[Federal Register Volume 65, Number 242 (Friday, December 15, 2000)]
[Rules and Regulations]
[Pages 78746-78819]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 00-31728]



[[Page 78745]]

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

Part VI





Environmental Protection Agency





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



40 CFR Part 799



Toxic Substances Control Act Test Guidelines; Final Rule

  Federal Register / Vol. 65, No. 242 / Friday, December 15, 2000 / 
Rules and Regulations  

[[Page 78746]]


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

ENVIRONMENTAL PROTECTION AGENCY

40 CFR Part 799

[OPPTS-42211; FRL-6551-2]
RIN 2070-AD16


Toxic Substances Control Act Test Guidelines

AGENCY: Environmental Protection Agency (EPA).

ACTION: Final rule.

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

SUMMARY: This rule establishes 17 new Toxic Substances Control Act 
(TSCA) health effects test guidelines in the Code of Federal 
Regulations (CFR). Establishment of these guidelines provides a series 
of standardized test procedures and is necessary to ensure enforceable 
test standards in test rules promulgated under section 4 of TSCA. 
Codification of this series of TSCA test guidelines does not by itself 
impose obligations upon any person. Obligations are only imposed when 
these guidelines are cross-referenced in a test rule promulgated under 
section 4 of TSCA. The TSCA test guidelines are based on the harmonized 
test guidelines in the unified library for test guidelines issued by 
the Office of Prevention, Pesticides and Toxic Substances (OPPTS) for 
use in testing chemical substances to develop data for submission to 
EPA under TSCA, the Federal Food, Drug, and Cosmetic Act (FFDCA), and 
the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA). The 
process for developing and amending the harmonized test guidelines 
includes broad public participation and extensive involvement of the 
scientific community.

DATES: This rule is effective on December 15, 2000.

FOR FURTHER INFORMATION CONTACT: For general information contact: 
Barbara Cunningham, Director, Office of Program Management and 
Evaluation, Office of Pollution Prevention and Toxics (7401), 
Environmental Protection Agency, 1200 Pennsylvania Ave., NW., 
Washington, DC 20460; telephone number: (202) 554-1404; e-mail address: 
[email protected].
    For technical information regarding this action or related 
activities contact: Chemical Information and Testing Branch, Chemical 
Control Division, Office of Pollution Prevention and Toxics (7405), 
Environmental Protection Agency, 1200 Pennsylvania Ave., NW., 
Washington, DC 20460; telephone number: (202) 260-8130; e-mail address: 
[email protected].

SUPPLEMENTARY INFORMATION: This final rule establishes 17 new TSCA test 
guidelines in the series of TSCA test guidelines established in 40 CFR 
part 799.

I. General Information

A. Does this Action Apply to Me?

    You may be particularly interested in this action if you 
manufacture (defined by statute to include import) or process a 
chemical substance that could become the subject of a proposed test 
rule under TSCA section 4. This action does not, however, impose any 
obligations on anyone until the test guidelines are incorporated in a 
future test rule that would be proposed under TSCA section 4. 
Therefore, entities potentially affected by this action may include, 
but are not limited to:

 
------------------------------------------------------------------------
                                                          Examples of
         Type of Entity                 NAICS            Potentially
                                                      Affected Entities
------------------------------------------------------------------------
 Chemical Manufacturers or        325, 32411          Persons who
 Importers                                            manufacture
                                                      (defined by
                                                      statute to include
                                                      import) one or
                                                      more of the
                                                      subject chemical
                                                      substances.
 Chemical Processors              325, 32411          Persons who
                                                      process one or
                                                      more of the
                                                      subject chemical
                                                      substances.
------------------------------------------------------------------------

    This listing is not intended to be exhaustive, but rather provides 
a guide for readers regarding entities likely to be affected by this 
action. The North American Industrial Classification System (NAICS) 
codes have been provided to assist you and others in determining 
whether or not this action might apply to certain entities. If you have 
any questions regarding the applicability of this action to a 
particular entity, consult the technical information contact listed 
under FOR FURTHER INFORMATION CONTACT.

B. How Can I Get Additional Information, Including Copies of this 
Document and Other Related Documents?

    1. Electronically. You may obtain electronic copies of this 
document, and certain other related documents that might be available 
electronically, from the EPA Internet Home Page at http://www.epa.gov/. 
To access this document, on the Home Page select ``Laws and 
Regulations'' and then look up the entry for this document under the 
``Federal Register--Environmental Documents.'' You can also go directly 
to the Federal Register listings at http://www.epa.gov/fedrgstr/.
    2. In person. The Agency has established an official record for 
this action under docket control number OPPTS-42211. The official 
record consists of the documents specifically referenced in this 
action, any public comments received during an applicable comment 
period, and other information related to this action, including any 
information claimed as confidential business information (CBI). This 
official record includes the documents that are physically located in 
the docket, as well as the documents that are referenced in those 
documents. The public version of the official record does not include 
any information claimed as CBI. The public version of the official 
record, which includes printed, paper versions of any electronic 
comments submitted during an applicable comment period, is available 
for inspection in the TSCA Nonconfidential Information Center, North 
East Mall Rm. B-607, Waterside Mall, 401 M St., SW., Washington, DC. 
The Center is open from noon to 4 p.m., Monday through Friday, 
excluding legal holidays. The telephone number for the Center is (202) 
260-7099.

II. Background

A. What are Test Guidelines?

    Test guidelines are a standardized set of test procedures or 
protocols organized by health effect or other testing endpoint. These 
guidelines present generally formulated procedures for laboratory 
testing of an effect or characteristic deemed important for the 
evaluation of health and environmental hazards of a chemical. These 
guidelines are designed to, when followed, produce data which are 
accurate, reliable, and reproducible. Such data are necessary for the 
regulatory programs under TSCA.
    In adding these 17 TSCA test guidelines to the existing series of 
11 TSCA test guidelines, EPA recognizes concerns have been expressed 
about animal testing. EPA is committed to avoiding unnecessary or 
duplicative animal testing. As part of this commitment, the Agency 
plays an

[[Page 78747]]

important role in the Federal Interagency Coordinating Committee on the 
Validation of Alternative Methods (ICCVAM) (http://iccvam.niehs.nih.gov/home.htm) whose goals are: (1) To encourage the 
reduction of the number of animals used in testing; (2) to seek 
opportunities to replace test methods requiring animals with 
alternative test methods when acceptable alternative methods are 
available; and (3) to refine existing test methods to optimize animal 
use when there is no substitute for animal testing. Further, where 
testing is needed to develop scientifically adequate data, the Agency 
is committed to reducing the number of animals used for testing, 
including, whenever possible, by incorporating in vitro (non-animal) 
test methods or other alternative approaches that have been 
scientifically validated and have received regulatory acceptance. EPA 
considers these goals and commitments to be important considerations in 
developing health effects data; however, they must be balanced with the 
essential need to conduct scientifically sound chemical hazard/risk 
assessments in support of the Agency's mission to protect human health 
and the environment.
    The completion of this series of 28 TSCA test guidelines in part 
799 provides EPA with a range of guidelines available for cross-
referencing in TSCA actions. Several of these guidelines include in 
their design elimination of animals or reduction in the number of 
animals needed to conduct the tests. Some of the methods are designed 
to only develop data on chemical/physical properties. In addition, one 
of the guidelines involves the development of metabolism and 
pharmacokinetics data which could facilitate route-to-route 
extrapolations to existing (e.g., oral route) data and thus involve 
fewer test animals as compared to developing new data by, for example, 
the inhalation route. EPA believes that using these test guidelines 
will result in use of fewer test animals when it becomes necessary to 
conduct testing to fill identified data needs and will yield 
scientifically sound data.

B. What are TSCA Test Guidelines?

    TSCA test guidelines are guidelines which were established to meet 
the regulatory needs of TSCA, particularly the needs of the TSCA 
section 4 testing program. The TSCA section 4 testing program is a 
regulatory program which is based on the promulgation of rules 
requiring certain persons identified in the rule, usually manufacturers 
and processors of the chemical to conduct testing of the chemical 
specified in the rule. Section 4(b)(1)(B) of TSCA specifically requires 
that test rules promulgated under the section 4 include ``standards for 
the development of test data for such substance or mixture * * *.'' 
These ``standards for the development of test data'' specify how the 
study is to be conducted, what data will be collected, and how the data 
will be analyzed. Each test rule must specify such ``test standards'' 
which contain specifications for testing. Section 4(b)(1) of TSCA 
describes the elements which must be described in these test standards.
    The Agency has found that most of these elements can be 
standardized into the common set of protocols which EPA defines as 
``test guidelines.'' These guidelines are organized by testing 
endpoint. The test rule itself can add or subtract to the requirements 
of the test guidelines in order to meet the unique testing 
circumstances for the particular chemical substance.

C. How are TSCA Test Guidelines Used?

    The Agency uses this system of standardized guidelines, organized 
by testing endpoint and codified in a subpart of this part for use in 
cross-referencing in a TSCA section 4 action. When a section 4 test 
rule is promulgated, the test rule cross-references the appropriate 
TSCA test guideline for the bulk of the testing requirements. In this 
context, the public is given notice of, and an opportunity to comment 
on, these guidelines as they are applied in chemical-specific test 
rules. This approach eliminates the need to repeat the same test 
specifications for each substance-specific test rule since most of the 
specifications for testing do not change across substances. The test 
specifications in a guideline can be varied, when necessary, to the 
specific requirements of a test rule by language in the test rule 
itself.

D. Where Did the TSCA Test Guidelines Come From?

    The TSCA test guidelines series were first promulgated in 1985 (50 
FR 39252, September 27, 1985) and were established in 40 CFR parts 795 
through 798. The Agency has over time amended and improved these 
guidelines (52 FR 19072, May 20, 1987) and in some cases revoked those 
guidelines which had not been cross-referenced in any test rules (60 FR 
31917, June 19, 1995) (FRL-4955-2)).
    In 1991, EPA began an effort to blend the testing guidance and 
requirements that existed in the Office of Pollution Prevention and 
Toxics (OPPT) appearing in 40 CFR parts 795 through 798, the Office of 
Pesticides Programs (OPP) guidelines which appeared in publications of 
the National Technical Information Service (NTIS), and the guidelines 
published by the international Organization for Economic Cooperation 
and Development (OECD). The product of this effort would be one set of 
guidelines which would be thus blended or ``harmonized.'' These 
harmonized guidelines would then be made available to the EPA, other 
government agencies, and the public through the World Wide Web 
(Internet) and would be accessible by anyone with a personal computer 
and the ability to connect to the Internet. The EPA Internet web site 
would be the site and publication source for the ``OPPTS Harmonized 
Guidelines'' at http://www.epa.gov/opptsfrs/home/guidelin.htm.
    In addition, EPA has published three new OPPTS harmonized test 
guidelines for three health effects end points. These three guidelines 
(with their OPPTS harmonized guideline reference) are: (1) Repeated 
dose 28-day oral toxicity study in rodents (OPPTS 870.3050), (2) 
Reproduction/developmental toxicity screening test (OPPTS 870.3550), 
and (3) Combined repeat dose toxicity study with the reproduction/
developmental toxicity screening test (OPPTS 870.3650). Their 
publication was announced in the Federal Register of July 13, 2000 (65 
FR 43329) (FRL-6393-5), with the guidelines available from the EPA 
Internet web site at http://www.epa.gov/opptsfrs/home/guidelin.htm. By 
adopting these combined testing guidelines, which incorporate more than 
one endpoint, the Agency is acknowledging the desirability of reducing 
costs and numbers of animals required to meet the Agency's testing 
needs. EPA recommends the use of combined protocols wherever feasible 
to meet data development requirements.

E. How were these OPPTS Harmonized Test Guidelines Developed?

    The OPPTS harmonized test guidelines for health effects endpoints 
were first drafted by EPA scientists for specific testing endpoints. 
These drafts were reviewed by other EPA experts and, in some instances, 
presented at domestic and international colloquia in order to solicit 
the views of recognized experts and the regulated community. These 
draft harmonized guidelines were made available on the Internet as 
public drafts and a notice was published in the Federal Register of 
June 20, 1996 (61 FR 31522) (FRL-5367-7) announcing the availability of 
these draft guidelines soliciting public comment.

[[Page 78748]]

    After review of the public drafts, EPA published the final OPPTS 
harmonized guidelines for the health effects endpoints on the Internet 
and announced their availability to the public in the Federal Register 
of August 5, 1998 (63 FR 41845) (FRL-5740-1). EPA published the 
rationale for the changes made in finalizing the June 1996 OPPTS 
``Public Draft'' guidelines to the August 1998 OPPTS ``Final'' 
guidelines in a document entitled ``Overview and Summary of Changes 
made in the Harmonization of OPPTS 870 Toxicology Guidelines with OECD 
Guidelines'' (which is available at http://www.epa.gov/opptsfrs/home/guidelin.htm).

F. What is Done to Make TSCA Test Guidelines From the OPPTS Harmonized 
Test Guidelines?

    Harmonization has resulted in significantly improved guidelines. 
However, creating a single set of guidelines which can be used by both 
OPP, in its administration of the Federal Insecticide, Fungicide, and 
Rodenticide Act (FIFRA), and the Federal Food, Drug and Cosmetic Act 
(FFDCA), and the Office of Pollution Prevention and Toxics (OPPT), 
which administers TSCA presents certain challenges. Under FIFRA, test 
guidelines are used in an interactive process between the Agency and 
registrants seeking registration of pesticides or food residue 
tolerances. Flexibility to tailor required testing to individual 
circumstances is critical, and the Agency has considerable discretion 
to determine whether submitted test results are adequate to support the 
requested action. Under this scheme, registrants have an intrinsic 
motivation to conduct well-grounded testing. Thus, pesticide testing 
protocols tend to have few absolute requirements specifying the details 
of the conduct of the testing.
    Under section 4 of TSCA, on the other hand, the Agency imposes 
prescriptive test requirements using notice and comment rulemaking. 
Rules promulgated under section 4 of TSCA must specify classes of 
affected parties and specify the standards to be followed by these 
parties in conducting the required testing. In contrast to FIFRA, the 
Agency does not interact with companies on an individual basis in 
designing the testing requirements.
    TSCA section 4 rulemakings typically take years to complete. 
Without initiating another rulemaking process, the Agency has the 
ability to require further testing only if the tests were not conducted 
in accordance with the procedures specified in the test rule. In 
addition, the Agency has an alternative process of negotiating TSCA 
testing requirements via enforceable consent agreements (ECAs), but 
these agreements require the consent of all the parties involved. Under 
TSCA section 4 enforceable test standards, much in the conduct of these 
test protocols is left to the judgment of those professionals 
conducting the testing. EPA believes that certain provisions must be 
mandatory whenever the guidelines are cross-referenced in specific test 
rules.
    Therefore, the Agency has used the OPPTS harmonized test guidelines 
developed using the public notice and comment process to create the 
TSCA-specific test guidelines which are the subject of this rule. TSCA 
section 4 test rules now cross-reference only the part 799 guidelines 
rather than the older, non-harmonized guidelines established in 40 CFR 
parts 795 through 798 mostly in 1985. The only significant difference 
between the TSCA test guidelines and the OPPTS harmonized test 
guidelines is that certain discretionary procedures in the OPPTS 
harmonized test guidelines are made mandatory (i.e., the guideline 
states that they ``must'' be carried out) in order to ensure the 
enforcibility of the test standard.
    EPA promulgated the first set of guidelines in the new part 799 
guidelines series in a Federal Register document published on August 
15, 1997 (62 FR 43820) (FRL-5719-5). Eleven health effects guidelines 
were published, including those for inhalation toxicity, developmental 
toxicity, reproductive effects, carcinogenicity, mutagenicity, and 
immunotoxicity. EPA amended 7 of these 11 guidelines in a Federal 
Register document published on June 30, 1999 (64 FR 35072) (FRL-6067-
4). These amendments reflected changes made to the corresponding OPPTS 
harmonized guideline.

III. What Action is Being Taken?

    EPA is adding 17 new health effects test guidelines to 40 CFR part 
799. These 17 new guidelines are listed in the following table 1 with 
the OPPTS harmonized guideline from which it was developed:

                                 Table 1
------------------------------------------------------------------------
                                            Original OPPTS harmonized
New TSCA test guideline name (and cite)     guideline name (and cite)
------------------------------------------------------------------------
TSCA partition coefficient (n-octanol/   Partition coefficient (n-
 water) shake flask method (799.6755).    octanol/H2O) shake flask
                                          method (830.7550)
TSCA partition coefficient (n-octanol/   Partition coefficient (n-
 water), generator column method          octanol/H2O), generator column
 (799.6756).                              method (830.7560)
TSCA water solubility: Column elution    Water solubility: Column
 method; shake flask method (799.6784).   elution method; shake flask
                                          method (830.7840)
TSCA water solubility, generator column  Water solubility, generator
 method (799.6786).                       column method (830.7860)
TSCA acute oral toxicity (799.9110)....  Acute oral toxicity (870.1100)
TSCA acute dermal toxicity (799.9120)..  Acute dermal toxicity
                                          (870.1200)
TSCA acute inhalation toxicity           Acute inhalation toxicity
 (799.9130).                              (870.1300)
TSCA repeated dose 28-day oral toxicity  Repeated dose 28-day oral
 study in rodents (799.9305).             toxicity study in rodents
                                          (870.3050)
TSCA 90-day oral toxicity in rodents     90-day oral toxicity in rodents
 (799.9310).                              (870.3100)
TSCA 90-day dermal toxicity (799.9325).  90-day dermal toxicity
                                          (870.3250)
TSCA reproduction/developmental          Reproduction/developmental
 toxicity screening test (799.9355).      toxicity screening test
                                          (870.3550)
TSCA combined repeated dose toxicity     Combined repeated dose toxicity
 study with the reproduction/             study with the reproduction/
 developmental toxicity screening test    developmental toxicity
 (799.9365).                              screening test (870.3650)
TSCA chronic toxicity (799.9410).......  Chronic toxicity (870.4100)
TSCA combined chronic toxicity/          Combined chronic toxicity/
 carcinogenicity (799.9430).              carcinogenicity (870.4300)
TSCA in vitro mammalian chromosome        In vitro mammalian chromosome
 aberration test (799.9537).              aberration test (870.5375)
TSCA developmental neurotoxicity         Developmental neurotoxicity
 (799.9630).                              (870.6300)
TSCA metabolism and pharmacokinetics     Metabolism and pharmacokinetics
 (799.9748).                              (870.7485)
------------------------------------------------------------------------


[[Page 78749]]

IV. How are the New TSCA Test Guidelines Different From the OPPTS 
Harmonized Test Guideline From Which they were Derived?

    EPA developed the TSCA test guideline from the original OPPTS test 
guideline shown in the right column of table 1 in Unit III. In keeping 
with the policy of using a unified set of test guidelines across OPPTS, 
only minimal changes were made to the OPPTS harmonized guidelines in 
the development of the TSCA guidelines. These minimal changes consisted 
of deleting references to FIFRA in the TSCA guidelines, where it was 
believed by the Agency to be irrelevant to the purpose of the TSCA test 
guideline, and to specify those provisions of the guidelines which the 
Agency believed must be made mandatory in order to ensure the integrity 
of any data produced by the test.
    EPA sumarizes below, guideline-by-guideline, the changes the Agency 
made to the OPPTS harmonized test guideline in developing the TSCA test 
guideline.

A. Section 799.6755 TSCA Partition Coefficient (n-octanol/water), Shake 
Flask Method

    1. EPA deleted references to FIFRA.
    2. EPA made several grammatical changes.
    3. EPA deleted references which were unavailable.

B. Section 799.6756 TSCA Partition Coefficient (n-octanol/water), 
Generator Column Method

    1. EPA deleted references to FIFRA.
    2. EPA made several grammatical changes.
    3. EPA deleted references which were unavailable.

C. Section 799.6784 TSCA Water Solubility; Column Elution Method; Shake 
Flask Method

    1. EPA deleted references to FIFRA.
    2. EPA made several grammatical changes.
    3. EPA deleted references which were unavailable.

D. Section 799.6786 TSCA, Water Solubility, Generator Column Method

    1. EPA deleted references to FIFRA.
    2. EPA made several grammatical changes.
    3. EPA deleted references which were unavailable.

E. Section 799.9110 TSCA Acute Oral Toxicity

    1. EPA clarified those provisions describing alternative acute 
testing procedures. EPA acknowledges that both the current OPPTS 
harmonized guideline and international consideration of acute toxicity 
guidelines are in a period of transition toward specifying reduced 
animal testing requirements.
    2. EPA deleted references to FIFRA and discussions of pesticides.
    3. EPA added ``musts'' to those requirements deemed critical to the 
successful production of scientifically-valid data for Agency risk 
assessment purposes.

F. Section 799.9120 TSCA Acute Dermal Toxicity

    1. EPA deleted references to FIFRA and discussions of pesticides.
    2. EPA added ``musts'' to those requirements deemed critical to the 
successful production of scientifically-valid data for Agency risk 
assessment purposes.

G. Section 799.9130 TSCA Acute Inhalation Toxicity

    1. EPA deleted references to FIFRA and discussions of pesticides.
    2. EPA added ``musts'' to those requirements deemed critical to the 
successful production of scientifically-valid data for Agency risk 
assessment purposes.
    3. EPA revised and reorganized certain narrative sections for 
consistency with the comparable sections in the previously-promulgated 
40 CFR 799.9135 (TSCA acute inhalation toxicity with histopathology).

H. Section 799.9305 TSCA Repeated Dose 28-day Oral Toxicity Study in 
Rodents

    1. EPA deleted references to FIFRA and discussions of pesticides.
    2. EPA made editorial changes to text to ensure consistency with 
the TSCA series of guidelines.

I. Section 799.9310 TSCA 90-day Oral Toxicity in Rodents

    1. EPA deleted references to FIFRA and discussions of pesticides.
    2. EPA added ``musts'' to those requirements deemed critical to the 
successful production of scientifically-valid data for Agency risk 
assessment purposes.
    3. EPA removed the included neurotoxicity testing provisions in 
paragraphs (e)(8)(ii) through (e)(8)(v) because TSCA practice is to 
specify the more detailed neurotoxicity testing provisions of 40 CFR 
799.9620.

J. Section 799.9325 TSCA 90-day Dermal Toxicity

    1. EPA deleted references to FIFRA and discussions of pesticides.
    2. EPA added ``musts'' to those requirements deemed critical to the 
successful production of scientifically-valid data for Agency risk 
assessment purposes.
    3. EPA removed the included neurotoxicity testing provisions in 
paragraphs (e)(9)(ii) through (e)(9)(v) because TSCA practice is to 
specify the more detailed neurotoxicity testing provisions of 40 CFR 
799.9620.
    4. EPA deleted references which were unavailable.

K. Section 799.9355 TSCA Reproduction/Developmental Toxicity Screening 
Test

    1. EPA deleted references to FIFRA and discussions of pesticides.
    2. EPA made editorial changes to text to ensure consistency with 
the TSCA series of guidelines.

L. Section 799.9365 TSCA Combined Repeated Dose Toxicity Study With the 
Reproduction/Developmental Toxicity Screening Test

    1. EPA deleted references to FIFRA and discussions of pesticides.
    2. EPA made editorial changes to text to ensure consistency with 
the TSCA series of guidelines.

M. Section 799.9410 TSCA Chronic Toxicity

    1. EPA deleted references to FIFRA and discussions of pesticides.
    2. EPA removed recommendations for the use of particular non-rodent 
species.

N. Section 799.9430 TSCA Combined Chronic Toxicity/Carcinogenicity

    1. EPA deleted references to FIFRA and discussions of pesticides.
    2. EPA added ``musts'' to those requirements deemed critical to the 
successful production of scientifically-valid data for Agency risk 
assessment purposes.
    3. EPA removed the included neurotoxicity testing provisions in 
paragraphs (e)(7)(ii)through (e)(7)(v) because TSCA practice is to 
specify the more detailed neurotoxicity testing provisions of 40 CFR 
799.9620.
    4. EPA deleted references which were unavailable.
    5. EPA added a new provision (paragraph (e)(5)(ii)(J)) requiring 
that care be taken when the physical and chemical properties of the 
test substance show a low flash point or is otherwise known or thought 
to be explosive.

O. Section 799.9537 TSCA in vitro Mammalian Chromosome Aberration Test

    1. EPA deleted references to FIFRA.
    2. EPA made several provisions mandatory by specifying ``must'' 
instead of ``should.''

[[Page 78750]]

    3. EPA clarified the regulatory text in citing particular 
references in the standard.

P. Section 799.9630 TSCA Developmental Neurotoxicity

    1. EPA deleted references to FIFRA.
    2. EPA made several provisions mandatory by specifying ``must'' 
instead of ``should.''

Q. Section 799.9748 TSCA Metabolism and Pharmacokinetics

    1. EPA deleted references to FIFRA.
    2. EPA made several provisions mandatory by specifying ``must'' 
instead of ``should.''

V. Regulatory Assessment Requirements

A. Why is this Action Being Issued as a Final Rule?

    EPA is publishing this action as a final rule without prior notice 
and an opportunity to comment because the Agency believes that 
providing notice and an opportunity to comment is unnecessary. The test 
guidelines codified in this document by themselves have no substantive 
effect on any person until and unless the test guidelines are 
incorporated in a test rule promulgated under TSCA section 4. Before 
any such test rule is promulgated, EPA will provide notice and an 
opportunity to comment on the incorporation of a particular test 
guideline into a specific test rule. In addition, the process for 
developing and amending the harmonized test guidelines includes broad 
public participation and extensive involvement of the scientific 
community. EPA therefore finds that there is ``good cause'' under 
section 553(b)(3)(B) of the Administrative Procedure Act (APA) (5 
U.S.C. 553(b)(3)(B)) to codify these test guidelines without prior 
notice and comment, and that this rule may be made effective 
immediately, without a 30 day delay, pursuant to 5 U.S.C. 553(d)(3).

B. Do the Regulatory Assessment Requirements Apply to this Action?

    No. As indicated previously, this final rule does not impose any 
requirements. It only incorporates test guidelines into the TSCA series 
of test guidelines that are published in the CFR and which would be 
considered for potential incorporation in a future test rule that would 
be proposed under TSCA section 4. At which time potentially affected 
entities are afforded an opportunity to comment on the incorporation of 
a particular test guideline into a specific test rule.
    As such, this is not a ``significant regulatory action'' that 
requires review by the Office of Management and Budget (OMB) under 
Executive Order 12866, entitled Regulatory Planning and Review (58 FR 
51735, October 4, 1993).
    Since this action is not ``economically significant'' as defined by 
section 3(f) of Executive Order 12866, this action is not subject to 
Executive Order 13045, entitled Protection of Children from 
Environmental Health Risks and Safety Risks (62 FR 19885, April 23, 
1997).
    This action will not result in environmental justice related issues 
and does not, therefore, require special consideration under Executive 
Order 12898, entitled Federal Actions to Address Environmental Justice 
in Minority Populations and Low-Income Populations (59 FR 7629, 
February 16, 1994).
    Since the Agency has made a ``good cause'' finding that this action 
is not subject to notice-and-comment requirements under the APA or any 
other statute (see Unit V.A.), this action is not subject to the 
regulatory flexibility provisions of the Regulatory Flexibility Act 
(RFA) (5 U.S.C. 601 et seq.), or to sections 202 and 205 of the 
Unfunded Mandates Reform Act of 1995 (UMRA) (Public Law 104-4). In 
addition, this action does not significantly or uniquely affect small 
governments or impose a significant intergovernmental mandate, as 
described in sections 203 and 204 of UMRA. Nor does this action 
significantly or uniquely affect the communities of tribal governments 
as specified by Executive Order 13084, entitled Consultation and 
Coordination with Indian Tribal Governments (63 FR 27655, May 10, 
1998). This rule will not have substantial direct effects on the 
States, on the relationship between the national government and the 
States, or on the distribution of power and responsibilities among the 
various levels of government, as specified in Executive Order 13132, 
entitled Federalism (64 FR 43255, August 10, 1999).
    This rule does not contain any information collection requirements 
that require review and approval by OMB pursuant to the Paperwork 
Reduction Act of 1995 (PRA) (44 U.S.C. 3501 et seq.).
    In issuing this rule, EPA has taken the necessary steps to 
eliminate drafting errors and ambiguity, minimize potential litigation, 
and provide a clear legal standard for affected conduct, as required by 
section 3 of Executive Order 12988, entitled Civil Justice Reform (61 
FR 4729, February 7, 1996).
    EPA has also complied with Executive Order 12630, entitled 
Governmental Actions and Interference with Constitutionally Protected 
Property Rights (53 FR 8859, March 15, 1988), by examining the takings 
implications of this rule in accordance with the ``Attorney General's 
Supplemental Guidelines for the Evaluation of Risk and Avoidance of 
Unanticipated Takings'' issued under the Executive Order.

C. Are there Any Applicable Voluntary Consensus Standards?

    No. Section 12(d) of the National Technology Transfer and 
Advancement Act of 1995 (NTTAA), Public Law 104-113, section 12(d) (15 
U.S.C. 272 note), directs EPA to use voluntary consensus standards in 
its regulatory activities unless to do so would be inconsistent with 
applicable law or otherwise impractical. Voluntary consensus standards 
are technical standards (e.g., materials specifications, test methods, 
sampling procedures, business practices, etc.) that are developed or 
adopted by voluntary consensus standards bodies. The NTTAA requires EPA 
to provide an explanation to Congress, through OMB, when the Agency 
decides not to use available and applicable voluntary consensus 
standards when the NTTAA directs the Agency to do so.
    As indicated earlier, this final rule does not impose any 
obligations on anyone until the test guidelines are incorporated in a 
test rule promulgated under TSCA section 4. Before any such test rule 
is promulgated, EPA will provide notice and an opportunity to comment 
on the incorporation of a particular test guideline into that specific 
test rule, including the availability of applicable voluntary consent 
standards.
    In addition, although the NTTAA requirements do not specifically 
apply to the issuance of the harmonized test guidelines, EPA has sought 
comments on the availability of applicable voluntary consensus 
standards that should be considered during the development of future 
rules under TSCA. This allows the Agency to consider such standards 
during the development of the harmonized test guidelines, upon which 
the TSCA test guidelines are based.

VI. Submission to Congress and the Comptroller General

    The Congressional Review Act, 5 U.S.C. 801 et seq., as added by the 
Small Business Regulatory Enforcement Fairness Act of 1996, generally 
provides that before a rule may take effect, the agency promulgating 
the rule must submit a rule report, which includes a

[[Page 78751]]

copy of the rule, to each House of the Congress and to the Comptroller 
General of the United States. Section 808 allows the issuing agency to 
make a good cause finding that notice and public procedure is 
impracticable, unnecessary or contrary to the public interest. This 
determination must be supported by a brief statement. 5 U.S.C. 808(2). 
EPA has made such a good cause finding for this final rule, and 
established an effective date of December 15, 2000. Pursuant to 5 
U.S.C. 808(2), this determination is supported by the brief statement 
in Unit V.A. EPA will submit a report containing this final rule and 
other required information to the U.S. Senate, the U.S. House of 
Representatives, and the Comptroller General of the United States prior 
to publication of the rule in the Federal Register. This is not a 
``major rule'' as defined by 5 U.S.C. 804(2).

List of Subjects in 40 CFR Part 799

    Environmental protection, Chemicals, Hazardous substances, Health, 
Reporting and recordkeeping requirements.

    Dated: November 27, 2000.
Susan H. Wayland,
Acting Assistant Administrator for Prevention, Pesticides and Toxic 
Substances.

    Therefore, 40 CFR part 799 is amended as follows:

PART 799--[AMENDED]

    1. The authority citation for part 799 continues to read as 
follows:

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

    2. A new subpart E, consisting of Secs. 799.6755 to 799.6786 is 
added to read as follows:
Subpart E--Product Properties Test Guidelines
Sec.
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.

Subpart E--Product Properties Test Guidelines


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 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  105       (4.6  x
                                          104 - 2.8  x  105)
Hexachlorobenzene (OECD)...............      3.6  x  105       (1.1  x
                                          105 - 8.3  x  105)
o-Dichlorobenzene European Economic          5.1  x  103       (1.5  x
 Community (EEC).                         103 - 2.3  x  104)
Dibutyl phthalate (EEC)................      1.3  x  104       (1.7  x
                                          103 - 2.8  x  104)
Trichloroethylene (OECD)...............      2.0  x  103       (5.2 x
                                          102 - 3.7 x 103)
Urea (OECD)............................      6.2 x 10-2       (2.0 x 10-
                                          2--2.4 x 10-1)
------------------------------------------------------------------------
\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

[[Page 78752]]

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   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.
    (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 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

[[Page 78753]]

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).
    (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 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

[[Page 78754]]

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 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 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
                                                               -----------------------------------------------------------------------
                           Chemical                                           Generator                           Hansch                  Recommended
                                                                 Hansch and     Column    Banerjee\2\    Other      and    Hawker and       log10Kow
                                                                   Leo\1\       Method                  values    Leo\3\   Connell\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

[[Page 78755]]

 
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.
\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.


[[Page 78756]]

    (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 
106).
    (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 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 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

[[Page 78757]]

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 30 x 0.6 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
[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 L loop, use n = 5; for a 50 
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 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 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

[[Page 78758]]

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 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

[[Page 78759]]

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 
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 
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 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.

[[Page 78760]]

    (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.
    (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 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

[[Page 78761]]

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-    6
                                                                                                       2
                                                   25      9.96  x  10-3    1.19  x  10-3 to 2.31  x  10-    7
                                                                                                       2
-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
----------------------------------------------------------------------------------------------------------------

    (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:

[[Page 78762]]

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:

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 78763]]

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 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).....................   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 78765]]

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.
    (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

[[Page 78766]]

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.
    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/103 g or parts per million (ppm); those expressed in 
micrograms per liter (g/L) are approximately equal to 
10-6 g/103 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/cm3). 
The density of a solution is the mass of a unit volume of the solution 
and suitable units of measurement are g/cm3.
    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 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 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

[[Page 78767]]

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.

    (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 is 
enclosed in a water jacket for temperature control as shown in the 
following figure 2:

[[Page 78768]]

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 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 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
  340.5 g/kilogram (kg) and the pH 8.00.2. 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:

[[Page 78769]]

Figure 3--Schematic of HPLC--Generator Column Flow System
[GRAPHIC] [TIFF OMITTED] TR15DE00.057

    (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 L loop, use n = 5; for a 50 
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 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:
[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%

[[Page 78770]]

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 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 
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

[[Page 78771]]

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.
    (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.

Subpart H--[Amended]

    3. Sections 799.9110, 799.9120, 799.9130, 799.9305, 799.9310, 
799.9325, 799.9355, 799.9365, 799.9410, 799.9430, 799.9537, 799.9630, 
and 799.9748 are added to subpart H to read as follows:


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

[[Page 78772]]

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 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 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.

[[Page 78773]]

    (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 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 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.

[[Page 78774]]

    (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 
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.


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, 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.

[[Page 78775]]

    (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 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

[[Page 78776]]

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 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.
    (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.


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.

[[Page 78777]]

    (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 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 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 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

[[Page 78778]]

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 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 
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 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 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-

[[Page 78779]]

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; 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

[[Page 78780]]

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).
    (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.


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 
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

[[Page 78781]]

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 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.
    (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

[[Page 78782]]

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 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.
    (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.

[[Page 78783]]

    (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 H.A., MacPhail R.C. (1991). Interlaboratory Comparison 
of Motor Activity Experiments: Implication for Neurotoxicological 
Assessments. Neurotoxicology and Teratology, 13:599-609.


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

[[Page 78784]]

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.
    (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 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

[[Page 78785]]

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 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.
    (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

[[Page 78786]]

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.
    (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)


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).
    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

[[Page 78787]]

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 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 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 78788]]

    (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) 
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.

[[Page 78789]]

    (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, 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 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.


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.
    (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 Secs. 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,

[[Page 78790]]

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 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 water using the 
procedures described for this study, produces no observable toxic 
effects and if toxicity would not be expected based upon data from

[[Page 78791]]

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.
    [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.

[[Page 78792]]

    (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 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., 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.

[[Page 78793]]

    (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]


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 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 Secs. 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, 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

[[Page 78794]]

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 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.
    (iii) Animals in a satellite group scheduled for follow-up 
observations, if

[[Page 78795]]

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.
    (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.

[[Page 78796]]

    (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 
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 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

[[Page 78797]]

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.
    (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 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.


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

[[Page 78798]]

(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 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.
    (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

[[Page 78799]]

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, 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 L for the mouse and less than 300 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 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

[[Page 78800]]

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 
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 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 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

[[Page 78801]]

enzymes, (such as alanine aminotransferase, aspartate aminotransferase, 
alkaline phosphatase, sorbitol dehydrogenase, or 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 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.

[[Page 78802]]

    (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.
    (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 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.


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

[[Page 78803]]

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 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%.
    (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

[[Page 78804]]

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.
    (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 L for the mouse and less than 300 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 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

[[Page 78805]]

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 
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 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 
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, 
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

[[Page 78806]]

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.
    (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:

[[Page 78807]]

    (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) 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 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.


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 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

[[Page 78808]]

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 to the test 
substance, they are treated with a metaphase-arresting substance (e.g., 
Colcemid 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 -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.
    (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 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

[[Page 78809]]

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 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 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 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

[[Page 78810]]

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 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. 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).

[[Page 78811]]

    (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).


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 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.

[[Page 78812]]

    (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.
    (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 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

[[Page 78813]]

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 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

[[Page 78814]]

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 
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.
    (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

[[Page 78815]]

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. 
15:213-218 (1985).
    (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).


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 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 14C 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

[[Page 78816]]

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.
    (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 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

[[Page 78817]]

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.
    (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.

[[Page 78818]]

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, 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 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.

[[Page 78819]]

    (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 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.

[FR Doc. 00-31728 Filed 12-14-00; 8:45 am]
BILLING CODE 6560-50-F