from the laboratory performing the test 
that demonstrate the sensitivity of the procedures being used. 
Historical data may be used if all essential aspects of the experimental 
protocol are the same. Historical control data can be critical in the 
interpretation of study findings. The Agency encourages submission of 
such data to facilitate the rapid and complete review of the 
significance of effects seen.
    (2) Results. The following information shall be arranged by test 
group dose level.
    (i) In tabular form, data for each animal shall be provided showing:
    (A) Its identification number.
    (B) Its body weight and score on each sign at each observation time, 
the time and cause of death (if appropriate), total session activity 
counts, and intrasession subtotals for each day measured.

[[Page 452]]

    (ii) Summary data for each group must include:
    (A) The number of animals at the start of the test.
    (B) The number of animals showing each observation score at each 
observation time.
    (C) The mean and standard deviation for each continuous endpoint at 
each observation time.
    (D) Results of statistical analyses for each measure, where 
appropriate.
    (iii) All neuropathological observations shall be recorded and 
arranged by test groups. This data may be presented in the following 
recommended format:
    (A) Description of lesions for each animal. For each animal, data 
must be submitted showing its identification (animal number, sex, 
treatment, dose, and duration), a list of structures examined as well as 
the locations, nature, frequency, and severity of lesions. Inclusion of 
photomicrographs is strongly recommended for demonstrating typical 
examples of the type and severity of the neuropathological alterations 
observed. Any diagnoses derived from neurological signs and lesions 
including naturally occurring diseases or conditions, should be 
recorded.
    (B) Counts and incidence of neuropathological alterations by test 
group. Data should be tabulated to show:
    (1) The number of animals used in each group and the number of 
animals in which any lesion was found.
    (2) The number of animals affected by each different type of lesion, 
the locations, frequency, and average grade of each type of lesion.
    (3) Evaluation of data. The findings from the screening battery 
should be evaluated in the context of preceding and/or concurrent 
toxicity studies and any correlated functional and histopathological 
findings. The evaluation shall include the relationship between the 
doses of the test substance and the presence or absence, incidence and 
severity, of any neurotoxic effects. The evaluation shall include 
appropriate statistical analyses, for example, parametric tests for 
continuous data and nonparametric tests for the remainder. Choice of 
analyses should consider tests appropriate to the experimental design, 
including repeated measures. There may be many acceptable ways to 
analyze data.
    (g) References. For additional background information on this test 
guideline, the following references should be consulted. These 
references are available for inspection at the TSCA Nonconfidential 
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M 
St., SW., Washington, DC, 12 noon to 4 p.m., Monday through Friday, 
except legal holidays.
    (1) Bennet, H.S. et al. Science and art in the preparing tissues 
embedded in plastic for light microscopy, with special reference to 
glycol methacrylate, glass knives and simple stains. Stain Technology. 
51:71-97 (1976).
    (2) Di Sant Agnese, P.A. and De Mesy Jensen, K. Dibasic staining of 
large epoxy sections and application to surgical pathology. American 
Journal of Clinical Pathology. 81:25-29 (1984).
    (3) Edwards, P.M. and Parker, V.H. A simple, sensitive and objective 
method for early assessment of acrylamide neuropathy in rats. Toxicology 
and Applied Pharmacology. 40:589-591 (1977).
    (4) Finger, F.W. Ed. Myers, R.D. Measuring Behavioral Activity. Vol. 
2. Methods in Psychobiology (Academic, NY, 1972) pp.1-19.
    (5) Gad, S. A neuromuscular screen for use in industrial toxicology. 
Journal of Toxicology and Environmental Health. 9:691-704 (1982).
    (6) Irwin, S. Comprehensive observational assessment: Ia. A 
systematic quantitative procedure for assessing the behavioral 
physiological state of the mouse. Psychopharmacologia. 13:222-257 
(1968).
    (7) Kinnard, E.J. and Watzman, N. Techniques utilized in the 
evaluation of psychotropic drugs on animals activity. Journal of 
Pharmaceutical Sciences. 55:995-1012 (1966).
    (8) Meyer, O.A. et al. A method for the routine assessment of fore- 
and hindlimb grip strength of rats and mice. Neurobehavioral Toxicology. 
1:233-236 (1979).
    (9) Moser V.C. et al. Comparison of chlordimeform and carbaryl using 
a functional observational battery. Fundamental and Applied Toxicology. 
11:189-206 (1988).

[[Page 453]]

    (10) O'Callaghan, J.P. Quantification of glial fibrillary acidic 
protein: Comparison of slot-immunobinding assays with a novel sandwich 
ELISA. Neurotoxicology and Teratology. 13:275-281 (1991).
    (11) Pender, M.P. A simple method for high resolution light 
microscopy of nervous tissue. Journal of Neuroscience Methods. 15:213-
218 (1985).
    (12) Reiter, L.W. Use of activity measures in behavioral toxicology. 
Environmental Health Perspectives. 26:9-20 (1978).
    (13) Reiter, L.W. and MacPhail, R.C. Motor activity: A survey of 
methods with potential use in toxicity testing. Neurobehavorial 
Toxicology. 1--Supplement. 1:53-66 (1979).
    (14) Robbins, T.W. Eds. Iversen, L.L., Iverson, D.S., and Snyder, 
S.H. A critique of the methods available for the measurement of 
spontaneous motor activity. Vol 7. Handbook of Psychopharmacology 
(Plenum, NY, 1977) pp. 37-82.

[62 FR 43824, Aug. 15, 1997, as amended at 64 FR 35080, June 30, 1999]



Sec. 799.9630  TSCA developmental neurotoxicity.

    (a) Scope--(1) Applicability. This section is intended to meet the 
testing requirements under section 4 of the Toxic Substances Control Act 
(TSCA).
    (2) Source. The source material used in developing this TSCA test 
guideline is the OPPTS harmonized test guideline 870.6300 (August 1998).
    (b) Purpose. In the assessment and evaluation of the toxic 
characteristics of a chemical substance or mixture (test substance), 
determination of the potential for developmental neurotoxicity is 
important. This study is designed to develop data on the potential 
functional and morphological hazards to the nervous system which may 
arise in the offspring from exposure of the mother during pregnancy and 
lactation.
    (c) Principle of the test method. The test substance is administered 
to several groups of pregnant animals during gestation and early 
lactation, one dose level being used per group. Offspring are randomly 
selected from within litters for neurotoxicity evaluation. The 
evaluation includes observations to detect gross neurologic and 
behavioral abnormalities, determination of motor activity, response to 
auditory startle, assessment of learning, neuropathological evaluation, 
and brain weights. This protocol may be used as a separate study, as a 
followup to a standard developmental toxicity and/or adult neurotoxicity 
study, or as part of a two-generation reproduction study, with 
assessment of the offspring conducted on the second (F2) generation.
    (d) Test procedure--(1) Animal selection--(i) Species and strain. 
Testing must be performed in the rat. Because of its differences in 
timing of developmental events compared to strains that are more 
commonly tested in other developmental and reproductive toxicity 
studies, it is preferred that the Fischer 344 strain not be used. If a 
sponsor wishes to use the Fischer 344 rat or a mammalian species other 
than the rat, ample justification/reasoning for this selection must be 
provided.
    (ii) Age. Young adult (nulliparous females) animals must be used.
    (iii) Sex. Pregnant female animals must be used at each dose level.
    (iv) Number of animals. (A) The objective is for a sufficient number 
of pregnant rats to be exposed to the test substance to ensure that an 
adequate number of offspring are produced for neurotoxicity evaluation. 
At least 20 litters are recommended at each dose level.
    (B) On postnatal day 4, the size of each litter should be adjusted 
by eliminating extra pups by random selection to yield, as nearly as 
possible, four male and four females per litter. Whenever the number of 
pups of either sex prevents having four of each sex per litter, partial 
adjustment (for example, five males and three females) is permitted. 
Testing is not appropriate for litters of less than seven pups. 
Elimination of runts only is not appropriate. Individual pups should be 
identified uniquely after standardization of litters. A method that may 
be used for identification can be found under paragraph (f)(1) of this 
section.
    (v) Assignment of animals for behavioral tests, brain weights, and 
neuropathological evaluations. After standardization of litters, one 
male or

[[Page 454]]

one female from each litter (total of 10 males and 10 females per dose 
group) must be randomly assigned to one of the following tests: Motor 
activity, auditory startle, and learning and memory, in weanling and 
adult animals. On postnatal day 11, either 1 male or 1 female pup from 
each litter (total of 10 males and 10 females per dose group) must be 
sacrificed. Brain weights must be measured in all of these pups and, of 
these pups, six per sex per dose must be selected for neuropathological 
evaluation. At the termination of the study, either 1 male or 1 female 
from each litter (total of 10 males and 10 females per dose group) must 
be sacrificed and brain weights must be measured. An additional group of 
six animals per sex per dose group (one male or one female per litter) 
must be sacrificed at the termination of the study for neuropathological 
evaluation.
    (2) Control group. A concurrent control group is required. This 
group must be a sham-treated group or, if a vehicle is used in 
administering the test substance, a vehicle control group. The vehicle 
must neither be developmentally toxic nor have effects on reproduction. 
Animals in the control group must be handled in an identical manner to 
test group animals.
    (3) Dose levels and dose selection. (i) At least three dose levels 
of the test substance plus a control group (vehicle control, if a 
vehicle is used) must be used.
    (ii) If the test substance has been shown to be developmentally 
toxic either in a standard developmental toxicity study or in a pilot 
study, the highest dose level must be the maximum dose which will not 
induce in utero or neonatal death or malformations sufficient to 
preclude a meaningful evaluation of neurotoxicity.
    (iii) If a standard developmental toxicity study has not been 
conducted, the highest dose level, unless limited by the physicochemical 
nature or biological properties of the substance, must induce some overt 
maternal toxicity, but must not result in a reduction in weight gain 
exceeding 20 percent during gestation and lactation.
    (iv) The lowest dose should not produce any grossly observable 
evidence of either maternal or developmental neurotoxicity.
    (v) The intermediate doses must be equally spaced between the 
highest and lowest doses used.
    (4) Dosing period. Day 0 of gestation is the day on which a vaginal 
plug and/or sperm are observed. The dosing period must cover the period 
from day 6 of gestation through day 10 postnatally. Dosing should not 
occur on the day of parturition in those animals who have not completely 
delivered their offspring.
    (5) Administration of the test substance. The test substance or 
vehicle must be administered orally. Other routes of administration may 
be acceptable, on a case-by-case basis, with ample justification/
reasoning for this selection. The test substance or vehicle must be 
administered based on the most recent weight determination.
    (6) Observation of dams. (i) A gross examination of the dams must be 
made at least once each day before daily treatment.
    (ii) Ten dams per group must be observed outside the home cage at 
least twice during the gestational dosing period (days 6-21) and twice 
during the lactational dosing period (days 1-10) for signs of toxicity. 
The animals must be observed by trained technicians who are unaware of 
the animals' treatment, using standardized procedures to maximize 
interobserver reliability. Where possible, it is advisable that the same 
observer be used to evaluate the animals in a given study. If this is 
not possible, some demonstration of interobserver reliability is 
required.
    (iii) During the treatment and observation periods under paragraph 
(d)(6)(ii) of this section, observations must include:
    (A) Assessment of signs of autonomic function, including but not 
limited to:
    (1) Ranking of the degree of lacrimation and salivation, with a 
range of severity scores from none to severe.
    (2) Presence or absence of piloerection and exophthalmus.
    (3) Ranking or count of urination and defecation, including polyuria 
and diarrhea.

[[Page 455]]

    (4) Pupillary function such as constriction of the pupil in response 
to light or a measure of pupil size.
    (5) Degree of palpebral closure, e.g., ptosis.
    (B) Description, incidence, and severity of any convulsions, 
tremors, or abnormal movements.
    (C) Description and incidence of posture and gait abnormalities.
    (D) Description and incidence of any unusual or abnormal behaviors, 
excessive or repetitive actions (stereotypies), emaciation, dehydration, 
hypotonia or hypertonia, altered fur appearance, red or crusty deposits 
around the eyes, nose, or mouth, and any other observations that may 
facilitate interpretation of the data.
    (iv) Signs of toxicity must be recorded as they are observed, 
including the time of onset, degree, and duration.
    (v) Animals must be weighed at least weekly and on the day of 
delivery and postnatal days 11 and 21 (weaning) and such weights must be 
recorded.
    (vi) The day of delivery of litters must be recorded and considered 
as postnatal day 0.
    (7) Study conduct--(i) Observation of offspring. (A) All offspring 
must be examined cage-side at least daily for gross signs of mortality 
or morbidity.
    (B) A total of 10 male offspring and 10 female offspring per dose 
group must be examined outside the cage for signs of toxicity on days 4, 
11, 21, 35, 45, and 60. The offspring must be observed by trained 
technicians, who are unaware of the treatment being used, using 
standardized procedures to maximize interobserver reliability. Where 
possible, it is advisable that the same observer be used to evaluate the 
animals in a given study. If this is not possible, some demonstration of 
interobserver reliability is required. At a minimum, the end points 
outlined in paragraph (d)(6)(iii) of this section must be monitored as 
appropriate for the developmental stage being observed.
    (C) Any gross signs of toxicity in the offspring must be recorded as 
they are observed, including the time of onset, degree, and duration.
    (ii) Developmental landmarks. Live pups must be counted and each pup 
within a litter must be weighed individually at birth or soon 
thereafter, and on postnatal days 4, 11, 17, and 21 and at least once 
every 2 weeks thereafter. The age of vaginal opening and preputial 
separation must be determined. General procedures for these 
determinations may be found in paragraphs (f)(1) and (f)(11) of this 
section.
    (iii) Motor activity. Motor activity must be monitored specifically 
on postnatal days 13, 17, 21, and 60 (+2 days). Motor activity must be 
monitored by an automated activity recording apparatus. The device must 
be capable of detecting both increases and decreases in activity, (i.e., 
baseline activity as measured by the device must not be so low as to 
preclude detection of decreases nor so high as to preclude detection of 
increases in activity). Each device must be tested by standard 
procedures to ensure, to the extent possible, reliability of operation 
across devices and across days for any one device. In addition, 
treatment groups must be balanced across devices. Each animal must be 
tested individually. The test session must be long enough for motor 
activity to approach asymptotic levels by the last 20 percent of the 
session for nontreated control animals. All sessions must have the same 
duration. Treatment groups must be counter-balanced across test times. 
Activity counts must be collected in equal time periods of no greater 
than 10 minutes duration. Efforts must be made to ensure that variations 
in the test conditions are minimal and are not systematically related to 
treatment. Among the variables that can affect motor activity are sound 
level, size and shape of the test cage, temperature, relative humidity, 
light conditions, odors, use of home cage or novel test cage, and 
environmental distractions. Additional information on the conduct of a 
motor activity study may be obtained in Sec. 799.9620.
    (iv) Auditory startle test. An auditory startle habituation test 
should be performed on the offspring around the time of weaning and 
around day 60. Day of testing should be counterbalanced across treated 
and control groups. Details on the conduct of this testing may be 
obtained under paragraph (f)(1) of this section. In performing the 
auditory startle task, the mean response amplitude on each

[[Page 456]]

block of 10 trials (5 blocks of 10 trials per session on each day of 
testing) must be made. While use of prepulse inhibition is not a 
requirement, it is highly recommended. Details on the conduct of this 
test may be obtained in paragraph (f)(10) of this section.
    (v) Learning and memory tests. A test of associative learning and 
memory should be conducted around the time of weaning and around day 60. 
Day of testing should be counterbalanced across treated and control 
groups. The same or separate tests may be used at these two stages of 
development. Some flexibility is allowed in the choice of tests for 
learning and memory in weanling and adult rats. However, the tests must 
be designed to fulfill two criteria. First, learning must be assessed 
either as a change across several repeated learning trials or sessions, 
or, in tests involving a single trial, with reference to a condition 
that controls for nonassociative effects of the training experience. 
Second, the tests must include some measure of memory (short-term or 
long-term) in addition to original learning (acquisition). If the tests 
of learning and memory reveal an effect of the test compound, it may be 
in the best interest of the sponsor to conduct additional tests to rule 
out alternative interpretations based on alterations in sensory, 
motivational, and/or motor capacities. In addition to the above two 
criteria, it is recommended that the test of learning and memory be 
chosen on the basis of its demonstrated sensitivity to the class of 
compound under investigation, if such information is available in the 
literature. In the absence of such information, examples of tests that 
could be made to meet the above criteria include: Delayed-matching-to-
position, as described for the adult rat (see paragraph (f)(3) of this 
section) and for the infant rat (see paragraph (f)(9) of this section); 
olfactory conditioning, as described in paragraph (f)(13) of this 
section; and acquisition and retention of schedule-controlled behavior 
(see paragraphs (f)(4) and (f)(5) of this section). Additional tests for 
weanling rats are described under paragraphs (f)(20) and (f)(12) of this 
section, and for adult rats under paragraph (f)(16) of this section.
    (vi) Neuropathology. Neuropathological evaluation must be conducted 
on animals on postnatal day 11 and at the termination of the study. At 
11 days of age, one male or female pup must be removed from each litter 
such that equal numbers of male and female offspring are removed from 
all litters combined. Of these, six male and six female pups per dose 
group will be sacrificed for neuropathological analysis. The pups will 
be sacrificed by exposure to carbon dioxide and immediately thereafter 
the brains should be removed, weighed, and immersion-fixed in an 
appropriate aldehyde fixative. The remaining animals will be sacrificed 
in a similar manner and immediately thereafter their brains removed and 
weighed. At the termination of the study, one male or one female from 
each litter will be sacrificed by exposure to carbon dioxide and 
immediately thereafter the brain must be removed and weighed. In 
addition, six animals per sex per dose group (one male or female per 
litter) must be sacrificed at the termination of the study for 
neuropathological evaluation. Neuropathological analysis of animals 
sacrificed at the termination of the study must be performed in 
accordance with Sec. 799.9620. Neuropathological evaluation of animals 
sacrificed on postnatal day 11 and at termination of the study must 
include a qualitative analysis and semiquantitative analysis as well as 
simple morphometrics.
    (A) Fixation and processing of tissue samples for postnatal day 11 
animals. Immediately following removal, the brain must be weighed and 
immersion fixed in an appropriate aldehyde fixative. The brains must be 
postfixed and processed according to standardized published histological 
protocols such as those discussed in references listed under paragraphs 
(f)(6), (f)(14), (f)(17), and (f)(21) of this section. Paraffin 
embedding is acceptable but plastic embedding is preferred and 
recommended. Tissue blocks and slides must be appropriately identified 
when stored. Histological sections must be stained for hematoxylin and 
eosin, or a similar stain according to standard published protocols such 
as those discussed in references listed under paragraphs (f)(2), 
(f)(18), and (f)(23) of this section. For

[[Page 457]]

animals sacrificed at the termination of the study, methods for fixation 
and processing of tissue samples are provided in Sec. 
799.9620(e)(7)(iv)(A).
    (B) Qualitative analysis. The purposes of the qualitative 
examination are threefold--to identify regions within the nervous system 
exhibiting evidence of neuropathological alterations, to identify types 
of neuropathological alterations resulting from exposure to the test 
substance, and to determine the range of severity of the 
neuropathological alterations. Representative histological sections from 
the tissue samples should be examined microscopically by an 
appropriately trained pathologist for evidence of neuropathological 
alterations. The following stepwise procedure is recommended for the 
qualitative analysis. First, sections from the high dose group are 
compared with those of the control group. If no evidence of 
neuropathological alterations is found in animals of the high dose 
group, no further analysis is required. If evidence of neuropathological 
alterations are found in the high dose group, then animals from the 
intermediate and low dose group are examined. Subject to professional 
judgment and the kind of neuropathological alterations observed, it is 
recommended that additional methods such as Bodian's or Bielchowsky's 
silver methods and/or immunohistochemistry for glial fibrillary acid 
protein be used in conjunction with more standard stains to determine 
the lowest dose level at which neuropathological alterations are 
observed. Evaluations of postnatal day 11 pups is described in 
paragraphs (d)(7)(vi)(B)(1) and (d)(7)(vi)(B)(2) of this section. For 
animals sacrificed at the termination of the study, the regions to be 
examined and the types of alterations that must be assessed are 
identified in Sec. 799.9620(e)(7)(iv)(B).
    (1) Regions to be examined. The brains should be examined for any 
evidence of treatment-related neuropathological alterations and adequate 
samples should be taken from all major brain regions (e.g., olfactory 
bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, 
hypothalamus, midbrain (tectum, tegmentum, and cerebral peduncles), 
brainstem and cerebellum) to ensure a thorough examination.
    (2) Types of alterations. Guidance for neuropathological examination 
for indications of developmental insult to the brain can be found in 
paragraphs (f)(8) and (f)(22) of this section. In addition to more 
typical kinds of cellular alterations (e.g., neuronal vacuolation, 
degeneration, necrosis) and tissue changes (e.g., astrocytic 
proliferation, leukocytic infiltration, and cystic formation) particular 
emphasis should be paid to structural changes indicative of 
developmental insult including but not restricted to:
    (i) Gross changes in the size or shape of brain regions such as 
alterations in the size of the cerebral hemispheres or the normal 
pattern of foliation of the cerebellum.
    (ii) The death of neuronal precursors, abnormal proliferation, or 
abnormal migration, as indicated by pyknotic cells or ectopic neurons, 
or gross alterations in regions with active proliferative and migratory 
zones, alterations in transient developmental structures (e.g., the 
external germinal zone of the cerebellum, see paragraph (f)(15) of this 
section).
    (iii) Abnormal differentiation, while more apparent with special 
stains, may also be indicated by shrunken and malformed cell bodies.
    (iv) Evidence of hydrocephalus, in particular enlargement of the 
ventricles, stenosis of the cerebral aqueduct and general thinning of 
the cerebral hemispheres.
    (C) Subjective diagnosis. If any evidence of neuropathological 
alterations is found in the qualitative examination, then a subjective 
diagnosis will be performed for the purpose of evaluating dose-response 
relationships. All regions of the brain exhibiting any evidence of 
neuropathological changes must be included in this analysis. Sections of 
each region from all dose groups will be coded as to treatment and 
examined in randomized order. The frequency of each type and the 
severity of each lesion will be recorded. After all sections from all 
dose groups including all regions have been rated, the code will be 
broken and statistical analyses performed to evaluate dose-response 
relationships. For each type of dose related

[[Page 458]]

lesion observed, examples of different ranges of severity must be 
described. The examples will serve to illustrate a rating scale, such as 
1+, 2+, and 3+ for the degree of severity ranging from very slight to 
very extensive.
    (D) Simple morphometric analysis. Since the disruption of 
developmental processes is sometimes more clearly reflected in the rate 
or extent of growth of particular brain regions, some form of 
morphometric analysis must be performed on postnatal day 11 and at the 
termination of the study to assess the structural development of the 
brain. At a minimum, this would consist of a reliable estimate of the 
thickness of major layers at representative locations within the 
neocortex, hippocampus, and cerebellum. For guidance on such 
measurements see Rodier and Gramann under paragraph (f)(19) of this 
section.
    (e) Data collection, reporting, and evaluation. The following 
specific information must be reported:
    (1) Description of test system and test methods. A description of 
the general design of the experiment should be provided. This must 
include:
    (i) A detailed description of the procedures used to standardize 
observations and procedures as well as operational definitions for 
scoring observations.
    (ii) Positive control data from the laboratory performing the test 
that demonstrate the sensitivity of the procedures being used. These 
data do not have to be from studies using prenatal exposures. However, 
the laboratory must demonstrate competence in evaluation of effects in 
neonatal animals perinatally exposed to chemicals and establish test 
norms for the appropriate age group.
    (iii) Procedures for calibrating and ensuring the equivalence of 
devices and the balancing of treatment groups in testing procedures.
    (iv) A short justification explaining any decisions involving 
professional judgement.
    (2) Results. The following information must be arranged by each 
treatment and control group:
    (i) In tabular form, data for each animal must be provided showing:
    (A) Its identification number and the litter from which it came.
    (B) Its body weight and score on each developmental landmark at each 
observation time.
    (C) Total session activity counts and intrasession subtotals on each 
day measured.
    (D) Auditory startle response amplitude per session and intrasession 
amplitudes on each day measured.
    (E) Appropriate data for each repeated trial (or session) showing 
acquisition and retention scores on the tests of learning and memory on 
each day measured.
    (F) Time and cause of death (if appropriate); any neurological signs 
observed; a list of structures examined as well as the locations, 
nature, frequency, and extent of lesions; and brain weights.
    (ii) The following data should also be provided, as appropriate:
    (A) Inclusion of photomicrographs demonstrating typical examples of 
the type and extent of the neuropathological alterations observed is 
recommended.
    (B) Any diagnoses derived from neurological signs and lesions, 
including naturally occurring diseases or conditions, should also be 
recorded.
    (iii) Summary data for each treatment and control group must 
include:
    (A) The number of animals at the start of the test.
    (B) The body weight of the dams during gestation and lactation.
    (C) Litter size and mean weight at birth.
    (D) The number of animals showing each abnormal sign at each 
observation time.
    (E) The percentage of animals showing each abnormal sign at each 
observation time.
    (F) The mean and standard deviation for each continuous endpoint at 
each observation time. These will include body weight, motor activity 
counts, auditory startle responses, performance in learning and memory 
tests, regional brain weights and whole brain weights (both absolute and 
relative).
    (G) The number of animals in which any lesion was found.

[[Page 459]]

    (H) The number of animals affected by each different type of lesion, 
the location, frequency and average grade of each type of lesion for 
each animal.
    (I) The values of all morphometric measurements made for each animal 
listed by treatment group.
    (3) Evaluation of data. An evaluation of test results must be made. 
The evaluation must include the relationship between the doses of the 
test substance and the presence or absence, incidence, and extent of any 
neurotoxic effect. The evaluation must include appropriate statistical 
analyses. The choice of analyses must consider tests appropriate to the 
experimental design and needed adjustments for multiple comparisons. The 
evaluation must include the relationship, if any, between observed 
neuropathological and behavioral alterations.
    (f) References. For additional background information on this test 
guideline, the following references should be consulted. These 
references are available for inspection at the TSCA Nonconfidential 
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M 
St., SW., Washington, DC, 12 noon to 4 p.m., Monday through Friday, 
except legal holidays.
    (1) Adams, J., Buelke-Sam, J., Kimmel, C.A., Nelson, C.J., Reiter, 
L.W., Sobotka, T.J., Tilson, H.A., and Nelson, B.K. Collaborative 
behavioral teratolgy study: Protocol design and testing 
procedures.Neurobehavioral Toxicology and Teratology 7:579-586 (1985).
    (2) Bennett, H.S., Wyrick, A.D., Lee, S.W., and McNeil, J.H. Science 
and art in preparing tissues embedded in plastic for light microscopy, 
with special reference to glycol methacrylate, glass knives and simple 
stains. Stain Technology 51:71-97 (1976).
    (3) Bushnell, P.J. Effects of delay, intertrial interval, delay 
behavior and trimethyltin on spatial delayed response in rats. 
Neurotoxicology and Teratology 10:237-244 (1988).
    (4) Campbell, B.A. and Haroutunian, V. Effects of age on long-term 
memory: Retention of fixed interval responding. Journal of Gerontology 
36:338-341 (1981).
    (5) Cory-Slechta, D.A., Weiss, B., and Cox, C. Delayed behavioral 
toxicity of lead with increasing exposure concentration. Toxicology and 
Applied Pharmacology 71:342-352 (1983).
    (6) Di Sant Agnese, P. A. and De Mesy Jensen, K.L. Dibasic staining 
of large epoxy tissue sections and application to surgical pathology. 
American Journal of Clinical Pathology 81:25-29 (1984).
    (7) U.S. Environmental Protection Agency. Neurotoxicity Screening 
Battery. In: Pesticide Assessment Guidelines, Subdivision F, Addendum 
10. EPA 540/09-91-123. NTIS PB 91-154617 (1991).
    (8) Friede, R. L. Developmental Neuropathology. Springer-Verlag, New 
York. pp. 1-23, 297-313, 326-351 (1975).
    (9) Green, R.J. and Stanton, M.E. Differential ontogeny of working 
memory and reference memory in the rat. Behavioral Neuroscience 103:98-
105 (1989).
    (10) Ison, J.R. Reflex modification as an objective test for sensory 
processing following toxicant exposure. Neurobehavioral Toxicology and 
Teratology 6:437-445 (1984).
    (11) Korenbrot, C.C., Huhtaniemi, I.T., and Weiner, R.I. Preputial 
separation as an external sign of pubertal development in the male rat. 
Biology of Reproduction 17:298-303 (1977).
    (12) Krasnegor, N.A., Blass, E.M., Hofer, M.A., and Smotherman, W.P. 
(eds.) Perinatal Development: A Psychobiological Perspective. Academic 
Press, Orlando. pp.11-37, 145-167. (1987).
    (13) Kucharski, D. and Spear, N.E. Conditioning of aversion to an 
odor paired with peripheral shock in the developing rat. Developmental 
Psychobiology 17:465-479 (1984).
    (14) Luna, L. G. (editor). Manual of Histologic Staining Methods of 
the Armed Forces Institute of Pathology. (Third Edition). McGraw-Hill, 
New York. pp. 1-31 (1968).
    (15) Miale, I. L. and Sidman, R.L. An autoradiographic analysis of 
histogenesis in the mouse cerebellum. Experimental Neurology. 4:277-296 
(1961).
    (16) Miller, D.B. and Eckerman, D.A. Learning and memory measures. 
In: Neurobehavioral Toxicology, Z. Annau (ed). Johns Hopkins University 
Press, Baltimore. pp. 94-149 (1986).
    (17) Pender, M.P. A simple method for high resolution light 
microscopy of nervous tissue. Journal of Neuroscience Methods. 15:213-
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    (18) Ralis, H.M., Beesley, R.A., and Ralis, Z.A. Techniques in 
Neurohistology. Butterworths, London. pp. 57-145 (1973).
    (19) Rodier, P.M. and Gramann, W.J. Morphologic effects of 
interference with cell proliferation in the early fetal period. 
Neurobehavioral Toxicology 1:129-135 (1979).
    (20) Spear, N.E. and Campbell, B.A. (eds.) Ontogeny of Learning and 
Memory. Erlbaum, New Jersey. pp. 101-133, 157-224 (1979).
    (21) Spencer, P.S., Bischoff, M.C., and Schaumburg, H.H. 
Neuropathological methods for the detection of neurotoxic disease. In: 
Experimental and Clinical Neurotoxicology. Spencer, P.S. and Schaumburg, 
H.H. (eds.). Williams and Wilkins, Baltimore. pp. 743-757 (1980).
    (22) Suzuki, K. Special vulnerabilities of the developing nervous 
system to toxic substances. In: Experimental and Clinical 
Neurotoxicology. Spencer, P.S. and Schaumburg, H.H. (eds.). Williams and 
Wilkins, Baltimore. pp. 48-61 (1980). (23) Luna, L.G. (ed.). Manual of 
Histologic Staining Methods of the Armed Forces Institute of Pathology. 
(Third Edition). McGraw-Hill, New York. pp. 32-46 (1968).

[65 FR 78811, Dec. 15, 2000]



Sec. 799.9748  TSCA metabolism and pharmacokinetics

    (a) Scope. (1) This section is intended to meet the testing 
requirements under section 4 of the Toxic Substances Control Act (TSCA). 
(1) Testing of the disposition of a test substance is designed to obtain 
adequate information on its absorption, distribution, biotransformation, 
and excretion and to aid in understanding the mechanism of toxicity. 
Basic pharmacokinetic parameters determined from these studies will also 
provide information on the potential for accumulation of the test 
substance in tissues and/or organs and the potential for induction of 
biotransformation as a result of exposure to the test substance. These 
data can be used to assess the adequacy and relevance of the 
extrapolation of animal toxicity data (particularly chronic toxicity 
and/or carcinogenicity data) to human risk assessment.
    (2) Metabolism data can also be used to assist in determining 
whether animal toxicity studies have adequately addressed any toxicity 
concerns arising from exposure to plant metabolites, and in the setting 
of tolerances, if any, for those metabolites in raw agricultural 
commodities.
    (b) Source. The source material used in developing this TSCA test 
guideline is the Office of Prevention, Pesticides and Toxic Substances 
(OPPTS) harmonized test guideline 870.7485 (August 1998, final 
guideline). This source is available at the address in paragraph (h) of 
this section.
    (c) Definitions. The following definitions apply to this section.
    Metabolism (biotransformation) is the sum of the processes by which 
a foreign chemical is subjected to chemical change by living organisms.
    LOEL is the lowest observable effects level.
    NOEL is the no observable effects level.
    Pharmacokinetics is the quantitation and determination of the time 
course and dose dependency of the absorption, distribution, 
biotransformation, and excretion of chemicals.
    (d) Good laboratory practice standards. The pharmacokinetics and 
metabolism tests outlined in this guideline must conform to the 
laboratory practices stipulated in 40 CFR Part 792--Good Laboratory 
Practice Standards.
    (e) Test Procedures. Test procedures presented below utilize a tier 
system to minimize the use of resources and to allow flexibility in the 
conduct of metabolism studies. The proposed tier system consists of a 
basic data set (Tier 1) and additional studies (Tier 2). These 
additional studies may be requested based upon the existing toxicology 
data base and/or the results of Tier 1 testing which are found to impact 
upon the risk assessment process. For Tier 1 testing, the oral route 
will typically be required; however, if the use pattern results in other 
types of exposure, other routes (dermal and/or inhalation) may be 
required for initial testing of the disposition of a chemical substance. 
The registrant should justify the route of exposure to the Agency. 
Complete descriptions of the test procedures for these other routes of 
exposure can be found in paragraph (i) of

[[Page 461]]

this section. Except in unusual circumstances, the tiered approach to 
metabolism testing should apply to all listed routes of exposure.
    (1) Pilot studies. The use of pilot studies is recommended and 
encouraged for the selection of experimental conditions for the 
pharmacokinetics and metabolism studies (mass balance, analytical 
procedures, dose-finding, excretion of CO2, etc.).
    (2) Animal selection--(i) Species. The rat must normally be used for 
testing because it has been used extensively for metabolic and 
toxicological studies. The use of other or additional species may be 
required if critical toxicology studies demonstrate evidence of 
significant toxicity in these species or if metabolism is shown to be 
more relevant to humans in the test species.
    (ii) Strain. Adult animals of the strain used or proposed to be used 
for the determination of adverse health effects associated with the test 
substance.
    (3) Material to be tested--(i) Test substance. (A) A radiolabeled 
test substance using \14\C should be used for all material balance and 
metabolite identification aspects of the study. Other radioactive and 
stable isotopes may be used, particularly if the element is responsible 
for or is a part of the toxic portion of the compound. If it can be 
demonstrated that the material balance and metabolite identification 
requirements can be met using unlabeled test substance, then 
radiolabeled compound need not be used. If possible, the radiolabel 
should be located in a core portion of the molecule which is 
metabolically stable (it is not exchangeable, is not removed 
metabolically as CO2, and does not become part of the one-
carbon pool of the organism). Labeling of multiple sites of the molecule 
may be necessary to follow the metabolic fate of the compound.
    (B) The label should follow the test compound and/or its major 
metabolites until excreted. The radiopurity of the radioactive test 
substance shall be the highest attainable for a particular test 
substance (ideally it should be greater than 95%) and reasonable effort 
should be made to identify impurities present at or above 2%. The 
purity, along with the identity of major impurities which have been 
identified, shall be reported. For other segments of the study, 
nonradioactive test substance may be used if it can be demonstrated that 
the analytical specificity and sensitivity of the method used with 
nonradioactive test substance is equal to or greater than that which 
could be obtained with the radiolabeled test substance. The radioactive 
and nonradioactive test substances shall be analyzed using an 
appropriate method to establish purity and identity. Additional guidance 
will be provided in chemical specific test rules to assist in the 
definition and specifications of test substances composed of mixtures 
and methods for determination of purity.
    (ii) Administration of test substance. Test substance should be 
dissolved or suspended homogeneously in a vehicle usually employed for 
acute administration. A rationale for the choice of vehicle should be 
provided. The customary method of administration will be by oral gavage; 
however, administration by gelatin capsule or as a dietary mixture may 
be advantageous in specific situations. Verification of the actual dose 
administered to each animal should be provided.
    (4) Tier testing. (i) The multiplicity of metabolic parameters that 
impact the outcome of toxicological evaluations preclude the use of a 
universal study design for routine toxicological evaluation of a test 
substance. The usefulness of a particular study design depends upon the 
biological activity of a compound and circumstances of exposure. For 
these reasons, a tiered system is proposed for evaluation of the 
metabolism/kinetic properties of a test substance.
    (ii) The first tier data set is a definitive study by the 
appropriate route of exposure conducted in male rats to determine the 
routes and rate of excretion and to identify excreted metabolites. First 
tier data will also provide basic information for additional testing 
(Tier 2) if such testing is considered necessary. In the majority of 
cases, Tier 1 data are expected to satisfy regulatory requirements for 
biotransformation and pharmacokinetic data on test chemicals.

[[Page 462]]

    (iii) Second tier testing describes a variety of metabolism/kinetic 
experiments which address specific questions based on the existing 
toxicology data base and/or those results of Tier 1 testing impacting 
significantly on the risk assessment process. For conduct of these 
studies, individualized protocols may be necessary. Protocols for these 
studies, if required, can be developed as a cooperative effort between 
Agency and industry scientists.
    (f) Tier 1 data requirements (minimum data set). At this initial 
level of testing, biotransformation and pharmacokinetic data from a 
single low dose group will be required. This study will determine the 
rate and routes of excretion and the type of metabolites generated.
    (1) Number and sex of animals. A minimum of four male young adult 
animals must be used for Tier 1 testing. The use of both sexes may be 
required in cases where there is evidence to support significant sex-
related differences in toxicity.
    (2) Dose selection. (i) A single dose is required for each route of 
exposure. The dose should be nontoxic, but high enough to allow for 
metabolite identification in excreta. If no other toxicity data are 
available for selection of the low dose, a dose identified as a fraction 
of the LD50 (as determined from acute toxicity studies) may 
be used. The magnitude of the dose used in Tier 1 studies should be 
justified in the final report.
    (ii) For test substances of low toxicity a maximum dose of 1,000 mg/
kg should be used; chemical-specific considerations may necessitate a 
higher maximum dose and will be addressed in specific test rules.
    (3) Measurements--(i) Excretion. (A) Data obtained from this section 
(percent recovery of administered dose from urine, feces, and expired 
air) will be used to determine the rate and extent of excretion of test 
chemical, to assist in establishing mass balance, and will be used in 
conjunction with pharmacokinetic parameters to determine the extent of 
absorption. The quantities of radioactivity eliminated in the urine, 
feces, and expired air shall be determined separately at appropriate 
time intervals.
    (B) If a pilot study has shown that no significant amount of 
radioactivity is excreted in expired air, then expired air need not be 
collected in the definitive study.
    (C) Each animal must be placed in a separate metabolic unit for 
collection of excreta (urine, feces and expired air). At the end of each 
collection period, the metabolic units must be rinsed with appropriate 
solvent to ensure maximum recovery of radiolabel. Excreta collection 
must be terminated at 7 days, or after at least 90% of the administered 
dose has been recovered, whichever occurs first. The total quantities of 
radioactivity in urine must be determined at 6, 12, and 24 hours on day 
1 of collection, and daily thereafter until study termination, unless 
pilot studies suggest alternate or additional time points for 
collection. The total quantities of radioactivity in feces should be 
determined on a daily basis beginning at 24 hours post-dose, and daily 
thereafter until study termination. The collection of CO2 and 
other volatile materials may be discontinued when less than 1% of the 
administered dose is found in the exhaled air during a 24-hour 
collection period.
    (ii) Tissue distribution. At the termination of the Tier 1 study, 
the following tissues should be collected and stored frozen: Liver, fat, 
gastrointestinal tract, kidney, spleen, whole blood, and residual 
carcass. If it is determined that a significant amount of the 
administered dose is unaccounted for in the excreta, then data on the 
percent of the total (free and bound) radioactive dose in these tissues 
as well as residual carcass will be requested. Additional tissues must 
be included if there is evidence of target organ toxicity from 
subchronic or chronic toxicity studies. For other routes of exposure, 
specific tissues may also be required, such as lungs in inhalation 
studies and skin in dermal studies. Certain techniques currently at 
various stages of development, e.g., quantitative whole-body 
autoradiography, may prove useful in determining if a test substance 
concentrates in certain organs or in determining a specific pattern of 
distribution within a given tissue. The use of such techniques is 
encouraged, but not

[[Page 463]]

required, and may be employed to limit the number of tissues collected 
to those shown to contain a measurable amount of radioactivity.
    (iii) Metabolism. Excreta must be collected for identification and 
quantitation of unchanged test substance and metabolites as described in 
paragraph (f)(3)(i) of this section. Pooling of excreta to facilitate 
metabolite identification within a given dose group is acceptable. 
Profiling of metabolites from each time period is recommended. However, 
if lack of sample and/or radioactivity precludes this, pooling of urine 
as well as pooling of feces across several time points is acceptable. 
Appropriate qualitative and quantitative methods must be used to assay 
urine, feces, and expired air from treated animals. Reasonable efforts 
should be made to identify all metabolites present at 5% or greater of 
the administered dose and to provide a metabolic scheme for the test 
chemical. Compounds which have been characterized in excreta as 
comprising 5% or greater of the administered dose should be identified. 
If identification at this level is not possible, a justification/
explanation should be provided in the final report. Identification of 
metabolites representing less than 5% of the administered dose might be 
requested if such data are needed for risk assessment of the test 
chemical. Structural confirmation should be provided whenever possible. 
Validation of the methods used in metabolite identification should be 
included.
    (g) Tier 2 data requirements. Studies at the Tier 2 level are 
designed to answer questions about the disposition of test chemicals 
based on the existing toxicology data base and/or results of Tier 1 
testing which may have a significant impact on the risk assessment for 
the test chemical. Such studies may address questions regarding 
absorption, persistence, or distribution of the test chemical, or a 
definitive alteration in the metabolic profile occurring with dose which 
may be of toxicological concern. At the Tier 2 level, only those studies 
which address a specific concern are required, and if required must be 
conducted according to mutual agreement between the registrant and the 
Agency. Flexibility will be allowed in the design of specific 
experiments as warranted by technological advances in this field.
    (1) Absorption. (i) If the extent of absorption cannot be 
established from Tier 1 studies, or where greater than 20% of the 
administered dose is present in feces, a study to determine the extent 
of absorption will be required. This can be accomplished either through 
intravenous administration of test material and measurement of 
radioactivity in excreta or after oral administration of test material 
and measurement of radioactivity in bile.
    (ii) For the intravenous study, a single dose (not to exceed the 
oral dose used in Tier 1) of test chemical using an appropriate vehicle 
should be administered in a suitable volume (e.g., 1 mL/kg) at a 
suitable site to at least three male rats (both sexes might be used if 
warranted). The disposition of the test chemical should be monitored for 
oral dosing as outlined in paragraph (f)(3)(i) of this section. 
Metabolite identification will not be required for this study.
    (iii) If a biliary excretion study is chosen the oral route of 
administration may be requested. In this study, the bile ducts of at 
least three male rats (or of both sexes, if warranted) should be 
appropriately cannulated and a single dose of the test chemical should 
be administered to these rats. Following administration of the test 
chemical, excretion of radioactivity in bile should be monitored as long 
as necessary to determine if a significant percentage of the 
administered dose is excreted via this route.
    (2) Tissue distribution time course. (i) A time course of tissue 
distribution in selected tissues may be required to aid in the 
determination of a possible mode of toxic action. This concern may arise 
from evidence of extended half-life or possible accumulation of 
radioactivity in specific tissues. The selection of tissues for this 
type of study will be based upon available evidence of target organ 
toxicity and/or carcinogenicity, and the number of time points required 
will be based upon pharmacokinetic information obtained from Tier 1 
data. Flexibility will be allowed in the selection of time points to be 
studied.

[[Page 464]]

    (ii) For this type of study, three rats per time point will be 
administered an appropriate oral dose of test chemical, and the time 
course of distribution monitored in selected tissues. Only one sex may 
be required, unless target organ toxicity is observed in sex-specific 
organs. Assessment of tissue distribution will be made using appropriate 
techniques for assessment of total amount distributed to tissue and for 
assessment of metabolite distribution.
    (3) Plasma kinetics. The purpose of this experiment is to obtain 
estimates of basic pharmacokinetic parameters (half-life, volume of 
distribution, absorption rate constant, area under the curve) for the 
test substance. Kinetic data may be required if the data can be used to 
resolve issues about bioavailability and to clarify whether clearance is 
saturated in a dose-dependent fashion. For this experiment a minimum of 
three rats per group is required. At least two doses will be required, 
usually the NOEL and LOEL from the critical toxicology study. Following 
administration of test substance, samples should be obtained from each 
animal at suitable time points appropriate sampling methodology. Total 
radioactivity present (or total amount of chemical, for nonradioactive 
materials) should be analyzed in whole blood and plasma using 
appropriate methods, and the blood/plasma ratio should be calculated.
    (4) Induction. (i) Studies addressing possible induction of 
biotransformation may be requested under one or more of the following 
conditions:
    (A) Available evidence indicates a relationship between induced 
metabolism and enhanced toxicity.
    (B) The available toxicity data indicate a nonlinear relationship 
between dose and metabolism.
    (C) The results of Tier 1 metabolite identification studies show 
identification of a potentially toxic metabolite.
    (D) Induction can plausibly be invoked as a factor in such effects 
where status may depend on the level of inducible enzymes present. 
Several in vivo and in vitro methods are available for assessment of 
enzyme induction, and the experiments which best address the issue at 
hand can be determined between Agency and industry scientists. If 
induction is demonstrated, the relationship of this phenomenon to 
toxicity observed from subchronic and/or chronic toxicity studies will 
need to be addressed.
    (ii) [Reserved]
    (iii) If toxicologically significant alterations in the metabolic 
profile of the test chemical are observed through either in vitro or in 
vivo experiments, characterization of the enzyme(s) involved (for 
example, Phase I enzymes such as isozymes of the Cytochrome P450-
dependent mono-oxygenase system, Phase II enzymes such as isozymes of 
sulfotransferase or uridine diphosphate glucuronosyl transferase, or any 
other relevant enzymes) may be requested. This information will help 
establish the relevance of the involved enzyme(s) to human risk, as it 
is known that certain isozymes are present in animal species which are 
not present in humans, and vice versa.
    (5) Physiologically-based modeling. Traditional methods of modeling 
have been used to determine kinetic parameters associated with drug and 
xenobiotic disposition, but have assumed a purely mathematical construct 
of mammalian organisms in their operation. On the other hand, more 
recent models which take into account the physiological processes of the 
animal have been used with success in defining biological determinants 
of chemical disposition as well as the relationship between tissue dose 
and tissue response. These so-called physiologically-based models, also 
allow for cross-species extrapolation which is often necessary in the 
risk-assessment process. The use of physiologically-based modeling as an 
experimental tool for addressing specific issues related to 
biotransformation and pharmacokinetics of a test substance is 
encouraged. Information as derived from physiologically-based modeling 
experiments may aid in the comparison of biotransformation and 
pharmacokinetics of a test substance between animal species and humans, 
and in the assessment of risk under specific exposure conditions. At the 
discretion of the Agency, or by mutual agreement,

[[Page 465]]

results of physiologically based pharmacokinetic (PBPK) studies with 
parent compound may be submitted in lieu of other studies, if it is 
determined that such data would provide adequate information to satisfy 
this guideline.
    (h) Reporting of study results. In addition to the reporting 
requirements specified under EPA Good Laboratory Practice Standards at 
40 CFR part 792, subpart J, the completed study (Tier 1 or Tier 2) 
should be presented in the following format:
    (1) Title/cover page. Title page and additional requirements 
(requirements for data submission, good laboratory practice, statements 
of data confidentiality claims and quality assurance) if relevant to the 
study report, should precede the content of the study formatted below. 
These requirements are to be found in 40 CFR parts 790, 792, and 799.
    (2) Table of contents. A concise listing must precede the body of 
the report, containing all essential elements of the study and the page 
and table number where the element is located in the final report of the 
study. Essential elements of the table of contents should include a 
summary, an introduction, the materials and methods section, results, 
discussion/conclusions, references, tables, figures, appendices, and key 
subsections as deemed appropriate. The table of contents should include 
the page number of each of these elements.
    (3) Body of the report. The body of the report must include 
information required under this section, organized into sections and 
paragraphs as follows:
    (i) Summary. This section of the study report must contain a summary 
and analysis of the test results and a statement of the conclusions 
drawn from the analysis. This section should highlight the nature and 
magnitude of metabolites, tissue residue, rate of clearance, 
bioaccumulation potential, sex differences, etc. The summary should be 
presented in sufficient detail to permit independent evaluation of the 
findings.
    (ii) Introduction. This section of the report should include the 
objectives of the study, guideline references, regulatory history, if 
any, and a rationale.
    (iii) Materials and methods. This section of the report must include 
detailed descriptions of all elements including:
    (A) Test substance. (1) This section should include identification 
of the test substance--chemical name, molecular structure, qualitative 
and quantitative determination of its chemical composition, and type and 
quantities of any impurities whenever possible.
    (2) This section should also include information on physical 
properties including physical state, color, gross solubility and/or 
partition coefficient, and stability.
    (3) The type or description of any vehicle, diluents, suspending 
agents, and emulsifiers or other materials used in administering the 
test substance should be stated.
    (4) If the test substance is radiolabeled, information on the 
following should be included in this subsection: The type of 
radionuclide, position of label, specific activity, and radiopurity.
    (B) Test animals. This section should include information on the 
test animals, including: Species, strain, age at study initiation, sex, 
body weight, health status, and animal husbandry.
    (C) Methods. This subsection should include details of the study 
design and methodology used. It should include a description of:
    (1) How the dosing solution was prepared and the type of solvent, if 
any, used.
    (2) Number of treatment groups and number of animals per group.
    (3) Dosage levels and volume.
    (4) Route of administration.
    (5) Frequency of dosing.
    (6) Fasting period (if used).
    (7) Total radioactivity per animal.
    (8) Animal handling.
    (9) Sample collection.
    (10) Sample handling.
    (11) Analytical methods used for separation.
    (12) Quantitation and identification of metabolites.
    (13) Other experimental measurements and procedures employed 
(including validation of test methods for metabolite analysis).
    (D) Statistical analysis. If statistical analysis is used to analyze 
the study findings, then sufficient information on

[[Page 466]]

the method of analysis and the computer program employed should be 
included so that an independent reviewer/statistician can reevaluate and 
reconstruct the analysis. Presentation of models should include a full 
description of the model to allow independent reconstruction and 
validation of the model.
    (iv) Results. All data should be summarized and tabulated with 
appropriate statistical evaluation and placed in the text of this 
section. Radioactivity counting data should be summarized and presented 
as appropriate for the study, typically as disintegrations per minute 
and microgram or milligram equivalents, although other units may be 
used. Graphic illustrations of the findings, reproduction of 
representative chromatographic and spectrometric data, and proposed 
metabolic pathways and molecular structure of metabolites should be 
included in this section. In addition the following information is to be 
included in this section if applicable:
    (A) Justification for modification of exposure conditions, if 
applicable.
    (B) Justification for selection of dose levels for pharmacokinetic 
and metabolism studies.
    (C) Description of pilot studies used in the experimental design of 
the pharmacokinetic and metabolism studies, if applicable.
    (D) Quantity and percent recovery of radioactivity in urine, feces, 
and expired air, as appropriate. For dermal studies, include recovery 
data for treated skin, skin washes, and residual radioactivity in the 
covering apparatus and metabolic unit as well as results of the dermal 
washing study.
    (E) Tissue distribution reported as percent of administered dose and 
microgram equivalents per gram of tissue.
    (F) Material balance developed from each study involving the assay 
of body tissues and excreta.
    (G) Plasma levels and pharmacokinetic parameters after 
administration by the relevant routes of exposure.
    (H) Rate and extent of absorption of the test substance after 
administration by the relevant routes of exposure.
    (I) Quantities of the test substance and metabolites (reported as 
percent of the administered dose) collected in excreta.
    (J) Individual animal data.
    (v) Discussion and conclusions. (A) In this section the author(s) 
should:
    (1) Provide a plausible explanation of the metabolic pathway for the 
test chemical.
    (2) Emphasize species and sex differences whenever possible.
    (3) Discuss the nature and magnitude of metabolites, rates of 
clearance, bioaccumulation potential, and level of tissue residues as 
appropriate.
    (B) The author(s) should be able to derive a concise conclusion that 
can be supported by the findings of the study.
    (vi) Optional sections. The authors may include additional sections 
such as appendices, bibliography, tables, etc.
    (i) Alternate routes of exposure for Tier 1 testing--(1) Dermal--(i) 
Dermal treatment. One (or more if needed) dose levels of the test 
substance must be used in the dermal portion of the study. The low dose 
level should be selected in accordance with paragraph (f)(2) of this 
section. The dermal doses must be dissolved, if necessary, in a suitable 
vehicle and applied in a volume adequate to deliver the doses. Shortly 
before testing, fur is to be clipped from the dorsal area of the trunk 
of the test animals. Shaving may be employed, but it should be carried 
out approximately 24 hour before the test. When clipping or shaving the 
fur, care should be taken to avoid abrading the skin, which could alter 
its permeability. Approximately 10% of the body surface should be 
cleared for application of the test substance. With highly toxic 
substances, the surface area covered may be less than approximately 10%, 
but as much of the area as possible is to be covered with a thin and 
uniform film. The same nominal treatment surface area must be used for 
all dermal test groups. The dosed areas are to be protected with a 
suitable covering which is secured in place. The animals must be housed 
separately.
    (ii) Dermal washing study. (A) A washing experiment must be 
conducted to assess the removal of the applied dose of the test 
substance by washing the treated skin area with a mild soap and water. A 
single dose must be applied to

[[Page 467]]

two animals in accordance with paragraph (f)(2) of this section. After 
application (2 to 5 minutes) the treated areas of the animals must be 
washed with a mild soap and water. The amounts of test substance 
recovered in the washes must be determined to assess the effectiveness 
of removal by washing.
    (B) Unless precluded by corrosiveness, the test substance must be 
applied and kept on the skin for a minimum of 6 hours. At the time of 
removal of the covering, the treated area must be washed following the 
procedure as outlined in the dermal washing study. Both the covering and 
the washes must be analyzed for residual test substance. At the 
termination of the studies, each animal must be sacrificed and the 
treated skin removed. An appropriate section of treated skin must be 
analyzed to determine residual radioactivity.
    (2) Inhalation. A single (or more if needed) concentration of test 
substance must be used in this portion of the study. The concentration 
should be selected in accordance with paragraph (f)(2) of this section. 
Inhalation treatments are to be conducted using a ``nose-cone'' or 
``head-only'' apparatus to prevent absorption by alternate routes of 
exposure. If other inhalation exposure conditions are proposed for use 
in a chemical-specific test rule, justification for the modification 
must be documented. A single exposure over a defined period must be used 
for each group--a typical exposure is 4-6 hours.

[65 FR 78815, Dec. 15, 2000]



Sec. 799.9780  TSCA immunotoxicity.

    (a) Scope. This section is intended to meet the testing requirements 
under section 4 of TSCA. This section is intended to provide information 
on suppression of the immune system which might occur as a result of 
repeated exposure to a test chemical. While some information on 
potential immunotoxic effects may be obtained from hematology, lymphoid 
organ weights and histopathology (usually done as part of routine 
toxicity testing), there are data which demonstrate that these endpoints 
alone are not sufficient to predict immunotoxicity (Luster et al., 1992, 
1993 see paragraphs (j)(8) and (j)(9) of this section). Therefore, the 
tests described in this section are intended to be used along with data 
from routine toxicity testing, to provide more accurate information on 
risk to the immune system. The tests in this section do not represent a 
comprehensive assessment of immune function.
    (b) Source. The source material used in developing this TSCA test 
guideline is the OPPTS harmonized test guideline 870.7800 (June 1996 
Public Draft). This source is available at the address in paragraph (j) 
of this section.
    (c) Definitions. The following definitions apply to this section.
    Antibodies or immunoglobulins (Ig) are part of a large family of 
glycoprotein molecules. They are produced by B cells in response to 
antigens, and bind specifically to the eliciting antigen. The different 
classes of immunoglobulins involved in immunity are IgG, IgA, IgM, IgD, 
and IgE. Antibodies are found in extracellular fluids, such as serum, 
saliva, milk, and lymph. Most antibody responses are T cell-dependent, 
that is, functional T and B lymphocytes, as well as antigen-presenting 
cells (usually macrophages), are required for the production of 
antibodies.
    Cluster of differentiation (CD) refers to molecules expressed on the 
cell surface. These molecules are useful as distinct CD molecules are 
found on different populations of cells of the immune system. Antibodies 
against these cell surface markers (e.g., CD4, CD8) are used to identify 
and quantitate different cell populations.
    Immunotoxicity refers to the ability of a test substance to suppress 
immune responses that could enhance the risk of infectious or neoplastic 
disease, or to induce inappropriate stimulation of the immune system, 
thus contributing to allergic or autoimmune disease. This section only 
addresses potential immune suppression.
    Natural Killer (NK) cells are large granular lymphocytes which 
nonspecifically lyse cells bearing tumor or viral antigens. NK cells are 
up-regulated soon after infection by certain microorganisms, and are 
thought to represent the first line of defense against viruses and 
tumors.

[[Page 468]]

    T and B cells are lymphocytes which are activated in response to 
specific antigens (foreign substances, usually proteins). B cells 
produce antigen-specific antibodies (see the definition for ``antibodies 
or immunoglobulins''), and subpopulations of T cells are frequently 
needed to provide help for the antibody response. Other types of T cell 
participate in the direct destruction of cells expressing specific 
foreign (tumor or infectious agent) antigens on the cell surface.
    (d) Principles of the test methods. (1) In order to obtain data on 
the functional responsiveness of major components of the immune system 
to a T cell dependent antigen, sheep red blood cells (SRBC), rats and/or 
mice \1\ shall be exposed to the test and control substances for at 
least 28 days. \2\ The animals shall be immunized by intravenous or 
intraperitoneal injection of SRBCs approximately 4 days (depending on 
the strain of animal) prior to the end of the exposure. At the end of 
the exposure period, either the plaque forming cell (PFC) assay or an 
enzyme linked immunosorbent assay (ELISA) shall be performed to 
determine the effects of the test substance on the splenic anti-SRBC 
(IgM) response or serum anti-SRBC IgM levels, respectively.
---------------------------------------------------------------------------

    \1\ If absorption/distribution/metabolism/excretion (ADME) data are 
similar between species, then either rats or mice may be used for the 
test compound in question. If such data are lacking, both species should 
be used.
    \2\ Because there is a fairly rapid turnover of many of the cells in 
the immune system, 28 days is considered sufficient for the purposes of 
the anti-SRBC tests.
---------------------------------------------------------------------------

    (2) In the event the test substance produces significant suppression 
of the anti-SRBC response, expression of phenotypic markers for major 
lymphocyte populations (total T and total B), and T cell subpopulations 
(T helpers (CD\4\) and T cytotoxic/suppressors (CD\8\)), as assessed by 
flow cytometry, may be performed to determine the effects of the test 
substance on either splenic or peripheral-blood lymphocyte populations 
and T cell subpopulations. When this study is performed, the appropriate 
monoclonal antibodies for the species being tested should be used. If 
the test substance has no significant effect on the anti-SRBC assay, a 
functional test for NK cells may be performed to test for a chemical's 
effect on non-specific immunity. \3\ For tests performed using cells or 
sera from blood (ELISA or flow cytometry), it is not necessary to 
destroy the animals, since immunization with SRBCs at 28 days is not 
expected to markedly affect the results of other assays included in 
subchronic or longer-term studies (these tests are discussed in the 
reference under paragraph (j)(7) of this section). The necessity to 
perform either a quantitative analysis of the effects of a chemical on 
the numbers of cells in major lymphocyte populations and T Cell 
subpopulations by flow cytometry, or a splenic NK cell activity assay to 
assess the effects of the test compound on non-specific immunity shall 
be determined on a case-by-case basis, depending upon the outcome of the 
anti-SRBC assay.
---------------------------------------------------------------------------

    \3\ When these optional tests are included, the phenotypic or NK 
cell analyses may be performed at 28 days of exposure, or at a later 
timepoint if ADME data suggest that a longer exposure is more 
appropriate.
---------------------------------------------------------------------------

    (e) Limit test. If a test at one dose level of at least 1,000 mg/kg 
body weight (or 2 mg/L for inhalation route of exposure) using the 
procedures described for this study produces no observable toxic effects 
or if toxic effects would not be expected based upon data of 
structurally related compounds, then a full study using three dose 
levels might not be necessary. Expected human exposure may indicate the 
need for a higher dose level.
    (f) Test procedures--(1) Animal selection--(i) Species and strain. 
These tests are intended for use in rats and/or mice. Commonly used 
laboratory strains shall be employed. \4\ All test animals shall be free 
of pathogens, internal and external parasites. Females shall be 
nulliparous and nonpregnant. The species, strain, and source of the 
animals shall be identified.
---------------------------------------------------------------------------

    \4\ The study director shall be aware of strain differences in 
response to SRBC. For example, if the 
B6C3F1 hybrid mouse is used in the PFC 
assay, a response of 800-1,000 PFC/10\6\ spleen cells in control mice 
should be the minimally acceptable PFC response.

---------------------------------------------------------------------------

[[Page 469]]

    (ii) Age/weight. (A) Young, healthy animals shall be employed. At 
the commencement of the study, the weight variation of the animals used 
shall not exceed 20% of the mean weight for each 
sex.
    (B) Dosing shall begin when the test animals are between 6 and 8 
weeks old.
    (iii) Sex. Either sex may be used in the study; if one sex is known 
or believed to be more sensitive to the test compound, then that sex 
shall be used.
    (iv) Numbers. (A) At least eight animals shall be included in each 
dose and control group. The number of animals tested shall yield 
sufficient statistical power to detect a 20% change based upon the 
interanimal variation which may be encountered in these assays.
    (B) To avoid bias, the use of adequate randomization procedures for 
the proper allocation of animals to test and control groups is required.
    (C) Each animal shall be assigned a unique identification number. 
Dead animals, their preserved organs and tissues, and microscopic slides 
shall be identified by reference to the animal's unique number.
    (v) Husbandry. (A) Animals may be group-caged by sex, but the number 
of animals per cage shall not interfere with clear observation of each 
animal. The biological properties of the test substance or toxic effects 
(e.g., morbidity, excitability) may indicate a need for individual 
caging.
    (B) The temperature of the experimental animal rooms shall be at 22 
3 [deg]C.
    (C) The relative humidity of the experimental animal rooms shall be 
between 30 and 70%.
    (D) Where lighting is artificial, the sequence shall be 12 hrs 
light, 12 hrs dark.
    (E) Control and test animals shall be maintained on the same type of 
bedding and receive feed from the same lot. The feed shall be analyzed 
to assure adequacy of nutritional requirements of the species tested and 
for impurities that might influence the outcome of the test. Rodents 
shall be fed and watered ad libitum with food replaced at least weekly.
    (F) The study shall not be initiated until the animals have been 
allowed an adequate period of acclimatization or quarantine to 
environmental conditions. The period of acclimatization shall be at 
least 1 week in duration.
    (2) Control and test substances. (i) The test substance shall be 
dissolved or suspended in a suitable vehicle. Ideally, if a vehicle or 
diluent is needed, it shall not elicit toxic effects or substantially 
alter the chemical or toxicological properties of the test substance. It 
is recommended that an aqueous solution should be used. If solubility is 
a problem a solution in oil may be used. Other vehicles may be 
considered, but only as a last resort.
    (ii) One lot of the test substance shall be used, if possible, 
throughout the duration of the study, and the research sample shall be 
stored under conditions that maintain its purity and stability. Prior to 
the initiation of the study, there shall be a characterization of the 
test substance, including the purity of the test compound and if 
technically feasible, the name and quantities of any known contaminants 
and impurities.
    (iii) If the test or positive control substance is to be 
incorporated into feed or another vehicle, the period during which the 
test substance is stable in such a mixture shall be determined prior to 
the initiation of the study. Its homogeneity and concentration shall 
also be determined prior to the initiation of the study and periodically 
during the study. Statistically randomized samples of the mixture shall 
be analyzed to ensure that proper mixing, formulation, and storage 
procedures are being followed, and that the appropriate concentration of 
the test or control substance is contained in the mixture.
    (3) Control groups. (i) A concurrent, vehicle-treated control group 
is required.
    (ii) A separate untreated control group is required if the toxicity 
of the vehicle is unknown.
    (iii) A positive control group with a known immunosuppressant (e.g., 
cyclophosphamide) shall be included in the study. A group of at least 
eight animals shall be given the immunosuppressive chemical.
    (4) Dose levels. (i) In repeated-dose toxicity tests, it is 
desirable to have a

[[Page 470]]

dose-response relationship and a no observed immunotoxic effect level. 
Therefore, at least three dose levels and a negative control shall be 
used, unless a limit test is performed as specified under paragraph (e) 
of this section.
    (ii) The highest dose level shall not produce significant stress, 
malnutrition, or fatalities, but ideally should produce some measurable 
sign of general toxicity (e.g., a 10% loss of body weight).
    (iii) The lowest dose level ideally shall not produce any evidence 
of immunotoxicity.
    (5) Administration of the test substance. (i) The test substance, 
vehicle, or positive control substance shall be administered for at 
least 28 days for the anti-SRBC assay. The route of administration of 
the test material will usually be oral; however, this shall be 
determined by the likely route of occupational or indoor exposure. 
Therefore, under certain conditions, the dermal or inhalation route of 
exposure may be more relevant for the study. All animals shall be dosed 
by the same method during the entire experimental period.
    (ii) If the test substance is administered by gavage, the animals 
are dosed with the test substance ideally on a 7-days-per-week basis. 
However, based primarily on practical considerations, dosing by gavage 
on a 5-days-per-week basis shall be acceptable. If the test substance is 
administered in the drinking water, or mixed directly into the diet, 
then exposure shall be on a 7-days-per-week basis.
    (A) For substances of low toxicity, it is important to ensure that 
when administered in the diet, the quantities of the test substance 
involved do not interfere with normal nutrition. When the test substance 
is administered in the diet, either a constant dietary concentration in 
parts per million (ppm) or a constant dose level in terms of the 
animal's body weight shall be used; the alternative used should be 
specified.
    (B) For a substance administered by gavage, the dose shall be given 
at approximately the same time each day, and adjusted at intervals 
(weekly for mice, twice per week for rats) to maintain a constant dose 
level in terms of the animal's body weight.
    (iii) If the test substance is administered dermally, use paragraphs 
(f)(5)(iii)(A) through (f)(5)(iii)(D) of this section.
    (A) Dose levels and dose selection. (1) In this test, it is 
desirable to determine a dose-response relationship as well as a NOEL. 
Therefore, at least three dose levels plus a control and, where 
appropriate, a vehicle control (corresponding to the concentration of 
vehicle at the highest dose level) group should be used. Doses should be 
spaced appropriately to produce test groups with a range of toxic 
effects. The data should be sufficient to produce a dose-response curve.
    (2) The highest dose level should elicit signs of toxicity but not 
produce severe skin irritation or an incidence of fatality which would 
prevent a meaningful evaluation. If application of the test substance 
produces severe skin irritation, the concentration may be reduced, 
although this may result in a reduction in, or absence of, other toxic 
effects at the high dose level. If the skin has been badly damaged early 
in the study, it may be necessary to terminate the study and undertake a 
new one at lower concentrations.
    (3) The intermediate dose levels should be spaced to produce a 
gradation of toxic effects.
    (4) The lowest dose level should not produce any evidence of toxic 
effects.
    (B) Preparation of animal skin. Shortly before testing, fur should 
be clipped from not less than 10% of the body surface area for 
application of the test substance. In order to dose approximately 10% of 
the body surface, the area starting at the scapulae (shoulders) to the 
wing of the ileum (hipbone) and half-way down the flank on each side of 
the animal should be shaved. Shaving should be carried out approximately 
24 hrs before dosing. Repeated clipping or shaving is usually needed at 
approximately weekly intervals. When clipping or shaving the fur, care 
should be taken to avoid abrading the skin which could alter its 
permeability.
    (C) Preparation of test substance. (1) Liquid test substances are 
generally used undiluted, except as indicated in paragraph 
(f)(5)(iii)(A)(2) of this section.

[[Page 471]]

    (2) Solids should be pulverized when possible. The substance should 
be moistened sufficiently with water or, when necessary, a suitable 
vehicle to ensure good contact with the skin. When a vehicle is used, 
the influence of the vehicle on toxicity of, and penetration of the skin 
by, the test substance should be taken into account.
    (3) The volume of application should be kept constant, e.g. less 
than 300 
                       SUBCHAPTERS S-T [RESERVED]



[[Page 477]]



                              FINDING AIDS




  --------------------------------------------------------------------

  A list of CFR titles, subtitles, chapters, subchapters and parts and 
an alphabetical list of agencies publishing in the CFR are included in 
the CFR Index and Finding Aids volume to the Code of Federal Regulations 
which is published separately and revised annually.

  Table of CFR Titles and Chapters
  Alphabetical List of Agencies Appearing in the CFR
  List of CFR Sections Affected

[[Page 479]]



                    Table of CFR Titles and Chapters




                      (Revised as of July 1, 2009)

                      Title 1--General Provisions

         I  Administrative Committee of the Federal Register 
                (Parts 1--49)
        II  Office of the Federal Register (Parts 50--299)
        IV  Miscellaneous Agencies (Parts 400--500)

                    Title 2--Grants and Agreements

            Subtitle A--Office of Management and Budget Guidance 
                for Grants and Agreements
         I  Office of Management and Budget Governmentwide 
                Guidance for Grants and Agreements (Parts 100--
                199)
        II  Office of Management and Budget Circulars and Guidance 
                (200--299)
            Subtitle B--Federal Agency Regulations for Grants and 
                Agreements
       III  Department of Health and Human Services (Parts 300-- 
                399)
        VI  Department of State (Parts 600--699)
      VIII  Department of Veterans Affairs (Parts 800--899)
        IX  Department of Energy (Parts 900--999)
        XI  Department of Defense (Parts 1100--1199)
       XII  Department of Transportation (Parts 1200--1299)
      XIII  Department of Commerce (Parts 1300--1399)
       XIV  Department of the Interior (Parts 1400--1499)
        XV  Environmental Protection Agency (Parts 1500--1599)
     XVIII  National Aeronautics and Space Administration (Parts 
                1880--1899)
      XXII  Corporation for National and Community Service (Parts 
                2200--2299)
     XXIII  Social Security Administration (Parts 2300--2399)
      XXIV  Housing and Urban Development (Parts 2400--2499)
       XXV  National Science Foundation (Parts 2500--2599)
      XXVI  National Archives and Records Administration (Parts 
                2600--2699)
     XXVII  Small Business Administration (Parts 2700--2799)
    XXVIII  Department of Justice (Parts 2800--2899)
      XXXI  Institute of Museum and Library Services (Parts 3100--
                3199)
     XXXII  National Endowment for the Arts (Parts 3200--3299)
    XXXIII  National Endowment for the Humanities (Parts 3300--
                3399)

[[Page 480]]

      XXXV  Export-Import Bank of the United States (Parts 3500--
                3599)
    XXXVII  Peace Corps (Parts 3700--3799)

                Title 3--The PresidentCHAPNO

            Presidential Documents
         I  Executive Office of the President (Parts 100--199)

                           Title 4--Accounts

         I  Government Accountability Office (Parts 1--99)

                   Title 5--Administrative Personnel

         I  Office of Personnel Management (Parts 1--1199)
        II  Merit Systems Protection Board (Parts 1200--1299)
       III  Office of Management and Budget (Parts 1300--1399)
         V  The International Organizations Employees Loyalty 
                Board (Parts 1500--1599)
        VI  Federal Retirement Thrift Investment Board (Parts 
                1600--1699)
      VIII  Office of Special Counsel (Parts 1800--1899)
        IX  Appalachian Regional Commission (Parts 1900--1999)
        XI  Armed Forces Retirement Home (Parts 2100--2199)
       XIV  Federal Labor Relations Authority, General Counsel of 
                the Federal Labor Relations Authority and Federal 
                Service Impasses Panel (Parts 2400--2499)
        XV  Office of Administration, Executive Office of the 
                President (Parts 2500--2599)
       XVI  Office of Government Ethics (Parts 2600--2699)
       XXI  Department of the Treasury (Parts 3100--3199)
      XXII  Federal Deposit Insurance Corporation (Parts 3200--
                3299)
     XXIII  Department of Energy (Parts 3300--3399)
      XXIV  Federal Energy Regulatory Commission (Parts 3400--
                3499)
       XXV  Department of the Interior (Parts 3500--3599)
      XXVI  Department of Defense (Parts 3600-- 3699)
    XXVIII  Department of Justice (Parts 3800--3899)
      XXIX  Federal Communications Commission (Parts 3900--3999)
       XXX  Farm Credit System Insurance Corporation (Parts 4000--
                4099)
      XXXI  Farm Credit Administration (Parts 4100--4199)
    XXXIII  Overseas Private Investment Corporation (Parts 4300--
                4399)
      XXXV  Office of Personnel Management (Parts 4500--4599)
        XL  Interstate Commerce Commission (Parts 5000--5099)
       XLI  Commodity Futures Trading Commission (Parts 5100--
                5199)
      XLII  Department of Labor (Parts 5200--5299)
     XLIII  National Science Foundation (Parts 5300--5399)
       XLV  Department of Health and Human Services (Parts 5500--
                5599)

[[Page 481]]

      XLVI  Postal Rate Commission (Parts 5600--5699)
     XLVII  Federal Trade Commission (Parts 5700--5799)
    XLVIII  Nuclear Regulatory Commission (Parts 5800--5899)
         L  Department of Transportation (Parts 6000--6099)
       LII  Export-Import Bank of the United States (Parts 6200--
                6299)
      LIII  Department of Education (Parts 6300--6399)
       LIV  Environmental Protection Agency (Parts 6400--6499)
        LV  National Endowment for the Arts (Parts 6500--6599)
       LVI  National Endowment for the Humanities (Parts 6600--
                6699)
      LVII  General Services Administration (Parts 6700--6799)
     LVIII  Board of Governors of the Federal Reserve System 
                (Parts 6800--6899)
       LIX  National Aeronautics and Space Administration (Parts 
                6900--6999)
        LX  United States Postal Service (Parts 7000--7099)
       LXI  National Labor Relations Board (Parts 7100--7199)
      LXII  Equal Employment Opportunity Commission (Parts 7200--
                7299)
     LXIII  Inter-American Foundation (Parts 7300--7399)
      LXIV  Merit Systems Protection Board (Parts 7400--7499)
       LXV  Department of Housing and Urban Development (Parts 
                7500--7599)
      LXVI  National Archives and Records Administration (Parts 
                7600--7699)
     LXVII  Institute of Museum and Library Services (Parts 7700--
                7799)
    LXVIII  Commission on Civil Rights (Parts 7800--7899)
      LXIX  Tennessee Valley Authority (Parts 7900--7999)
      LXXI  Consumer Product Safety Commission (Parts 8100--8199)
    LXXIII  Department of Agriculture (Parts 8300--8399)
     LXXIV  Federal Mine Safety and Health Review Commission 
                (Parts 8400--8499)
     LXXVI  Federal Retirement Thrift Investment Board (Parts 
                8600--8699)
    LXXVII  Office of Management and Budget (Parts 8700--8799)
     XCVII  Department of Homeland Security Human Resources 
                Management System (Department of Homeland 
                Security--Office of Personnel Management) (Parts 
                9700--9799)
      XCIX  Department of Defense Human Resources Management and 
                Labor Relations Systems (Department of Defense--
                Office of Personnel Management) (Parts 9900--9999)

                      Title 6--Domestic Security

         I  Department of Homeland Security, Office of the 
                Secretary (Parts 0--99)

                         Title 7--Agriculture

            Subtitle A--Office of the Secretary of Agriculture 
                (Parts 0--26)

[[Page 482]]

            Subtitle B--Regulations of the Department of 
                Agriculture
         I  Agricultural Marketing Service (Standards, 
                Inspections, Marketing Practices), Department of 
                Agriculture (Parts 27--209)
        II  Food and Nutrition Service, Department of Agriculture 
                (Parts 210--299)
       III  Animal and Plant Health Inspection Service, Department 
                of Agriculture (Parts 300--399)
        IV  Federal Crop Insurance Corporation, Department of 
                Agriculture (Parts 400--499)
         V  Agricultural Research Service, Department of 
                Agriculture (Parts 500--599)
        VI  Natural Resources Conservation Service, Department of 
                Agriculture (Parts 600--699)
       VII  Farm Service Agency, Department of Agriculture (Parts 
                700--799)
      VIII  Grain Inspection, Packers and Stockyards 
                Administration (Federal Grain Inspection Service), 
                Department of Agriculture (Parts 800--899)
        IX  Agricultural Marketing Service (Marketing Agreements 
                and Orders; Fruits, Vegetables, Nuts), Department 
                of Agriculture (Parts 900--999)
         X  Agricultural Marketing Service (Marketing Agreements 
                and Orders; Milk), Department of Agriculture 
                (Parts 1000--1199)
        XI  Agricultural Marketing Service (Marketing Agreements 
                and Orders; Miscellaneous Commodities), Department 
                of Agriculture (Parts 1200--1299)
       XIV  Commodity Credit Corporation, Department of 
                Agriculture (Parts 1400--1499)
        XV  Foreign Agricultural Service, Department of 
                Agriculture (Parts 1500--1599)
       XVI  Rural Telephone Bank, Department of Agriculture (Parts 
                1600--1699)
      XVII  Rural Utilities Service, Department of Agriculture 
                (Parts 1700--1799)
     XVIII  Rural Housing Service, Rural Business-Cooperative 
                Service, Rural Utilities Service, and Farm Service 
                Agency, Department of Agriculture (Parts 1800--
                2099)
        XX  Local Television Loan Guarantee Board (Parts 2200--
                2299)
      XXVI  Office of Inspector General, Department of Agriculture 
                (Parts 2600--2699)
     XXVII  Office of Information Resources Management, Department 
                of Agriculture (Parts 2700--2799)
    XXVIII  Office of Operations, Department of Agriculture (Parts 
                2800--2899)
      XXIX  Office of Energy Policy and New Uses, Department of 
                Agriculture (Parts 2900--2999)
       XXX  Office of the Chief Financial Officer, Department of 
                Agriculture (Parts 3000--3099)
      XXXI  Office of Environmental Quality, Department of 
                Agriculture (Parts 3100--3199)

[[Page 483]]

     XXXII  Office of Procurement and Property Management, 
                Department of Agriculture (Parts 3200--3299)
    XXXIII  Office of Transportation, Department of Agriculture 
                (Parts 3300--3399)
     XXXIV  Cooperative State Research, Education, and Extension 
                Service, Department of Agriculture (Parts 3400--
                3499)
      XXXV  Rural Housing Service, Department of Agriculture 
                (Parts 3500--3599)
     XXXVI  National Agricultural Statistics Service, Department 
                of Agriculture (Parts 3600--3699)
    XXXVII  Economic Research Service, Department of Agriculture 
                (Parts 3700--3799)
   XXXVIII  World Agricultural Outlook Board, Department of 
                Agriculture (Parts 3800--3899)
       XLI  [Reserved]
      XLII  Rural Business-Cooperative Service and Rural Utilities 
                Service, Department of Agriculture (Parts 4200--
                4299)
         L  Rural Business-Cooperative Service, Rurual Housing 
                Service, and Rural Utilities Service, Department 
                of Agriculture (Parts 5000--5099)

                    Title 8--Aliens and Nationality

         I  Department of Homeland Security (Immigration and 
                Naturalization) (Parts 1--499)
         V  Executive Office for Immigration Review, Department of 
                Justice (Parts 1000--1399)

                 Title 9--Animals and Animal Products

         I  Animal and Plant Health Inspection Service, Department 
                of Agriculture (Parts 1--199)
        II  Grain Inspection, Packers and Stockyards 
                Administration (Packers and Stockyards Programs), 
                Department of Agriculture (Parts 200--299)
       III  Food Safety and Inspection Service, Department of 
                Agriculture (Parts 300--599)

                           Title 10--Energy

         I  Nuclear Regulatory Commission (Parts 0--199)
        II  Department of Energy (Parts 200--699)
       III  Department of Energy (Parts 700--999)
         X  Department of Energy (General Provisions) (Parts 
                1000--1099)
      XIII  Nuclear Waste Technical Review Board (Parts 1303--
                1399)
      XVII  Defense Nuclear Facilities Safety Board (Parts 1700--
                1799)
     XVIII  Northeast Interstate Low-Level Radioactive Waste 
                Commission (Parts 1800--1899)

[[Page 484]]

                      Title 11--Federal Elections

         I  Federal Election Commission (Parts 1--9099)
        II  Election Assistance Commission (Parts9400--9499)

                      Title 12--Banks and Banking

         I  Comptroller of the Currency, Department of the 
                Treasury (Parts 1--199)
        II  Federal Reserve System (Parts 200--299)
       III  Federal Deposit Insurance Corporation (Parts 300--399)
        IV  Export-Import Bank of the United States (Parts 400--
                499)
         V  Office of Thrift Supervision, Department of the 
                Treasury (Parts 500--599)
        VI  Farm Credit Administration (Parts 600--699)
       VII  National Credit Union Administration (Parts 700--799)
      VIII  Federal Financing Bank (Parts 800--899)
        IX  Federal Housing Finance Board (Parts 900--999)
        XI  Federal Financial Institutions Examination Council 
                (Parts 1100--1199)
       XII  Federal Housing Finance Agency (Parts 1200--1299)
       XIV  Farm Credit System Insurance Corporation (Parts 1400--
                1499)
        XV  Department of the Treasury (Parts 1500--1599)
      XVII  Office of Federal Housing Enterprise Oversight, 
                Department of Housing and Urban Development (Parts 
                1700--1799)
     XVIII  Community Development Financial Institutions Fund, 
                Department of the Treasury (Parts 1800--1899)

               Title 13--Business Credit and Assistance

         I  Small Business Administration (Parts 1--199)
       III  Economic Development Administration, Department of 
                Commerce (Parts 300--399)
        IV  Emergency Steel Guarantee Loan Board (Parts 400--499)
         V  Emergency Oil and Gas Guaranteed Loan Board (Parts 
                500--599)

                    Title 14--Aeronautics and Space

         I  Federal Aviation Administration, Department of 
                Transportation (Parts 1--199)
        II  Office of the Secretary, Department of Transportation 
                (Aviation Proceedings) (Parts 200--399)
       III  Commercial Space Transportation, Federal Aviation 
                Administration, Department of Transportation 
                (Parts 400--499)
         V  National Aeronautics and Space Administration (Parts 
                1200--1299)
        VI  Air Transportation System Stabilization (Parts 1300--
                1399)

[[Page 485]]

                 Title 15--Commerce and Foreign Trade

            Subtitle A--Office of the Secretary of Commerce (Parts 
                0--29)
            Subtitle B--Regulations Relating to Commerce and 
                Foreign Trade
         I  Bureau of the Census, Department of Commerce (Parts 
                30--199)
        II  National Institute of Standards and Technology, 
                Department of Commerce (Parts 200--299)
       III  International Trade Administration, Department of 
                Commerce (Parts 300--399)
        IV  Foreign-Trade Zones Board, Department of Commerce 
                (Parts 400--499)
       VII  Bureau of Industry and Security, Department of 
                Commerce (Parts 700--799)
      VIII  Bureau of Economic Analysis, Department of Commerce 
                (Parts 800--899)
        IX  National Oceanic and Atmospheric Administration, 
                Department of Commerce (Parts 900--999)
        XI  Technology Administration, Department of Commerce 
                (Parts 1100--1199)
      XIII  East-West Foreign Trade Board (Parts 1300--1399)
       XIV  Minority Business Development Agency (Parts 1400--
                1499)
            Subtitle C--Regulations Relating to Foreign Trade 
                Agreements
        XX  Office of the United States Trade Representative 
                (Parts 2000--2099)
            Subtitle D--Regulations Relating to Telecommunications 
                and Information
     XXIII  National Telecommunications and Information 
                Administration, Department of Commerce (Parts 
                2300--2399)

                    Title 16--Commercial Practices

         I  Federal Trade Commission (Parts 0--999)
        II  Consumer Product Safety Commission (Parts 1000--1799)

             Title 17--Commodity and Securities Exchanges

         I  Commodity Futures Trading Commission (Parts 1--199)
        II  Securities and Exchange Commission (Parts 200--399)
        IV  Department of the Treasury (Parts 400--499)

          Title 18--Conservation of Power and Water Resources

         I  Federal Energy Regulatory Commission, Department of 
                Energy (Parts 1--399)
       III  Delaware River Basin Commission (Parts 400--499)
        VI  Water Resources Council (Parts 700--799)

[[Page 486]]

      VIII  Susquehanna River Basin Commission (Parts 800--899)
      XIII  Tennessee Valley Authority (Parts 1300--1399)

                       Title 19--Customs Duties

         I  Bureau of Customs and Border Protection, Department of 
                Homeland Security; Department of the Treasury 
                (Parts 0--199)
        II  United States International Trade Commission (Parts 
                200--299)
       III  International Trade Administration, Department of 
                Commerce (Parts 300--399)
        IV  Bureau of Immigration and Customs Enforcement, 
                Department of Homeland Security (Parts 400--599)

                     Title 20--Employees' Benefits

         I  Office of Workers' Compensation Programs, Department 
                of Labor (Parts 1--199)
        II  Railroad Retirement Board (Parts 200--399)
       III  Social Security Administration (Parts 400--499)
        IV  Employees Compensation Appeals Board, Department of 
                Labor (Parts 500--599)
         V  Employment and Training Administration, Department of 
                Labor (Parts 600--699)
        VI  Employment Standards Administration, Department of 
                Labor (Parts 700--799)
       VII  Benefits Review Board, Department of Labor (Parts 
                800--899)
      VIII  Joint Board for the Enrollment of Actuaries (Parts 
                900--999)
        IX  Office of the Assistant Secretary for Veterans' 
                Employment and Training Service, Department of 
                Labor (Parts 1000--1099)

                       Title 21--Food and Drugs

         I  Food and Drug Administration, Department of Health and 
                Human Services (Parts 1--1299)
        II  Drug Enforcement Administration, Department of Justice 
                (Parts 1300--1399)
       III  Office of National Drug Control Policy (Parts 1400--
                1499)

                      Title 22--Foreign Relations

         I  Department of State (Parts 1--199)
        II  Agency for International Development (Parts 200--299)
       III  Peace Corps (Parts 300--399)
        IV  International Joint Commission, United States and 
                Canada (Parts 400--499)
         V  Broadcasting Board of Governors (Parts 500--599)
       VII  Overseas Private Investment Corporation (Parts 700--
                799)
        IX  Foreign Service Grievance Board (Parts 900--999)

[[Page 487]]

         X  Inter-American Foundation (Parts 1000--1099)
        XI  International Boundary and Water Commission, United 
                States and Mexico, United States Section (Parts 
                1100--1199)
       XII  United States International Development Cooperation 
                Agency (Parts 1200--1299)
      XIII  Millenium Challenge Corporation (Parts 1300--1399)
       XIV  Foreign Service Labor Relations Board; Federal Labor 
                Relations Authority; General Counsel of the 
                Federal Labor Relations Authority; and the Foreign 
                Service Impasse Disputes Panel (Parts 1400--1499)
        XV  African Development Foundation (Parts 1500--1599)
       XVI  Japan-United States Friendship Commission (Parts 
                1600--1699)
      XVII  United States Institute of Peace (Parts 1700--1799)

                          Title 23--Highways

         I  Federal Highway Administration, Department of 
                Transportation (Parts 1--999)
        II  National Highway Traffic Safety Administration and 
                Federal Highway Administration, Department of 
                Transportation (Parts 1200--1299)
       III  National Highway Traffic Safety Administration, 
                Department of Transportation (Parts 1300--1399)

                Title 24--Housing and Urban Development

            Subtitle A--Office of the Secretary, Department of 
                Housing and Urban Development (Parts 0--99)
            Subtitle B--Regulations Relating to Housing and Urban 
                Development
         I  Office of Assistant Secretary for Equal Opportunity, 
                Department of Housing and Urban Development (Parts 
                100--199)
        II  Office of Assistant Secretary for Housing-Federal 
                HousingCommissioner, Department of Housing and 
                Urban Development (Parts 200--299)
       III  Government National Mortgage Association, Department 
                of Housing and Urban Development (Parts 300--399)
        IV  Office of Housing and Office of Multifamily Housing 
                Assistance Restructuring, Department of Housing 
                and Urban Development (Parts 400--499)
         V  Office of Assistant Secretary for Community Planning 
                and Development, Department of Housing and Urban 
                Development (Parts 500--599)
        VI  Office of Assistant Secretary for Community Planning 
                and Development, Department of Housing and Urban 
                Development (Parts 600--699) [Reserved]
       VII  Office of the Secretary, Department of Housing and 
                Urban Development (Housing Assistance Programs and 
                Public and Indian Housing Programs) (Parts 700--
                799)

[[Page 488]]

      VIII  Office of the Assistant Secretary for Housing--Federal 
                Housing Commissioner, Department of Housing and 
                Urban Development (Section 8 Housing Assistance 
                Programs, Section 202 Direct Loan Program, Section 
                202 Supportive Housing for the Elderly Program and 
                Section 811 Supportive Housing for Persons With 
                Disabilities Program) (Parts 800--899)
        IX  Office of Assistant Secretary for Public and Indian 
                Housing, Department of Housing and Urban 
                Development (Parts 900--1699)
         X  Office of Assistant Secretary for Housing--Federal 
                Housing Commissioner, Department of Housing and 
                Urban Development (Interstate Land Sales 
                Registration Program) (Parts 1700--1799)
       XII  Office of Inspector General, Department of Housing and 
                Urban Development (Parts 2000--2099)
        XX  Office of Assistant Secretary for Housing--Federal 
                Housing Commissioner, Department of Housing and 
                Urban Development (Parts 3200--3899)
       XXV  Neighborhood Reinvestment Corporation (Parts 4100--
                4199)

                           Title 25--Indians

         I  Bureau of Indian Affairs, Department of the Interior 
                (Parts 1--299)
        II  Indian Arts and Crafts Board, Department of the 
                Interior (Parts 300--399)
       III  National Indian Gaming Commission, Department of the 
                Interior (Parts 500--599)
        IV  Office of Navajo and Hopi Indian Relocation (Parts 
                700--799)
         V  Bureau of Indian Affairs, Department of the Interior, 
                and Indian Health Service, Department of Health 
                and Human Services (Part 900)
        VI  Office of the Assistant Secretary-Indian Affairs, 
                Department of the Interior (Parts 1000--1199)
       VII  Office of the Special Trustee for American Indians, 
                Department of the Interior (Parts 1200--1299)

                      Title 26--Internal Revenue

         I  Internal Revenue Service, Department of the Treasury 
                (Parts 1--899)

           Title 27--Alcohol, Tobacco Products and Firearms

         I  Alcohol and Tobacco Tax and Trade Bureau, Department 
                of the Treasury (Parts 1--399)
        II  Bureau of Alcohol, Tobacco, Firearms, and Explosives, 
                Department of Justice (Parts 400--699)

                   Title 28--Judicial Administration

         I  Department of Justice (Parts 0--299)

[[Page 489]]

       III  Federal Prison Industries, Inc., Department of Justice 
                (Parts 300--399)
         V  Bureau of Prisons, Department of Justice (Parts 500--
                599)
        VI  Offices of Independent Counsel, Department of Justice 
                (Parts 600--699)
       VII  Office of Independent Counsel (Parts 700--799)
      VIII  Court Services and Offender Supervision Agency for the 
                District of Columbia (Parts 800--899)
        IX  National Crime Prevention and Privacy Compact Council 
                (Parts 900--999)
        XI  Department of Justice and Department of State (Parts 
                1100--1199)

                            Title 29--Labor

            Subtitle A--Office of the Secretary of Labor (Parts 
                0--99)
            Subtitle B--Regulations Relating to Labor
         I  National Labor Relations Board (Parts 100--199)
        II  Office of Labor-Management Standards, Department of 
                Labor (Parts 200--299)
       III  National Railroad Adjustment Board (Parts 300--399)
        IV  Office of Labor-Management Standards, Department of 
                Labor (Parts 400--499)
         V  Wage and Hour Division, Department of Labor (Parts 
                500--899)
        IX  Construction Industry Collective Bargaining Commission 
                (Parts 900--999)
         X  National Mediation Board (Parts 1200--1299)
       XII  Federal Mediation and Conciliation Service (Parts 
                1400--1499)
       XIV  Equal Employment Opportunity Commission (Parts 1600--
                1699)
      XVII  Occupational Safety and Health Administration, 
                Department of Labor (Parts 1900--1999)
        XX  Occupational Safety and Health Review Commission 
                (Parts 2200--2499)
       XXV  Employee Benefits Security Administration, Department 
                of Labor (Parts 2500--2599)
     XXVII  Federal Mine Safety and Health Review Commission 
                (Parts 2700--2799)
        XL  Pension Benefit Guaranty Corporation (Parts 4000--
                4999)

                      Title 30--Mineral Resources

         I  Mine Safety and Health Administration, Department of 
                Labor (Parts 1--199)
        II  Minerals Management Service, Department of the 
                Interior (Parts 200--299)
       III  Board of Surface Mining and Reclamation Appeals, 
                Department of the Interior (Parts 300--399)
        IV  Geological Survey, Department of the Interior (Parts 
                400--499)

[[Page 490]]

       VII  Office of Surface Mining Reclamation and Enforcement, 
                Department of the Interior (Parts 700--999)

                 Title 31--Money and Finance: Treasury

            Subtitle A--Office of the Secretary of the Treasury 
                (Parts 0--50)
            Subtitle B--Regulations Relating to Money and Finance
         I  Monetary Offices, Department of the Treasury (Parts 
                51--199)
        II  Fiscal Service, Department of the Treasury (Parts 
                200--399)
        IV  Secret Service, Department of the Treasury (Parts 
                400--499)
         V  Office of Foreign Assets Control, Department of the 
                Treasury (Parts 500--599)
        VI  Bureau of Engraving and Printing, Department of the 
                Treasury (Parts 600--699)
       VII  Federal Law Enforcement Training Center, Department of 
                the Treasury (Parts 700--799)
      VIII  Office of International Investment, Department of the 
                Treasury (Parts 800--899)
        IX  Federal Claims Collection Standards (Department of the 
                Treasury--Department of Justice) (Parts 900--999)

                      Title 32--National Defense

            Subtitle A--Department of Defense
         I  Office of the Secretary of Defense (Parts 1--399)
         V  Department of the Army (Parts 400--699)
        VI  Department of the Navy (Parts 700--799)
       VII  Department of the Air Force (Parts 800--1099)
            Subtitle B--Other Regulations Relating to National 
                Defense
       XII  Defense Logistics Agency (Parts 1200--1299)
       XVI  Selective Service System (Parts 1600--1699)
      XVII  Office of the Director of National Intelligence (Parts 
                1700--1799)
     XVIII  National Counterintelligence Center (Parts 1800--1899)
       XIX  Central Intelligence Agency (Parts 1900--1999)
        XX  Information Security Oversight Office, National 
                Archives and Records Administration (Parts 2000--
                2099)
       XXI  National Security Council (Parts 2100--2199)
      XXIV  Office of Science and Technology Policy (Parts 2400--
                2499)
     XXVII  Office for Micronesian Status Negotiations (Parts 
                2700--2799)
    XXVIII  Office of the Vice President of the United States 
                (Parts 2800--2899)

               Title 33--Navigation and Navigable Waters

         I  Coast Guard, Department of Homeland Security (Parts 
                1--199)
        II  Corps of Engineers, Department of the Army (Parts 
                200--399)

[[Page 491]]

        IV  Saint Lawrence Seaway Development Corporation, 
                Department of Transportation (Parts 400--499)

                          Title 34--Education

            Subtitle A--Office of the Secretary, Department of 
                Education (Parts 1--99)
            Subtitle B--Regulations of the Offices of the 
                Department of Education
         I  Office for Civil Rights, Department of Education 
                (Parts 100--199)
        II  Office of Elementary and Secondary Education, 
                Department of Education (Parts 200--299)
       III  Office of Special Education and Rehabilitative 
                Services, Department of Education (Parts 300--399)
        IV  Office of Vocational and Adult Education, Department 
                of Education (Parts 400--499)
         V  Office of Bilingual Education and Minority Languages 
                Affairs, Department of Education (Parts 500--599)
        VI  Office of Postsecondary Education, Department of 
                Education (Parts 600--699)
       VII  Office of Educational Research and Improvmeent, 
                Department of Education [Reserved]
        XI  National Institute for Literacy (Parts 1100--1199)
            Subtitle C--Regulations Relating to Education
       XII  National Council on Disability (Parts 1200--1299)

                          Title 35 [Reserved]

             Title 36--Parks, Forests, and Public Property

         I  National Park Service, Department of the Interior 
                (Parts 1--199)
        II  Forest Service, Department of Agriculture (Parts 200--
                299)
       III  Corps of Engineers, Department of the Army (Parts 
                300--399)
        IV  American Battle Monuments Commission (Parts 400--499)
         V  Smithsonian Institution (Parts 500--599)
        VI  [Reserved]
       VII  Library of Congress (Parts 700--799)
      VIII  Advisory Council on Historic Preservation (Parts 800--
                899)
        IX  Pennsylvania Avenue Development Corporation (Parts 
                900--999)
         X  Presidio Trust (Parts 1000--1099)
        XI  Architectural and Transportation Barriers Compliance 
                Board (Parts 1100--1199)
       XII  National Archives and Records Administration (Parts 
                1200--1299)
        XV  Oklahoma City National Memorial Trust (Parts 1500--
                1599)
       XVI  Morris K. Udall Scholarship and Excellence in National 
                Environmental Policy Foundation (Parts 1600--1699)

[[Page 492]]

             Title 37--Patents, Trademarks, and Copyrights

         I  United States Patent and Trademark Office, Department 
                of Commerce (Parts 1--199)
        II  Copyright Office, Library of Congress (Parts 200--299)
       III  Copyright Royalty Board, Library of Congress (Parts 
                301--399)
        IV  Assistant Secretary for Technology Policy, Department 
                of Commerce (Parts 400--499)
         V  Under Secretary for Technology, Department of Commerce 
                (Parts 500--599)

           Title 38--Pensions, Bonuses, and Veterans' Relief

         I  Department of Veterans Affairs (Parts 0--99)

                       Title 39--Postal Service

         I  United States Postal Service (Parts 1--999)
       III  Postal Regulatory Commission (Parts 3000--3099)

                  Title 40--Protection of Environment

         I  Environmental Protection Agency (Parts 1--1099)
        IV  Environmental Protection Agency and Department of 
                Justice (Parts 1400--1499)
         V  Council on Environmental Quality (Parts 1500--1599)
        VI  Chemical Safety and Hazard Investigation Board (Parts 
                1600--1699)
       VII  Environmental Protection Agency and Department of 
                Defense; Uniform National Discharge Standards for 
                Vessels of the Armed Forces (Parts 1700--1799)

          Title 41--Public Contracts and Property Management

            Subtitle B--Other Provisions Relating to Public 
                Contracts
        50  Public Contracts, Department of Labor (Parts 50-1--50-
                999)
        51  Committee for Purchase From People Who Are Blind or 
                Severely Disabled (Parts 51-1--51-99)
        60  Office of Federal Contract Compliance Programs, Equal 
                Employment Opportunity, Department of Labor (Parts 
                60-1--60-999)
        61  Office of the Assistant Secretary for Veterans' 
                Employment and Training Service, Department of 
                Labor (Parts 61-1--61-999)
            Chapters 62--100 [Reserved]
            Subtitle C--Federal Property Management Regulations 
                System
       101  Federal Property Management Regulations (Parts 101-1--
                101-99)
       102  Federal Management Regulation (Parts 102-1--102-299)
            Chapters 103--104 [Reserved]
       105  General Services Administration (Parts 105-1--105-999)

[[Page 493]]

       109  Department of Energy Property Management Regulations 
                (Parts 109-1--109-99)
       114  Department of the Interior (Parts 114-1--114-99)
       115  Environmental Protection Agency (Parts 115-1--115-99)
       128  Department of Justice (Parts 128-1--128-99)
            Chapters 129--200 [Reserved]
            Subtitle D--Other Provisions Relating to Property 
                Management [Reserved]
            Subtitle E--Federal Information Resources Management 
                Regulations System [Reserved]
            Subtitle F--Federal Travel Regulation System
       300  General (Parts 300-1--300-99)
       301  Temporary Duty (TDY) Travel Allowances (Parts 301-1--
                301-99)
       302  Relocation Allowances (Parts 302-1--302-99)
       303  Payment of Expenses Connected with the Death of 
                Certain Employees (Part 303-1--303-99)
       304  Payment of Travel Expenses from a Non-Federal Source 
                (Parts 304-1--304-99)

                        Title 42--Public Health

         I  Public Health Service, Department of Health and Human 
                Services (Parts 1--199)
        IV  Centers for Medicare & Medicaid Services, Department 
                of Health and Human Services (Parts 400--499)
         V  Office of Inspector General-Health Care, Department of 
                Health and Human Services (Parts 1000--1999)

                   Title 43--Public Lands: Interior

            Subtitle A--Office of the Secretary of the Interior 
                (Parts 1--199)
            Subtitle B--Regulations Relating to Public Lands
         I  Bureau of Reclamation, Department of the Interior 
                (Parts 200--499)
        II  Bureau of Land Management, Department of the Interior 
                (Parts 1000--9999)
       III  Utah Reclamation Mitigation and Conservation 
                Commission (Parts 10000--10010)

             Title 44--Emergency Management and Assistance

         I  Federal Emergency Management Agency, Department of 
                Homeland Security (Parts 0--399)
        IV  Department of Commerce and Department of 
                Transportation (Parts 400--499)

[[Page 494]]

                       Title 45--Public Welfare

            Subtitle A--Department of Health and Human Services 
                (Parts 1--199)
            Subtitle B--Regulations Relating to Public Welfare
        II  Office of Family Assistance (Assistance Programs), 
                Administration for Children and Families, 
                Department of Health and Human Services (Parts 
                200--299)
       III  Office of Child Support Enforcement (Child Support 
                Enforcement Program), Administration for Children 
                and Families, Department of Health and Human 
                Services (Parts 300--399)
        IV  Office of Refugee Resettlement, Administration for 
                Children and Families, Department of Health and 
                Human Services (Parts 400--499)
         V  Foreign Claims Settlement Commission of the United 
                States, Department of Justice (Parts 500--599)
        VI  National Science Foundation (Parts 600--699)
       VII  Commission on Civil Rights (Parts 700--799)
      VIII  Office of Personnel Management (Parts 800--899) 
                [Reserved]
         X  Office of Community Services, Administration for 
                Children and Families, Department of Health and 
                Human Services (Parts 1000--1099)
        XI  National Foundation on the Arts and the Humanities 
                (Parts 1100--1199)
       XII  Corporation for National and Community Service (Parts 
                1200--1299)
      XIII  Office of Human Development Services, Department of 
                Health and Human Services (Parts 1300--1399)
       XVI  Legal Services Corporation (Parts 1600--1699)
      XVII  National Commission on Libraries and Information 
                Science (Parts 1700--1799)
     XVIII  Harry S. Truman Scholarship Foundation (Parts 1800--
                1899)
       XXI  Commission on Fine Arts (Parts 2100--2199)
     XXIII  Arctic Research Commission (Part 2301)
      XXIV  James Madison Memorial Fellowship Foundation (Parts 
                2400--2499)
       XXV  Corporation for National and Community Service (Parts 
                2500--2599)

                          Title 46--Shipping

         I  Coast Guard, Department of Homeland Security (Parts 
                1--199)
        II  Maritime Administration, Department of Transportation 
                (Parts 200--399)
       III  Coast Guard (Great Lakes Pilotage), Department of 
                Homeland Security (Parts 400--499)
        IV  Federal Maritime Commission (Parts 500--599)

                      Title 47--Telecommunication

         I  Federal Communications Commission (Parts 0--199)

[[Page 495]]

        II  Office of Science and Technology Policy and National 
                Security Council (Parts 200--299)
       III  National Telecommunications and Information 
                Administration, Department of Commerce (Parts 
                300--399)

           Title 48--Federal Acquisition Regulations System

         1  Federal Acquisition Regulation (Parts 1--99)
         2  Defense Acquisition Regulations System, Department of 
                Defense (Parts 200--299)
         3  Department of Health and Human Services (Parts 300--
                399)
         4  Department of Agriculture (Parts 400--499)
         5  General Services Administration (Parts 500--599)
         6  Department of State (Parts 600--699)
         7  Agency for International Development (Parts 700--799)
         8  Department of Veterans Affairs (Parts 800--899)
         9  Department of Energy (Parts 900--999)
        10  Department of the Treasury (Parts 1000--1099)
        12  Department of Transportation (Parts 1200--1299)
        13  Department of Commerce (Parts 1300--1399)
        14  Department of the Interior (Parts 1400--1499)
        15  Environmental Protection Agency (Parts 1500--1599)
        16  Office of Personnel Management, Federal Employees 
                Health Benefits Acquisition Regulation (Parts 
                1600--1699)
        17  Office of Personnel Management (Parts 1700--1799)
        18  National Aeronautics and Space Administration (Parts 
                1800--1899)
        19  Broadcasting Board of Governors (Parts 1900--1999)
        20  Nuclear Regulatory Commission (Parts 2000--2099)
        21  Office of Personnel Management, Federal Employees 
                Group Life Insurance Federal Acquisition 
                Regulation (Parts 2100--2199)
        23  Social Security Administration (Parts 2300--2399)
        24  Department of Housing and Urban Development (Parts 
                2400--2499)
        25  National Science Foundation (Parts 2500--2599)
        28  Department of Justice (Parts 2800--2899)
        29  Department of Labor (Parts 2900--2999)
        30  Department of Homeland Security, Homeland Security 
                Acquisition Regulation (HSAR) (Parts 3000--3099)
        34  Department of Education Acquisition Regulation (Parts 
                3400--3499)
        51  Department of the Army Acquisition Regulations (Parts 
                5100--5199)
        52  Department of the Navy Acquisition Regulations (Parts 
                5200--5299)
        53  Department of the Air Force Federal Acquisition 
                Regulation Supplement [Reserved]

[[Page 496]]

        54  Defense Logistics Agency, Department of Defense (Parts 
                5400--5499)
        57  African Development Foundation (Parts 5700--5799)
        61  General Services Administration Board of Contract 
                Appeals (Parts 6100--6199)
        63  Department of Transportation Board of Contract Appeals 
                (Parts 6300--6399)
        99  Cost Accounting Standards Board, Office of Federal 
                Procurement Policy, Office of Management and 
                Budget (Parts 9900--9999)

                       Title 49--Transportation

            Subtitle A--Office of the Secretary of Transportation 
                (Parts 1--99)
            Subtitle B--Other Regulations Relating to 
                Transportation
         I  Pipeline and Hazardous Materials Safety 
                Administration, Department of Transportation 
                (Parts 100--199)
        II  Federal Railroad Administration, Department of 
                Transportation (Parts 200--299)
       III  Federal Motor Carrier Safety Administration, 
                Department of Transportation (Parts 300--399)
        IV  Coast Guard, Department of Homeland Security (Parts 
                400--499)
         V  National Highway Traffic Safety Administration, 
                Department of Transportation (Parts 500--599)
        VI  Federal Transit Administration, Department of 
                Transportation (Parts 600--699)
       VII  National Railroad Passenger Corporation (AMTRAK) 
                (Parts 700--799)
      VIII  National Transportation Safety Board (Parts 800--999)
         X  Surface Transportation Board, Department of 
                Transportation (Parts 1000--1399)
        XI  Research and Innovative Technology Administration, 
                Department of Transportation [Reserved]
       XII  Transportation Security Administration, Department of 
                Homeland Security (Parts 1500--1699)

                   Title 50--Wildlife and Fisheries

         I  United States Fish and Wildlife Service, Department of 
                the Interior (Parts 1--199)
        II  National Marine Fisheries Service, National Oceanic 
                and Atmospheric Administration, Department of 
                Commerce (Parts 200--299)
       III  International Fishing and Related Activities (Parts 
                300--399)
        IV  Joint Regulations (United States Fish and Wildlife 
                Service, Department of the Interior and National 
                Marine Fisheries Service, National Oceanic and 
                Atmospheric Administration, Department of 
                Commerce); Endangered Species Committee 
                Regulations (Parts 400--499)

[[Page 497]]

         V  Marine Mammal Commission (Parts 500--599)
        VI  Fishery Conservation and Management, National Oceanic 
                and Atmospheric Administration, Department of 
                Commerce (Parts 600--699)

                      CFR Index and Finding Aids

            Subject/Agency Index
            List of Agency Prepared Indexes
            Parallel Tables of Statutory Authorities and Rules
            List of CFR Titles, Chapters, Subchapters, and Parts
            Alphabetical List of Agencies Appearing in the CFR

[[Page 499]]





           Alphabetical List of Agencies Appearing in the CFR




                      (Revised as of July 1, 2009)

                                                  CFR Title, Subtitle or 
                     Agency                               Chapter

Administrative Committee of the Federal Register  1, I
Advanced Research Projects Agency                 32, I
Advisory Council on Historic Preservation         36, VIII
African Development Foundation                    22, XV
  Federal Acquisition Regulation                  48, 57
Agency for International Development              22, II
  Federal Acquisition Regulation                  48, 7
Agricultural Marketing Service                    7, I, IX, X, XI
Agricultural Research Service                     7, V
Agriculture Department                            5, LXXIII
  Agricultural Marketing Service                  7, I, IX, X, XI
  Agricultural Research Service                   7, V
  Animal and Plant Health Inspection Service      7, III; 9, I
  Chief Financial Officer, Office of              7, XXX
  Commodity Credit Corporation                    7, XIV
  Cooperative State Research, Education, and      7, XXXIV
       Extension Service
  Economic Research Service                       7, XXXVII
  Energy, Office of                               2, IX; 7, XXIX
  Environmental Quality, Office of                7, XXXI
  Farm Service Agency                             7, VII, XVIII
  Federal Acquisition Regulation                  48, 4
  Federal Crop Insurance Corporation              7, IV
  Food and Nutrition Service                      7, II
  Food Safety and Inspection Service              9, III
  Foreign Agricultural Service                    7, XV
  Forest Service                                  36, II
  Grain Inspection, Packers and Stockyards        7, VIII; 9, II
       Administration
  Information Resources Management, Office of     7, XXVII
  Inspector General, Office of                    7, XXVI
  National Agricultural Library                   7, XLI
  National Agricultural Statistics Service        7, XXXVI
  Natural Resources Conservation Service          7, VI
  Operations, Office of                           7, XXVIII
  Procurement and Property Management, Office of  7, XXXII
  Rural Business-Cooperative Service              7, XVIII, XLII, L
  Rural Development Administration                7, XLII
  Rural Housing Service                           7, XVIII, XXXV, L
  Rural Telephone Bank                            7, XVI
  Rural Utilities Service                         7, XVII, XVIII, XLII, L
  Secretary of Agriculture, Office of             7, Subtitle A
  Transportation, Office of                       7, XXXIII
  World Agricultural Outlook Board                7, XXXVIII
Air Force Department                              32, VII
  Federal Acquisition Regulation Supplement       48, 53
Air Transportation Stabilization Board            14, VI
Alcohol and Tobacco Tax and Trade Bureau          27, I
Alcohol, Tobacco, Firearms, and Explosives,       27, II
     Bureau of
AMTRAK                                            49, VII
American Battle Monuments Commission              36, IV
American Indians, Office of the Special Trustee   25, VII
Animal and Plant Health Inspection Service        7, III; 9, I
Appalachian Regional Commission                   5, IX

[[Page 500]]

Architectural and Transportation Barriers         36, XI
     Compliance Board
Arctic Research Commission                        45, XXIII
Armed Forces Retirement Home                      5, XI
Army Department                                   32, V
  Engineers, Corps of                             33, II; 36, III
  Federal Acquisition Regulation                  48, 51
Benefits Review Board                             20, VII
Bilingual Education and Minority Languages        34, V
     Affairs, Office of
Blind or Severely Disabled, Committee for         41, 51
     Purchase From People Who Are
Broadcasting Board of Governors                   22, V
  Federal Acquisition Regulation                  48, 19
Census Bureau                                     15, I
Centers for Medicare & Medicaid Services          42, IV
Central Intelligence Agency                       32, XIX
Chief Financial Officer, Office of                7, XXX
Child Support Enforcement, Office of              45, III
Children and Families, Administration for         45, II, III, IV, X
Civil Rights, Commission on                       5, LXVIII; 45, VII
Civil Rights, Office for                          34, I
Coast Guard                                       33, I; 46, I; 49, IV
Coast Guard (Great Lakes Pilotage)                46, III
Commerce Department                               44, IV
  Census Bureau                                   15, I
  Economic Affairs, Under Secretary               37, V
  Economic Analysis, Bureau of                    15, VIII
  Economic Development Administration             13, III
  Emergency Management and Assistance             44, IV
  Federal Acquisition Regulation                  48, 13
  Fishery Conservation and Management             50, VI
  Foreign-Trade Zones Board                       15, IV
  Industry and Security, Bureau of                15, VII
  International Trade Administration              15, III; 19, III
  National Institute of Standards and Technology  15, II
  National Marine Fisheries Service               50, II, IV, VI
  National Oceanic and Atmospheric                15, IX; 50, II, III, IV, 
       Administration                             VI
  National Telecommunications and Information     15, XXIII; 47, III
       Administration
  National Weather Service                        15, IX
  Patent and Trademark Office, United States      37, I
  Productivity, Technology and Innovation,        37, IV
       Assistant Secretary for
  Secretary of Commerce, Office of                15, Subtitle A
  Technology, Under Secretary for                 37, V
  Technology Administration                       15, XI
  Technology Policy, Assistant Secretary for      37, IV
Commercial Space Transportation                   14, III
Commodity Credit Corporation                      7, XIV
Commodity Futures Trading Commission              5, XLI; 17, I
Community Planning and Development, Office of     24, V, VI
     Assistant Secretary for
Community Services, Office of                     45, X
Comptroller of the Currency                       12, I
Construction Industry Collective Bargaining       29, IX
     Commission
Consumer Product Safety Commission                5, LXXI; 16, II
Cooperative State Research, Education, and        7, XXXIV
     Extension Service
Copyright Office                                  37, II
Copyright Royalty Board                           37, III
Corporation for National and Community Service    2, XXII; 45, XII, XXV
Cost Accounting Standards Board                   48, 99
Council on Environmental Quality                  40, V
Court Services and Offender Supervision Agency    28, VIII
     for the District of Columbia
Customs and Border Protection Bureau              19, I
Defense Contract Audit Agency                     32, I
Defense Department                                5, XXVI; 32, Subtitle A; 
                                                  40, VII

[[Page 501]]

  Advanced Research Projects Agency               32, I
  Air Force Department                            32, VII
  Army Department                                 32, V; 33, II; 36, III, 
                                                  48, 51
  Defense Acquisition Regulations System          48, 2
  Defense Intelligence Agency                     32, I
  Defense Logistics Agency                        32, I, XII; 48, 54
  Engineers, Corps of                             33, II; 36, III
  Human Resources Management and Labor Relations  5, XCIX
       Systems
  National Imagery and Mapping Agency             32, I
  Navy Department                                 32, VI; 48, 52
  Secretary of Defense, Office of                 2, XI; 32, I
Defense Contract Audit Agency                     32, I
Defense Intelligence Agency                       32, I
Defense Logistics Agency                          32, XII; 48, 54
Defense Nuclear Facilities Safety Board           10, XVII
Delaware River Basin Commission                   18, III
District of Columbia, Court Services and          28, VIII
     Offender Supervision Agency for the
Drug Enforcement Administration                   21, II
East-West Foreign Trade Board                     15, XIII
Economic Affairs, Under Secretary                 37, V
Economic Analysis, Bureau of                      15, VIII
Economic Development Administration               13, III
Economic Research Service                         7, XXXVII
Education, Department of                          5, LIII
  Bilingual Education and Minority Languages      34, V
       Affairs, Office of
  Civil Rights, Office for                        34, I
  Educational Research and Improvement, Office    34, VII
       of
  Elementary and Secondary Education, Office of   34, II
  Federal Acquisition Regulation                  48, 34
  Postsecondary Education, Office of              34, VI
  Secretary of Education, Office of               34, Subtitle A
  Special Education and Rehabilitative Services,  34, III
       Office of
  Vocational and Adult Education, Office of       34, IV
Educational Research and Improvement, Office of   34, VII
Election Assistance Commission                    11, II
Elementary and Secondary Education, Office of     34, II
Emergency Oil and Gas Guaranteed Loan Board       13, V
Emergency Steel Guarantee Loan Board              13, IV
Employee Benefits Security Administration         29, XXV
Employees' Compensation Appeals Board             20, IV
Employees Loyalty Board                           5, V
Employment and Training Administration            20, V
Employment Standards Administration               20, VI
Endangered Species Committee                      50, IV
Energy, Department of                             5, XXIII; 10, II, III, X
  Federal Acquisition Regulation                  48, 9
  Federal Energy Regulatory Commission            5, XXIV; 18, I
  Property Management Regulations                 41, 109
Energy, Office of                                 7, XXIX
Engineers, Corps of                               33, II; 36, III
Engraving and Printing, Bureau of                 31, VI
Environmental Protection Agency                   2, XV; 5, LIV; 40, I, IV, 
                                                  VII
  Federal Acquisition Regulation                  48, 15
  Property Management Regulations                 41, 115
Environmental Quality, Office of                  7, XXXI
Equal Employment Opportunity Commission           5, LXII; 29, XIV
Equal Opportunity, Office of Assistant Secretary  24, I
     for
Executive Office of the President                 3, I
  Administration, Office of                       5, XV
  Environmental Quality, Council on               40, V
  Management and Budget, Office of                5, III, LXXVII; 14, VI; 
                                                  48, 99

[[Page 502]]

  National Drug Control Policy, Office of         21, III
  National Security Council                       32, XXI; 47, 2
  Presidential Documents                          3
  Science and Technology Policy, Office of        32, XXIV; 47, II
  Trade Representative, Office of the United      15, XX
       States
Export-Import Bank of the United States           2, XXXV; 5, LII; 12, IV
Family Assistance, Office of                      45, II
Farm Credit Administration                        5, XXXI; 12, VI
Farm Credit System Insurance Corporation          5, XXX; 12, XIV
Farm Service Agency                               7, VII, XVIII
Federal Acquisition Regulation                    48, 1
Federal Aviation Administration                   14, I
  Commercial Space Transportation                 14, III
Federal Claims Collection Standards               31, IX
Federal Communications Commission                 5, XXIX; 47, I
Federal Contract Compliance Programs, Office of   41, 60
Federal Crop Insurance Corporation                7, IV
Federal Deposit Insurance Corporation             5, XXII; 12, III
Federal Election Commission                       11, I
Federal Emergency Management Agency               44, I
Federal Employees Group Life Insurance Federal    48, 21
     Acquisition Regulation
Federal Employees Health Benefits Acquisition     48, 16
     Regulation
Federal Energy Regulatory Commission              5, XXIV; 18, I
Federal Financial Institutions Examination        12, XI
     Council
Federal Financing Bank                            12, VIII
Federal Highway Administration                    23, I, II
Federal Home Loan Mortgage Corporation            1, IV
Federal Housing Enterprise Oversight Office       12, XVII
Federal Housing Finance Agency                    12, XII
Federal Housing Finance Board                     12, IX
Federal Labor Relations Authority, and General    5, XIV; 22, XIV
     Counsel of the Federal Labor Relations 
     Authority
Federal Law Enforcement Training Center           31, VII
Federal Management Regulation                     41, 102
Federal Maritime Commission                       46, IV
Federal Mediation and Conciliation Service        29, XII
Federal Mine Safety and Health Review Commission  5, LXXIV; 29, XXVII
Federal Motor Carrier Safety Administration       49, III
Federal Prison Industries, Inc.                   28, III
Federal Procurement Policy Office                 48, 99
Federal Property Management Regulations           41, 101
Federal Railroad Administration                   49, II
Federal Register, Administrative Committee of     1, I
Federal Register, Office of                       1, II
Federal Reserve System                            12, II
  Board of Governors                              5, LVIII
Federal Retirement Thrift Investment Board        5, VI, LXXVI
Federal Service Impasses Panel                    5, XIV
Federal Trade Commission                          5, XLVII; 16, I
Federal Transit Administration                    49, VI
Federal Travel Regulation System                  41, Subtitle F
Fine Arts, Commission on                          45, XXI
Fiscal Service                                    31, II
Fish and Wildlife Service, United States          50, I, IV
Fishery Conservation and Management               50, VI
Food and Drug Administration                      21, I
Food and Nutrition Service                        7, II
Food Safety and Inspection Service                9, III
Foreign Agricultural Service                      7, XV
Foreign Assets Control, Office of                 31, V
Foreign Claims Settlement Commission of the       45, V
     United States
Foreign Service Grievance Board                   22, IX
Foreign Service Impasse Disputes Panel            22, XIV
Foreign Service Labor Relations Board             22, XIV
Foreign-Trade Zones Board                         15, IV
Forest Service                                    36, II

[[Page 503]]

General Services Administration                   5, LVII; 41, 105
  Contract Appeals, Board of                      48, 61
  Federal Acquisition Regulation                  48, 5
  Federal Management Regulation                   41, 102
  Federal Property Management Regulations         41, 101
  Federal Travel Regulation System                41, Subtitle F
  General                                         41, 300
  Payment From a Non-Federal Source for Travel    41, 304
       Expenses
  Payment of Expenses Connected With the Death    41, 303
       of Certain Employees
  Relocation Allowances                           41, 302
  Temporary Duty (TDY) Travel Allowances          41, 301
Geological Survey                                 30, IV
Government Accountability Office                  4, I
Government Ethics, Office of                      5, XVI
Government National Mortgage Association          24, III
Grain Inspection, Packers and Stockyards          7, VIII; 9, II
     Administration
Harry S. Truman Scholarship Foundation            45, XVIII
Health and Human Services, Department of          2, III; 5, XLV; 45, 
                                                  Subtitle A,
  Centers for Medicare & Medicaid Services        42, IV
  Child Support Enforcement, Office of            45, III
  Children and Families, Administration for       45, II, III, IV, X
  Community Services, Office of                   45, X
  Family Assistance, Office of                    45, II
  Federal Acquisition Regulation                  48, 3
  Food and Drug Administration                    21, I
  Human Development Services, Office of           45, XIII
  Indian Health Service                           25, V
  Inspector General (Health Care), Office of      42, V
  Public Health Service                           42, I
  Refugee Resettlement, Office of                 45, IV
Homeland Security, Department of                  6, I
  Coast Guard                                     33, I; 46, I; 49, IV
  Coast Guard (Great Lakes Pilotage)              46, III
  Customs and Border Protection Bureau            19, I
  Federal Emergency Management Agency             44, I
  Human Resources Management and Labor Relations  5, XCVII
       Systems
  Immigration and Customs Enforcement Bureau      19, IV
  Immigration and Naturalization                  8, I
  Transportation Security Administration          49, XII
HOPE for Homeowners Program, Board of Directors   24, XXIV
     of
Housing and Urban Development, Department of      2, XXIV; 5, LXV; 24, 
                                                  Subtitle B
  Community Planning and Development, Office of   24, V, VI
       Assistant Secretary for
  Equal Opportunity, Office of Assistant          24, I
       Secretary for
  Federal Acquisition Regulation                  48, 24
  Federal Housing Enterprise Oversight, Office    12, XVII
       of
  Government National Mortgage Association        24, III
  Housing--Federal Housing Commissioner, Office   24, II, VIII, X, XX
       of Assistant Secretary for
  Housing, Office of, and Multifamily Housing     24, IV
       Assistance Restructuring, Office of
  Inspector General, Office of                    24, XII
  Public and Indian Housing, Office of Assistant  24, IX
       Secretary for
  Secretary, Office of                            24, Subtitle A, VII
Housing--Federal Housing Commissioner, Office of  24, II, VIII, X, XX
     Assistant Secretary for
Housing, Office of, and Multifamily Housing       24, IV
     Assistance Restructuring, Office of
Human Development Services, Office of             45, XIII
Immigration and Customs Enforcement Bureau        19, IV
Immigration and Naturalization                    8, I
Immigration Review, Executive Office for          8, V
Independent Counsel, Office of                    28, VII

[[Page 504]]

Indian Affairs, Bureau of                         25, I, V
Indian Affairs, Office of the Assistant           25, VI
     Secretary
Indian Arts and Crafts Board                      25, II
Indian Health Service                             25, V
Industry and Security, Bureau of                  15, VII
Information Resources Management, Office of       7, XXVII
Information Security Oversight Office, National   32, XX
     Archives and Records Administration
Inspector General
  Agriculture Department                          7, XXVI
  Health and Human Services Department            42, V
  Housing and Urban Development Department        24, XII
Institute of Peace, United States                 22, XVII
Inter-American Foundation                         5, LXIII; 22, X
Interior Department
  American Indians, Office of the Special         25, VII
       Trustee
  Endangered Species Committee                    50, IV
  Federal Acquisition Regulation                  48, 14
  Federal Property Management Regulations System  41, 114
  Fish and Wildlife Service, United States        50, I, IV
  Geological Survey                               30, IV
  Indian Affairs, Bureau of                       25, I, V
  Indian Affairs, Office of the Assistant         25, VI
       Secretary
  Indian Arts and Crafts Board                    25, II
  Land Management, Bureau of                      43, II
  Minerals Management Service                     30, II
  National Indian Gaming Commission               25, III
  National Park Service                           36, I
  Reclamation, Bureau of                          43, I
  Secretary of the Interior, Office of            2, XIV; 43, Subtitle A
  Surface Mining and Reclamation Appeals, Board   30, III
       of
  Surface Mining Reclamation and Enforcement,     30, VII
       Office of
Internal Revenue Service                          26, I
International Boundary and Water Commission,      22, XI
     United States and Mexico, United States 
     Section
International Development, United States Agency   22, II
     for
  Federal Acquisition Regulation                  48, 7
International Development Cooperation Agency,     22, XII
     United States
International Fishing and Related Activities      50, III
International Joint Commission, United States     22, IV
     and Canada
International Organizations Employees Loyalty     5, V
     Board
International Trade Administration                15, III; 19, III
International Trade Commission, United States     19, II
Interstate Commerce Commission                    5, XL
Investment Security, Office of                    31, VIII
James Madison Memorial Fellowship Foundation      45, XXIV
Japan-United States Friendship Commission         22, XVI
Joint Board for the Enrollment of Actuaries       20, VIII
Justice Department                                2, XXVII; 5, XXVIII; 28, 
                                                  I, XI; 40, IV
  Alcohol, Tobacco, Firearms, and Explosives,     27, II
       Bureau of
  Drug Enforcement Administration                 21, II
  Federal Acquisition Regulation                  48, 28
  Federal Claims Collection Standards             31, IX
  Federal Prison Industries, Inc.                 28, III
  Foreign Claims Settlement Commission of the     45, V
       United States
  Immigration Review, Executive Office for        8, V
  Offices of Independent Counsel                  28, VI
  Prisons, Bureau of                              28, V
  Property Management Regulations                 41, 128
Labor Department                                  5, XLII
  Benefits Review Board                           20, VII
  Employee Benefits Security Administration       29, XXV
  Employees' Compensation Appeals Board           20, IV
  Employment and Training Administration          20, V

[[Page 505]]

  Employment Standards Administration             20, VI
  Federal Acquisition Regulation                  48, 29
  Federal Contract Compliance Programs, Office    41, 60
       of
  Federal Procurement Regulations System          41, 50
  Labor-Management Standards, Office of           29, II, IV
  Mine Safety and Health Administration           30, I
  Occupational Safety and Health Administration   29, XVII
  Public Contracts                                41, 50
  Secretary of Labor, Office of                   29, Subtitle A
  Veterans' Employment and Training Service,      41, 61; 20, IX
       Office of the Assistant Secretary for
  Wage and Hour Division                          29, V
  Workers' Compensation Programs, Office of       20, I
Labor-Management Standards, Office of             29, II, IV
Land Management, Bureau of                        43, II
Legal Services Corporation                        45, XVI
Library of Congress                               36, VII
  Copyright Office                                37, II
  Copyright Royalty Board                         37, III
Local Television Loan Guarantee Board             7, XX
Management and Budget, Office of                  5, III, LXXVII; 14, VI; 
                                                  48, 99
Marine Mammal Commission                          50, V
Maritime Administration                           46, II
Merit Systems Protection Board                    5, II, LXIV
Micronesian Status Negotiations, Office for       32, XXVII
Millenium Challenge Corporation                   22, XIII
Mine Safety and Health Administration             30, I
Minerals Management Service                       30, II
Minority Business Development Agency              15, XIV
Miscellaneous Agencies                            1, IV
Monetary Offices                                  31, I
Morris K. Udall Scholarship and Excellence in     36, XVI
     National Environmental Policy Foundation
Museum and Library Services, Institute of         2, XXXI
National Aeronautics and Space Administration     2, XVIII; 5, LIX; 14, V
  Federal Acquisition Regulation                  48, 18
National Agricultural Library                     7, XLI
National Agricultural Statistics Service          7, XXXVI
National and Community Service, Corporation for   45, XII, XXV
National Archives and Records Administration      2, XXVI; 5, LXVI; 36, XII
  Information Security Oversight Office           32, XX
National Capital Planning Commission              1, IV
National Commission for Employment Policy         1, IV
National Commission on Libraries and Information  45, XVII
     Science
National Council on Disability                    34, XII
National Counterintelligence Center               32, XVIII
National Credit Union Administration              12, VII
National Crime Prevention and Privacy Compact     28, IX
     Council
National Drug Control Policy, Office of           21, III
National Endowment for the Arts                   2, XXXII
National Endowment for the Humanities             2, XXXIII
National Foundation on the Arts and the           45, XI
     Humanities
National Highway Traffic Safety Administration    23, II, III; 49, V
National Imagery and Mapping Agency               32, I
National Indian Gaming Commission                 25, III
National Institute for Literacy                   34, XI
National Institute of Standards and Technology    15, II
National Intelligence, Office of Director of      32, XVII
National Labor Relations Board                    5, LXI; 29, I
National Marine Fisheries Service                 50, II, IV, VI
National Mediation Board                          29, X
National Oceanic and Atmospheric Administration   15, IX; 50, II, III, IV, 
                                                  VI
National Park Service                             36, I
National Railroad Adjustment Board                29, III
National Railroad Passenger Corporation (AMTRAK)  49, VII

[[Page 506]]

National Science Foundation                       2, XXV; 5, XLIII; 45, VI
  Federal Acquisition Regulation                  48, 25
National Security Council                         32, XXI
National Security Council and Office of Science   47, II
     and Technology Policy
National Telecommunications and Information       15, XXIII; 47, III
     Administration
National Transportation Safety Board              49, VIII
Natural Resources Conservation Service            7, VI
Navajo and Hopi Indian Relocation, Office of      25, IV
Navy Department                                   32, VI
  Federal Acquisition Regulation                  48, 52
Neighborhood Reinvestment Corporation             24, XXV
Northeast Interstate Low-Level Radioactive Waste  10, XVIII
     Commission
Nuclear Regulatory Commission                     5, XLVIII; 10, I
  Federal Acquisition Regulation                  48, 20
Occupational Safety and Health Administration     29, XVII
Occupational Safety and Health Review Commission  29, XX
Offices of Independent Counsel                    28, VI
Oklahoma City National Memorial Trust             36, XV
Operations Office                                 7, XXVIII
Overseas Private Investment Corporation           5, XXXIII; 22, VII
Patent and Trademark Office, United States        37, I
Payment From a Non-Federal Source for Travel      41, 304
     Expenses
Payment of Expenses Connected With the Death of   41, 303
     Certain Employees
Peace Corps                                       22, III
Pennsylvania Avenue Development Corporation       36, IX
Pension Benefit Guaranty Corporation              29, XL
Personnel Management, Office of                   5, I, XXXV; 45, VIII
  Human Resources Management and Labor Relations  5, XCIX
       Systems, Department of Defense
  Human Resources Management and Labor Relations  5, XCVII
       Systems, Department of Homeland Security
  Federal Acquisition Regulation                  48, 17
  Federal Employees Group Life Insurance Federal  48, 21
       Acquisition Regulation
  Federal Employees Health Benefits Acquisition   48, 16
       Regulation
Pipeline and Hazardous Materials Safety           49, I
     Administration
Postal Regulatory Commission                      5, XLVI; 39, III
Postal Service, United States                     5, LX; 39, I
Postsecondary Education, Office of                34, VI
President's Commission on White House             1, IV
     Fellowships
Presidential Documents                            3
Presidio Trust                                    36, X
Prisons, Bureau of                                28, V
Procurement and Property Management, Office of    7, XXXII
Productivity, Technology and Innovation,          37, IV
     Assistant Secretary
Public Contracts, Department of Labor             41, 50
Public and Indian Housing, Office of Assistant    24, IX
     Secretary for
Public Health Service                             42, I
Railroad Retirement Board                         20, II
Reclamation, Bureau of                            43, I
Refugee Resettlement, Office of                   45, IV
Relocation Allowances                             41, 302
Research and Innovative Technology                49, XI
     Administration
Rural Business-Cooperative Service                7, XVIII, XLII, L
Rural Development Administration                  7, XLII
Rural Housing Service                             7, XVIII, XXXV, L
Rural Telephone Bank                              7, XVI
Rural Utilities Service                           7, XVII, XVIII, XLII, L
Saint Lawrence Seaway Development Corporation     33, IV
Science and Technology Policy, Office of          32, XXIV
Science and Technology Policy, Office of, and     47, II
   National Security Council
[[Page 507]]

Secret Service                                    31, IV
Securities and Exchange Commission                17, II
Selective Service System                          32, XVI
Small Business Administration                     2, XXVII; 13, I
Smithsonian Institution                           36, V
Social Security Administration                    2, XXIII; 20, III; 48, 23
Soldiers' and Airmen's Home, United States        5, XI
Special Counsel, Office of                        5, VIII
Special Education and Rehabilitative Services,    34, III
     Office of
State Department                                  2, VI; 22, I; 28, XI
  Federal Acquisition Regulation                  48, 6
Surface Mining and Reclamation Appeals, Board of  30, III
Surface Mining Reclamation and Enforcement,       30, VII
     Office of
Surface Transportation Board                      49, X
Susquehanna River Basin Commission                18, VIII
Technology Administration                         15, XI
Technology Policy, Assistant Secretary for        37, IV
Technology, Under Secretary for                   37, V
Tennessee Valley Authority                        5, LXIX; 18, XIII
Thrift Supervision Office, Department of the      12, V
     Treasury
Trade Representative, United States, Office of    15, XX
Transportation, Department of                     2, XII; 5, L
  Commercial Space Transportation                 14, III
  Contract Appeals, Board of                      48, 63
  Emergency Management and Assistance             44, IV
  Federal Acquisition Regulation                  48, 12
  Federal Aviation Administration                 14, I
  Federal Highway Administration                  23, I, II
  Federal Motor Carrier Safety Administration     49, III
  Federal Railroad Administration                 49, II
  Federal Transit Administration                  49, VI
  Maritime Administration                         46, II
  National Highway Traffic Safety Administration  23, II, III; 49, V
  Pipeline and Hazardous Materials Safety         49, I
       Administration
  Saint Lawrence Seaway Development Corporation   33, IV
  Secretary of Transportation, Office of          14, II; 49, Subtitle A
  Surface Transportation Board                    49, X
  Transportation Statistics Bureau                49, XI
Transportation, Office of                         7, XXXIII
Transportation Security Administration            49, XII
Transportation Statistics Bureau                  49, XI
Travel Allowances, Temporary Duty (TDY)           41, 301
Treasury Department                               5, XXI; 12, XV; 17, IV; 
                                                  31, IX
  Alcohol and Tobacco Tax and Trade Bureau        27, I
  Community Development Financial Institutions    12, XVIII
       Fund
  Comptroller of the Currency                     12, I
  Customs and Border Protection Bureau            19, I
  Engraving and Printing, Bureau of               31, VI
  Federal Acquisition Regulation                  48, 10
  Federal Claims Collection Standards             31, IX
  Federal Law Enforcement Training Center         31, VII
  Fiscal Service                                  31, II
  Foreign Assets Control, Office of               31, V
  Internal Revenue Service                        26, I
  Investment Security, Office of                  31, VIII
  Monetary Offices                                31, I
  Secret Service                                  31, IV
  Secretary of the Treasury, Office of            31, Subtitle A
  Thrift Supervision, Office of                   12, V
Truman, Harry S. Scholarship Foundation           45, XVIII
United States and Canada, International Joint     22, IV
     Commission
United States and Mexico, International Boundary  22, XI
     and Water Commission, United States Section
Utah Reclamation Mitigation and Conservation      43, III
     Commission
Veterans Affairs Department                       2, VIII; 38, I
  Federal Acquisition Regulation                  48, 8

[[Page 508]]

Veterans' Employment and Training Service,        41, 61; 20, IX
     Office of the Assistant Secretary for
Vice President of the United States, Office of    32, XXVIII
Vocational and Adult Education, Office of         34, IV
Wage and Hour Division                            29, V
Water Resources Council                           18, VI
Workers' Compensation Programs, Office of         20, I
World Agricultural Outlook Board                  7, XXXVIII

[[Page 509]]



List of CFR Sections Affected



All changes in this volume of the Code of Federal Regulations that were 
made by documents published in the Federal Register since January 1, 
2001, are enumerated in the following list. Entries indicate the nature 
of the changes effected. Page numbers refer to Federal Register pages. 
The user should consult the entries for chapters and parts as well as 
sections for revisions.
Title 40 was established at 36 FR 12213, June 29, 1971. For the period 
before January 1, 2001, see the ``List of CFR Sections Affected, 1964-
1972, 1973-1985, and 1986-2000'', published in ten separate volumes.

                                  2001

                       (No regulations published)

                                  2002

                       (No regulations published)

                                  2003

                       (No regulations published)

                                  2004

40 CFR
                                                                   69 FR
                                                                    Page
Title 40 Nomenclature change.......................................18803
Chapter I
799.5115 Added.....................................................22436

                                  2005

40 CFR
                                                                   70 FR
                                                                    Page
Chapter I
799.5025 Table amended......................................39629, 39636

                                  2006

40 CFR
                                                                   71 FR
                                                                    Page
Chapter I
790.5 (b) amended..................................................33642
799.5 Amended......................................................33642
799.19 Revised.....................................................66245
799.5085 Added.....................................................13730
    (j) Table 2 amended............................................71062
799.5115 (h)(5)(vii)(A) and (j) Table 2 amended....................18654

                                  2007

                       (No regulations published)

                                  2008

                       (No regulations published)

                                  2009

  (No regulations published from January 1, 2009, through July 1, 2009)


                                  [all]