[Federal Register Volume 62, Number 158 (Friday, August 15, 1997)]
[Rules and Regulations]
[Pages 43820-43864]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 97-21413]



[[Page 43819]]

_______________________________________________________________________

Part III





Environmental Protection Agency





_______________________________________________________________________



40 CFR Part 799



Toxic Substances Control Act Test Guidelines; Final Rule

  Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules 
and Regulations  



[[Page 43820]]


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ENVIRONMENTAL PROTECTION AGENCY

40 CFR Part 799

[OPPTS-42193; FRL-5719-5]
RIN 2070-AB76


Toxic Substances Control Act Test Guidelines

AGENCY: Environmental Protection Agency (EPA).

ACTION: Final rule.

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

SUMMARY: This rule establishes 11 Toxic Substances Control Act (TSCA) 
health effects test guidelines in the Code of Federal Regulations 
(CFR). Establishment of these guidelines 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.

DATES: This rule is effective on August 15, 1997.

FOR FURTHER INFORMATION CONTACT: Susan Hazen, Director, Environmental 
Assistance Division (7408), Office of Pollution Prevention and Toxics, 
Environmental Protection Agency, Rm. E-543B, 401 M St., SW., 
Washington, DC 20460; telephone: (202) 554-1404; TDD: (202) 554-0551; 
e-mail: TSCA-H[email protected]. For specific information 
regarding this action or related activities, please contact Roger 
Nelson, Chemical Control Division, OPPT; telephone: (202) 260-8163; e-
mail: [email protected].

SUPPLEMENTARY INFORMATION: This final rule establishes a new series of 
TSCA test guidelines in the CFR.

I. Introduction

    Section 4(b)(1)(B) of TSCA requires that test rules promulgated 
under the authority of TSCA section 4 include ``standards for the 
development of test data for such substance or mixture * * *.'' Test 
rules promulgated under TSCA section 4 must specify the standards for 
the development of data. Standards established in test rules for the 
development of data must specify how the study is to be conducted, what 
data will be collected, and how the data will be analyzed. The Agency 
has found that these specifications to a large degree can be 
standardized into a common set of protocols, or, as the Agency terms 
them, ``guidelines.'' These guidelines are organized by testing 
endpoint. Each test standard can modify these guidelines as needed for 
an individual test substance.
    The Agency uses a system where standardized guidelines are 
organized by testing endpoint and codified in a subpart of this part. 
When a test rule is promulgated, the test standard specified in the 
test rule cross-references the guideline for the bulk of the testing 
requirements. In this context, the public is given notice of, and an 
opportunity to comment on, the 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.
    In 1985, the Agency established a set of TSCA test guidelines in 40 
CFR parts 795 through 798 (50 FR 39252, September 27, 1985). These 
guidelines were established as standardized protocols for laboratory 
testing of an effect or characteristic deemed important for the 
evaluation of health or environmental hazards of a chemical. 
Standardized guidelines are necessary for the establishment of 
enforceable test standards in test rules promulgated under section 4 of 
TSCA.
    The Agency has over time amended and improved these guidelines (52 
FR 19072, May 20, 1987). In order to reduce the text of the CFR, the 
Agency deleted those guidelines which had not been cited in any test 
rules (60 FR 31917, June 19, 1995 (FRL-4955-2)).

II. OPPTS Harmonized Test Guidelines

    EPA is undertaking a comprehensive modification, or harmonization, 
of its pesticides and toxics guidelines for testing of health effects, 
environmental effects, and chemical fate. The rationale for this 
harmonization is to incorporate state of the art science, and to 
minimize variations among the protocols contained in:
    1. Test guidelines developed by the EPA Office of Pesticide 
Programs (OPP), which appeared in publications of the National 
Technical Information Service.
    2. The series of TSCA test guidelines established in 1985, which 
are contained in 40 CFR parts 795, 796, 797, and 798.
    3. Guidelines published by the Organization for Economic 
Cooperation and Development (OECD).
    Harmonization operates as follows: EPA scientists develop 
guidelines (or modify existing guidelines) for specific endpoints. The 
new or rewritten guidelines are reviewed by other Agency experts and, 
in some instances, presented at domestic and international colloquia to 
solicit the views of recognized experts and the regulated community. 
The draft harmonized guidelines are made available as public drafts. A 
notice is published in the Federal Register announcing their 
availability and soliciting public comment.
    Seven of the 11 health effects test guidelines that are being 
codified in subpart H of 40 CFR part 799 have their origin in this 
harmonization process. A notice was published in the Federal Register 
of June 20, 1996, (61 FR 31522 (FRL-5367-7)) announcing the 
availability of the proposed test guidelines for Series 870--Health 
Effects Test Guidelines and soliciting public comment. Comments were 
received, and a meeting of the Agency's Federal Insecticide, Fungicide, 
and Rodenticide Act (FIFRA) Scientific Advisory Panel (SAP) was held on 
October 29 and 30, 1996. The SAP, an advisory committee consisting of 
scientific experts both inside and outside the U.S. Government, 
reviewed the guidelines and made comments. The Agency reviewed these 
comments in developing the harmonized health effects guidelines.
    Four of the 11 guidelines (Sec. Sec. 799.9510, 799.9530, 799.9538, 
and 799.9539) were initially developed by the OECD.

III. TSCA 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 FIFRA and the Federal Food, Drug and 
Cosmetic Act (FFDCA), and the Office of Pollution Prevention and Toxics 
(OPPT), which administers TSCA presented 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.
    By contrast the Agency is required under section 4 of TSCA to 
impose prescriptive test requirements by notice and comment rulemaking. 
Rules

[[Page 43821]]

promulgated under section 4 of TSCA specify classes of affected 
parties, usually manufacturers and processors of the chemical being 
specified for testing, rather than interacting with companies on an 
individual basis. These 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 health effects 
test guidelines developed using the public notice and comment process 
described in Unit II. of this preamble as well as certain OECD 
guidelines to create the TSCA-specific test guidelines which are the 
subject of this rule. Future TSCA section 4 test rules will cross-
reference part 799 guidelines rather than the older, 1985 non-
harmonized guidelines in 40 CFR parts 795 through 798. The only 
significant difference between the TSCA test guidelines and the OPPTS 
harmonized test guidelines is that certain recommended procedures in 
the OPPTS harmonized test guidelines are made mandatory (i.e., the 
guideline states that they ``shall'' be carried out).

IV. Codification in 40 CFR Part 799

    The Agency had originally planned not to publish the guidelines in 
the CFR, but to instead make the guidelines available via other means 
(such as the Internet) and reference the guidelines in specific test 
rules using the incorporation by reference procedures provided by 5 
U.S.C. 552(a)(1)(E) and 1 CFR part 51. In the Federal Register document 
proposing the TSCA section 4 test rule for 21 hazardous air pollutant 
substances (HAPs) (61 FR 33178, 33187, June 26, 1996 (FRL-4869-1)), the 
Agency stated that it was considering using incorporation by reference. 
Subsequently, however, the Director of the Office of Federal Register 
advised EPA that the planned TSCA section 4 process for guideline 
incorporation was not eligible for incorporation by reference under 1 
CFR part 51. Therefore, the Agency finds it necessary to codify a 
separate set of TSCA test guidelines into the CFR. As discussed in this 
preamble, the TSCA guidelines are essentially those resulting from the 
harmonization process with minor changes to promote enforceability. EPA 
has elected to codify these new guidelines in part 799 so as to 
distinguish them from the pre-harmonization guidelines in 40 CFR parts 
795 through 798.
    These guidelines will be placed in a new subpart H of part 799. In 
addition, EPA plans to reserve additional subparts of part 799 for test 
guidelines, so that the structure of part 799 would be as follows:

    Subpart A--General Provisions
    Subpart B--Specific Chemical Test Rules
    Subpart C--Testing Consent Orders
    Subpart D--Multichemical Test Rules
    Subpart E--G [Reserved]
    Subpart H--Health Effects Test Guidelines

    The TSCA test guidelines currently in 40 CFR parts 795 through 798 
will be retained for so long as there exist test rules whose data 
reimbursement periods under TSCA section 4(c) have not expired and 
which cross-reference the guidelines.
    This table identifies the TSCA test guideline number with its 
comparable OPPTS harmonized test guideline public draft.

 Table 1.--TSCA Test Guidelines Cross-Referenced to the OPPTS Harmonized
                             Test Guidelines                            
------------------------------------------------------------------------
                                                       OPPTS harmonized 
         Guideline title              TSCA 40 CFR       test guideline  
                                        section         (public draft)  
------------------------------------------------------------------------
TSCA acute inhalation toxicity    799.9135..........  870.1350          
 with histopathology.                                                   
TSCA subchronic inhalation        799.9346..........  870.3465          
 toxicity.                                                              
TSCA prenatal developmental       799.9370..........  870.3700          
 toxicity.                                                              
TSCA reproduction and fertility   799.9380..........  870.3800          
 effects.                                                               
TSCA carcinogenicity............  799.9420..........  870.4200          
TSCA bacterial reverse mutation   \1\799.9510.......  \1\OECD 471       
 test.                                                and 472           
TSCA in vitro mammalian cell      \1\799.9530.......  \1\OECD 476       
 gene mutation test.                                                    
TSCA mammalian bone marrow        \1\799.9538.......  \1\OECD 475       
 chromosomal aberration test.                                           
TSCA mammalian erythrocyte        \1\799.9539.......  \1\OECD 474       
 micronucleus test.                                                     
TSCA neurotoxicity screening      799.9620..........  870.6200          
 battery.                                                               
TSCA immunotoxicity.............  799.9780..........  870.7800          
------------------------------------------------------------------------
\1\The four TSCA genetic toxicity testing guidelines were adopted from  
  the OECD guideline series and not the OPPTS public drafts.            

    Codification of these guidelines does not itself impose any 
obligations on any person. Obligations are imposed only when the 
guidelines are cross-referenced in individual TSCA section 4 
rulemakings. When cross-referenced in such test rules, the pertinent 
TSCA guidelines serve as test standards for only these particular 
section 4 rules. EPA may propose modifications to the various 
guidelines as they are utilized for chemical-specific test rules. In 
each chemical-specific test rule, the proposed test standards and any 
modifications thereto will be subject to public notice and comment.

V. Guideline by Guideline Discussion

    In this unit is a summary of the significant changes made to the 11 
harmonized guidelines proposed on June 20, 1996, which are being 
published in this document.

A. Section 799.9135 TSCA Acute Inhalation Toxicity with Histopathology

    1. EPA dropped the requirement for a 1-hour (hr) exposure test. The 
Agency recognizes that such a technically difficult test would not be 
likely to yield useful information due to complicating factors such as 
biological rhythms and inapplicability to insoluble or chemically 
inactive particulates. Instead, EPA is requiring a 4-hr exposure point 
with a trigger for an 8-hr exposure point. Test sponsors have

[[Page 43822]]

the option to extrapolate from shorter-term exposures.
    2. EPA dropped the requirement for performing histopathology in all 
animals and substituted a triggered approach (wherein gross pathology 
will be performed only when the frequency and severity of adverse 
effects for dosed animals are greater than those for control animals in 
the study).
    3. EPA dropped the requirement of a breathing zone purity 
determination as unnecessary since the Agency now believes that 
standard inhalation toxicology will provide the purity measurement of 
the test substances.
    4. EPA requires only a single control group in some circumstances. 
If both 4- and 8-hr exposures are being conducted in the study, then 
there would be a single control at the 8-hr exposure provided adequate 
historical control data show no changes in histopathology or 
bronchoalveolar lavage between controls for these test periods. If the 
8-hr exposure is being performed as a result of the 4-hr trigger, there 
would need to be control groups for both 4- and 8-hr exposure groups.
    5. EPA redefined the test exposure to 4 hrs of exposure to the 
target concentration as defined by an average of plus or minus 5% for 
gases and plus or minus 11% for particles. This redefinition 
establishes exposure tolerances, which better assures known test 
concentration than the original provision which only allowed for test 
exposure after the test chamber reached equilibrium.
    6. EPA now distinguishes air change requirements between nose-only 
exposure (300 milliter (mL)/minutes (min)/animal) and whole-body 
exposure (at least 12 to 15 air changes per hr).
    7. EPA changed its description of the respiratory histopathology 
requirements to ensure that inflated state and fixed pressure with 
infusion fixation are used to prepare the lungs for examination.
    8. EPA added the requirement to specify the anatomical location 
where the four sections are to be taken for nasal histopathology.

B. Section 799.9346 TSCA Subchronic Inhalation Toxicity

    1. EPA changed the terms used for certain weekly observations from 
``motor activity'' to ``level of activity'' and from ``grip strength'' 
to ``altered strength'' to reinforce the point that these observations 
need not be automated.
    2. ``Dose'' and ``dose level'' were changed to ``concentration'' 
and ``dosing'' was changed to ``exposure'' to reflect that this is an 
inhalation study.

C. Section 799.9370 TSCA Prenatal Developmental Toxicity

    EPA made no significant changes to this guideline.

D. Section 799.9380 TSCA Reproduction and Fertility Effects

    1. EPA added the requirement for a triggered quantitative 
evaluation of primordial follicles from qualitative evidence of a 
possible treatment-related effect. While the Agency recognizes that 
there are issues concerning the validity of existing methods used to 
screen ovarian-primordial follicle counts, the Agency believes that the 
necessity to identify early senescence in females outweighs these 
concerns. EPA considers data about the effects of chemical substances 
on effects such as early female senescence to be essential to 
protecting human health.
    2. EPA reduced the requirement for taking organ weights for pups 
already opened for necropsy. The guideline only requires organ weight 
data from one randomly selected pup/sex/litter rather than the three 
pups specified in the public draft. The Agency believes that collection 
of organ weight data from one pup/sex/litter rather than three will 
reduce burdens without compromising the ability to detect a treatment-
related effect on brain, spleen, or thymus weight. The random selection 
is to be made from the population of pups already opened for necropsy.
    3. EPA reduced the requirement that 20 adult animals per sex per 
exposure group be examined for histopathology to 10 animals (randomly 
chosen) per sex per exposure group. This reduction was made because 
there would be little additional statistical value in examining more 
than 10 animals per sex per group. Since the guideline still requires 
that gross necropsy and organ weight data be collected for all parental 
animals and that the weighed organs be preserved, questions about 
interpretation of marginal histopathological effects can be resolved by 
evaluation of the tissues from these animals.
    4. EPA dropped the requirement of histopathology of developmental 
anomalies observed macroscopically in F1 and F2 weanlings. Since the 
intent of this requirement was to confirm the nature of the lesions 
already identified macroscopically, the Agency believes that the added 
value of the information would not be worth the cost of the evaluation.

E. Section 799.9420 TSCA Carcinogenicity

    1. EPA revised the guideline to allow 5-day per week dosing for 
both gavage and capsule administration. This change was made to 
eliminate the disparity between the original 7-day specification for 
capsules and 5 days for gavage since there was no justification for 
this disparity.
    2. EPA changed the terms used for certain weekly observations from 
``motor activity'' to ``level of activity'' and from ``grip strength'' 
to ``altered strength'' to reinforce the point that these observations 
need not be automated.
    3. The requirement for the immunotoxicity screen has been deleted. 
The Agency agreed that the immunotoxicity screen conducted at study 
termination would provide little meaningful information on the 
potential toxicity of the chemical on the immune function system due to 
the geriatric changes in the animals.
    4. EPA deleted the requirement for the weighing of spleens because 
their weight would be unacceptably variable due to the amount of blood 
lost during the exsanguination process. (The weighing of spleens is 
still a requirement in the immunotoxicity guideline).

F. Genetic Toxicity Testing

    1. Section 799.9510 TSCA Bacterial Reverse Mutation Test.

    2. Section 799.9530 TSCA In Vitro Mammalian Cell Gene Mutation 
Test.

    3. Section 799.9538 TSCA Mammalian Bone Marrow Chromosomal 
Aberration Test.

    4. Section 799.9539 TSCA Mammalian Erythrocyte Micronucleus Test.
    EPA is incorporating these genetic toxicity guidelines directly 
from the OECD versions. The Agency made format changes in order to 
ensure consistency with the TSCA test guidelines format. The Agency 
actively participated in international discussions regarding the 
development of these guidelines. EPA participated in the review of the 
OECD drafts. EPA believes that because these OECD guidelines were 
developed with international scientific input through the OECD 
guideline development process, they provide state-of-the-art guidance 
which is equivalent to and more broadly accepted than that in the OPPTS 
harmonized test guidelines public drafts published on June 20, 1996. 
The process EPA used in developing the four TSCA genetic toxicity test 
guidelines is described in reference 5 of Unit VI. of this preamble.

[[Page 43823]]

G. Section 799.9620 TSCA Neurotoxicity Screening Battery

    EPA made no significant changes to the public draft of this 
guideline although EPA made two clarifications to address SAP concerns. 
Clarifications to the positive control treatment were made to indicate 
that such testing need not be done as frequently as every 12 months. 
Examples were eliminated to clarify EPA's position that permanently 
injurious chemicals are not necessary, though EPA continues to believe 
that chemical exposures are appropriate

H. Section 799.9780 TSCA Immunotoxicity

    1. EPA incorporated the recommendation of the SAP that the 
requirement for flow cytometric analysis of lymphocyte and Natural 
Killer (NK) cell phenotypes be eliminated. A test for the primary 
antibody (IgM) response to sheep red blood cell (PFC) or enzyme linked 
immunosorbent assay (ELISA) would still be required. The guideline now 
sets the required exposure time for the anti-sheep red blood cells 
(SRBC) assay at 28 days, thus providing information on the effects of 
the test material on non-specific immunity.
    2. EPA adopted the SAP recommendation to delete the ``optional 
immunotoxicity screen'' because lymphocyte phenotyping by flow 
cytometry should be an option.
    3. EPA added the requirement that appropriate species-specific 
monoclonal antibodies be used in the phenotyping assay. The Agency 
accepts the SAP recommendation that this will allow sufficient 
flexibility to allow for future advances in flow cytometry and antibody 
marker technology.
    4. EPA adopted the SAP recommendation that a minimum of eight 
animals per treatment group be used in order to yield a sufficient 
statistical power to detect a 20% change based upon the inter-animal 
variation usually encountered in these assays.
    5. EPA added the intraperitoneal route of exposure to the guideline 
in response to the SAP comment that this is an acceptable method for 
immunization with SRBCs.
    6. EPA adopted the SAP recommendation that testing laboratories 
need not perform a positive control after every experiment. Instead, it 
is sufficient to include this control every 6 months or whenever new 
reagents are titrated.

VI. Public Record

    The official record for this rulemaking, as well as the public 
version, has been established for this rulemaking under docket control 
number OPPTS-42193 (including comments and data submitted 
electronically). This record contains the basic information considered 
by EPA in developing this rule. EPA will supplement this record as 
necessary.
    A public version of this record, including printed, paper versions 
of electronic comments, which does not include any information claimed 
as Confidential Business Information (CBI), is available for inspection 
from 12 noon to 4 p.m., Monday through Friday, except legal holidays. 
The public record is located in the TSCA Nonconfidential Information 
Center, Rm. NE-B607, 401 M St., SW., Washington, DC 20460.
    The record includes the following information:
    1. Public drafts of seven OPPTS harmonized health effects 
guidelines.
    2. Four OECD genetic toxicity test guidelines.
    3. References contained in TSCA health effects test guidelines 
promulgated in this document.
    4. Final report of the FIFRA Scientific Advisory Panel meeting, 
held October 29-30, 1996.
    5. USEPA. Memorandum, Angela Auletta to Roger Nelson. HAPs Rule: 
OECD Process for Update of Genetic Toxicity Test Guidelines. March 10, 
1997.

VII. Regulatory Assessment Requirements

A. Waiver of Notice of Proposed Rulemaking and Delay in Effective Date

    Because the test guidelines codified in this document have no 
substantive effect on any person without further rulemaking, and such 
rulemaking would be conducted under public notice and comment 
procedures, EPA finds that public notice and comment are unnecessary 
for this action. Thus, this rule may be promulgated without prior 
opportunity for public notice and comment, pursuant to the 
Administrative Procedure Act, 5 U.S.C. 553(b)(3)(B), and may be made 
effective immediately, without a 30-day delay, pursuant to 5 U.S.C. 
553(d)(3).

B. Executive Order 12866, Executive Order 12898, and Executive Order 
13045

    This action is not subject to Executive Order 12866 (58 FR 51735, 
October 4, 1993) since, as explained in Units I. and IV. of this 
preamble, the guidelines are not intended to have the force and effect 
of law until they are cross-referenced in future test rules through 
public notice and comment procedures that establish those rules. For 
the same reason, this action is not considered under Executive Order 
12898 (59 FR 7629, February 16, 1994) as having a disproportionately 
high and adverse human health or environmental effect on minority 
populations and low-income populations. In addition, the action is not 
subject to Executive Order 13045 ``Protection of Children From 
Environmental Health Risks and Safety Risk'' (62 FR 19885, April 23, 
1997) since it is neither economically significant under Executive 
Order 12866 nor does it concern an environmental health risk or safety 
risk that an agency has reason to believe may disproportionately affect 
children.

C. Paperwork Reduction Act

    This rule does not contain information collection requirements that 
necessitate the approval of OMB under the Paperwork Reduction Act of 
1980 (44 U.S.C. 3501 et seq.).

D. Regulatory Flexibility Act

    The guidelines codified in this document do not constitute a rule 
for which EPA must publish a general notice of proposed rulemaking 
under 5 U.S.C. 553(b). Therefore, sections 603 and 604 of the 
Regulatory Flexibility Act, 5 U.S.C. 603 and 604 do not apply to this 
action.

E. Unfunded Mandates Reform Act

    Title II of the Unfunded Mandates Reform Act of 1995 (UMRA), Pub. 
L. 104-4, which establishes requirements for Federal agencies to assess 
the effects of certain regulatory actions on State, local, and tribal 
governments and the private sector, does not apply. This action 
contains neither a private sector nor an intergovernmental mandate 
because it does not impose an enforceable duty on anyone. Furthermore, 
a written statement is not required under section 202 of UMRA because 
section 202 only applies to rules for which a general notice of 
proposed rulemaking was published, and no such notice was issued for 
this rule.

F. Submission to Congress and the General Accounting Office

    This action is not a major rule as defined by 5 U.S.C. 804(2). 
Pursuant to 5 U.S.C. 801(a)(1)(A), EPA has submitted a report 
containing this rule and other required information to the U.S. Senate, 
the U.S. House of Representatives, and the Comptroller General of the 
General Accounting Office prior to its publication in today's Federal 
Register.

[[Page 43824]]

List of Subjects in 40 CFR Part 799

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

    Dated: August 7, 1997.

Lynn R. Goldman,

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. By adding a new paragraph (d) to Sec. 799.1 to read as follows:


 Sec. 799.1  Scope and purpose.

      *      *      *      *      *
    (d) This part contains certain TSCA test guidelines which are 
cross-referenced in the test rules contained in this part.
    3. By adding and reserving subparts E through G.
    4. By adding a new subpart H, consisting of Sec. Sec. 799.9135-
799.9780, to read as follows:

Subpart H--Health Effects Test Guidelines

799.9135  TSCA acute inhalation toxicity with histopathology.
799.9346  TSCA subchronic inhalation toxicity.
799.9370  TSCA prenatal developmental toxicity.
799.9380  TSCA reproduction and fertility effects.
799.9420  TSCA carcinogenicity.
799.9510  TSCA bacterial reverse mutation test.
799.9530  TSCA in vitro mammalian cell gene mutation test.
799.9538  TSCA mammalian bone marrow chromosomal aberration test.
799.9539  TSCA mammalian erythrocyte micronucleus test.
799.9620  TSCA neurotoxicity screening battery.
799.9780  TSCA immunotoxicity.

Subpart H--Health Effects Test Guidelines


Sec. 799.9135  TSCA acute inhalation toxicity with histopathology.

    (a) Scope. This section is intended to meet the testing 
requirements under section 4 of the Toxic Substances Control Act 
(TSCA). In the assessment and evaluation of the potential human health 
effects of chemical substances, it is appropriate to test for acute 
inhalation toxic effects. The goals of this test are to characterize 
the exposure-response relationship for sensitive endpoints following 
acute exposure and to characterize toxicologic response following acute 
high exposures. The latter is of particular concern in relation to 
spills and other accidental releases. This testing is designed to 
determine the gross pathology and histopathology resulting from acute 
inhalation exposure to a substance. Because toxic effects on the 
respiratory tract are of particular concern following inhalation 
exposure, several indicators of respiratory toxicity consisting of 
histopathology on fixed tissue and evaluation of cellular and 
biochemical parameters in bronchoalveolar lavage fluid should be 
employed. The respiratory histopathology consists of specialized 
techniques to preserve tissues of the respiratory tract in order to 
allow detailed microscopic examination to identify adverse effects of 
chemical substances on this organ system. The bronchoalveolar lavage is 
designed to be a rapid screening test to provide an early indicator of 
pulmonary toxicity by examining biochemical and cytologic endpoints of 
material from the lungs of animals exposed to potentially toxic 
chemical substances. These acute tests are designed to assess the 
relationship, if any, between the animals' exposure to the test 
substance and to demonstrate relationship between the animals' exposure 
and the incidence and severity of observed abnormalities, including 
gross or histopathologic lesions, body weight changes, effects on 
mortality, and any other toxic effects. These acute tests are not 
intended to provide a complete evaluation of the toxicologic effects of 
a substance, and additional functional and morphological evaluations 
may be necessary to assess completely the potential effects produced by 
a chemical substance. Additional tests may include longer-term 
exposures, or more in-depth evaluation of specific organ systems as 
indicated by signs of toxicity following acute exposure.
    (b) Source. This a new section developed by the United States 
Environmental Protection Agency.
    (c) Definitions. The following definitions apply to this section.
    Aerodynamic diameter (dae) refers to the size of 
particles. It is the diameter of a sphere of unit density that behaves 
aerodynamically (has the same settling velocity in air) as the particle 
of the test substance. It is used to compare particles of different 
size, shape, and density, and to predict where in the respiratory tract 
such particles may be primarily deposited.
    Exposure response is the relationship between the exposure 
concentration and the measured toxic response, whether expressed as a 
group mean ( standard deviation) in the case of a 
continuous variable or as incidence in the case of a quantal variable. 
This definiton should not preclude the exploration of other dose 
metrics in establishing this relationship.
    Geometric standard deviation (GSD) is a dimensionless number equal 
to the ratio between the mass median aerodynamic diameter (MMAD) and 
either 84% or 16% of the diameter size distribution (e.g., MMAD = 2 
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).
    Inhalability is the ratio of the number concentration of particles 
of a certain aerodynamic diameter, dae, that are inspired 
through the nose or mouth to the number concentration of the same 
dae present in the inspired volume of ambient air. In 
humans, inhalability can exceed 15 m dae, whereas 
inhalability dramatically decreases for particles above 4 m 
dae in small laboratory animals.
    Lower respiratory tract consists of those structures of the 
respiratory tract below the larynx.
     Mass geometric mean aerodynamic diameter or the mass median 
aerodynamic diameter (MMAD) is the calculated aerodynamic diameter that 
divides the particles of an aerosol (a gaseous suspension of fine 
liquid or solid particles) in half, based on the weight of the 
particles. By weight, 50% of the particles will be larger than the MMAD 
and 50% of the particles will be smaller than the MMAD.
    Particle regional deposition is the fraction of inhaled particles 
that deposits in the specific region of the respiratory tract. The 
major mechanisms of particle deposition in the respiratory tract 
include impaction, sedimentation, diffusion, interception, and 
electrostatic precipitation. The deposition mechanism that is dominant 
for a given region depends on the respiratory tract architecture and 
ventilation rate of the species and the aerosol particle size and 
distribution. The respiratory tract in both humans and various 
experimental mammals can be divided into three regions on the basis of 
structure, size, and function:
    (1) The extrathoracic region or upper respiratory tract that 
includes the nose, mouth, nasopharynx, oropharynx, laryngopharynx, and 
larynx.
    (2) The tracheobronchial region that includes the trachea, bronchi, 
and

[[Page 43825]]

bronchioles (including the terminal bronchioles).
    (3) The alveolar region that includes the respiratory bronchioles 
(if present in the species), alveolar ducts, alveolar sacs, and 
alveoli.
    Respiratory effects are any adverse effects on the structure or 
functions of the respiratory system related to exposure to a chemical 
substance.
    Target organ is any organ found to be a target of toxicity in the 
4-hour (hr) high concentration group as a result of:
    (1) The initial histopathologic examination (respiratory tract, 
liver, kidney, gross lesions); or
    (2) The retrospective histopathologic examination of archived 
organs triggered by their identification as targets of toxicity in a 
90-day study.
    Toxic effects are any adverse changes (a change that is 
statistically and biologically significant) in the structure or 
function of an experimental animal as a result of exposure to a 
chemical substance.
    Upper respiratory tract consists of those structures of the 
respiratory tract above and including the larynx.
    (d) Principle of the test method. The test substance shall be 
administered to several groups of experimental animals; one 
concentration level and duration being used per group. Bronchoalveolar 
lavage shall be used to evaluate early effects on the respiratory 
system by examining changes in the content of the lavage fluid of the 
lung. At 24 hrs following exposure, the animals shall be sacrificed and 
necropsied, and tissue samples from the respiratory tract and other 
major organs will be prepared for microscopic examination. The exposure 
levels at which significant toxic effects on the respiratory organ 
system are produced are compared to those levels that produce other 
toxic effects. As triggered by the results of the 4-hr test, additional 
exposure periods of 1 hr and 8 hrs will be required to determine the 
effect of exposure time on the toxicity observed. A 1-hr exposure study 
can be elected as an option to provide data suitable for risk 
assessment for very short duration exposures as may occur from chemical 
releases. In the absence of adequate toxicological data for 1-hr 
exposure, the Agency will extrapolate to shorter-term exposures from 
the 4-hr data on the basis of concentration alone. This is a 
conservative method of extrapolation, consistent with general Agency 
methods for deriving criteria for short-term exposure from longer-term 
studies (a concentration x time extrapolation would result in higher 
concentration for a shorter duration).
    (e) Test procedures--(1) Animal selection--(i) Species. In general, 
the laboratory rat and mouse should be used. Under some circumstances, 
other species, such as the hamster or guinea pig, may be more 
appropriate, and if these or other species are used, justification 
should be provided.
    (ii) Strain. If rats and mice are used, the use of the F344 rat and 
the B6C3F1 mouse is preferred to facilitate comparison with existing 
data.
    (iii) Age. Young adults shall be used. The weight variation of 
animals used in a test should not exceed  20% of the mean 
weight for each species.
    (iv) Sex. Equal numbers of animals of each sex shall be used for 
each concentration level. The females shall be nulliparous and 
nonpregnant.
    (v) Health status. Body weight and feed consumption are not 
sufficient indicators of the health status of animals prior to 
initiating an inhalation toxicity study. Prior to initiating the study, 
animals shall be monitored for known viral and bacterial respiratory 
pathogens determined by conventional microbiological assays (e.g., 
serology). The animals shall be free from pathogens at the start of 
exposure.
    (2) Number of animals. At least five males and five females shall 
be used in each concentration/duration and control group. Animals shall 
be randomly assigned to treatment and control groups.
    (3) Control groups. The control group shall be a sham-treated 
group. Except for treatment with the test substance, animals in the 
control group shall be handled in a manner identical to the test-group 
animals. Where a vehicle is used to help generate an appropriate 
concentration of the substance in the atmosphere, a vehicle control 
group shall be used. If the 4- and 8-hr exposure studies are conducted 
concurrently, a concurrent 8-hr sham-exposed control group may serve as 
the control group for both the 4-hr and the 8-hr exposure studies, 
provided there is adequate historical control data showing no changes 
in histopathology or bronchoalveolar lavage of controls exposed for 4 
and 8 hrs. Similarly, if the optional 1-hr exposure study is conducted 
concurrently with the 4- and/or 8-hr study, the concurrent control 
group for those studies may also be used for the 1-hr study, provided 
adequate historical control data show no changes in histopathology or 
bronchoalveolar lavage between controls exposed for these time periods.
    (4) Concentration level and concentration selection. For the 4-hr 
study, at least three concentrations shall be used in addition to the 
control group. Ideally, the data generated from the test should be 
sufficient to produce an exposure-response curve. The concentrations 
can either be linearly or logarithmically spaced depending on the 
anticipated steepness of the concentration-response curve. A rationale 
for concentration selection should be provided to indicate that the 
selected concentrations will maximally support detection of 
concentration-response relationship. The high concentration should be 
clearly toxic or a limit concentration, but should not result in an 
incidence of fatalities that would preclude a meaningful evaluation of 
the data. The lowest concentration should define a no-observed-adverse-
effects level (NOAEL).
    (i) Limit concentration. For aerosols and particles, the high 
concentrations need not be greater than 2 mg/L, or concentrations that 
cannot maintain a particle size distribution having an MMAD between 1 
and 4 m (i.e., a particle size that permits inhalability and 
deposition throughout the respiratory tract). For fibers, the bivariate 
distribution of length and diameter must ensure inhalability. For gases 
and vapors, the concentrations need not be greater than 50,000 ppm or 
50% of the lower explosive limit, whichever is lower. If a test at an 
aerosol or particulate exposure of 2 mg/L (actual concentration of 
respirable substance) for 4 hrs or, where this is not feasible, the 
maximum attainable concentration, using the procedures described for 
this study, produces no observable toxic effects, then a full study 
using three concentrations will not be necessary. Similarly, if a test 
at a gas or vapor exposure of 50,000 ppm or 50% of the lower explosive 
limit, whichever is lower, produces no observable toxic effects, then a 
full study using three concentrations will not be necessary.
    (ii) 8-Hr study and optional 1-hr study. If the 8-hr study is 
triggered, three concentrations shall be tested. These concentrations 
should allow for the determination of an effect level and a NOAEL. If 
the option to perform a 1-hr study is elected, three concentrations 
shall be selected and tested in a similar manner.
    (5) Inhalation exposure. Animals can be exposed to the substance by 
either a nose-only procedure or in a whole-body exposure chamber.
    (i) Inhalation chambers. The animals shall be tested in inhalation 
equipment designed to sustain a dynamic airflow for nose-only exposures 
of at least 300 ml/minute/animal or an airflow for whole-body exposures 
of at least 12 to 15 air changes per hr and ensure an adequate oxygen 
content of at least 19% and an evenly distributed exposure

[[Page 43826]]

atmosphere. Where a whole-body chamber is used, its design shall 
minimize crowding by providing individual caging. As a general rule, to 
ensure stability of a chamber atmosphere, the total ``volume'' of the 
test animals should not exceed 5% of the volume of the test chamber.
    (ii) Environmental conditions. The temperature at which the test is 
performed shall be maintained at 22  deg.C ( 2  deg.C). 
Ideally, the relative humidity should be maintained between 40% and 
60%, but in certain instances (e.g., tests using water as a vehicle), 
this may not be practical.
    (iii) Exposure periodicity. For acute testing, the exposure design 
shall enable 4 hrs of exposure to the target concentrations, as defined 
by an average of  5% for gases and vapors and  
15% for particles and aerosols. If triggered by the results of the 4-hr 
exposure, additional testing shall be conducted in a comparable manner 
using an 8-hr exposure period.
    (6) Physical measurements. Measurements or monitoring shall be made 
of the following:
    (i) Chemical purity of the test material shall be analyzed.
    (ii) The rate of airflow shall be monitored continuously, but shall 
be recorded at least every 30 minutes.
    (iii) The actual concentrations of the test substance shall be 
measured in the breathing zone. During the exposure period, the actual 
concentrations of the test substance shall be held as constant as 
practical, monitored continuously or intermittently depending on the 
method of analysis, and recorded at least at the beginning, at an 
intermediate time, and at the end of the exposure period. Well-
established and published monitoring methods should be used where 
available. If no standard methods are available, then accuracy and 
precision information must be supplied.
    (iv) During the development of the generating system, appropriate 
particle size analysis shall be performed to establish the stability of 
the aerosol. During exposure, analysis should be conducted as often as 
necessary to determine the consistency of particle size distribution. 
The particle size distribution shall have an MMAD between 1 and 4 
m. The particle size of hygroscopic materials shall be small 
enough when dry to assure that the size of the particle at saturation 
will still have an MMAD between 1 and 4 m. Characterization 
for fibers shall include the bivariate distribution of length and 
diameter; this distribution must ensure inhalability.
    (v) If the test substance is present in a mixture, the mass and 
composition of the entire mixture, as well as the principal compound, 
shall be measured.
    (vi) Temperature and humidity shall be monitored continuously, but 
shall be recorded at least every 30 minutes.
    (7) Food and water during exposure period. Food shall be withheld 
during exposure. Water may also be withheld in certain cases.
    (8) Observation period. The bronchoalveolar lavage and respiratory 
pathology shall be conducted 24 hrs following exposure to allow 
expression of signs of toxicity. There is concern that some latency 
time will be required to allow migration of cells and macromolecules 
into the lungs following exposure, and that some pathology may require 
macromolecular synthesis or degradation before cell damage develops.
    (9) Gross pathology. (i) All animals shall be subjected to a full 
gross necropsy which includes examination of orifices and the cranial, 
thoracic, and abdominal cavities and their contents.
    (ii) At least the lungs, liver, kidneys, adrenals, brain, and 
gonads shall be weighed wet, as soon as possible after dissection to 
avoid drying.
    (iii) The following organs and tissues, or representative samples 
thereof, shall be preserved in a suitable medium for possible future 
histopathological examination: All gross lesions; brain-including 
sections of medulla/pons; cerebellar cortex and cerebral cortex; 
pituitary; thyroid/parathyroid; thymus; heart; sternum with bone 
marrow; salivary glands; liver; spleen; kidneys; adrenals; pancreas; 
gonads; accessory genital organs (epididymis, prostrate, and, if 
present, seminal vesicles); aorta; skin; gall bladder (if present); 
esophagus; stomach; duodenum; jejunum; ileum; cecum; colon; rectum; 
urinary bladder; representative lymph nodes; thigh musculature; 
peripheral nerve; spinal cord at three levels cervical, midthoracic, 
and lumbar; and eyes. Respiratory tract tissues shall also be preserved 
in a suitable medium.
    (10) Histopathology. The following histopathology shall be 
performed:
    (i) Full histopathology shall be performed on the respiratory 
tract, liver and kidney of all animals in the control and high 
concentration groups. The histopathology of the respiratory tract is 
described under paragraph (e)(11) of this section.
    (ii) All gross lesions which differ from controls in frequency, 
distribution, type, or severity in all concentration groups.
    (iii) Target organs in all animals, as indicated by the 
observations in the high concentration group in this study. 
Histopathologic examination of target organs in animals at all 
concentration levels (rather than only to the extent necessary to 
define the NOAEL) can support the application of exposure-response 
analyses such as the benchmark concentration approach.
    (iv) Archived organs identified as targets of toxicity from results 
of the 90-day study (if a 90-day study is required for this substance) 
should be elevated in high concentration animals of the 4-hr acute 
study to determine if they are also targets of acute toxicity.
    (11) Respiratory tract histopathology. (i) Representative sections 
of the respiratory tract shall be examined histologically. These shall 
include the trachea, major conducting airways, alveolar region, 
terminal and respiratory bronchioles (if present), alveolar ducts and 
sacs, and interstitial tissues.
    (ii) Care shall be taken that the method used to kill the animal 
does not result in damage to the tissues of the upper or lower 
respiratory tract. The lungs shall be infused with a fixative while in 
an inflated state of fixed pressure.
    (iii) The upper respiratory tract shall be examined for 
histopathologic lesions. This examination shall use a minimum of four 
sections located as specified under paragraphs (e)(11)(iii)(A) through 
(e)(11)(iii)(D) of this section. An evaluation of the nasal vestibule 
shall be conducted. The method described by the reference under 
paragraph (h)(11) of this section should be given consideration. The 
use of additional sections shall be left to the discretion of the study 
pathologist, but consideration should be given to additional sections 
as recommended in the reference under paragraph (h)(8) of this section 
to ensure adequate evaluation of the entire upper respiratory tract, 
particularly the nasopharyngeal meatus. The following transverse 
sections shall be examined:
    (A) Immediately posterior to the upper incisor teeth.
    (B) At the incisor papilla.
    (C) At the second palatal ridge.
    (D) At the level of the first upper molar teeth.
    (iv) The laryngeal mucosa shall be examined for histopathologic 
changes. Sections of the larynx to be examined include the epithelium 
covering the base of the epiglottis, the ventral pouch, and the medial 
surfaces of the vocal processes of the arytenoid cartilages.
    (12) Bronchoalveolar lavage. (i) Animals can be exposed to the 
substance by either a nose-only procedure or in a whole-body exposure 
chamber.
    (ii) Care should be taken that the method used to kill the animal 
results in minimum changes in the fluid of the lungs of the test 
animals.

[[Page 43827]]

    (iii) At the appropriate time, the test animals shall be killed and 
the heart-lung including trachea removed in bloc. Alternatively, lungs 
can be lavaged in situ. If the study will not be compromised, one lobe 
of the lungs may be used for lung lavage while the other is fixed for 
histologic evaluation. The lungs should be lavaged using physiological 
saline. The lavages shall consist of two washes, each of which consists 
of approximately 80% (e.g., 5 ml in rats and 1 ml in mice) of the total 
lung volume. Additional washes merely tend to reduce the concentrations 
of the material collected. The lung lavage fluid shall be stored on ice 
at 5  deg.C until assayed.
    (iv) The following parameters shall be determined in the lavage 
fluid as indicators of cellular damage in the lungs: total protein, 
cell count, and percent leukocytes. In addition, a phagocytosis assay 
shall be performed to determine macrophage activity. Assay methods 
described in the references under paragraphs (h)(1) and (h)(3) of this 
section may be used.
    (13) Combined protocol. The tests described may be combined with 
any other toxicity study, as long as none of the requirements of either 
are violated by the combination.
    (f) Triggered testing. If no adverse effects are seen in the 4-hr 
study as compared with controls, no further testing is necessary. If 
the 4-hr study shows positive effects in histopathology or the 
bronchoalveolar lavage, an 8-hr study shall be conducted. Only those 
tissues showing positive results in the 4-hr study must be pursued in 
the follow-up 8-hr study. Similarly, if the option to perform a 1-hr 
study is exercised, only those tissues showing positive results in the 
4-hr study shall be pursued.
    (g) Data reporting and evaluation. The final test report shall 
include the following information:
    (1) Description of equipment and test methods. A description of the 
general design of the experiment and any equipment used shall be 
provided.
    (i) Description of exposure apparatus, including design, type, 
dimensions, source of air, system for generating particles, aerosols, 
gasses, and vapors, method of conditioning air, treatment of exhaust 
air, and the method of housing animals in a test chamber.
    (ii) Description of the equipment for measuring temperature, 
humidity, and particulate aerosol concentration and size.
    (iii) Exposure data shall be tabulated and presented with mean 
values and measure of variability (e.g., standard deviation) and should 
include:
    (A) Chemical purity of the test material.
    (B) Airflow rates through the inhalation equipment.
    (C) Temperature and humidity of air.
    (D) Nominal concentration (total amount of test substance fed into 
the inhalation equipment divided by the volume of air).
    (E) Actual concentration in test breathing zone.
    (F) Particle size distribution (e.g., MMAD with GSD) and the 
bivariate distribution of fiber length and diameter, where appropriate.
    (2) Results--(i) General group animal data. The following 
information shall be arranged by test group exposure level.
    (A) Number of animals exposed.
    (B) Number of animals dying.
    (C) Number of animals showing overt signs of toxicity.
    (D) Pre- and post-exposure body weight change in animals, and 
weight change during the observation period.
    (ii) Counts and incidence of gross alterations observed at necropsy 
in the test and control groups. Data shall be tabulated to show:
    (A) The number of animals used in each group and the number of 
animals in which any gross lesions were found.
    (B) The number of animals affected by each different type of 
lesion, and the locations and frequency of each type of lesion.
    (iii) Counts and incidence of general histologic alterations in the 
test group. Data shall be tabulated to show:
    (A) The number of animals used in each group and the number of 
animals in which any histopathologic lesions were found.
    (B) The number of animals affected by each different type of 
lesion, and the locations, frequency, and average grade of each type of 
lesion.
    (iv) Counts and incidence of respiratory histopathologic 
alterations by the test group. Data shall be tabulated to show:
    (A) The number of animals used in each group and the number of 
animals in which any histopathologic lesions were found.
    (B) The number of animals affected by each different type of 
lesion, and the locations, frequency, and average grade of each type of 
lesion.
    (v) Results of the bronchoalveolar lavage study. Data shall be 
tabulated to show:
    (A) The amount of administered lavage fluid and recovered lavage 
fluid for each test animal.
    (B) The magnitude of change of biochemical and cytologic indices in 
lavage fluids at each test concentration for each animal.
    (C) Results shall be quantified as amount of constituent/mL of 
lavage fluid. This assumes that the amount of lavage fluid recovered is 
a representative sample of the total lavage fluid.
    (3) Evaluation of data. The findings from this acute study should 
be evaluated in the context of preceding and/or concurrent toxicity 
studies and any correlated functional findings. The evaluation shall 
include the relationship between the concentrations of the test 
substance and the presence or absence, incidence, and severity of any 
effects. The evaluation should include appropriate statistical 
analyses, for example, parametric tests for continuous data and non-
parametric tests for the remainder. Choice of analyses should consider 
tests appropriate to the experimental design, including repeated 
measures. The report must include concentration-response curves for the 
bronchoalveolar lavage and tables reporting observations at each 
concentration level for necropsy findings and gross, general, and 
respiratory system histopathology.
    (h) Reference. For additional background information on this test 
guideline, the following references should be consulted. These 
references are available for inspection at the TSCA Nonconfidential 
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M 
St., SW., Washington, DC, 12 noon to 4 p.m., Monday through Friday, 
except legal holidays.
    (1) Burleson, G.R., Fuller, L.B., Menache, M.G., and Graham, J.A. 
Poly (I): poly (C)-enhanced alveolar peritoneal macrophage 
phagocytosis: Quantification by a new method utilizing fluorescent 
beads. Proceedings of the Society of Experimental Biology and Medicine. 
184:468-476 (1987).
    (2) Gardner, D.E., Crapo, J.D., and McClellan, R.O. (Eds.) 
Toxicology of the Lung. (Raven Press, New York, 1993) pp. i-xii, 1-30.
    (3) Gilmour, G.I., and Selgrade, M.K. A comparison of the pulmonary 
defenses against streptococcal infection in rats and mice following O3 
exposure: Differences in disease susceptibility and neutrophil 
recruitment. Toxicology and Applied Pharmacology. 123:211-218 (1993).
    (4) Henderson, R.F., Benson, J.M., Hahn, F.F., Hobbs, C.H., Jones, 
R.K., Mauderly, J.L., McClellan, R.O., and Pickrell, J.A. New 
approaches for the evaluation of pulmonary toxicity: Bronchoalveolar 
lavage fluid analysis. Fundamental and Applied Toxicology. 5:451-458 
(1985).

[[Page 43828]]

    (5) Henderson, R.F. Use of bronchoalveolar lavage to detect lung 
damage. Environmental Health Perspectives. 56:115-129 (1984).
    (6) Henderson, R.F., Rebar, A.H., Pickrell, J.A., and Newton, G.J. 
Early damage indicators in the lung. III. Biochemical and cytological 
response of the lung to inhaled metal salts. Toxicology and Applied 
Pharmacology. 50:123-136 (1979).
    (7) McClellan, R.O. and Henderson, R.F. (Eds.) Second edition. 
Concepts in Inhalation Toxicology. (Taylor and Francis, Washington, DC, 
1995) pp.i-xxiv, 1-24, 441-470.
    (8) Mery, S., Gross, E.A., Joyner, D.R., Godo, M., and Morgan, K.T. 
Nasal Diagrams: A Tool for Recording the Distribution of Nasal Lesions 
in Rats and Mice. Toxicologic Pathology. 22:353-372 (1994).
    (9) Phalen, R.F. (Ed) Methods in Inhalation Toxicology. (CRC Press, 
Boca Raton, FL, 1997) pp. i-xii, 1-12.
    (10) Renne, R.A., Gideon, K.M., Miller, R.A., Mellick, P.W., and 
Grumbein, S.L. Histologic methods and interspecies variations in the 
laryngeal histology of F344/N rats and B6C3F1 mice. Toxicology and 
Pathology. 20:44-51 (1992).
    (11) Young, J.T. Histopathologic examination of the rat nasal 
cavity. Fundamental and Applied Toxicology. 1:309-312 (1981).


Sec. 799.9346  TSCA subchronic inhalation toxicity.

    (a) Scope This section is intended to meet the testing requirements 
under section 4 of TSCA. In the assessment and evaluation of the toxic 
characteristics of a gas, volatile substance, or aerosol/particulate, 
determination of subchronic inhalation toxicity may be carried out 
after initial information on toxicity has been obtained by acute 
testing. The subchronic inhalation study has been designed to permit 
the determination of the no-observed-effect-level (NOEL) and toxic 
effects associated with continuous or repeated exposure to a test 
substance for a period of 90 days. This study is not capable of 
determining those effects that have a long latency period for 
development (e.g., carcinogenicity and life shortening). Extrapolation 
from the results of this study to humans is valid only to a limited 
degree. It can, however, provide useful information on health hazards 
likely to arise from repeated exposures by the inhalation route over a 
limited period of time. It will provide information on target organs 
and the possibilities of accumulation, and can be of use in selecting 
concentration levels for chronic studies and establishing safety 
criteria for human exposure. Hazards of inhaled substances are 
influenced by the inherent toxicity and by physical factors such as 
volatility and particle size.
    (b) Source. The source material used in developing this TSCA test 
guideline is the OPPTS harmonized test guideline 870.3465 (June 1996 
Public Draft). This source is available at the address in paragraph (h) 
of this section.
    (c) Definitions. The following definitions apply to this section.
    Aerodynamic equivalent diameter is defined as the diameter of a 
unit density sphere having the same terminal settling velocity as the 
particle in question, whatever its size, shape, and density. It is used 
to predict where in the respiratory tract such particles may be 
deposited.
    Concentration in a subchronic inhalation study is the amount of 
test substance administered via inhalation for a period of 90-days. 
Concentration is expressed as weight of the test substance per unit 
volume of air (milligrams per liter or parts per million).
    Cumulative toxicity is the adverse effects of repeated exposures 
occurring as a result of prolonged action on, or increased 
concentration of the administered test substance or its metabolites in 
susceptible tissues.
    Inhalable diameter refers to that aerodynamic diameter of a 
particle which is considered to be inhalable for the organism. It is 
used to refer to particles which are capable of being inhaled and may 
be deposited anywhere within the respiratory tract
    Mass median aerodynamic diameter (MMAD) is the median aerodynamic 
diameter and along with the geometric standard deviation (GSD) is used 
to describe the particle size distribution of any aerosol statistically 
based on the weight and size of the particles. Fifty percent of the 
particles by weight will be smaller than the median diameter and 50% of 
the particles will be larger.
    No-observed-effect-level (NOEL) is the maximum concentration used 
in a study which produces no adverse effects.
    Subchronic inhalation toxicity is the adverse effects occurring as 
a result of the repeated daily exposure of experimental animals to a 
chemical by inhalation for part (approximately 10%) of a life span.
    (d) Limit test. The exposure is at a concentration of 1 mg/L or 
greater (expected human exposure may indicate the need for a higher 
concentration), where such concentration is not possible due to 
physical or chemical properties of the test substance, or where the 
maximum attainable concentration produces no observable toxic effects. 
A full study using three concentrations may not be necessary.
    (e) Test procedures--(1) Animal selection--(i) Species and strain. 
A mammalian species shall be used for testing. A variety of rodent 
species may be used, although the rat is the preferred species. 
Commonly used laboratory strains should be employed. If another 
mammalian species is used, the tester shall provide justification/
reasoning for its selection.
    (ii) Age/weight. (A) Testing should be started with young healthy 
animals as soon as possible after weaning and acclimatization.
    (B) Exposure shall commence no later than 8 weeks of age.
    (C) At the commencement of the study the weight variation of 
animals used shall not exceed  20% of the mean weight for 
each sex.
    (iii) Sex. (A) Equal numbers of animals of each sex shall be used 
at each concentration.
    (B) Females shall be nulliparous and nonpregnant.
    (iv) Numbers. (A) At least 20 rodents (10 females and 10 males) 
should be used for each test group. If another mammalian species is 
selected (e.g. dog, rabbit, or nonhuman primate), at least eight 
animals per group (four males and four females) shall be used.
    (B) If interim sacrifices are planned, the number of animals shall 
be increased by the number of animals scheduled to be sacrificed before 
the completion of the study.
    (C) To avoid bias, the use of adequate randomization procedures for 
the proper allocation of animals to test and control groups is 
required.
    (D) Each animal shall be assigned a unique identification number. 
Dead animals, their preserved organs and tissues, and microscopic 
slides shall be identified by reference to the animal's unique number.
    (v) Husbandry. (A) Animals may be group-caged by sex, but the 
number of animals per cage must not interfere with clear observation of 
each animal. The biological properties of the test substance or toxic 
effects (e.g., morbidity, excitability) may indicate a need for 
individual caging. Animals must be housed individually in inhalation 
chambers during exposure to aerosols.
    (B) The temperature of the experimental animal rooms should be at 
22 3  deg.C.
    (C) The relative humidity of the experimental animal rooms should 
be 30-70%.

[[Page 43829]]

    (D) Where lighting is artificial, the sequence should be 12 h 
light/12 h dark.
    (E) Control and test animals should be fed from the same batch and 
lot. The feed should be analyzed to assure adequacy of nutritional 
requirements of the species tested and for impurities that might 
influence the outcome of the test. Animals should be fed and watered ad 
libitum with food replaced at least weekly. For nonrodents feeding 
should be at least daily and water ad libitum.
    (F) The study should not be initiated until animals have been 
allowed a period of acclimatization/quarantine.
    (2) Control and test substances. (i) Whenever it is necessary to 
formulate the test substance with a vehicle for aerosol generation, the 
vehicle ideally should not elicit toxic effects or substantially alter 
the chemical or toxicological properties of the test substance.
    (ii) One lot of the test substance should be used, if possible 
throughout the duration of the study, and the research sample should be 
stored under conditions that maintain its purity and stability. Prior 
to the initiation of the study, there should be a characterization of 
the test substance, including the purity of the test substance and, if 
technically feasible, the name and quantities of unknown contaminants 
and impurities.
    (3) Control groups. A concurrent control group is required. This 
group shall be an untreated or sham-treated control group. Except for 
treatment with the test substance, animals in the control group shall 
be handled in a manner identical to the test group animals. Where a 
vehicle other than water is used to generate a substance, a vehicle 
control group should be used. If the toxic properties of the vehicle 
are not known or cannot be made available, both untreated and vehicle 
control groups are required.
    (4) Satellite group. A satellite group of 20 animals (10 animals 
per sex) may be treated with the high concentration level for 90 days 
and observed for reversibility, persistence, or delayed occurrence of 
toxic effects for a post-treatment period of appropriate length, 
normally not less than 28 days. In addition, a control group of 20 
animals (10 animals of each sex) should be added to the satellite 
study.
    (5) Concentration levels and concentration selection. (i) In 
subchronic toxicity tests, it is desirable to have a concentration-
response relationship as well as a NOEL. Therefore, at least three 
concentration levels plus a control and, where appropriate, a vehicle 
control (corresponding to the concentration of vehicle at the highest 
exposure level) shall be used. Concentrations should be spaced 
appropriately to produce test groups with a range of toxic effects. The 
data should be sufficient to produce a concentration-response curve.
    (ii) The highest concentration should result in toxic effects but 
not produce an incidence of fatalities which would prevent a meaningful 
evaluation.
    (iii) The intermediate concentrations should be spaced to produce a 
gradation of toxic effects.
    (iv) The lowest concentration should produce no evidence of 
toxicity.
    (v) In the case of potentially explosive test substances, care 
should be taken to avoid generating explosive concentrations.
    (6) Administration of the test substance. Animals should be exposed 
to the test substance for 6 h per day on a 7-day per week basis for a 
period of at least 90 days. Based primarily on practical 
considerations, exposure for 6 h per day on a 5-day per week basis is 
acceptable.
    (7) Observation period. The animals should be observed for a period 
of 90 days. Animals in the satellite group (if used) scheduled for 
follow-up observations should be kept for at least 28 days further 
without treatment to assess reversibility.
    (8) Exposure specifications. (i) The animals shall be tested in 
dynamic inhalation equipment designed to sustain a minimum airflow of 
10 air changes per hr, an adequate oxygen content of at least 19%, and 
uniform conditions throughout the exposure chamber. Maintenance of 
slight negative pressure inside the chamber will prevent leakage of the 
test substance into the surrounding areas. It is not normally necessary 
to measure chamber oxygen concentration if airflow is adequate.
    (ii) The selection of a dynamic inhalation chamber should be 
appropriate for the test substance and test system. Where a whole body 
chamber is used to expose animals to an aerosol, individual housing 
must be used to minimize crowding of the test animals and maximize 
their exposure to the test substance. To ensure stability of a chamber 
atmosphere, the total volume occupied by the test animals shall not 
exceed 5% of the volume of the test chamber. It is recommended, but not 
required, that nose-only or head-only exposure be used for aerosol 
studies in order to minimize oral exposures due to animals licking 
compound off their fur. Heat stress should be minimized.
    (iii) The temperature at which the test is performed should be 
maintained at 22  2  deg.C. The relative humidity should be 
maintained between 40 and 60%, but in certain instances (e.g., use of 
water vehicle) this may not be practicable.
    (9) Physical measurements. Measurements or monitoring shall be made 
of the following:
    (i) The rate of airflow shall be monitored continuously but 
recorded at least three times during the exposure.
    (ii) The actual concentrations of the test substance shall be 
measured in the animal's breathing zone. During the exposure period, 
the actual concentrations of the test substance shall be held as 
constant as practicable and monitored continuously or intermittently 
depending on the method of analysis. Chamber concentration may be 
measured using gravimetric or analytical methods as appropriate. If 
trial run measurements are reasonably consistent ( 10% for 
liquid, aerosol, gas, or vapor;  20% for dry aerosol), then 
two measurements should be sufficient. If measurements are not 
consistent, three to four measurements should be taken. Whenever the 
test substance is a formulation, or it is necessary to formulate the 
test substance with a vehicle for aerosol generation, the analytical 
concentration must be reported for the total formulation, and not just 
for the active ingredient (AI). If, for example, a formulation contains 
10% AI and 90% inerts, a chamber analytical limit concentration of 2 
mg/L would consist of 0.2 mg/L of the AI. It is not necessary to 
analyze inert ingredients provided the mixture at the animal's 
breathing zone is analogous to the formulation; the grounds for this 
conclusion must be provided in the study report. If there is some 
difficulty in measuring chamber analytical concentration due to 
precipitation, nonhomogeneous mixtures, volatile components, or other 
factors, additional analyses of inert components may be necessary.
    (iii) During the development of the generating system, particle 
size analysis shall be performed to establish the stability of aerosol 
concentrations with respect to particle size. The MMAD particle size 
range should be between 1-3 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 
valves for each exposure level are within 10% of each other, then two 
measurements during the exposures should be sufficient. If pretest 
measurements are not within 10% of each other, three to four 
measurements should be taken.

[[Page 43830]]

    (iv) Temperature and humidity shall be monitored continuously and 
recorded at least three times during an exposure.
    (10) Feed and water during exposure period. Feed shall be withheld 
during exposure. Water may also be withheld during exposure.
    (11) Observation of animals. (i) During and following exposure, 
observations are made and recorded systematically; individual records 
should be maintained for each animal. It is not always possible to 
observe animals during exposure in a whole-body chamber.
    (ii) Observations shall be made at least once each day for 
morbidity and mortality. Appropriate actions should be taken to 
minimize loss of animals to the study (e.g., Necropsy or refrigeration 
of those animals found dead and isolation or sacrifice of weak or 
moribund animals).
    (iii) A careful clinical examination shall be made at least once 
weekly. Observations should be detailed and carefully recorded, 
preferably using explicitly defined scales. Observations should 
include, but not be limited to, evaluation of skin and fur, eyes and 
mucous membranes, respiratory and circulatory effects, autonomic 
effects such as salivation, central nervous system effects, including 
tremors and convulsions, changes in the level of activity, gait and 
posture, reactivity to handling or sensory stimuli, altered strength, 
and stereotypes or bizarre behavior (e.g., self-mutilation, walking 
backwards).
    (iv) Signs of toxicity should be recorded as they are observed 
including the time of onset, degree and duration.
    (v) Individual weights of animals shall be determined shortly 
before the test substance is administered, and weekly thereafter.
    (vi) Food consumption shall also be determined weekly if abnormal 
body weight changes are observed.
    (vii) Moribund animals should be removed and sacrificed when 
noticed and the time of death should be recorded as precisely as 
possible.
    (viii) At termination, all survivors in the treatment groups shall 
be sacrificed.
    (12) Clinical pathology. Hematology and clinical chemistry 
examinations shall be made on all animals, including controls, of each 
sex in each group for rodents and all animals when nonrodents are used 
as test animals. For rodents, the hematology and clinical chemistry 
parameters should be examined once prior to initiation of exposure and 
at terminal sacrifice. For nonrodents, the hematology and clinical 
chemistry parameters should be examined once prior to initiation of 
exposure, at monthly intervals or midway through the test period and at 
termination.
    (i) The recommended hematology parameters are: Hemoglobin and 
hematocrit concentrations, red blood cell count, white blood cell 
count, differential leukocyte count, platelet count, and a measure of 
clotting potential such as prothrombin time or thromboplastin time.
    (ii) Clinical chemistry parameters which are considered appropriate 
to all studies are electrolyte balance, carbohydrate metabolism, and 
liver and kidney function. Other determinations which may be necessary 
for an adequate toxicological evaluation include analyses of lipids, 
hormones, acid/base balance, methemoglobin and cholinesterase activity. 
Additional clinical biochemistry may be employed where necessary to 
extend the investigation of observed effects.The selection of specific 
tests will be influenced by observations on the mode of action of the 
substance and signs of clinical toxicity. Suggested blood clinical 
chemistry determinations:
    (A) Electrolytes.
    (1) Calcium.
    (2) Chloride.
    (3) Magnesium.
    (4) Inorganic phosphorus.
    (5) Potassium.
    (6) Sodium.
    (B) Enzymes.
    (1) Alkaline phosphatase.
    (2) Alanine aminotransferase.
    (3) Aspartate aminotransferase.
    (4) Gamma glutamyl transferase.
    (C) Other.
    (1) Albumin.
    (2) Blood creatinine.
    (3) Blood urea nitrogen.
    (4) Globulins.
    (5) Glucose (fasting).
    (6) Total bilirubin.
    (7) Total cholesterol.
    (8) Total serum protein.
    (iii) Urinalysis is not recommended on a routine basis, but only 
when there is an indication based on expected or observed toxicity.
    (13) Ophthalmological examination. Ophthalmological examinations 
shall be made on all animals prior to the administration of the test 
substance and on all high concentration and control groups at 
termination. If changes in the eyes are detected, all animals in the 
other concentration groups shall be examined.
    (14) Gross pathology. (i) All animals shall be subjected to a full 
gross necropsy which includes examination of the external surface of 
the body, all orifices and the cranial, thoracic, and abdominal 
cavities and their contents.
    (ii) At least the liver, kidneys, brain, and gonads shall be 
trimmed and weighed wet, as soon as possible after dissection to avoid 
drying.
    (iii) The following organs and tissues, or representative samples 
thereof, shall be preserved in a suitable medium for possible future 
histopathological examination:
    (A) Digestive system.
    (1) Salivary glands.
    (2) Esophagus.
    (3) Stomach.
    (4) Duodenum.
    (5) Jejunum.
    (6) Ileum.
    (7) Cecum.
    (8) Colon.
    (9) Rectum.
    (10) Liver.
    (11) Pancreas.
    (12) Gallbladder (dogs).
    (B) Nervous system.
    (1) Brain (multiple sections).
    (2) Pituitary.
    (3) Peripheral nerve(s).
    (4) Spinal cord (three levels).
    (5) Eyes (retina, optic nerve).
    (C) Glandular system.
    (1) Adrenals.
    (2) Parathyroids.
    (3) Thyroids.
    (D) Respiratory system.
    (1) Trachea.
    (2) Lung.
    (3) Pharynx.
    (4) Larynx.
    (5) Nose.
    (E) Cardiovascular/hematopoietic system.
    (1) Aorta (thoracic).
    (2) Heart.
    (3) Bone marrow.
    (4) Lymph nodes.
    (5) Spleen.
    (6) Thymus.
    (F) Urogenital system.
    (1) Kidneys.
    (2) Urinary bladder.
    (3) Prostate.
    (4) Testes.
    (5) Epididymides.
    (6) Seminal vesicle(s).
    (7) Uterus.
    (8) Ovaries.
    (G) Other.
    (1) Lacrimal gland.
    (2) Mammary gland.
    (3) Skin.
    (4) Skeletal muscle.
    (5) All gross lesions and masses.
    (6) Sternum and/or femur.
    (15) Histopathology. (i) The following histopathology shall be 
performed:
    (A) Full histopathology on the respiratory tract and other organs 
and tissues, listed under paragraph (e)(15)(iii) of this section, of 
all animals in the control and high exposure groups

[[Page 43831]]

and all animals that died or were killed during the study.
    (B) All gross lesions in all animals.
    (C) Target organs in all animals.
    (D) Lungs, liver and kidneys of all animals. Special attention to 
examination of the respiratory tract should be made for evidence of 
infection as this provides a convenient assessment of the state of 
health of the animals.
    (E) When a satellite group is used, histopathology shall be 
performed on tissues and organs identified as showing effects in the 
treated groups.
    (ii) If excessive early deaths or other problems occur in the high 
exposure group compromising the significance of the data, the next 
concentration should be examined for complete histopathology.
    (iii) An attempt should be made to correlate gross observations 
with microscopic findings.
    (iv) Tissues and organs designated for microscopic examination 
should be fixed in 10% buffered formalin or a recognized suitable 
fixative as soon as necropsy is performed and no less than 48 hrs prior 
to trimming. Tissues should be trimmed to a maximum thickness of 0.4 cm 
for processing.
    (f) Data and reporting--(1) Treatment of results. (i) Data shall be 
summarized in tabular form, showing for each test group the number of 
animals at the start of the test, the number of animals showing 
lesions, the types of lesions, and the percentage of animals displaying 
each type of lesion.
    (ii) All observed results (quantitative and qualitative) should be 
evaluated by an appropriate statistical method. Any generally accepted 
statistical method may be used; the statistical methods including 
significance criteria should be selected during the design of the 
study.
    (2) Evaluation of study results. The findings of the subchronic 
inhalation toxicity study should be evaluated in conjunction with the 
findings of preceding studies and considered in terms of the observed 
toxic effects and the necropsy and histopathological findings. The 
evaluation will include the relationship between the concentration of 
the test substance and duration of exposure, and the presence or 
absence, the incidence and severity, of abnormalities, including 
behavioral and clinical abnormalities, gross lesions, identified target 
organs, body weight changes, effects on mortality and any other general 
or specific toxic effects. A properly conducted subchronic test should 
provide a satisfactory estimation of a no-effect level. It also can 
indicate the need for an additional longer-term study and provide 
information on the selection of concentrations.
    (3) Test report. In addition to reporting requirements specified 
under 40 CFR part 792, subpart J, the following specific information 
shall be reported. Both individual and summary data should be 
presented.
    (i) Test substance characterization shall include:
    (A) Chemical identification.
    (B) Lot or batch number.
    (C) Physical properties.
    (D) Purity/impurities.
    (E) Identification and composition of any vehicle used.
    (ii) Test system information shall include:
    (A) Species and strain of animals used and rationale for selection 
if other than that recommended.
    (B) Age, sex, and body weight.
    (C) Test environment including cage conditions, ambient 
temperature, humidity, and light/dark periods.
    (iii) Test procedure information shall include:
    (A) Method of randomization used.
    (B) Full description of experimental design and procedure.
    (C) Exposure regimen including concentration levels, methods, and 
volume.
    (D) Description of test conditions; the following exposure 
conditions shall be reported:
    (1) Description of exposure apparatus including design, type, 
volume, source of air, system for generating aerosols, method of 
conditioning air, treatment of exhaust air and the method of housing 
the animals in a test chamber.
    (2) The equipment for measuring temperature, humidity, and 
particulate aerosol concentrations and size should be described.
    (E) Exposure data shall be tabulated and presented with mean values 
and a measure of variability (e.g., standard deviation) and include:
    (1) Airflow rates through the inhalation equipment.
    (2) Temperature and humidity of air.
    (3) Actual (analytical or gravimetric) concentration in the 
breathing zone.
    (4) Nominal concentration (total amount of test substance fed into 
the inhalation equipment divided by volume of air).
    (5) Particle size distribution, calculated mass median aerodynamic 
diameter (MMAD) and geometric standard deviation (GSD).
    (6) Explanation as to why the desired chamber concentration and/or 
particle size could not be achieved (if applicable) and the efforts 
taken to comply with this aspect of the section.
    (iv) Test results information shall include:
    (A) Group animal data. Tabulation of toxic response data by 
species, strain, sex and exposure level for:
    (1) Number of animals exposed.
    (2) Number of animals showing signs of toxicity.
    (3) Number of animals dying.
    (B) Individual animal data. Data should be presented as summary 
(group mean) as well as for individual animals.
    (1) Time of death during the study or whether animals survived to 
termination.
    (2) Time of observation of each abnormal sign and its subsequent 
course.
    (3) Body weight data.
    (4) Feed consumption data, when collected.
    (5) Results of ophthalmological examination, when performed.
    (6) Results of hematological tests performed. .
    (7) Results of clinical chemistry tests performed.
    (8) Results of urinalysis tests performed.
    (9) Necropsy findings, including absolute and relative organ weight 
data.
    (10) Detailed description of all histopathological findings.
    (11) Statistical treatment of results, where appropriate.
    (g) Quality control. A system shall be developed and maintained to 
assure and document adequate performance of laboratory staff and 
equipment. The study shall be conducted in compliance with 40 CFR Part 
792--Good Laboratory Practice Standards.
    (h) References. For additional background information on this test 
guideline, the following references should be consulted. These 
references are available for inspection at the TSCA Nonconfidential 
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M 
St., SW., Washington, DC, 12 noon to 4 p.m., Monday through Friday, 
except legal holidays.
    (1) Cage, J.C. Ed. Paget, G.E. Experimental Inhalation Toxicology, 
Methods in Toxicology. (F.A. Davis Co., Philadelphia, PA, 1970) pp. 
258-277.
    (2) Casarett, L.J. and Doull. Chapter 9. Toxicology: The Basic 
Science of Poisons (New York: Macmillan Publishing Co., Inc., 1975).
    (3) U.S. Environmental Protection Agency, Office of Pesticide 
Programs, Health Effects Division. Interim policy for particle size and 
limit concentration issues in inhalation toxicity studies (February 1, 
1994).
    (4) MacFarland, H.N. Ed. Hayes, W.J. Vol. 7. Respiratory 
Toxicology, Essays in Toxicology. (Academic Press, New York, NY, 1976) 
pp. 121-154.

[[Page 43832]]

    (5) Organisation for Economic Co-operation and Development. 
Guidelines for testing of chemicals, section 4-health effects, part 
413. Subchronic Inhalation Toxicity Studies (Paris, 1981).


Sec. 799.9370  TSCA prenatal developmental toxicity.

    (a) Scope This section is intended to meet the testing requirements 
under section 4 of TSCA. This guideline for developmental toxicity 
testing is designed to provide general information concerning the 
effects of exposure on the pregnant test animal and on the developing 
organism; this may include death, structural abnormalities, or altered 
growth and an assessment of maternal effects. For information on 
testing for functional deficiencies and other postnatal effects, the 
guidelines for the two-generation reproductive toxicity study and the 
developmental neurotoxicity study should be consulted.
    (b) Source. The source material used in developing this TSCA test 
guideline is the OPPTS harmonized test guideline 870.3700 (February 
1996 Public Draft). This source is available at the address in 
paragraph (h) of this section.
    (c) Good laboratory practice standards. The study shall be 
conducted in compliance with 40 CFR Part 792--Good Laboratory Practice 
Standards.
    (d) Principle of the test method. The test substance is 
administered to pregnant animals at least from implantation to one day 
prior to the expected day of parturition. Shortly before the expected 
date of delivery, the pregnant females are terminated, the uterine 
contents are examined, and the fetuses are processed for visceral and 
skeletal evaluation.
    (e) Test procedures--(1) Animal selection--(i) Species and strain. 
It is recommended that testing be performed in the most relevant 
species, and that laboratory species and strains which are commonly 
used in prenatal developmental toxicity testing be employed. The 
preferred rodent species is the rat and the preferred non-rodent 
species is the rabbit.
    (ii) Age. Young adult animals shall be used.
    (iii) Sex. Nulliparous female animals shall be used at each dose 
level. Animals should be mated with males of the same species and 
strain, avoiding the mating of siblings, if parentage is known. Day 0 
in the test is the day on which a vaginal plug and/or sperm are 
observed in the rodent or that insemination is performed or observed in 
the rabbit.
    (iv) Number of animals. Each test and control group shall contain a 
sufficient number of animals to yield approximately 20 animals with 
implantation sites at necropsy.
    (2) Administration of test and control substances--(i) Dose levels 
and dose selection. (A) At least three-dose levels and a concurrent 
control shall be used. Healthy animals shall be randomly assigned to 
the control and treatment groups, in a manner which results in 
comparable mean body weight values among all groups. The dose levels 
should be spaced to produce a gradation of toxic effects. Unless 
limited by the physical/chemical nature or biological properties of the 
test substance, the highest dose shall be chosen with the aim to induce 
some developmental and/or maternal toxicity but not death or severe 
suffering. In the case of maternal mortality, this should not be more 
than approximately 10%. The intermediate dose levels should produce 
minimal observable toxic effects. The lowest dose level should not 
produce any evidence of either maternal or developmental toxicity 
(i.e., the no-observed-adverse-effect level, NOAEL) or should be at or 
near the limit of detection for the most sensitive endpoint. Two- or 
four-fold intervals are frequently optimal for spacing the dose levels, 
and the addition of a fourth test group is often preferable to using 
very large intervals (e.g., more than a factor of 10) between dosages.
    (B) It is desirable that additional information on metabolism and 
pharmacokinetics of the test substance be available to demonstrate the 
adequacy of the dosing regimen. This information should be available 
prior to testing.
    (C) The highest dose tested need not exceed 1,000 mg/kg/day by oral 
or dermal administration, or 2 mg/L (or the maximum attainable 
concentration) by inhalation, unless potential human exposure data 
indicate the need for higher doses. If a test performed at the limit 
dose level, using the procedures described for this study, produces no 
observable toxicity and if an effect would not be expected based upon 
data from structurally related compounds, then a full study using 
three-dose levels may not be considered necessary.
    (ii) Control group. (A) A concurrent control group shall be used. 
This group shall be a sham-treated control group or a vehicle-control 
group if a vehicle is used in administering the test substance.
    (B) The vehicle control group should receive the vehicle in the 
highest volume used.
    (C) If a vehicle or other additive is used to facilitate dosing, 
consideration should be given to the following characteristics: Effects 
on the absorption, distribution, metabolism, or retention of the test 
substance; effects on the chemical properties of the test substance 
which may alter its toxic characteristics; and effects on the food or 
water consumption or the nutritional status of the animals.
    (iii) Route of administration. (A) The test substance or vehicle is 
usually administered orally by intubation.
    (B) If another route of administration is used, for example, when 
the route of administration is based upon the principal route of 
potential human exposure, the tester shall provide justification and 
reasoning for its selection, and appropriate modifications may be 
necessary. Care should be taken to minimize stress on the maternal 
animals. For materials administered by inhalation, whole-body exposure 
is preferable to nose-only exposure due to the stress of restraint 
required for nose-only exposure.
    (C) The test substance shall be administered at approximately the 
same time each day.
    (D) When administered by gavage or dermal application, the dose to 
each animal shall be based on the most recent individual body weight 
determination.
    (iv) Dosing schedule. At minimum, the test substance shall be 
administered daily from implantation to the day before cesarean section 
on the day prior to the expected day of parturition. Alternatively, if 
preliminary studies do not indicate a high potential for 
preimplantation loss, treatment may be extended to include the entire 
period of gestation, from fertilization to approximately 1 day prior to 
the expected day of termination.
    (f) Observation of animals--(1) Maternal. (i) Each animal shall be 
observed at least once daily, considering the peak period of 
anticipated effects after dosing. Mortality, moribundity, pertinent 
behavioral changes, and all signs of overt toxicity shall be recorded 
at this cageside observation. In addition, thorough physical 
examinations shall be conducted at the same time maternal body weights 
are recorded.
    (ii) Animals shall be weighed on day 0, at termination, and at 
least at 3-day intervals during the dosing period.
    (iii) Food consumption shall be recorded on at least 3-day 
intervals, preferably on days when body weights are recorded.
    (iv) (A) Females shall be terminated immediately prior to the 
expected day of delivery.
    (B) Females showing signs of abortion or premature delivery prior 
to scheduled termination shall be killed

[[Page 43833]]

and subjected to a thorough macroscopic examination.
    (v) At the time of termination or death during the study, the dam 
shall be examined macroscopically for any structural abnormalities or 
pathological changes which may have influenced the pregnancy. 
Evaluation of the dams during cesarean section and subsequent fetal 
analyses should be conducted without knowledge of treatment group in 
order to minimize bias.
    (vi) (A) Immediately after termination or as soon as possible after 
death, the uteri shall be removed and the pregnancy status of the 
animals ascertained. Uteri that appear nongravid shall be further 
examined (e.g. by ammonium sulfide staining) to confirm the nonpregnant 
status.
    (B) Each gravid uterus (with cervix) shall be weighed. Gravid 
uterine weights should not be obtained from dead animals if autolysis 
or decomposition has occurred.
    (C) The number of corpora lutea shall be determined for pregnant 
animals.
    (D) The uterine contents shall be examined for embryonic or fetal 
deaths and the number of viable fetuses. The degree of resorption shall 
be described in order to help estimate the relative time of death of 
the conceptus.
    (2) Fetal. (i) The sex and body weight of each fetus shall be 
determined.
    (ii) Each fetus shall be examined for external anomalies.
    (iii) Fetuses shall be examined for skeletal and soft tissue 
anomalies (e.g. variations and malformations or other categories of 
anomalies as defined by the performing laboratory).
    (A) For rodents, approximately one-half of each litter shall be 
prepared by standard techniques and examined for skeletal alterations, 
preferably bone and cartilage. The remainder shall be prepared and 
examined for soft tissue anomalies, using appropriate serial sectioning 
or gross dissection techniques. It is also acceptable to examine all 
fetuses by careful dissection for soft tissue anomalies followed by an 
examination for skeletal anomalies.
    (B) For rabbits, all fetuses shall be examined for both soft tissue 
and skeletal alterations. The bodies of these fetuses should be 
evaluated by careful dissection for soft-tissue anomalies, followed by 
preparation and examination for skeletal anomalies. An adequate 
evaluation of the internal structures of the head, including the eyes, 
brain, nasal passages, and tongue, should be conducted for at least 
half of the fetuses.
    (g) Data and reporting--(1) Treatment of results. Data shall be 
reported individually and summarized in tabular form, showing for each 
test group the types of change and the number of dams, fetuses, and 
litters displaying each type of change.
    (2) Evaluation of study results. The following shall be provided:
    (i) Maternal and fetal test results, including an evaluation of the 
relationship, or lack thereof, between the exposure of the animals to 
the test substance and the incidence and severity of all findings.
    (ii) Criteria used for categorizing fetal external, soft tissue, 
and skeletal anomalies.
    (iii) When appropriate, historical control data to enhance 
interpretation of study results. Historical data (on litter incidence 
and fetal incidence within litter), when used, should be compiled, 
presented, and analyzed in an appropriate and relevant manner. In order 
to justify its use as an analytical tool, information such as the dates 
of study conduct, the strain and source of the animals, and the vehicle 
and route of administration should be included.
    (iv) Statistical analysis of the study findings should include 
sufficient information on the method of analysis, so that an 
independent reviewer/statistician can reevaluate and reconstruct the 
analysis. In the evaluation of study data, the litter should be 
considered the basic unit of analysis.
    (v) In any study which demonstrates an absence of toxic effects, 
further investigation to establish absorption and bioavailability of 
the test substance should be considered.
    (3) Test report. In addition to the reporting requirements as 
specified under 40 CFR part 792, subpart J, the following specific 
information shall be reported. Both individual and summary data should 
be presented.
    (i) Species and strain.
    (ii) Maternal toxic response data by dose, including but not 
limited to:
    (A) The number of animals at the start of the test, the number of 
animals surviving, the number pregnant, and the number aborting.
    (B) Day of death during the study or whether animals survived to 
termination.
    (C) Day of observation of each abnormal clinical sign and its 
subsequent course.
    (D) Body weight and body weight change data, including body weight 
change adjusted for gravid uterine weight.
    (E) Food consumption and, if applicable, water consumption data.
    (F) Necropsy findings, including gravid uterine weight.
    (iii) Developmental endpoints by dose for litters with implants, 
including:
    (A) Corpora lutea counts.
    (B) Implantation data, number and percent of live and dead fetuses, 
and resorptions (early and late).
    (C) Pre- and postimplantation loss calculations.
    (iv) Developmental endpoints by dose for litters with live fetuses, 
including:
    (A) Number and percent of live offspring.
    (B) Sex ratio.
    (C) Fetal body weight data, preferably by sex and with sexes 
combined.
    (D) External, soft tissue, and skeletal malformation and variation 
data. The total number and percent of fetuses and litters with any 
external, soft tissue, or skeletal alteration, as well as the types and 
incidences of individual anomalies, should be reported.
    (v) The numbers used in calculating all percentages or indices.
    (vi) Adequate statistical treatment of results.
    (vii) A copy of the study protocol and any amendments should be 
included.
    (h) References. For additional background information on this test 
guideline, the following references should be consulted. These 
references are available for inspection at the TSCA Nonconfidential 
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M 
St., SW., Washington, DC, 12 noon to 4 p.m., Monday through Friday, 
except legal holidays.
    (1) Aliverti, V.L. et al. The extent of fetal ossification as an 
index of delayed development in teratogenicity studies in the rat. 
Teratology. 20:237-242 (1979).
    (2) Barrow, M.V. and W.J. Taylor. A rapid method for detecting 
malformations in rat fetuses. Journal of Morphology 127:291-306 (1969).
    (3) Burdi, A.R. Toluidine blue-alizarin red S staining of cartilage 
and bone in whole-mount skeltons in vitro. Stain Technolology. 40:45-48 
(1965).
    (4) Edwards, J.A. Ed. Woolam,D.H.M. The external development of the 
rabbit and rat embryo. Vol. 3. Advances in Teratology (Academic, NY, 
1968).
    (5) Fritz, H. Prenatal ossification in rabbits as indicative of 
fetal maturity. Teratology. 11:313-320 (1974).
    (6) Fritz, H. and Hess, R. Ossification of the rat and mouse 
skeleton in the perinatal period. Teratology. 3:331-338 (1970).
    (7) Gibson, J.P. et al. Use of the rabbit in teratogenicity 
studies. Toxicology and Applied Pharmacology. 9:398-408 (1966).
    (8) Inouye, M. Differential staining of cartilage and bone in fetal 
mouse skeleton by alcian blue and alizarin red S. Congenital Anomalies. 
16(3):171-173 (1976).

[[Page 43834]]

    (9) Igarashi, E. et al. Frequence of spontaneous axial skeletal 
variations detected by the double staining technique for ossified and 
cartilaginous skeleton in rat fetuses. Congenital Anomalies. 32:381-391 
(1992).
    (10) Kimmel, C.A. et al. Skeletal development following heat 
exposure in the rat. Teratology. 47:229-242 (1993).
    (11) Kimmel, C.A. and Francis, E.Z. Proceedings of the workshop on 
the acceptability and interpretation of dermal developmental toxicity 
studies. Fundamental and Applied Toxicology. 14:386-398 (1990).
    (12) Kimmel, C.A. and C. Trammell. A rapid procedure for routine 
double staining of cartilage and bone in fetal and adult animals. Stain 
Technology. 56:271-273 (1981).
    (13) Kimmel, C.A. and Wilson, J.G. Skeletal deviation in rats: 
malformations or variations? Teratology. 8:309-316 (1973).
    (14) Marr, M.C. et al. Comparison of single and double staining for 
evaluation of skeletal development: the effects of ethylene glycol (EG) 
in CD rats. Teratology. 37:476 (1988).
    (15) Marr, M.C. et al. Developmental stages of the CD (Sprague-
Dawley) rat skeleton after maternal exposure to ethylene glycol. 
Teratology. 46:169-181 (1992).
    (16) McLeod, M.J. Differential staining of cartilage and bone in 
whole mouse fetuses by Alcian blue and alizarin red S. Teratology. 
22:299-301 (1980).
    (17) Monie, I.W. et al. Dissection procedures for rat fetuses 
permitting alizarin red staining of skeleton and histological study of 
viscera. Supplement to Teratology Workshop Manual. pp. 163-173 (1965).
    (18) Organisation for Economic Co-operation and Development, No. 
414: Teratogenicity, Guideline for Testing of Chemicals. [C(83)44 
(Final)] (1983).
    (19) Salewski (Koeln), V.E. Faerbermethode zum makroskopischen 
nachweis von implantations stellen am uterus der ratte. Naunyn-
Schmeidebergs Archiv fur Pharmakologie und Experimentelle Pathologie. 
247:367 (1964).
    (20) Spark, C. and Dawson,A.B. The order and time of appearance of 
centers of ossification in the fore and hind limbs of the albino rat, 
with special reference to the possible influence of the sex factor. 
American Journal of Anatomy. 41:411-445 (1928).
    (21) Staples, R.E. Detection of visceral alterations in mammalian 
fetuses. Teratology. 9(3):A37-A38 (1974).
    (22) Staples, R.E. and Schnell, V.L. Refinements in rapid clearing 
technique in the KOH--alizarin red S method for fetal bone. Stain 
Technology. 39:61-63 (1964).
    (23) Strong, R.M. The order time and rate of ossification of the 
albino rat (mus norvegicus albinus) skeleton. American Journal of 
Anatomy. 36: 313-355 (1928).
    (24) Stuckhardt, J.L. and Poppe, S.M. Fresh visceral examination of 
rat and rabbit fetuses used in teratogenicity testing. Teratogenesis, 
Carcinogenesis, and Mutagenesis. 4:181-188 (1984).
    (25) Van Julsingha, E.B. and Bennett,C.G. Eds. Neubert, D., Merker, 
H.J., and Kwasigroch, T.E. A dissecting procedure for the detection of 
anomalies in the rabbit foetal head. Methods in Prenatal Toxicology 
(University of Chicago, Chicago, IL, 1977) pp. 126-144 .
    (26) Whitaker, J. and Dix, D.M. Double-staining for rat foetus 
skeletons in teratological studies. Laboratory Animals. 13:309-310 
(1979).
    (27) Wilson, J.G. Eds. Wilson, J.G. and Warkany, J. Embryological 
considerations in teratology. Teratology: Principles and Techniques 
(University of Chicago, Chicago, IL, 1965) pp. 251-277.


Sec. 799.9380  TSCA reproduction and fertility effects.

    (a) Scope. This section is intended to meet the testing 
requirements under section 4 of the TSCA. This section is for two-
generation reproduction testing and is designed to provide general 
information concerning the effects of a test substance on the integrity 
and performance of the male and female reproductive systems, including 
gonadal function, the estrous cycle, mating behavior, conception, 
gestation, parturition, lactation, and weaning, and on the growth and 
development of the offspring. The study may also provide information 
about the effects of the test substance on neonatal morbidity, 
mortality, target organs in the offspring, and preliminary data on 
prenatal and postnatal developmental toxicity and serve as a guide for 
subsequent tests. Additionally, since the study design includes in 
utero as well as postnatal exposure, this study provides the 
opportunity to examine the susceptibility of the immature/neonatal 
animal.
    (b) Source. The source material used in developing this TSCA test 
guideline is the OPPTS harmonized test guideline 870.3800 (February 
1996 Public Draft). This source is available at the address in 
paragraph (g) of this section.
    (c) Good laboratory practice standards. The study shall be 
conducted in compliance with 40 CFR Part 792--Good Laboratory Practice 
Standards.
    (d) Principle of the test method. The test substance is 
administered to parental (P) animals prior to and during their mating, 
during the resultant pregnancies, and through the weaning of their F1 
offspring. The substance is then administered to selected F1 offspring 
during their growth into adulthood, mating, and production of an F2 
generation, until the F2 generation is weaned.
    (e) Test procedures--(1) Animal selection--(i) Species and strain. 
The rat is the most commonly used species for testing. If another 
mammalian species is used, the tester shall provide justification/
reasoning for its selection, and appropriate modifications will be 
necessary. Healthy parental animals, which have been acclimated to 
laboratory conditions for at least 5 days and have not been subjected 
to previous experimental procedures, should be used. Strains of low 
fecundity shall not be used.
    (ii) Age. Parental (P) animals shall be 5 to 9 weeks old at the 
start of dosing. The animals of all test groups should be of uniform 
weight, age, and parity as nearly as practicable, and should be 
representative of the species and strain under study.
    (iii) Sex. (A) For an adequate assessment of fertility, both males 
and females shall be studied.
    (B) The females shall be nulliparous and nonpregnant.
    (iv) Number of animals. Each control group shall contain a 
sufficient number of mating pairs to yield approximately 20 pregnant 
females. Each test group shall contain a similar number of mating 
pairs.
    (v) Identification of animals. Each animal shall be assigned a 
unique identification number. For the P generation, this should be done 
before dosing starts. For the F1 generation, this should be done for 
animals selected for mating; in addition, records indicating the litter 
of origin shall be maintained for all selected F1 animals.
    (2) Administration of test and control substances--(i) Dose levels 
and dose selection. (A) At least three-dose levels and a concurrent 
control shall be used. Healthy animals should be randomly assigned to 
the control and treatment groups, in a manner which results in 
comparable mean body weight values among all groups. The dose levels 
should be spaced to produce a gradation of toxic effects. Unless 
limited by the physical/chemical nature or biological properties of the 
test substance, the highest dose should be chosen with the aim to 
induce some reproductive and/or systemic toxicity but not death or 
severe suffering. In the case of parental

[[Page 43835]]

mortality, this should not be more than approximately 10%. The 
intermediate dose levels should produce minimal observable toxic 
effects. The lowest dose level should not produce any evidence of 
either systemic or reproductive toxicity (i.e., the no-observed-
adverse-effect level, NOAEL) or should be at or near the limit of 
detection for the most sensitive endpoint. Two- or four-fold intervals 
are frequently optimal for spacing the dose levels, and the addition of 
a fourth test group is often preferable to using very large intervals 
(e.g., more than a factor of 10) between dosages.
    (B) It is desirable that additional information on metabolism and 
pharmacokinetics of the test substance be available to demonstrate the 
adequacy of the dosing regimen. This information should be available 
prior to testing.
    (C) The highest dose tested should not exceed 1,000 mg/kg/day (or 
20,000 ppm in the diet), unless potential human exposure data indicate 
the need for higher doses. If a test performed at the limit dose level, 
using the procedures described for this study, produces no observable 
toxicity and if an effect would not be expected based upon data from 
structurally related compounds, then a full study using three dose 
levels may not be considered necessary.
    (ii) Control group. (A) A concurrent control group shall be used. 
This group shall be an untreated or sham treated group or a vehicle-
control group if a vehicle is used in administering the test substance.
    (B) If a vehicle is used in administering the test substance, the 
control group shall receive the vehicle in the highest volume used.
    (C) If a vehicle or other additive is used to facilitate dosing, 
consideration should be given to the following characteristics: Effects 
on the absorption, distribution, metabolism, or retention of the test 
substance; effects on the chemical properties of the test substance 
which may alter its toxic characteristics; and effects on the food or 
water consumption or the nutritional status of the animals.
    (D) If a test substance is administered in the diet and causes 
reduced dietary intake or utilization, the use of a pair-fed control 
group may be considered necessary.
    (iii) Route of administration. (A) The test substance is usually 
administered by the oral route (diet, drinking water, or gavage).
    (B) If administered by gavage or dermal application, the dosage 
administered to each animal prior to mating and during gestation and 
lactation shall be based on the individual animal body weight and 
adjusted weekly at a minimum.
     (C) If another route of administration is used, for example, when 
the route of administration is based upon the principal route of 
potential human exposure, the tester should provide justification and 
reasoning for its selection, and appropriate modifications may be 
necessary. Care should be taken to minimize stress on the maternal 
animals and their litters during gestation and lactation.
    (D) All animals should be dosed by the same method during the 
appropriate experimental period.
    (iv) Dosing schedule. (A) The animals should be dosed with the test 
substance on a 7-days-a-week basis.
    (B) Daily dosing of the parental (P) males and females shall begin 
when they are 5 to 9 weeks old. Daily dosing of the F1 males and 
females shall begin at weaning. For both sexes (P and F1), dosing shall 
be continued for at least 10 weeks before the mating period.
    (C) Daily dosing of the P and F1 males and females shall continue 
until termination.
    (3) Mating procedure--(i) Parental. (A) For each mating, each 
female shall be placed with a single randomly selected male from the 
same dose level (1:1 mating) until evidence of copulation is observed 
or either 3 estrous periods or 2 weeks has elapsed. Animals should be 
separated as soon as possible after evidence of copulation is observed. 
If mating has not occurred after 2 weeks or 3 estrous periods, the 
animals should be separated without further opportunity for mating. 
Mating pairs should be clearly identified in the data.
    (B) Vaginal smears shall be collected daily and examined for all 
females during mating, until evidence of copulation is observed.
    (C) Each day, the females shall be examined for presence of sperm 
or vaginal plugs. Day 0 of pregnancy is defined as the day a vaginal 
plug or sperm are found.
    (ii) F1 mating. For mating the F1 offspring, at least one male and 
one female should be randomly selected from each litter for mating with 
another pup of the same dose level but different litter, to produce the 
F2 generation.
    (iii) Second mating. In certain instances, such as poor 
reproductive performance in the controls, or in the event of treatment-
related alterations in litter size, the adults may be remated to 
produce an F1b or F2b litter. If production of a second litter is 
deemed necessary in either generation, the dams should be remated 
approximately 1-2 weeks following weaning of the last F1a or F2a 
litter.
    (iv) Special housing. After evidence of copulation, animals that 
are presumed to be pregnant shall be caged separately in delivery or 
maternity cages. Pregnant animals shall be provided with nesting 
materials when parturition is near.
    (v) Standardization of litter sizes. (A) Animals should be allowed 
to litter normally and rear their offspring to weaning. Standardization 
of litter sizes is optional.
    (B) If standardization is performed, the following procedure should 
be used. On day 4 after birth, the size of each litter may be adjusted 
by eliminating extra pups by random selection to yield, as nearly as 
possible, four males and four females per litter or five males and five 
females per litter. Selective elimination of pups, i.e. based upon body 
weight, is not appropriate. Whenever the number of male or female pups 
prevents having four (or five) of each sex per litter, partial 
adjustment (for example, five males and three females, or four males 
and six females) is acceptable. Adjustments are not appropriate for 
litters of eight pups or less.
    (4) Observation of animals--(i) Parental. (A) Throughout the test 
period, each animal shall be observed at least once daily, considering 
the peak period of anticipated effects after dosing. Mortality, 
moribundity, pertinent behavioral changes, signs of difficult or 
prolonged parturition, and all signs of overt toxicity shall be 
recorded at this cageside examination. In addition, thorough physical 
examinations should be conducted weekly on each animal.
    (B) Parental animals (P and F1) shall be weighed on the first day 
of dosing and weekly thereafter. Parental females (P and F1) should be 
weighed at a minimum on approximately gestation days 0, 7, 14, and 21, 
and during lactation on the same days as the weighing of litters.
    (C) During the premating and gestation periods, food consumption 
shall be measured weekly at a minimum. Water consumption should be 
measured weekly at a minimum if the test substance is administered in 
the water.
    (D) Estrous cycle length and normality should be evaluated by 
vaginal smears for all P and F1 females during a minimum of 3 weeks 
prior to mating and throughout cohabitation; care should be taken to 
prevent the induction of pseudopregnancy.
    (E) For all P and F1 males at termination, sperm from one testis 
and one epididymis shall be collected for

[[Page 43836]]

enumeration of homogenization-resistant spermatids and cauda epididymal 
sperm reserves, respectively. In addition, sperm from the cauda 
epididymis (or vas deferens) should be collected for evaluation of 
sperm motility and sperm morphology.
    (1) The total number of homogenization-resistant testicular sperm 
and cauda epididymal sperm should be enumerated. The method described 
in the reference under paragraph (g)(8) of this section may be used. 
Cauda sperm reserves can be derived from the concentration and volume 
of sperm in the suspension used to complete the qualitative 
evaluations, and the number of sperm recovered by subsequent mincing 
and/or homogenizing of the remaining cauda tissue. Enumeration in only 
control and high-dose P and F1 males may be performed unless treatment-
related effects are observed; in that case, the lower dose groups 
should also be evaluated.
    (2) An evaluation of epididymal (or vas deferens) sperm motility 
should be performed. Sperm should be recovered while minimizing damage 
(the evaluation techniques as described in the reference under 
paragraph (g)(8) of this section may be used), and the percentage of 
progressively motile sperm should be determined either subjectively or 
objectively. For objective evaluations, an acceptable counting chamber 
of sufficient depth can be used to effectively combine the assessment 
of motility with sperm count and sperm morphology. When computer-
assisted motion analysis is performed, the derivation of progressive 
motility relies on user-defined thresholds for average path velocity 
and straightness or linear index. If samples are videotaped, or images 
otherwise recorded, at the time of necropsy, subsequent analysis of 
only control and high-dose P and F1 males may be performed unless 
treatment-related effects are observed; in that case, the lower dose 
groups should also be evaluated. In the absence of a video or digital 
image, all samples in all treatment groups should be analyzed at 
necropsy.
    (3) A morphological evaluation of an epididymal (or vas deferens) 
sperm sample shall be performed. Sperm (at least 200 per sample) should 
be examined as fixed, wet preparations (the techniques for such 
examinations is described in the references under paragraphs (g)(4) and 
(g)(8) of this section may be used) and classified as either normal 
(both head and midpiece/tail appear normal) or abnormal. Examples of 
morphologic sperm abnormalities would include fusion, isolated heads, 
and misshapen heads and/or tails. Evaluation of only control and high-
dose P and F1 males may be performed unless treatment-related effects 
are observed; in that case, the lower dose groups should also be 
evaluated.
    (ii) Offspring. (A) Each litter should be examined as soon as 
possible after delivery (lactation day 0) to establish the number and 
sex of pups, stillbirths, live births, and the presence of gross 
anomalies. Pups found dead on day 0 should be examined for possible 
defects and cause of death.
    (B) Live pups should be counted, sexed, and weighed individually at 
birth, or soon thereafter, at least on days 4, 7, 14, and 21 of 
lactation, at the time of vaginal patency or balanopreputial 
separation, and at termination.
    (C) The age of vaginal opening and preputial separation should be 
determined for F1 weanlings selected for mating. If there is a 
treatment-related effect in F1 sex ratio or sexual maturation, 
anogenital distance should be measured on day 0 for all F2 pups.
    (5) Termination schedule. (i) All P and F1 adult males and females 
should be terminated when they are no longer needed for assessment of 
reproductive effects.
    (ii) F1 offspring not selected for mating and all F2 offspring 
should be terminated at comparable ages after weaning.
    (6) Gross necropsy. (i) At the time of termination or death during 
the study, all parental animals (P and F1) and when litter size permits 
at least three pups per sex per litter from the unselected F1 weanlings 
and the F2 weanlings shall be examined macroscopically for any 
structural abnormalities or pathological changes. Special attention 
shall be paid to the organs of the reproductive system.
    (ii) Dead pups or pups that are terminated in a moribund condition 
should be examined for possible defects and/or cause of death.
    (iii) At the time of necropsy, a vaginal smear should be examined 
to determine the stage of the estrous cycle. The uteri of all cohabited 
females should be examined, in a manner which does not compromise 
histopathological evaluation, for the presence and number of 
implantation sites.
    (7) Organ weights. (i) At the time of termination, the following 
organs of all P and F1 parental animals shall be weighed:
    (A) Uterus (with oviducts and cervix), ovaries.
    (B) Testes, epididymides (total weights for both and cauda weight 
for either one or both), seminal vesicles (with coagulating glands and 
their fluids), and prostate.
    (C) Brain, pituitary, liver, kidneys, adrenal glands, spleen, and 
known target organs.
    (ii) For F1 and F2 weanlings that are examined macroscopically, the 
following organs shall be weighed for one randomly selected pup per sex 
per litter.
    (A) Brain.
    (B) Spleen and thymus.
    (8) Tissue preservation. The following organs and tissues, or 
representative samples thereof, shall be fixed and stored in a suitable 
medium for histopathological examination.
    (i) For the parental (P and F1) animals:
    (A) Vagina, uterus with oviducts, cervix, and ovaries.
    (B) One testis (preserved in Bouins fixative or comparable 
preservative), one epididymis, seminal vesicles, prostate, and 
coagulating gland.
    (C) Pituitary and adrenal glands.
    (D) Target organs, when previously identified, from all P and F1 
animals selected for mating.
    (E) Grossly abnormal tissue.
    (ii) For F1 and F2 weanlings selected for macroscopic examination: 
Grossly abnormal tissue and target organs, when known.
    (9) Histopathology--(i) Parental animals. Full histopathology of 
the organs listed under paragraph (e)(8)(i) of this section shall be 
performed for ten randomly chosen high dose and control P and F1 
animals per sex, for those animals that were selected for mating. 
Organs demonstrating treatment-related changes shall also be examined 
for the remainder of the high-dose and control animals and for all 
parental animals in the low- and mid-dose groups. Additionally, 
reproductive organs of the low- and mid-dose animals suspected of 
reduced fertility, e.g., those that failed to mate, conceive, sire, or 
deliver healthy offspring, or for which estrous cyclicity or sperm 
number, motility, or morphology were affected, shall be subjected to 
histopathological evaluation. Besides gross lesions such as atrophy or 
tumors, testicular histopathological examination should be conducted in 
order to to identify treatment-related effects such as retained 
spermatids, missing germ cell layers or types, multinucleated giant 
cells, or sloughing of spermatogenic cells into the lumen. Examination 
of the intact epididymis should include the caput, corpus, and cauda, 
which can be accomplished by evaluation of a longitudinal section, and 
should be conducted in order to identify such lesions as sperm 
granulomas, leukocytic

[[Page 43837]]

infiltration (inflammation), aberrant cell types within the lumen, or 
the absence of clear cells in the cauda epididymal epithelium. The 
postlactational ovary should contain primordial and growing follicles 
as well as the large corpora lutea of lactation. Histopathological 
examination should detect qualitative depletion of the primordial 
follicle population. A quantitative evaluation of primordial follicles 
should be conducted for all F1 females if any of the following 
treatment-related findings were observed:
    (A) Reductions in ovarian weight and abnormal ovarian 
histopathology findings, e.g., follicular cysts or qualitative evidence 
of a reduced population of primordial follicles.
    (B) Abnormal estrous cyclicity and female infertility.
    (C) Depletion of testicular spermatid counts in F1 males and 
evidence of germ cell depletion in testicular histopathology 
evaluations.
    (ii) Examination of ovarian sections. If a quantitative evaluation 
is performed, ten ovarian sections shall be taken at least 100 
m apart from the inner third of each ovary. Examination should 
include enumeration of the total number of primordial and antral 
follicles from these 20 sections (the technique for this histological 
assessment as described in the reference under paragraph (g)(2) of this 
section may be used) for comparison with control ovaries.
    (iii) Weanlings. For F1 and F2 weanlings, histopathological 
examination of treatment-related abnormalities noted at macroscopic 
examination should be considered, if such evaluation were deemed 
appropriate and would contribute to the interpretation of the study 
data.
    (f) Data and reporting--(1) Treatment of results. Data shall be 
reported individually and summarized in tabular form, showing for each 
test group the types of change and the number of animals displaying 
each type of change.
    (2) Evaluation of study results. (i) An evaluation of test results, 
including the statistical analysis, shall be provided. This should 
include an evaluation of the relationship, or lack thereof, between the 
exposure of the animals to the test substance and the incidence and 
severity of all abnormalities.
    (ii) When appropriate, historical control data should be used to 
enhance interpretation of study results. Historical data, when used, 
should be compiled, presented, and analyzed in an appropriate and 
relevant manner. In order to justify its use as an analytical tool, 
information such as the dates of study conduct, the strain and source 
of the animals, and the vehicle and route of administration should be 
included.
    (iii) Statistical analysis of the study findings should include 
sufficient information on the method of analysis, so that an 
independent reviewer/statistician can reevaluate and reconstruct the 
analysis.
    (iv) In any study which demonstrates an absence of toxic effects, 
further investigation to establish absorption and bioavailability of 
the test substance should be considered.
    (3) Test report. In addition to the reporting requirements as 
specified under 40 CFR part 792, subpart J, the following specific 
information shall be reported. Both individual and summary data should 
be presented.
    (i) Species and strain.
    (ii) Toxic response data by sex and dose, including indices of 
mating, fertility, gestation, birth, viability, and lactation; 
offspring sex ratio; precoital interval, including the number of days 
until mating and the number of estrous periods until mating; and 
duration of gestation calculated from day 0 of pregnancy. The report 
should provide the numbers used in calculating all indices.
    (iii) Day (week) of death during the study or whether animals 
survived to termination; date (age) of litter termination.
    (iv) Toxic or other effects on reproduction, offspring, or 
postnatal growth.
    (v) Developmental milestone data (mean age of vaginal opening and 
preputial separation, and mean anogenital distance, when measured).
    (vi) Number of P and F1 females cycling normally and mean estrous 
cycle length.
    (vii) Day (week) of observation of each abnormal sign and its 
subsequent course.
    (viii) Body weight and body weight change data by sex for P, F1, 
and F2 animals.
    (ix) Food (and water, if applicable) consumption, food efficiency 
(body weight gain per gram of food consumed), and test material 
consumption for P and F1 animals, except for the period of 
cohabitation.
    (x) Total cauda epididymal sperm number, homogenization-resistant 
testis spermatid number, number and percent of progressively motile 
sperm, number and percent of morphologically normal sperm, and number 
and percent of sperm with each identified anomaly.
    (xi) Stage of the estrous cycle at the time of termination for P 
and F1 parental females.
    (xii) Necropsy findings.
    (xiii) Implantation data and postimplantation loss calculations for 
P and F1 parental females.
    (xiv) Absolute and adjusted organ weight data.
    (xv) Detailed description of all histopathological findings.
    (xvi) Adequate statistical treatment of results.
    (xvii) A copy of the study protocol and any amendments should be 
included.
    (g) References. For additional backgound information on this test 
guideline, the following references should be consulted. These 
references are available for inspection at the TSCA Nonconfidential 
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M 
St., SW., Washington, DC, 12 noon to 4 p.m., Monday through Friday, 
except legal holidays.
    (1) Gray, L.E. et al. A dose-response analysis of methoxychlor-
induced alterations of reproductive development and function in the 
rat. Fundamental and Applied Toxicology. 12:92-108 (1989).
    (2) Heindel, J.J. et al. Ed. Hirshfield, A.N. Histological 
assessment of ovarian follicle number in mice as a screen of ovarian 
toxicity. Growth Factors and the Ovary (Plenum, NY, 1989) pp. 421-426.
    (3) Korenbrot, C.C. et al. Preputial separation as an external sign 
of pubertal development in the male rat. Biology of Reproduction. 
17:298-303 (1977).
    (4) Linder, R.E. et al. Endpoints of spermatoxicity in the rat 
after short duration exposures to fourteen reproductive toxicants. 
Reproductive Toxicology. 6:491-505 (1992).
    (5) Manson, J.M. and Kang, Y.J. Ed. Hayes, A.W. Test methods for 
assessing female reproductive and developmental toxicology. Principles 
and Methods of Toxicology (Raven, NY, 1989).
    (6) Organisation for Economic Co-operation and Development, No. 
416: Two Generation Reproduction Toxicity Study, Guidelines for Testing 
of Chemicals. [C(83)44 (Final)] (1983).
    (7) Pederson, T. and Peters, H. Proposal for classification of 
oocytes and follicles in the mouse ovary. Journal of Reproduction and 
Fertility. 17:555-557 (1988).
    (8) Seed, J., Chapin, R.E. E.D. Clegg, L.A. Dostal, R.H. Foote, 
M.E. Hurtt, G.R. Klinefelter, S.L. Makris, S.D. Perreault, S. Schrader, 
D. Seyler, R. Sprando, K.A. Treinen, D.N.R. Veeramachaneni, and Wise, 
L.D. Methods for assessing sperm motility, morphology, and counts in 
the rat, rabbit, and dog: a consensus report. Reproductive Toxicology. 
10(3):237-244 (1996).

[[Page 43838]]

    (9) Smith, B.J. et al. Comparison of random and serial sections in 
assessment of ovarian toxicity. Reproductive Toxicology. 5:379-383 
(1991).
    (10) Thomas, J.A. Eds. M.O. Amdur, J. Doull, and C.D. Klaassen. 
Toxic responses of the reproductive system. Casarett and Doull's 
Toxicology (Pergamon, NY, 1991).
    (11) Working, P.K. and Hurtt, M. Computerized videomicrographic 
analysis of rat sperm motility. Journal of Andrology. 8:330-337 (1987).
    (12) Zenick, H. et al. Ed. Hayes, A.W. Assessment of male 
reproductive toxicity: a risk assessment approach. Principles and 
Methods of Toxicology (Raven, NY, 1994).


Sec. 799.9420  TSCA carcinogenicity.

    (a) Scope. This section is intended to meet the testing 
requirements under section 4 of TSCA. The objective of a long-term 
carcinogenicity study is to observe test animals for a major portion of 
their life span for development of neoplastic lesions during or after 
exposure to various doses of a test substance by an appropriate route 
of administration.
    (b) Source. The source material used in developing this TSCA test 
guideline is the OPPTS harmonized test guideline 870.4200 (June 1996 
Public Draft). This source is available at the address in paragraph (g) 
of this section.
    (c) Definitions. The following definitions apply to this section.
    Carcinogenicity is the development of neoplastic lesions as a 
result of the repeated daily exposure of experimental animals to a 
chemical by the oral, dermal, or inhalation routes of exposure.
    Cumulative toxicity is the adverse effects of repeated dose 
occurring as a result of prolonged action on, or increased 
concentration of, the administered test substance or its metabolites in 
susceptible tissues.
    Dose in a carcinogenicity study is the amount of test substance 
administered via the oral, dermal or inhalation routes for a period of 
up to 24 months. Dose is expressed as weight of the test substance 
(grams, milligrams) per unit body weight of test animal (milligram per 
kilogram), or as weight of the test substance in parts per million 
(ppm) in food or drinking water. When exposed via inhalation, dose is 
expressed as weight of the test substance per unit volume of air 
(milligrams per liter) or as parts per million.
    Target organ is any organ of a test animal showing evidence of an 
effect induced by a test substance.
    (d) Test procedures--(1) Animal selection--(i) Species and strain. 
Testing shall be performed on two mammalian species. Rats and mice are 
the species of choice because of their relatively short life spans, 
limited cost of maintenance, widespread use in pharmacological and 
toxicological studies, susceptibility to tumor induction, and the 
availability of inbred or sufficiently characterized strains. Commonly 
used laboratory strains shall be used. If other mammalian species are 
used, the tester shall provide justification/reasoning for their 
selection.
    (ii) Age/weight. (A) Testing shall be started with young healthy 
animals as soon as possible after weaning and acclimatization.
    (B) Dosing should generally begin no later than 8 weeks of age.
    (C) At commencement of the study, the weight variation of animals 
used shall not exceed  20% of the mean weight for each sex.
    (D) Studies using prenatal or neonatal animals may be recommended 
under special conditions.
    (iii) Sex. (A) Equal numbers of animals of each sex shall be used 
at each dose level.
    (B) Females shall be nulliparous and nonpregnant.
    (iv) Numbers. (A) At least 100 rodents (50 males and 50 females) 
shall be used at each dose level and concurrent control group.
     (B) If interim sacrifices are planned, the number shall be 
increased by the number of animals scheduled to be sacrificed during 
the course of the study.
    (C) For a meaningful and valid statistical evaluation of long term 
exposure and for a valid interpretation of negative results, the number 
of animals in any group should not fall below 50% at 15 months in mice 
and 18 months in rats. Survival in any group should not fall below 25% 
at 18 months in mice and 24 months in rats.
    (D) The use of adequate randomization procedures for the proper 
allocation of animals to test and control groups is required to avoid 
bias.
    (E) Each animal shall be assigned a unique identification number. 
Dead animals, their preserved organs and tissues, and microscopic 
slides shall be identified by reference to the unique numbers assigned.
    (v) Husbandry. (A) Animals may be group-caged by sex, but the 
number of animals per cage must not interfere with clear observation of 
each animal. The biological properties of the test substance or toxic 
effects (e.g., morbidity, excitability) may indicate a need for 
individual caging. Animals should be housed individually in dermal 
studies and during exposure in inhalation studies.
    (B) The temperature of the experimental animal rooms should be at 
22  3  deg.C.
    (C) The relative humidity of the experimental animal rooms should 
be 30 to 70%.
    (D) Where lighting is artificial, the sequence should be 12 h 
light/12 h dark.
    (E) Control and test animals should be fed from the same batch and 
lot. The feed should be analyzed to assure uniform distribution and 
adequacy of nutritional requirements of the species tested and for 
impurities that might influence the outcome of the test. Animals should 
be fed and watered ad libitum with food replaced at least weekly.
    (F) The study should not be initiated until animals have been 
allowed a period of acclimatization/quarantine to environmental 
conditions, nor should animals from outside sources be placed on test 
without an adequate period of quarantine.
    (2) Control and test substances. (i) Where necessary, the test 
substance is dissolved or suspended in a suitable vehicle. If a vehicle 
or diluent is needed, it should not elicit toxic effects itself. It is 
recommended that wherever possible the use of an aqueous solution be 
considered first, followed by consideration of solution in oil, and 
finally solution in other vehicles.
    (ii) One lot of the test substance should be used, if possible, 
throughout the duration of the study, and the research sample should be 
stored under conditions that maintain its purity and stability. Prior 
to the initiation of the study, there should be a characterization of 
the test substance, including the purity of the test compound, and, if 
possible, the name and quantities of contaminants and impurities.
    (iii) If the test or control substance is to be incorporated into 
feed or another vehicle, the period during which the test substance is 
stable in such a mixture should be determined prior to the initiation 
of the study. Its homogeneity and concentration should be determined 
prior to the initiation of the study and periodically during the study. 
Statistically randomized samples of the mixture should be analyzed to 
ensure that proper mixing, formulation, and storage procedures are 
being followed, and that the appropriate concentration of the test or 
control substance is contained in the mixture.
    (3) Control groups. A concurrent control group (50 males and 50 
females) is required. This group shall be

[[Page 43839]]

untreated or if a vehicle is used in administering the test substance, 
a vehicle control group. If the toxic properties of the vehicle are not 
known, both untreated and vehicle control groups are required.
    (4) Dose levels and dose selection. (i) For risk assessment 
purposes, at least three dose levels shall be used, in addition to the 
concurrent control group. Dose levels should be spaced to produce a 
gradation of effects. A rationale for the doses selected must be 
provided.
    (ii) The highest dose level should elicit signs of toxicity without 
substantially altering the normal life span due to effects other than 
tumors. The highest dose should be determined based on the findings 
from a 90-day study to ensure that the dose used is adequate to asses 
the carcinogenic potential of the test substance. Thus, the selection 
of the highest dose to be tested is dependent upon changes observed in 
several toxicological parameters in subchronic studies. The highest 
dose tested need not exceed 1,000 mg/kg/day.
    (iii) The intermediate-dose level should be spaced to produce a 
gradation of toxic effects.
    (iv) The lowest dose level should produce no evidence of toxicity.
    (v) For skin carcinogenicity studies, when toxicity to the skin is 
a determining factor, the highest dose selected should not destroy the 
functional integrity of the skin, the intermediate dose should be a 
minimally irritating dose, and the low dose should be the highest 
nonirritating dose.
    (vi) The criteria for selecting the dose levels for skin 
carcinogenicity studies, based on gross and histopathologic dermal 
lesions, are as follows:
    (A) Gross criteria for reaching the high dose:
    (1) Erythema (moderate).
    (2) Scaling.
    (3) Edema (mild).
    (4) Alopecia.
    (5) Thickening.
    (B) Histologic criteria for reaching the high dose:
    (1) Epidermal hyperplasia.
    (2) Epidermal hyperkeratosis.
    (3) Epidermal parakeratosis.
    (4) Adnexal atrophy/hyperplasia.
    (5) Fibrosis.
    (6) Spongiosis (minimal-mild).
    (7) Epidermal edema (minimal-mild).
    (8) Dermal edema (minimal-moderate).
    (9) Inflammation (moderate).
    (C) Gross criteria for exceeding the high dose:
    (1) Ulcers, fissures.
    (2) Exudate/crust (eschar).
    (3) nonviable (dead) tissues.
    (4) Anything leading to destruction of the functional integrity of 
the epidermis (e.g., caking, fissuring, open sores, eschar).
    (D) Histologic criteria for exceeding the high dose:
    (1) Crust (interfollicular and follicular).
    (2) Microulcer.
    (3) Degeneration/necrosis (mild to moderate).
    (4) Epidermal edema (moderate to marked).
    (5) Dermal edema (marked).
    (6) Inflammation (marked).
    (5) Administration of the test substance. The three main routes of 
administration are oral, dermal, and inhalation. The choice of the 
route of administration depends upon the physical and chemical 
characteristics of the test substance and the form typifying exposure 
in humans.
    (i) Oral studies. If the test substance is administered by gavage, 
the animals are dosed with the test substance on a 7-day per week basis 
for a period of at least 18 months for mice and hamsters and 24 months 
for rats. However, based primarily on practical considerations, dosing 
by gavage or via a capsule on a 5-day per week basis is acceptable. If 
the test substance is administered in the drinking water or mixed in 
the diet, then exposure should be on a 7-day per week basis.
    (ii) Dermal studies. (A) The animals should be treated with the 
test substance for at least 6 h/day on a 7-day per week basis for a 
period of at least 18 months for mice and hamsters and 24 months for 
rats. However, based primarily on practical considerations, application 
on a 5-day per week basis is acceptable. Dosing should be conducted at 
approximately the same time each day.
    (B) Fur should be clipped weekly from the dorsal area of the trunk 
of the test animals. Care should be taken to avoid abrading the skin 
which could alter its permeability. A minimum of 24 hrs should be 
allowed for the skin to recover before the next dosing of the animal.
    (C) The test substance shall be applied uniformly over a shaved 
area which is approximately 10% of the total body surface area. 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. The 
volume of application should be kept constant and should not exceed 100 
L for the mouse and 300 L for the rat; different 
concentrations of the test solution should be prepared for different 
dose levels. 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 
after application.
    (D) During the exposure period, the application site should not be 
covered when mice or hamsters are the species of choice. 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 and 
test substance and ensure that the animals cannot ingest the test 
substance.
    (iii) Inhalation studies. (A) The animals should be exposed to the 
test substance for 6 h/day on a 7-day per week basis, for a period of 
at least 18 months in mice and 24 months in rats. However, based 
primarily on practical considerations, exposure for 6 h/day on a 5-day 
per week basis is acceptable.
    (B) The animals shall be tested in dynamic inhalation equipment 
designed to sustain a minimum air flow of 10 air changes per hr, an 
adequate oxygen content of at least 19%, and uniform conditions 
throughout the exposure chamber. Maintenance of slight negative 
pressure inside the chamber will prevent leakage of the test substance 
into surrounding areas.
    (C) The selection of a dynamic inhalation chamber should be 
appropriate for the test substance and test system. Where a whole body 
chamber is used to expose animals to an aerosol, individual housing 
must be used to minimize crowding of the test animals and maximize 
their exposure to the test substance. To ensure stability of a chamber 
atmosphere, the total volume occupied by the test animals shall not 
exceed 5% of the volume of the test chamber. It is recommended, but not 
required, that nose-only or head-only exposure be used for aerosol 
studies in order to minimize oral exposures due to animals licking 
compound off their fur. Heat stress to the animals should be minimized.
    (D) The temperature at which the test is performed should be 
maintained at 22  2  deg.C. The relative humidity should be 
maintained between 40 to 60%, but in certain instances (e.g., tests of 
aerosols, use of water vehicle) this may not be practicable.
    (E) The rate of air flow shall be monitored continuously but 
recorded at least three times during exposure.

[[Page 43840]]

    (F) Temperature and humidity shall be monitored continuously but 
should be recorded at least every 30 minutes.
    (G) The actual concentrations of the test substance shall be 
measured in the breathing zone. During the exposure period, the actual 
concentrations of the test substance should be held as constant as 
practicable, monitored continuously or intermittently depending on the 
method of analysis. Chamber concentrations may be measured using 
gravimetric or analytical methods as appropriate. If trial run 
measurements are reasonably consistent ( 10% for liquid 
aerosol, gas, or dry aerosol), the two measurements should be 
sufficient. If measurements are not consistent, then three to four 
measurements should be taken.
    (H) During the development of the generating system, particle size 
analysis shall be performed to establish the stability of aerosol 
concentrations with respect to particle size. Measurement of 
aerodynamic particle size in the animals's breathing zone should be 
measured during a trial run. If median aerodynamic diameter (MMAD) 
values for each exposure level are within 10% of each other, then two 
measurements during the exposures should be sufficient. If pretest 
measurements are not within 10% of each other, three to four 
measurements should be taken. The MMAD particle size range should be 
between 1-3 m. The particle size of hygroscopic materials 
should be small enough to allow pulmonary deposition once the particles 
swell in the moist environment of the respiratory tract.
    (I) Feed shall be withheld during exposure. Water may also be 
withheld during exposure.
    (6) Observation period. It is necessary that the duration of the 
carcinogenicity study comprise the majority of the normal life span of 
the strain of animals used. This time period shall not be less than 24 
months for rats and 18 months for mice, and ordinarily not longer than 
30 months for rats and 24 months for mice. For longer time periods, and 
where any other species are used, consultation with the Agency in 
regard to the duration of the study is advised.
    (7) Observation of animals. (i) Observations shall be made at least 
once each day for morbidity and mortality. Appropriate actions should 
be taken to minimize loss of animals from the study (e.g., necropsy or 
refrigeration of those animals found dead and isolation or sacrifice of 
weak or moribund animals).
    (ii) A careful clinical examination shall be made at least once 
weekly. Observations should be detailed and carefully recorded, 
preferably using explicitly defined scales. Observations should 
include, but not be limited to, evaluation of skin and fur, eyes and 
mucous membranes, respiratory and circulatory effects, autonomic 
effects such as salivation, central nervous system effects, including 
tremors and convulsions, changes in the level of activity, gait and 
posture, reactivity to handling or sensory stimuli, altered strength 
and stereotypes or bizarre behavior (e.g., self-mutilation, walking 
backwards).
    (iii) Body weights shall be recorded individually for all animals; 
once a week during the first 13 weeks of the study and at least once 
every 4 weeks, thereafter, unless signs of clinical toxicity suggest 
more frequent weighing to facilitate monitoring of health status.
    (iv) When the test substance is administered in the feed or 
drinking water, measurements of feed or water consumption, 
respectively, should be determined weekly during the first 13 weeks of 
the study and then at approximately monthly intervals unless health 
status or body weight changes dictate otherwise.
    (v) Moribund animals shall be removed and sacrificed when noticed 
and the time of death should be recorded as precisely as possible. At 
the end of the study period, all survivors shall be sacrificed.
    (8) Clinical pathology. At 12 months, 18 months, and at terminal 
sacrifice, a blood smear shall be obtained from all animals. A 
differential blood count should be performed on blood smears from those 
animals in the highest dosage group and the controls from the terminal 
sacrifice. If these data, or data from the pathological examination 
indicate a need, then the 12- and 18-month blood smears should also be 
examined. Differential blood counts should be performed for the next 
lower groups if there is a major discrepancy between the highest group 
and the controls. If clinical observations suggest a deterioration in 
health of the animals during the study, a differential blood count of 
the affected animals shall be performed.
    (9) Gross necropsy. (i) A complete gross examination shall be 
performed on all animals, including those that died during the 
experiment or were killed in a moribund condition.
    (ii) The liver, lungs, kidneys, brain, and gonads should be trimmed 
and weighed wet as soon as possible after dissection to avoid drying. 
The organs should be weighed from interim sacrifice animals as well as 
from at least 10 animals per sex per group at terminal sacrifice.
    (iii) The following organs and tissues, or representative samples 
thereof, shall be preserved in a suitable medium for possible future 
histopathological examination.
    (A) Digestive system.
    (1) Salivary glands.
    (2) Esophagus.
    (3) Stomach.
    (4) Duodenum.
    (5) Jejunum.
    (6) Ileum.
    (7) Cecum.
    (8) Colon.
    (9) Rectum.
    (10) Liver.
    (11) Pancreas.
    (12) Gallbladder (mice).
    (13) Bile duct (rat).
    (B) Nervous system.
    (1) Brain (multiple sections).
    (2) Pituitary.
    (3) Peripheral nerves.
    (4) Spinal cord (three levels).
    (5) Eyes (retina, optic nerve).
    (C) Glandular system.
    (1) Adrenals.
    (2) Parathyroids.
    (3) Thyroids.
    (D) Respiratory system.
    (1) Trachea.
    (2) Lung.
    (3) Pharynx.
    (4) Larynx.
    (5) Nose (inhalation studies only).
    (E) Cardiovascular/hematopoietic system.
    (1) Aorta (thoracic).
    (2) Heart.
    (3) Bone marrow.
    (4) Lymph nodes.
    (5) Spleen.
    (6) Thymus.
    (F) Urogenital system.
    (1) Kidneys.
    (2) Urinary bladder.
    (3) Prostate.
    (4) Testes/epididymides.
    (5) Seminal vesicles.
    (6) Uterus.
    (7) Ovaries.
    (G) Other.
    (1) Lacrimal gland.
    (2) Mammary gland.
    (3) Skin.
    (4) Skeletal muscle.
    (5) All gross lesions and masses.
    (6) Sternum and/or femur.
    (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.

[[Page 43841]]

    (vi) Information from clinical pathology, and other in-life data 
should be considered before microscopic examination, since they may 
provide significant guidance to the pathologist.
    (10) Histopathology. (i) The following histopathology shall be 
performed:
    (A) Full histopathology on the organs and tissues under paragraph 
(d)(9) (iii) of this section of all animals in the control and high 
dose groups and all animals that died or were killed during the study.
    (B) All gross lesions in all animals.
    (C) Target organs in all animals.
    (D) Lungs, liver, and kidneys of all animals. Special attention to 
examination of the lungs of rodents should be made for evidence of 
infection since this provides an assessment of the state of health of 
the animals.
    (ii) If the results show substantial alteration of the animal's 
normal life span, the induction of effects that might affect a 
neoplastic response, or other effects that might compromise the 
significance of the data, the next lower dose levels shall be examined 
as described under paragraph (d)(11)(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 hrs prior 
to trimming. Tissues should be trimmed to a maximum thickness of 0.4 cm 
for processing.
    (e) Data and reporting--(1) Treatment of results. (i) Data shall be 
summarized in tabular form, showing for each test group the number of 
animals at the start of the test, the number of animals showing 
lesions, the types of lesions, and the percentage of animals displaying 
each type of lesion.
    (ii) All observed results (quantitative and qualitative) shall be 
evaluated by an appropriate statistical method. Any generally accepted 
statistical methods may be used; the statistical methods including 
significance criteria shall be selected during the design of the study.
    (2) Evaluation of study results. (i) The findings of a 
carcinogenicity study should be evaluated in conjunction with the 
findings of previous studies and considered in terms of the toxic 
effects, the necropsy and histopathological findings. The evaluation 
shall include the relationship between the dose of the test substance 
and the presence, incidence, and severity of abnormalities (including 
behavioral and clinical abnormalities), gross lesions, identified 
target organs, body weight changes, effects on mortality, and any other 
general or specific toxic effects.
    (ii) In any study which demonstrates an absence of toxic effects, 
further investigation to establish absorption and bioavailablity of the 
test substance should be considered.
    (iii) In order for a negative test to be acceptable, it must meet 
the following criteria: No more than 10% of any group is lost due to 
autolysis, cannibalism, or management problems; and survival in each 
group is no less than 50% at 15 months for mice and 18 months for rats. 
Survival should not fall below 25% at 18 months for mice and 24 months 
for rats.
    (iv) The use of historical control data from an appropriate time 
period from the same testing laboratory (i.e., the incidence of tumors 
and other suspect lesions normally occurring under the same laboratory 
conditions and in the same strain of animals employed in the test) is 
helpful for assessing the significance of changes observed in the 
current study.
    (3) Test report. (i) In addition to the reporting requirements as 
specified under 40 CFR part 792, subpart J, the following specific 
information shall be reported. Both individual and summary data should 
be presented.
    (A) Test substance characterization should include:
    (1) Chemical identification.
    (2) Lot or batch number.
    (3) Physical properties.
    (4) Purity/impurities.
    (5) Identification and composition of any vehicle used.
    (B) Test system should contain data on:
    (1) Species and strain of animals used and rationale for selection 
if other than that recommended.
    (2) Age including body weight data and sex
    (3) Test environment including cage conditions, ambient 
temperature, humidity, and light/dark periods.
    (C) Test procedure should include the following data:
    (1) Method of randomization used.
    (2) Full description of experimental design and procedure.
    (3) Dose regimen including levels, methods, and volume.
    (4) Test results--(i) Group animal data. Tabulation of toxic 
response data by species, strain, sex, and exposure level for:
    (A) Number of animals exposed.
    (B) Number of animals showing signs of toxicity.
    (C) Number of animals dying.
    (ii) Individual animal data. Data should be presented as summary 
(group mean) as well as for individual animals.
    (A) Time of death during the study or whether animals survived to 
termination.
    (B) Time of observation of each abnormal sign and its subsequent 
course.
    (C) Body weight data.
    (D) Feed and water consumption data, when collected.
    (E) Results of clinical pathology and immunotoxicity screen when 
performed.
    (F) Necropsy findings including absolute/relative organ weight 
data.
    (G) Detailed description of all histopathological findings.
    (H) Statistical treatment of results where appropriate.
    (I) Historical control data.
    (iii) Inhalation studies. In addition, for inhalation studies the 
following shall be reported:
    (A) Test conditions. The following exposure conditions shall be 
reported.
    (1) Description of exposure apparatus including design, type, 
dimensions, source of air, system for generating particulate and 
aerosols, method of conditioning air, treatment of exhaust air and the 
method of housing the animals in a test chamber.
    (2) The equipment for measuring temperature, humidity, and 
particulate aerosol concentrations and size should be described.
    (B) Exposure data. These shall be tabulated and presented with mean 
values and a measure of variability (e.g. standard deviation) and 
should include:
    (1) Airflow rates through the inhalation equipment.
    (2) Temperature and humidity of air.
    (3) Actual (analytical or gravimetric) concentration in the 
breathing zone.
    (4) Nominal concentration (total amount of test substance fed into 
the inhalation equipment divided by volume of air).
    (5) Particle size distribution, calculated MMAD and geometric 
standard deviation (GSD).
    (6) Explanation as to why the desired chamber concentration and/or 
particle size could not be achieved (if applicable) and the efforts 
taken to comply with this aspect of the sections.
    (f) Quality assurance. A system shall be developed and maintained 
to assure and document adequate performance of laboratory staff and 
equipment. The study shall be conducted in compliance with 40 CFR Part 
792--Good Laboratory Practice Standards.
    (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,

[[Page 43842]]

Rm. NE-B607, Environmental Protection Agency, 401 M St., SW., 
Washington, DC, 12 noon to 4 p.m., Monday through Friday, except legal 
holidays.
    (1) Benitz, K.F. Ed. Paget, G.E. Measurement of Chronic Toxicity. 
Methods of Toxicology (Blackwell, Oxford, 1970) pp. 82-131.
    (2) Fitzhugh, O.G. Chronic Oral Toxicity, Appraisal of the Safety 
of Chemicals in Foods, Drugs and Cosmetics. The Association of Food and 
Drug Officials of the United States. pp. 36-45 (1959, 3rd Printing 
1975).
    (3) Goldenthal, E.I. and D'Aguanno, W. Evaluation of Drugs, 
Appraisal of the Safety of Chemicals in Foods, Drugs, and Cosmetics. 
The Association of Food and Drug Officials of the United States. pp. 
60-67 (1959, 3rd Printing 1975).
    (4) Organisation for Economic Co-operation and Development. 
Guidelines for Testing of Chemicals, Section 4-Health Effects, Part 451 
Carcinogenicity Studies (Paris, 1981).
    (5) Page, N.P. Chronic Toxicity and Carcinogenicity Guidelines. 
Journal of Environmental Pathology and Toxicology. 11:161-182 (1977).
    (6) Page, N.P. Eds. Kraybill and Mehlman. Concepts of a Bioassay 
Program in Environmental Carcinogenesis. Vol.3. Advances in Modern 
Toxicology (Hemisphere, Washington, DC., 1977) pp. 87-171.
    (7) Sontag, J.M. et al. Guidelines for Carcinogen Bioassay in Small 
Rodents. NCI-CS-TR-1 United States Cancer Institute, Division of Cancer 
Control and Prevention, Carcinogenesis Bioassay Program (Bethesda, MD).


Sec. 799.9510  TSCA bacterial reverse mutation test.

    (a) Scope. This section is intended to meet the testing 
requirements under section 4 of TSCA.
    (1) The bacterial reverse mutation test uses amino-acid requiring 
strains of Salmonella typhimurium and Escherichia coli to detect point 
mutations, which involve substitution, addition or deletion of one or a 
few DNA base pairs. The principle of this bacterial reverse mutation 
test is that it detects mutations which revert mutations present in the 
test strains and restore the functional capability of the bacteria to 
synthesize an essential amino acid. The revertant bacteria are detected 
by their ability to grow in the absence of the amino acid required by 
the parent test strain.
    (2) Point mutations are the cause of many human genetic diseases 
and there is substantial evidence that point mutations in oncogenes and 
tumor suppressor genes of somatic cells are involved in tumor formation 
in humans and experimental animals. The bacterial reverse mutation test 
is rapid, inexpensive and relatively easy to perform. Many of the test 
strains have several features that make them more sensitive for the 
detection of mutations, including responsive DNA sequences at the 
reversion sites, increased cell permeability to large molecules and 
elimination of DNA repair systems or enhancement of error-prone DNA 
repair processes. The specificity of the test strains can provide some 
useful information on the types of mutations that are induced by 
genotoxic agents. A very large data base of results for a wide variety 
of structures is available for bacterial reverse mutation tests and 
well-established methodologies have been developed for testing 
chemicals with different physico-chemical properties, including 
volatile compounds.
    (b) Source. The source material used in developing this TSCA test 
guideline are the OECD replacement guidelines for 471 and 472 (February 
1997). This source is available at the address in paragraph (g) of this 
section.
    (c) Definitions. The following definitions apply to this section:
    A reverse mutation test in either Salmonella typhimurium or 
Escherichia coli detects mutation in an amino-acid requiring strain 
(histidine or tryptophan, respectively) to produce a strain independent 
of an outside supply of amino-acid.
    Base pair substitution mutagens are agents that cause a base change 
in DNA. In a reversion test this change may occur at the site of the 
original mutation, or at a second site in the bacterial genome.
    Frameshift mutagens are agents that cause the addition or deletion 
of one or more base pairs in the DNA, thus changing the reading frame 
in the RNA
    (d) Initial considerations. (1) The bacterial reverse mutation test 
utilizes prokaryotic cells, which differ from mammalian cells in such 
factors as uptake, metabolism, chromosome structure and DNA repair 
processes. Tests conducted in vitro generally require the use of an 
exogenous source of metabolic activation. In vitro metabolic activation 
systems cannot mimic entirely the mammalian in vivo conditions. The 
test therefore does not provide direct information on the mutagenic and 
carcinogenic potency of a substance in mammals.
    (2) The bacterial reverse mutation test is commonly employed as an 
initial screen for genotoxic activity and, in particular, for point 
mutation-inducing activity. An extensive data base has demonstrated 
that many chemicals that are positive in this test also exhibit 
mutagenic activity in other tests. There are examples of mutagenic 
agents which are not detected by this test; reasons for these 
shortcomings can be ascribed to the specific nature of the endpoint 
detected, differences in metabolic activation, or differences in 
bioavailability. On the other hand, factors which enhance the 
sensitivity of the bacterial reverse mutation test can lead to an 
overestimation of mutagenic activity.
    (3) The bacterial reverse mutation test may not be appropriate for 
the evaluation of certain classes of chemicals, for example highly 
bactericidal compounds (e.g. certain antibiotics) and those which are 
thought (or known) to interfere specifically with the mammalian cell 
replication system (e.g. some topoisomerase inhibitors and some 
nucleoside analogues). In such cases, mammalian mutation tests may be 
more appropriate.
    (4) Although many compounds that are positive in this test are 
mammalian carcinogens, the correlation is not absolute. It is dependent 
on chemical class and there are carcinogens that are not detected by 
this test because they act through other, non-genotoxic mechanisms or 
mechanisms absent in bacterial cells.
    (e) Test method--(1) Principle. (i) Suspensions of bacterial cells 
are exposed to the test substance in the presence and in the absence of 
an exogenous metabolic activation system. In the plate incorporation 
method, these suspensions are mixed with an overlay agar and plated 
immediately onto minimal medium. In the preincubation method, the 
treatment mixture is incubated and then mixed with an overlay agar 
before plating onto minimal medium. For both techniques, after 2 or 3 
days of incubation, revertant colonies are counted and compared to the 
number of spontaneous revertant colonies on solvent control plates.
    (ii) Several procedures for performing the bacterial reverse 
mutation test have been described. Among those commonly used are the 
plate incorporation method, the preincubation method, the fluctuation 
method, and the suspension method. Suggestions for modifications for 
the testing of gases or vapors are described in the reference in 
paragraph (g)(12) of this section.
    (iii) The procedures described in this section pertain primarily to 
the plate incorporation and preincubation methods. Either of them is 
acceptable for conducting experiments both with and without metabolic 
activation. Some compounds may be detected more

[[Page 43843]]

efficiently using the preincubation method. These compounds belong to 
chemical classes that include short chain aliphatic nitrosamines, 
divalent metals, aldehydes, azo-dyes and diazo compounds, pyrollizidine 
alkaloids, allyl compounds and nitro compounds. It is also recognized 
that certain classes of mutagens are not always detected using standard 
procedures such as the plate incorporation method or preincubation 
method. These should be regarded as ``special cases'' and it is 
strongly recommended that alternative procedures should be used for 
their detection. The following ``special cases'' could be identified 
(together with examples of procedures that could be used for their 
detection): azo-dyes and diazo compounds (alterative procedures are 
described in the references in paragraphs (g)(3), (g)(5), (g)(6), and 
(g)(13) of this section), gases and volatile chemicals (alterative 
procedures are described in the references in paragraphs (g)(12), 
(g)(14), (g)(15), and (g)(16) of this section), and glycosides 
(alterative procedures are described in the references in paragraphs 
(g)(17) and (g)(18) of this section). A deviation from the standard 
procedure needs to be scientifically justified.
    (2) Description--(i) Preparations--(A) Bacteria. (1) Fresh cultures 
of bacteria should be grown up to the late exponential or early 
stationary phase of growth (approximately 109 cells per ml). 
Cultures in late stationary phase should not be used. The cultures used 
in the experiment shall contain a high titre of viable bacteria. The 
titre may be demonstrated either from historical control data on growth 
curves, or in each assay through the determination of viable cell 
numbers by a plating experiment.
    (2) The culture temperature shall be 37 deg.C.
    (3) At least five strains of bacteria should be used. These should 
include four strains of S. typhimurium (TA1535; TA1537 or TA97a or 
TA97; TA98; and TA100) that have been shown to be reliable and 
reproducibly responsive between laboratories. These four S. typhimurium 
strains have GC base pairs at the primary reversion site and it is 
known that they may not detect certain oxidizing mutagens, cross-
linking agents, and hydrazines. Such substances may be detected by 
E.coli WP2 strains or S. typhimurium TA102 (see paragraph (g)(19) of 
this section) which have an AT base pair at the primary reversion site. 
Therefore the recommended combination of strains is:
    (i) S. typhimurium TA1535.
    (ii) S. typhimurium TA1537 or TA97 or TA97a.
    (iii) S. typhimurium TA98.
    (iv) S. typhimurium TA100.
    (v) E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. 
typhimurium TA102. In order to detect cross-linking mutagens it may be 
preferable to include TA102 or to add a DNA repair-proficient strain of 
E.coli [e.g. E.coli WP2 or E.coli WP2 (pKM101).]
    (4) Established procedures for stock culture preparation, marker 
verification and storage should be used. The amino-acid requirement for 
growth should be demonstrated for each frozen stock culture preparation 
(histidine for S. typhimurium strains, and tryptophan for E. coli 
strains). Other phenotypic characteristics should be similarly checked, 
namely: the presence or absence of R-factor plasmids where appropriate 
[i.e. ampicillin resistance in strains TA98, TA100 and TA97a or TA97, 
WP2 uvrA and WP2 uvrA (pKM101), and ampicillin + tetracycline 
resistance in strain TA102]; the presence of characteristic mutations 
(i.e. rfa mutation in S. typhimurium through sensitivity to crystal 
violet, and uvrA mutation in E. coli or uvrB mutation in S. 
typhimurium, through sensitivity to ultra-violet light). The strains 
should also yield spontaneous revertant colony plate counts within the 
frequency ranges expected from the laboratory's historical control data 
and preferably within the range reported in the literature.
    (B) Medium. An appropriate minimal agar (e.g. containing Vogel-
Bonner minimal medium E and glucose) and an overlay agar containing 
histidine and biotin or tryptophan, to allow for a few cell divisions, 
shall be used. The procedures described in the references under 
paragraphs (g)(1), (g)(2), and (g)(9) of this section may be used for 
this analysis.
    (C) Metabolic activation. Bacteria shall be exposed to the test 
substance both in the presence and absence of an appropriate metabolic 
activation system. The most commonly used system is a cofactor-
supplemented post-mitochondrial fraction (S9) prepared from the livers 
of rodents treated with enzyme-inducing agents such as Aroclor 1254 
(the system described in the references under paragraphs (g)(1) and 
(g)(2) of this section may be used) or a combination of phenobarbitone 
and -naphthoflavone (the system described in the references 
under paragraphs (g)(18), (g)(20), and (g)(21) of this section may be 
used). The post-mitochondrial fraction is usually used at 
concentrations in the range from 5 to 30% v/v in the S9-mix. The choice 
and condition of a metabolic activation system may depend upon the 
class of chemical being tested. In some cases it may be appropriate to 
utilize more than one concentration of post-mitochondrial fraction. For 
azo-dyes and diazo-compounds, using a reductive metabolic activation 
system may be more appropriate (the system described in the references 
under paragraphs (g)(6) and (g)(13) of this section may be used).
    (D) Test substance/preparation. Solid test substances should be 
dissolved or suspended in appropriate solvents or vehicles and diluted 
if appropriate prior to treatment of the bacteria. Liquid test 
substances may be added directly to the test systems and/or diluted 
prior to treatment. Fresh preparations should be employed unless 
stability data demonstrate the acceptability of storage.
    (ii) Test conditions--(A) Solvent/vehicle. The solvent/vehicle 
shall not be suspected of chemical reaction with the test substance and 
shall be compatible with the survival of the bacteria and the S9 
activity (see paragraph (g)(22) of this section). 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.
    (B) Exposure concentrations. (1) Amongst the criteria to be taken 
into consideration when determining the highest amount of test 
substance to be used are cytotoxicity and solubility in the final 
treatment mixture. It may be useful to determine toxicity and 
insolubility in a preliminary experiment. Cytotoxicity may be detected 
by a reduction in the number of revertant colonies, a clearing or 
diminution of the background lawn, or the degree of survival of treated 
cultures. The cytotoxicity of a substance may be altered in the 
presence of metabolic activation systems. Insolubility should be 
assessed as precipitation in the final mixture under the actual test 
conditions and evident to the unaided eye. The recommended maximum test 
concentration for soluble non-cytotoxic substances is 5 mg/plate or 5 
l/plate. For non-cytotoxic substances that are not soluble at 
5mg/plate or 5l/plate, one or more concentrations tested 
should be insoluble in the final treatment mixture. Test substances 
that are cytotoxic already below 5mg/plate or 5l/plate should 
be tested up to a cytotoxic concentration. The precipitate should not 
interfere with the scoring.
    (2) At least five different analyzable concentrations of the test 
substance shall be used with approximately half

[[Page 43844]]

log (i.e. 10) intervals between test points for an initial 
experiment. Smaller intervals may be appropriate when a concentration-
response is being investigated.
    (3) Testing above the concentration of 5 mg/plate or 5l/
plate may be considered when evaluating substances containing 
substantial amounts of potentially mutagenic impurities.
    (C) Controls. (1) Concurrent strain-specific positive and negative 
(solvent or vehicle) controls, both with and without metabolic 
activation, shall be included in each assay. Positive control 
concentrations that demonstrate the effective performance of each assay 
should be selected.
    (2)(i) For assays employing a metabolic activation system, the 
positive control reference substance(s) should be selected on the basis 
of the type of bacteria strains used. The following chemicals are 
examples of suitable positive controls for assays with metabolic 
activation:

                                                                        
------------------------------------------------------------------------
                 Chemical                              CAS No.          
------------------------------------------------------------------------
9,10-Dimethylanthracene...................  [CAS no. 781-43-1]          
7,12-Dimethylbenzanthracene...............  [CAS no. 57-97-6]           
Congo Red (for the reductive metabolic      [CAS no. 573-58-0]          
 activation method).                                                    
Benzo(a)pyrene............................  [CAS no. 50-32-8]           
Cyclophosphamide (monohydrate)............  [CAS no. 50-18-0]           
                                            [CAS no. 6055-19-2]         
2-Aminoanthracene.........................  [CAS no. 613-13-8]          
------------------------------------------------------------------------

    (ii) 2-Aminoanthracene should not be used as the sole indicator of 
the efficacy of the S9-mix. If 2-aminoanthracene is used, each batch of 
S9 should also be characterized with a mutagen that requires metabolic 
activation by microsomal enzymes, e.g., benzo(a)pyrene, 
dimethylbenzanthracene.
    (3) For assays performed without metabolic activation system, 
examples of strain-specific positive controls are:

                                                                        
------------------------------------------------------------------------
            Chemical                    CAS No.             Strain      
------------------------------------------------------------------------
(a) Sodium azide................  [CAS no. 26628-22-  TA1535 and TA100  
                                   8].                                  
(b) 2-Nitrofluorene.............  [CAS no. 607-57-8]  TA 98             
(c) 9-Aminoacridine or ICR 191..  [CAS no. 90-45-9]   TA1537, TA97 and  
                                   or.                 TA97a            
                                  [CAS no. 17070-45-                    
                                   0].                                  
(d) Cumene hydroperoxide........  [CAS no. 80-15-9].  TA102             
(e) Mitomycin C.................  [CAS no. 50-07-7].  WP2 uvrA and TA102
(f) N-Ethyl-N-nitro-N-            [CAS no. 70-25-7]   WP2, WP2 uvrA and 
 nitrosoguanidine or               or.                 WP2 uvrA (pKM101)
4-nitroquinoline 1-oxide........  [CAS no. 56-57-5].                    
(g) Furylfuramide (AF-2)........  [CAS no. 3688-53-   Plasmid-containing
                                   7].                 strains          
------------------------------------------------------------------------

    (4) Other appropriate positive control reference substances may be 
used. The use of chemical class-related positive control chemicals may 
be considered, when available.
    (5) Negative controls, consisting of solvent or vehicle alone, 
without test substance, and otherwise treated in the same way as the 
treatment groups, shall be included. In addition, untreated controls 
should also be used unless there are historical control data 
demonstrating that no deleterious or mutagenic effects are induced by 
the chosen solvent.
    (3) Procedure--(i) Treatment with test substance. (A) For the plate 
incorporation method, without metabolic activation, usually 0.05 ml or 
0.1 ml of the test solutions, 0.1 ml of fresh bacterial culture 
(containing approximately 108 viable cells) and 0.5 ml of 
sterile buffer are mixed with 2.0 ml of overlay agar. For the assay 
with metabolic activation, usually 0.5 ml of metabolic activation 
mixture containing an adequate amount of post-mitochondrial fraction 
(in the range from 5 to 30% v/v in the metabolic activation mixture) 
are mixed with the overlay agar (2.0 ml), together with the bacteria 
and test substance/test solution. The contents of each tube are mixed 
and poured over the surface of a minimal agar plate. The overlay agar 
is allowed to solidify before incubation.
    (B) For the preincubation method the test substance/test solution 
is preincubated with the test strain (containing approximately 
108 viable cells) and sterile buffer or the metabolic 
activation system (0.5 ml) usually for 20 min. or more at 30-37  deg.C 
prior to mixing with the overlay agar and pouring onto the surface of a 
minimal agar plate. Usually, 0.05 or 0.1 ml of test substance/test 
solution, 0.1 ml of bacteria, and 0.5 ml of S9-mix or sterile buffer, 
are mixed with 2.0 ml of overlay agar. Tubes should be aerated during 
pre-incubation by using a shaker.
    (C) For an adequate estimate of variation, triplicate plating 
should be used at each dose level. The use of duplicate plating is 
acceptable when scientifically justified. The occasional loss of a 
plate does not necessarily invalidate the assay.
    (D) Gaseous or volatile substances should be tested by appropriate 
methods, such as in sealed vessels (methods described in the references 
under paragraphs (g)(12), (g)(14), (g)(15), and (g)(16) of this section 
may be used).
    (ii) Incubation. All plates in a given assay shall be incubated at 
37  deg.C for 48-72 hrs. After the incubation period, the number of 
revertant colonies per plate is counted.
    (f) Data and reporting--(1) Treatment of results. (i) Data shall be 
presented as the number of revertant colonies per plate. The number of 
revertant colonies on both negative (solvent control, and

[[Page 43845]]

untreated control if used) and positive control plates shall also be 
given.
    (ii) Individual plate counts, the mean number of revertant colonies 
per plate and the standard deviation shall be presented for the test 
substance and positive and negative (untreated and/or solvent) 
controls.
    (iii) There is no requirement for verification of a clear positive 
response. Equivocal results shall be clarified by further testing 
preferably using a modification of experimental conditions. Negative 
results need to be confirmed on a case-by-case basis. In those cases 
where confirmation of negative results is not considered necessary, 
justification should be provided. Modification of study parameters to 
extend the range of conditions assessed should be considered in follow-
up experiments. Study parameters that might be modified include the 
concentration spacing, the method of treatment (plate incorporation or 
liquid preincubation), and metabolic activation conditions.
    (2) Evaluation and interpretation of results. (i) There are several 
criteria for determining a positive result, such as a concentration-
related increase over the range tested and/or a reproducible increase 
at one or more concentrations in the number of revertant colonies per 
plate in at least one strain with or without metabolic activation 
system. Biological relevance of the results should be considered first. 
Statistical methods may be used as an aid in evaluating the test 
results. However, statistical significance should not be the only 
determining factor for a positive response.
    (ii) A test substance for which the results do not meet the 
criteria described under paragraph (f)(2)(i) of this section is 
considered non-mutagenic in this test
    (iii) Although most experiments will give clearly positive or 
negative results, in rare cases the data set will preclude making a 
definite judgement about the activity of the test substance. Results 
may remain equivocal or questionable regardless of the number of times 
the experiment is repeated.
    (iv) Positive results from the bacterial reverse mutation test 
indicate that a substance induces point mutations by base substitutions 
or frameshifts in the genome of either Salmonella typhimurium and/or 
Escherichia coli. Negative results indicate that under the test 
conditions, the test substance is not mutagenic in the tested species.
    (3) Test report. In addition to the reporting requirements as 
specified under 40 CFR part 792, subpart J, the following specific 
information shall be reported. Both individual and summary data should 
be presented.
    (i) Test substance:
    (A) Identification data and CAS no., if known.
    (B) Physical nature and purity.
    (C) Physicochemical properties relevant to the conduct of the 
study.
    (D) Stability of the test substance, if known.
    (ii) Solvent/vehicle:
    (A) Justification for choice of solvent/vehicle.
    (B) Solubility and stability of the test substance in solvent/
vehicle, if known.
    (iii) Strains:
    (A) Strains used.
    (B) Number of cells per culture.
    (C) Strain characteristics.
    (iv) Test conditions:
    (A) Amount of test substance per plate (mg/plate or ml/plate) with 
rationale for selection of dose and number of plates per concentration.
    (B) Media used.
    (C) Type and composition of metabolic activation system, including 
acceptability criteria.
    (D) Treatment procedures.
    (v) Results:
    (A) Signs of toxicity.
    (B) Signs of precipitation.
    (C) Individual plate counts.
    (D) The mean number of revertant colonies per plate and standard 
deviation.
    (E) Dose-response relationship, where possible.
    (F) Statistical analyses, if any.
    (G) Concurrent negative (solvent/vehicle) and positive control 
data, with ranges, means and standard deviations.
    (H) Historical negative (solvent/vehicle) and positive control 
data, with e.g. ranges, means and standard deviations.
    (vi) Discussion of the results.
    (vii) Conclusion.
    (g) References. For additional background information on this test 
guideline, the following references should be consulted. These 
references are available for inspection at the TSCA Nonconfidential 
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M 
St., SW., Washington, DC, 12 noon to 4 p.m., Monday through Friday, 
except legal holidays.
    (1) Ames, B.N., McCann, J., and Yamasaki, E. Methods for Detecting 
Carcinogens and Mutagens With the Salmonella/Mammalian-Microsome 
Mutagenicity Test. Mutation Research. 31, 347-364 (1975).
    (2) Maron, D.M. and Ames, B.N. Revised Methods for the Salmonella 
Mutagenicity Test. Mutation Research. 113, 173-215 (1983).
    (3) Gatehouse, D., Haworth, S., Cebula, T., Gocke, E., Kier, L., 
Matsushima, T., Melcion, C., Nohmi, T., Venitt, S., and Zeiger, E. 
Recommendations for the Performance of Bacterial Mutation Assays. 
Mutation Research. 312, 217-233 (1994).
    (4) Kier, L.D., Brusick, D.J., Auletta, A.E., Von Halle, E.S., 
Brown, M.M., Simmon, V.F., Dunkel, V., McCann, J., Mortelmans, K., 
Prival, M., Rao, T.K., and Ray V. The Salmonella Typhimurium/Mammalian 
Microsomal Assay: A Report of the U.S. Environmental Protection Agency 
Gene-Tox Program. Mutation Research. 168, 69-240 (1986).
    (5) Yahagi, T., Degawa, M., Seino, Y.Y., Matsushima, T., Nagao, M., 
Sugimura, T., and Hashimoto, Y. Mutagenicity of Carcinogen Azo Dyes and 
Their Derivatives. Cancer Letters, 1. 91-96 (1975).
    (6) Matsushima, M., Sugimura, T., Nagao, M., Yahagi, T., Shirai, 
A., and Sawamura, M. Factors Modulating Mutagenicity Microbial Tests. 
Eds. Norpoth, K.H. and Garner, R.C. Short-Term Test Systems for 
Detecting Carcinogens (Springer, Berlin-Heidelberg-New York, 1980) pp. 
273-285.
    (7) Gatehouse, D.G., Rowland, I.R., Wilcox, P., Callender, R.D., 
and Foster, R. Bacterial Mutation Assays. Ed. Kirkland, D.J. Basic 
Mutagenicity Tests. UKEMS Part 1 Revised (Cambridge University Press, 
1990) pp. 13-61.
    (8) Aeschbacher, H.U., Wolleb, U., and Porchet, L.J. Liquid 
Preincubation Mutagenicity Test for Foods. Food Safety. 8, 167-177 
(1987).
    (9) Green, M.H.L., Muriel, W.J., and Bridges, B.A. Use of a 
Simplified Fluctuation Test to Detect Low Levels of Mutagens. Mutation 
Research. 38, 33-42 (1976).
    (10) Hubbard, S.A., Green, M.H.L., Gatehouse, D., and J.W. Bridges. 
The Fluctuation Test in Bacteria. 2nd Edition. Ed. Kilbey, B.J., 
Legator, M., Nichols, W., and Ramel C. Handbook of Mutagenicity Test 
Procedures (Elsevier, Amsterdam-New York-Oxford, 1984) pp. 141-161.
    (11) Thompson, E.D. and Melampy, P.J. An Examination of the 
Quantitative Suspension Assay for Mutagenesis With Strains of 
Salmonella Typhimurium. Environmental Mutagenesis. 3, 453-465 (1981).
    (12) Araki, A., Noguchi, T., Kato, F., and T. Matsushima. Improved 
Method for Mutagenicity Testing of Gaseous Compounds by Using a Gas 
Sampling Bag. Mutation Research. 307, 335-344 (1994).
    (13) Prival, M.J., Bell, S.J., Mitchell, V.D., Reipert, M.D., and 
Vaughn, V.L.

[[Page 43846]]

Mutagenicity of Benzidine and Benzidine-Congener Dyes and Selected 
Monoazo Dyes in a Modified Salmonella Assay. Mutation Research. 136, 
33-47 (1984).
    (14) Zeiger, E., Anderson, B. E., Haworth, S, Lawlor, T., and 
Mortelmans, K. Salmonella Mutagenicity Tests. V. Results from the 
Testing of 311 Chemicals. Environ. Mol. Mutagen. 19, 2-141 (1992).
    (15) Simmon, V., Kauhanen, K., and Tardiff, R.G. Mutagenic Activity 
of Chemicals Identified in Drinking Water. Ed. Scott, D., Bridges, B., 
and Sobels, F. Progress in Genetic Toxicology (Elsevier, Amsterdam, 
1977) pp. 249-258.
    (16) Hughes, T.J., Simmons, D.M., Monteith, I.G., and Claxton, L.D. 
Vaporization Technique to Measure Mutagenic Activity of Volatile 
Organic Chemicals in the Ames/Salmonella Assay. Environmental 
Mutagenesis. 9, 421-441 (1987).
    (17) Matsushima, T., Matsumoto, A., Shirai, M., Sawamura, M., and 
Sugimura, T. Mutagenicity of the Naturally Occurring Carcinogen Cycasin 
and Synthetic Methylazoxy Methane Conjugates in Salmonella Typhimurium. 
Cancer Research. 39, 3780-3782 (1979).
    (18) Tamura, G., Gold, C., Ferro-Luzzi, A., and Ames. B.N. 
Fecalase: A Model for Activation of Dietary Glycosides to Mutagens by 
Intestinal Flora. Proc. National Academy of Science. (USA, 1980) 77, 
4961-4965.
    (19) Wilcox, P., Naidoo, A., Wedd, D. J., and Gatehouse, D. G. 
Comparison of Salmonella Typhimurium TA 102 With Escherichia Coli WP2 
Tester Strains. Mutagenesis. 5, 285-291 (1990).
    (20) Matsushima, T., Sawamura, M., Hara, K., and Sugimura, T.  A 
Safe Substitute for Polychlorinated Biphenyls as an Inducer of 
Metabolic Activation Systems. Ed. F.J. de Serres et al. In Vitro 
Metabolic Activation in Mutagenesis Testing. (Elsevier, North Holland, 
1976) pp. 85-88.
    (21) Elliott, B.M., Combes, R.D., Elcombe, C.R., Gatehouse, D.G., 
Gibson, G.G., Mackay, J.M., and Wolf, R.C. Alternatives to Aroclor 
1254-Induced S9 in In Vitro Genotoxicity Assays. Mutagenesis. 7, 175-
177 (1992).
    (22) Maron, D., Katzenellenbogen, J., and Ames, B.N. Compatibility 
of Organic Solvents With the Salmonella/Microsome Test. Mutation 
Research. 88, 343-350 (1981).
    (23) Claxton, L.D., Allen, J., Auletta, A., Mortelmans, K., 
Nestmann, E., and Zeiger, E. Guide for the Salmonella Typhimurium/
Mammalian Microsome Tests for Bacterial Mutagenicity. Mutation 
Research. 189, 83-91 (1987).
    (24) Mahon, G.A.T., Green, M.H.L., Middleton, B., Mitchell, I., 
Robinson, W.D., and Tweats, D.J. Analysis of Data from Microbial Colony 
Assays. UKEMS Sub-Committee on Guidelines for Mutagenicity Testing Part 
II. Ed. Kirkland, D.J. Statistical Evaluation of Mutagenicity Test Data 
(Cambridge University Press, 1989) pp. 28-65.


Sec. 799.9530  TSCA in vitro mammalian cell gene mutation test.

    (a) Scope. This section is intended to meet the testing 
requirements under section 4 of TSCA. The in vitro mammalian cell gene 
mutation test can be used to detect gene mutations induced by chemical 
substances. Suitable cell lines include L5178Y mouse lymphoma cells, 
the CHO, AS52 and V79 lines of Chinese hamster cells, and TK6 human 
lymphoblastoid cells under paragraph (g)(1) of this section. In these 
cell lines the most commonly-used genetic endpoints measure mutation at 
thymidine kinase (TK) and hypoxanthine-guanine phosphoribosyl 
transferase (HPRT), and a transgene of xanthine-guanine phosphoribosyl 
transferase (XPRT). The TK, HPRT and XPRT mutation tests detect 
different spectra of genetic events. The autosomal location of TK and 
XPRT may allow the detection of genetic events (e.g. large deletions) 
not detected at the HPRT locus on X-chromosomes (For a discussion see 
the references in paragraphs (g)(2), (g)(3), (g)(4),(g)(5), and (g)(6) 
of this section).
    (b) Source. The source material used in developing this TSCA test 
guideline is the OECD guideline 476 (February 1997). This source is 
available at the address in paragraph (g) of this section.
    (c) Definitions. The following definitions apply to this section:
    Base pair substitution mutagens are substances which cause 
substitution of one or several base pairs in the DNA.
    Forward mutation is a gene mutation from the parental type to the 
mutant form which gives rise to an alteration or a loss of the 
enzymatic activity or the function of the encoded protein.
    Frameshift mutagens are substances which cause the addition or 
deletion of single or multiple base pairs in the DNA molecule.
    Mutant frequency is the number of mutant cells observed divided by 
the number of viable cells.
    Phenotypic expression time is a period during which unaltered gene 
products are depleted from newly mutated cells.
    Relative suspension growth is an increase in cell number over the 
expression period relative to the negative control.
    Relative total growth is an increase in cell number over time 
compared to a control population of cells; calculated as the product of 
suspension growth relative to the negative control times cloning 
efficiency relative to negative control.
    Survival is the cloning efficiency of the treated cells when plated 
at the end of the treatment period; survival is usually expressed in 
relation to the survival of the control cell population.
    Viability is the cloning efficiency of the treated cells at the 
time of plating in selective conditions after the expression period.
    (d) Initial considerations. (1) In the in vitro mammalian cell gene 
mutation test, cultures of established cell lines or cell strains can 
be used. The cells used are selected on the basis of growth ability in 
culture and stability of the spontaneous mutation frequency. Tests 
conducted in vitro generally require the use of an exogenous source of 
metabolic activation. This metabolic activation system cannot mimic 
entirely the mammalian in vivo conditions. Care should be taken to 
avoid conditions which would lead to results not reflecting intrinsic 
mutagenicity. Positive results which do not reflect intrinsic 
mutagenicity may arise from changes in pH, osmolality or high levels of 
cytotoxicity.
    (2) This test is used to screen for possible mammalian mutagens and 
carcinogens. Many compounds that are positive in this test are 
mammalian carcinogens; however, there is not a perfect correlation 
between this test and carcinogenicity. Correlation is dependent on 
chemical class and there is increasing evidence that there are 
carcinogens that are not detected by this test because they appear to 
act through other, non-genotoxic mechanisms or mechanisms absent in 
bacterial cells.
    (e)  Test method--(1) Principle. (i) Cells deficient in thymidine 
kinase (TK) due to the mutation TK+/- -
 TK-/- are resistant to the cytotoxic 
effects of the pyrimidine analogue trifluorothymidine (TFT). Thymidine 
kinase proficient cells are sensitive to TFT, which causes the 
inhibition of cellular metabolism and halts further cell division. Thus 
mutant cells are able to proliferate in the presence of TFT, whereas 
normal cells, which contain thymidine kinase, are not. Similarly, cells 
deficient in HPRT or XPRT are selected by resistance to 6-thioguanine 
(TG) or 8-azaguanine (AG). The properties of the test substance should 
be considered carefully if a base analogue or a compound related to the 
selective agent is tested in any of the mammalian cell gene mutation 
tests. For example, any suspected selective

[[Page 43847]]

toxicity by the test substance for mutant and non-mutant cells should 
be investigated. Thus, performance of the selection system/agent shall 
be confirmed when testing chemicals structurally related to the 
selective agent.
    (ii) Cells in suspension or monolayer culture shall be exposed to 
the test substance, both with and without metabolic activation, for a 
suitable period of time and subcultured to determine cytotoxicity and 
to allow phenotypic expression prior to mutant selection. Cytotoxicity 
is usually determined by measuring the relative cloning efficiency 
(survival) or relative total growth of the cultures after the treatment 
period. The treated cultures shall be maintained in growth medium for a 
sufficient period of time, characteristic of each selected locus and 
cell type, to allow near-optimal phenotypic expression of induced 
mutations. Mutant frequency is determined by seeding known numbers of 
cells in medium containing the selective agent to detect mutant cells, 
and in medium without selective agent to determine the cloning 
efficiency (viability). After a suitable incubation time, colonies 
shall be counted. The mutant frequency is derived from the number of 
mutant colonies in selective medium and the number of colonies in non-
selective medium.
    (2) Description--(i) Preparations--(A) Cells. (1) A variety of cell 
types are available for use in this test including subclones of L5178Y, 
CHO, CHO-AS52, V79, or TK6 cells. Cell types used in this test should 
have a demonstrated sensitivity to chemical mutagens, a high cloning 
efficiency and a stable spontaneous mutant frequency. Cells should be 
checked for mycoplasma contamination and should not be used if 
contaminated.
    (2) The test should be designed to have a predetermined sensitivity 
and power. The number of cells, cultures, and concentrations of test 
substance used should reflect these defined parameters. The parameters 
discussed in the reference under paragraph (g)(13) of this section may 
be used. The minimal number of viable cells surviving treatment and 
used at each stage in the test should be based on the spontaneous 
mutation frequency. A general guide is to use a cell number which is at 
least ten times the inverse of the spontaneous mutation frequency. 
However, it is recommended to utilize at least 106 cells. 
Adequate historical data on the cell system used should be available to 
indicate consistent performance of the test.
    (B) Media and culture conditions. Appropriate culture media and 
incubation conditions (culture vessels, temperature, CO2 
concentration and humidity) shall be used. Media should be chosen 
according to the selective systems and cell type used in the test. It 
is particularly important that culture conditions should be chosen that 
ensure optimal growth of cells during the expression period and colony 
forming ability of both mutant and non-mutant cells.
    (C) Preparation of cultures. Cells are propagated from stock 
cultures, seeded in culture medium and incubated at 37 deg.C. Prior to 
use in this test, cultures may need to be cleansed of pre-existing 
mutant cells.
    (D) Metabolic activation. Cells shall be exposed to the test 
substance both in the presence and absence of an appropriate metabolic 
activation system. The most commonly used system is a co-factor-
supplemented post-mitochondrial fraction (S9) prepared from the livers 
of rodents treated with enzyme-inducing agents such as Aroclor 1254 or 
a combination of phenobarbitone and -naphthoflavone. The post-
mitochondrial fraction is usually used at concentrations in the range 
from 1-10% v/v in the final test medium. The choice and condition of a 
metabolic activation system may depend upon the class of chemical being 
tested. In some cases it may be appropriate to utilize more than one 
concentration of post-mitochondrial fraction. A number of developments, 
including the construction of genetically engineered cell lines 
expressing specific activating enzymes, may provide the potential for 
endogenous activation. The choice of the cell lines used should be 
scientifically justified (e.g. by the relevance of the cytochrome P450 
isoenzyme to the metabolism of the test substance).
    (E) Test substance/preparations. Solid test substances should be 
dissolved or suspended in appropriate solvents or vehicles and diluted 
if appropriate prior to treatment of the cells. Liquid test substances 
may be added directly to the test systems and/or diluted prior to 
treatment. Fresh preparations should be employed unless stability data 
demonstrate the acceptability of storage.
    (ii) Test conditions--(A) Solvent/vehicle. The solvent/vehicle 
shall not be suspected of chemical reaction with the test substance and 
shall be compatible with the survival of the cells and the S9 activity. 
If other than well-known solvent/vehicles are used, their inclusion 
should be supported by data indicating their compatibility. It is 
recommended that wherever possible, the use of an aqueous solvent/
vehicle be considered first. When testing water-unstable substances, 
the organic solvents used should be free of water. Water can be removed 
by adding a molecular sieve.
    (B) Exposure concentrations. (1) Among the criteria to be 
considered when determining the highest concentration are cytotoxicity 
and solubility in the test system and changes in pH or osmolality.
    (2) Cytotoxicity should be determined with and without metabolic 
activation in the main experiment using an appropriate indicator of 
cell integrity and growth, such as relative cloning efficiency 
(survival) or relative total growth. It may be useful to determine 
cytotoxicity and solubility in a preliminary experiment.
    (3) At least four analyzable concentrations shall be used. Where 
there is cytotoxicity, these concentrations shall cover a range from 
the maximum to little or no toxicity; this will usually mean that the 
concentration levels should be separated by no more than a factor 
between 2 and 10. If the maximum concentration is based on 
cytotoxicity then it shall result in approximately 10-20% but not less 
than 10% relative survival (relative cloning efficiency) or relative 
total growth. For relatively non-cytotoxic compounds the maximum 
concentration should be 5 mg/ml, 5 l/ml, or 0.01 M, whichever 
is the lowest.
    (4) Relatively insoluble substances should be tested up to or 
beyond their limit of solubility under culture conditions. Evidence of 
insolubility should be determined in the final treatment medium to 
which cells are exposed. It may be useful to assess solubility at the 
beginning and end of the treatment, as solubility can change during the 
course of exposure in the test system due to presence of cells, S9, 
serum etc. Insolubility can be detected by using the unaided eye. The 
precipitate should not interfere with the scoring.
    (C) Controls. (1) Concurrent positive and negative (solvent or 
vehicle) controls both with and without metabolic activation shall be 
included in each experiment. When metabolic activation is used the 
positive control chemical shall be one that requires activation to give 
a mutagenic response.
    (2) Examples of positive control substances include:


[[Page 43848]]



                                                                                                                
----------------------------------------------------------------------------------------------------------------
    Metabolic Activation condition              Locus                   Chemical                 CAS No.        
----------------------------------------------------------------------------------------------------------------
Absence of exogenous metabolic         HPRT...................  Ethylmethanesulfonate..  [CAS no. 62-50-0]      
 activation                                                                                                     
                                                                Ethylnitrosourea.......  [CAS no. 759-73-9]     
                                       TK (small and large      Methylmethanesulfonate.  [CAS no. 66-27-3]      
                                        colonies).                                                              
                                       XPRT...................  Ethylmethanesulfonate..  [CAS no. 62-50-0]      
                                                                Ethylnitrosourea.......  [CAS no. 759-73-9]     
Presence of exogenous metabolic        HPRT...................  3-Methylcholanthrene...  [CAS no. 56-49-5]      
 activation.                                                                                                    
                                                                N-Nitrosodimethylamine.  [CAS no. 62-75-9]      
                                                                7,12-                    [CAS no. 57-97-6]      
                                                                 Dimethylbenzanthracene.                        
                                       TK (small and large      Cyclophosphamide         [CAS no. 50-18-0]      
                                        colonies).               (monohydrate).          [CAS no. 6055-19-2]    
                                                                Benzo(a)pyrene.........  [CAS no. 50-32-8]      
                                                                3-Methylcholanthrene...  [CAS no. 56-49-5]      
                                       XPRT...................  N-Nitrosodimethylamine   [CAS no. 62-75-9]      
                                                                 (for high levels of S-                         
                                                                 9).                                            
                                                                Benzo(a)pyrene.........  [CAS no. 50-32-8]      
----------------------------------------------------------------------------------------------------------------

    (3) Other appropriate positive control reference substances may be 
used, e.g., if a laboratory has a historical data base on 5-Bromo 2'-
deoxyuridine [CAS No. 59-14-3], this reference substance could be used 
as well. The use of chemical class-related positive control chemicals 
may be considered, when available.
    (4) Negative controls, consisting of solvent or vehicle alone in 
the treatment medium, and treated in the same way as the treatment 
groups shall be included. In addition, untreated controls should also 
be used unless there are historical control data demonstrating that no 
deleterious or mutagenic effects are induced by the chosen solvent.
    (3) Procedure--(i) Treatment with test substance. (A) Proliferating 
cells shall be exposed to the test substance both with and without 
metabolic activation. Exposure shall be for a suitable period of time 
(usually 3 to 6 hrs is effective). Exposure time may be extended over 
one or more cell cycles.
    (B) Either duplicate or single treated cultures may be used at each 
concentration tested. When single cultures are used, the number of 
concentrations should be increased to ensure an adequate number of 
cultures for analysis (e.g. at least eight analyzsable concentrations). 
Duplicate negative (solvent) control cultures should be used.
    (C) Gaseous or volatile substances should be tested by appropriate 
methods, such as in sealed culture vessels. Methods described in the 
references under paragraphs (g)(20) and (g)(21) of this section may be 
used.
    (ii) Measurement of survival, viability, and mutant frequency. (A) 
At the end of the exposure period, cells shall be washed and cultured 
to determine survival and to allow for expression of the mutant 
phenotype. Measurement of cytotoxicity by determining the relative 
cloning efficiency (survival) or relative total growth of the cultures 
is usually initiated after the treatment period.
    (B) Each locus has a defined minimum time requirement to allow near 
optimal phenotypic expression of newly induced mutants (HPRT and XPRT 
require at least 6-8 days, and TK at least 2 days). Cells are grown in 
medium with and without selective agent(s) for determination of numbers 
of mutants and cloning efficiency, respectively. The measurement of 
viability (used to calculate mutant frequency) is initiated at the end 
of the expression time by plating in non-selective medium.
    (C) If the test substance is positive in the L5178Y TK+/
- test, colony sizing should be performed on at least one of 
the test cultures (the highest positive concentration) and on the 
negative and positive controls. If the test substance is negative in 
the L5178Y TK+/- test, colony sizing should be 
performed on the negative and positive controls. In studies using 
TK6TK+/-, colony sizing may also be performed.
    (f) Data and reporting--(1) Treatment of results. (i) Data shall 
include cytotoxicity and viability determination, colony counts and 
mutant frequencies for the treated and control cultures. In the case of 
a positive response in the L5178Y TK+/- test, 
colonies are scored using the criteria of small and large colonies on 
at least one concentration of the test substance (highest positive 
concentration) and on the negative and positive control. The molecular 
and cytogenetic nature of both large and small colony mutants has been 
explored in detail and is discussed in the references under paragraphs 
(g)(22) and (g)(23) of this section. In the TK+/- 
test, colonies are scored using the criteria of normal growth (large) 
and slow growth (small) colonies (a scoring system similar to the one 
described in the reference under paragraph (g)(24) of this section may 
be used). Mutant cells that have suffered the most extensive genetic 
damage have prolonged doubling times and thus form small colonies. This 
damage typically ranges in scale from the losses of the entire gene to 
karyotypically visible chromosome aberrations. The induction of small 
colony mutants has been associated with chemicals that induce gross 
chromosome aberrations. Less seriously affected mutant cells grow at 
rates similar to the parental cells and form large colonies.
    (ii) Survival (relative cloning efficiencies) or relative total 
growth shall be given. Mutant frequency shall be expressed as number of 
mutant cells per number of surviving cells.
    (iii) Individual culture data shall be provided. Additionally, all 
data shall be summarized in tabular form.
    (iv) There is no requirement for verification of a clear positive 
response. Equivocal results shall be clarified by further testing 
preferably using a modification of experimental conditions. Negative 
results need to be confirmed on a case-by-case basis. In those cases 
where confirmation of negative results is not considered necessary, 
justification should be provided. Modification of study parameters to 
extend the range of conditions assessed should be considered in follow-
up experiments for either equivocal or negative results. Study 
parameters that might be modified include the concentration

[[Page 43849]]

spacing, and the metabolic activation conditions.
    (2) Evaluation and interpretation of results. (i) There are several 
criteria for determining a positive result, such as a concentration-
related, or a reproducible increase in mutant frequency. Biological 
relevance of the results should be considered first. Statistical 
methods may be used as an aid in evaluating the test results. 
Statistical significance should not be the only determining factor for 
a positive response.
    (ii) A test substance, for which the results do not meet the 
criteria described in paragraph (f)(2)(i) of this section is considered 
non-mutagenic in this system.
    (iii) Although most studies will give clearly positive or negative 
results, in rare cases the data set will preclude making a definite 
judgement about the activity of the test substance. Results may remain 
equivocal or questionable regardless of the number of times the 
experiment is repeated.
    (iv) Positive results for an in vitro mammalian cell gene mutation 
test indicate that the test substance induces gene mutations in the 
cultured mammalian cells used. A positive concentration-response that 
is reproducible is most meaningful. Negative results indicate that, 
under the test conditions, the test substance does not induce gene 
mutations in the cultured mammalian cells used.
    (3) Test report. The test report shall include the following 
information:
    (i) Test substance:
    (A) Identification data and CAS no., if known.
    (B) Physical nature and purity.
    (C) Physicochemical properties relevant to the conduct of the 
study.
    (D) Stability of the test substance.
    (ii) Solvent/vehicle:
    (A) Justification for choice of vehicle/solvent.
    (B) Solubility and stability of the test substance in solvent/
vehicle, if known.
    (iii) Cells:
    (A) Type and source of cells.
    (B) Number of cell cultures.
    (C) Number of cell passages, if applicable.
    (D) Methods for maintenance of cell cultures, if applicable.
    (E) Absence of mycoplasma.
    (iv) Test conditions:
    (A) Rationale for selection of concentrations and number of cell 
cultures including e.g., cytotoxicity data and solubility limitations, 
if available.
    (B) Composition of media, CO2 concentration.
    (C) Concentration of test substance.
    (D) Volume of vehicle and test substance added.
    (E) Incubation temperature.
    (F) Incubation time.
    (G) Duration of treatment.
    (H) Cell density during treatment.
    (I) Type and composition of metabolic activation system including 
acceptability criteria.
    (J) Positive and negative controls.
    (K) Length of expression period (including number of cells seeded, 
and subcultures and feeding schedules, if appropriate).
    (L) Selective agent(s).
    (M) Criteria for considering tests as positive, negative or 
equivocal.
    (N) Methods used to enumerate numbers of viable and mutant cells.
    (O) Definition of colonies of which size and type are considered 
(including criteria for ``small'' and ``large'' colonies, as 
appropriate).
    (v) Results:
    (A) Signs of toxicity.
    (B) Signs of precipitation.
    (C) Data on pH and osmolality during the exposure to the test 
substance, if determined.
    (D) Colony size if scored for at least negative and positive 
controls.
    (E) Laboratory's adequacy to detect small colony mutants with the 
L5178Y TK+/- system, where appropriate.
    (F) Dose-response relationship, where possible.
    (G) Statistical analyses, if any.
    (H) Concurrent negative (solvent/vehicle) and positive control 
data.
    (I) Historical negative (solvent/vehicle) and positive control data 
with ranges, means, and standard deviations.
    (J) Mutant frequency.
    (vi) Discussion of the results.
    (vii) Conclusion.
    (g) References. For additional background information on this test 
guideline, the following references should be consulted. These 
references are available for inspection at the TSCA Nonconfidential 
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M 
St., SW., Washington, DC, 12 noon to 4 p.m., Monday through Friday, 
except legal holidays.
    (1) Chu, E.H.Y. and Malling, H.V. Mammalian Cell Genetics. II. 
Chemical Induction of Specific Locus Mutations in Chinese Hamster Cells 
In Vitro, Proc. National Academy Science (USA, 1968) 61, 1306-1312.
    (2) Liber, H.L. and Thilly, W.G. Mutation Assay at the Thymidine 
Kinase Locus in Diploid Human Lymphoblasts. Mutation Research. 94, 467-
485 (1982).
    (3) Moore, M.M., Harrington-Brock, K., Doerr, C.L., and Dearfield, 
K.L. Differential Mutant Quantitation at the Mouse Lymphoma TK and CHO 
HGPRT Loci. Mutagenesis. 4, 394-403 (1989).
    (4) Aaron, C.S. and Stankowski, Jr., L.F. Comparison of the AS52/
XPRT and the CHO/HPRT Assays: Evaluation of Six Drug Candidates. 
Mutation Research. 223, 121-128 (1989).
    (5) Aaron, C.S., Bolcsfoldi, G., Glatt, H.R., Moore, M., Nishi, Y., 
Stankowski, L., Theiss, J., and Thompson, E. Mammalian Cell Gene 
Mutation Assays Working Group Report. Report of the International 
Workshop on Standardization of Genotoxicity Test Procedures. Mutation 
Research. 312, 235-239 (1994).
    (6) Scott, D., Galloway, S.M., Marshall, R.R., Ishidate, M., 
Brusick, D., Ashby, J., and Myhr, B.C. Genotoxicity Under Extreme 
Culture Conditions. A report from ICPEMC Task Group 9. Mutation 
Research. 257, 147-204 (1991).
    (7) Clive, D., McCuen, R., Spector, J.F.S., Piper, C., and 
Mavournin, K.H. Specific Gene Mutations in L5178Y Cells in Culture. A 
Report of the U.S. Environmental Protection Agency Gene-Tox Program. 
Mutation Research. 115, 225-251 (1983).
    (8) Li, A.P., Gupta, R.S., Heflich, R.H., and Wasson, J. S. A 
Review and Analysis of the Chinese Hamster Ovary/Hypoxanthine Guanine 
Phosphoribosyl Transferase System to Determine the Mutagenicity of 
Chemical Agents: A Report of Phase III of the U.S. Environmental 
Protection Agency Gene-Tox Program. Mutation Research. 196, 17-36 
(1988).
    (9) Li, A.P., Carver, J.H., Choy, W.N., Hsie, A.W., Gupta, R.S., 
Loveday, K.S., O'Neill, J.P., Riddle, J.C., Stankowski, Jr., L.F., and 
Yang, L.L. A Guide for the Performance of the Chinese Hamster Ovary 
Cell/Hypoxanthine-Guanine Phosphoribosyl Transferase Gene Mutation 
Assay. Mutation Research. 189, 135-141 (1987).
    (10) Liber, H.L., Yandell, D.W., and Little, J.B. A Comparison of 
Mutation Induction at the tk and hprt Loci in Human Lymphoblastoid 
Cells; Quantitative Differences are Due to an Additional Class of 
Mutations at the Autosomal TK Locus. Mutation Research. 216, 9-17 
(1989).
    (11) Stankowski, L.F. Jr., Tindall, K.R., and Hsie, A.W. 
Quantitative and Molecular Analyses of Ethyl Methanesulfonate- and ICR 
191-Induced Molecular Analyses of Ethyl Methanesulfonate- and ICR 191-
Induced Mutation in AS52 Cells. Mutation Reseach. 160, 133-147 (1986).
    (12) Turner, N.T., Batson, A.G., and Clive, D. Eds. Kilbey, B.J. et 
al. Procedures for the L5178Y/TK+/- > 
TK+/- Mouse Lymphoma Cell Mutagenicity Assay. 
Handbook of Mutagenicity Test Procedures (Elsevier

[[Page 43850]]

Science Publishers, New York, 1984) pp. 239-268.
    (13) Arlett, C.F., Smith, D.M., Clarke, G.M., Green, M.H.L., Cole, 
J., McGregor, D.B., and Asquith, J.C. Ed. Kirkland, D.J. Mammalian Cell 
Gene Mutation Assays Based Upon Colony Formation. Statistical 
Evaluation of Mutagenicity Test Data (Cambridge University Press, 1989) 
pp. 66-101.
    (14) Abbondandolo, A., Bonatti, S., Corti, G., Fiorio, R., 
Loprieno, N., and Mazzaccaro, A. Induction of 6-Thioguanine-Resistant 
Mutants in V79 Chinese Hamster Cells by Mouse-Liver Microsome-Activated 
Dimethylnitrosamine. Mutation Research. 46, 365-373 (1977).
    (15) Ames, B.N., McCann, J., and Yamasaki, E. Methods for Detecting 
Carcinogens and Mutagens with the Salmonella/Mammalian-Microsome 
Mutagenicity Test. Mutation Reseach. 31, 347-364 (1975).
    (16) Clive, D., Johnson, K.O., Spector, J.F.S., Batson, A.G., and 
Brown M.M.M. Validation and Characterization of the L5178Y/
TK+/- Mouse Lymphoma Mutagen Assay System. 
Mutation Reseach. 59, 61-108 (1979).
    (17) Maron, D.M. and Ames, B.N. Revised Methods for the Salmonella 
Mutagenicity Test. Mutation Reseach. 113, 173, 215 (1983).
    (18) Elliott, B.M., Combes, R.D., Elcombe, C.R., Gatehouse, D.G., 
Gibson, G.G., Mackay, J.M., and Wolf, R.C. Alternatives to Aroclor 
1254-Induced S9 in In Vitro Genotoxicity Assays. Mutagenesis. 7, 175-
177 (1992).
    (19) Matsushima, T., Sawamura, M., Hara, K., and Sugimura, T. A 
Safe Substitute for Polychlorinated Biphenyls as an Inducer of 
Metabolic Activation Systems. (Eds.) de Serres, F.J., Fouts, J.R., 
Bend, J.R., and Philpot, R.M. In Vitro Metabolic Activation in 
Mutagenesis Testing (Elsevier, North-Holland, 1976) pp. 85-88.
    (20) Krahn, D.F., Barsky, F.C., and McCooey, K.T. Eds. Tice, R.R., 
Costa, D.L., and Schaich, K.M. CHO/HGPRT Mutation Assay: Evaluation of 
Gases and Volatile Liquids. Genotoxic Effects of Airborne Agents (New 
York, Plenum, 1982) pp. 91-103.
    (21) Zamora, P.O., Benson, J.M., Li, A.P., and Brooks, A.L. 
Evaluation of an Exposure System Using Cells Grown on Collagen Gels for 
Detecting Highly Volatile Mutagens in the CHO/HGPRT Mutation Assay. 
Environmental Mutagenesis. 5, 795-801 (1983).
    (22) Applegate, M.L., Moore, M.M., Broder, C.B., Burrell, A., and 
Hozier, J.C. Molecular Dissection of Mutations at the Heterozygous 
Thymidine Kinase Locus in Mouse Lymphoma Cells. Proc. National Academy 
Science (USA, 1990) 87, 51-55.
    (23) Moore, M.M., Clive, D., Hozier, J.C., Howard, B.E., Batson, 
A.G., Turner, N.T., and Sawyer, J. Analysis of Trifluorothymidine-
Resistant (TFTr) Mutants of L5178Y/TK+/
- Mouse Lymphoma Cells. Mutation Research. 151, 161-174 
(1985).
    (24) Yandell, D.W., Dryja, T.P., and Little J.B. Molecular Genetic 
Analysis of Recessive Mutations at a Heterozygous Autosomal Locus in 
Human Cells. Mutation Research. 229, 89-102 (1990).
    (25) Moore, M.M. and Doerr, C.L. Comparison of Chromosome 
Aberration Frequency and Small-Colony TK-Deficient Mutant Frequency in 
L5178Y/TK+/- 3.7.2C Mouse Lymphoma Cells. 
Mutagenesis. 5, 609-614 (1990).


Sec. 799.9538  TSCA mammalian bone marrow chromosomal aberration test.

    (a) Scope. This section is intended to meet the testing 
requirements under section 4 of TSCA. The mammalian bone marrow 
chromosomal aberration test is used for the detection of structural 
chromosome aberrations induced by test compounds in bone marrow cells 
of animals, usually rodents. Structural chromosome aberrations may be 
of two types, chromosome or chromatid. An increase in polyploidy may 
indicate that a chemical has the potential to induce numerical 
aberrations. With the majority of chemical mutagens, induced 
aberrations are of the chromatid-type, but chromosome-type aberrations 
also occur. Chromosome mutations and related events are the cause of 
many human genetic diseases and there is substantial evidence that 
chromosome mutations and related events causing alterations in 
oncogenes and tumor suppressor genes are involved in cancer in humans 
and experimental systems.
    (b) Source. The source material used in developing this TSCA test 
guideline is the OECD guideline 475 (February 1997). This source is 
available at the address in paragraph (g) of this section.
    (c) Definitions. The following definitions apply to this section:
    Chromatid-type aberration is structural chromosome damage expressed 
as breakage of single chromatids or breakage and reunion between 
chromatids.
    Chromosome-type aberration is structural chromosome damage 
expressed as breakage, or breakage and reunion, of both chromatids at 
an identical site.
    Endoreduplication is a process in which after an S period of DNA 
replication, the nucleus does not go into mitosis but starts another S 
period. The result is chromosomes with 2,4,8,...chromatids.
    Gap is an achromatic lesion smaller than the width of one 
chromatid, and with minimum misalignment of the chromatids.
    Numerical aberration is a change in the number of chromosomes from 
the normal number characteristic of the animals utilized.
    Polyploidy is a multiple of the haploid chromosome number (n) other 
than the diploid number (i.e., 3n, 4n and so on).
    Structural aberration is a change in chromosome structure 
detectable by microscopic examination of the metaphase stage of cell 
division, observed as deletions and fragments, intrachanges or 
interchanges.
    (d) Initial considerations. (1) Rodents are routinely used in this 
test. Bone marrow is the target tissue in this test, since it is a 
highly vascularised tissue, and it contains a population of rapidly 
cycling cells that can be readily isolated and processed. Other species 
and target tissues are not the subject of this section.
    (2) This chromosome aberration test is especially relevant to 
assessing mutagenic hazard in that it allows consideration of factors 
of in vivo metabolism, pharmacokinetics and DNA-repair processes 
although these may vary among species and among tissues. An in vivo 
test is also useful for further investigation of a mutagenic effect 
detected by an in vitro test.
    (3) If there is evidence that the test substance, or a reactive 
metabolite, will not reach the target tissue, it is not appropriate to 
use this test.
    (e) Test method--(1) Principle. Animals are exposed to the test 
substance by an appropriate route of exposure and are sacrificed at 
appropriate times after treatment. Prior to sacrifice, animals are 
treated with a metaphase-arresting agent (e.g., colchicine or 
Colcemid). Chromosome preparations are then made from the 
bone marrow cells and stained, and metaphase cells are analyzed for 
chromosome aberrations.
    (2) Description--(i) Preparations--(A) Selection of animal species. 
Rats, mice and Chinese hamsters are commonly used, although any 
appropriate mammalian species may be used. Commonly used laboratory 
strains of young healthy adult animals should be employed. At the 
commencement of the study, the weight variation of animals should be 
minimal and not exceed  20% of the mean weight of each sex.
    (B) Housing and feeding conditions. The temperature in the 
experimental animal room should be 22 deg.C ( 3 deg.C).

[[Page 43851]]

Although the relative humidity should be at least 30% and preferably 
not exceed 70% other than during room cleaning, the aim should be 50-
60%. Lighting should be artificial, the sequence being 12 hrs light, 12 
hrs dark. For feeding, conventional laboratory diets may be used with 
an unlimited supply of drinking water. The choice of diet may be 
influenced by the need to ensure a suitable admixture of a test 
substance when administered by this method. Animals may be housed 
individually, or be caged in small groups of the same sex.
    (C) Preparation of the animals. Healthy young adult animals shall 
be randomly assigned to the control and treatment groups. Cages should 
be arranged in such a way that possible effects due to cage placement 
are minimized. The animals are identified uniquely. The animals are 
acclimated to the laboratory conditions for at least 5 days.
    (D) Preparation of doses. Solid test substances shall be dissolved 
or suspended in appropriate solvents or vehicles and diluted, as 
appropriate, prior to dosing of the animals. Liquid test substances may 
be dosed directly or diluted prior to dosing. Fresh preparations of the 
test substance should be employed unless stability data demonstrate the 
acceptability of storage.
    (ii) Test conditions--(A) Solvent/vehicle. The solvent/vehicle 
shall not produce toxic effects at the dose levels used, and shall not 
be suspected of chemical reaction with the test substance. If other 
than well-known solvents/vehicles are used, their inclusion should be 
supported with data indicating their compatibility. It is recommended 
that wherever possible, the use of an aqueous solvent/vehicle should be 
considered first.
    (B) Controls. (1) Concurrent positive and negative (solvent/
vehicle) controls shall be included for each sex in each test. Except 
for treatment with the test substance, animals in the control groups 
should be handled in an identical manner to the animals in the treated 
groups.
    (2) Positive controls shall produce structural chromosome 
aberrations in vivo at exposure levels expected to give a detectable 
increase over background. Positive control doses should be chosen so 
that the effects are clear but do not immediately reveal the identity 
of the coded slides to the reader. It is acceptable that the positive 
control be administered by a route different from the test substance 
and sampled at only a single time. The use of chemical class related 
positive control chemicals may be considered, when available. Examples 
of positive control substances include:

                                                                        
------------------------------------------------------------------------
                 Chemical                              CAS No.          
------------------------------------------------------------------------
Triethylenemelamine.......................  [CAS no. 51-18-3]           
Ethyl methanesulphonate...................  [CAS no. 62-50-0]           
Ethyl nitrosourea.........................  [CAS no. 759-73-9]          
Mitomycin C...............................  [CAS no. 50-07-7]           
Cyclophosphamide (monohydrate)............  [CAS no. 50-18-0]           
                                            [CAS no. 6055-19-2]         
------------------------------------------------------------------------

    (3) Negative controls, treated with solvent or vehicle alone, and 
otherwise treated in the same way as the treatment groups, shall be 
included for every sampling time, unless acceptable inter-animal 
variability and frequencies of cells with chromosome aberrations are 
available from historical control data. If single sampling is applied 
for negative controls, the most appropriate time is the first sampling 
time. In the absence of historical or published control data 
demonstrating that no deleterious or mutagenic effects are induced by 
the chosen solvent/vehicle, untreated controls shall be used .
    (3) Procedure--(i) Number and sex of animals. Each treated and 
control group shall include at least 5 analyzable animals per sex. If 
at the time of the study there are data available from studies in the 
same species and using the same route of exposure that demonstrate that 
there are no substantial differences in toxicity between sexes, then 
testing in a single sex will be sufficient. Where human exposure to 
chemicals may be sex-specific, as for example with some pharmaceutical 
agents, the test should be performed with animals of the appropriate 
sex.
    (ii) Treatment schedule. (A) Test substances are preferably 
administered as a single treatment. Test substances may also be 
administered as a split dose, i.e. two treatments on the same day 
separated by no more than a few hrs, to facilitate administering a 
large volume of material. Other dose regimens should be scientifically 
justified.
    (B) Samples shall be taken at two separate times following 
treatment on one day. For rodents, the first sampling interval is 1.5 
normal cell cycle length (the latter being normally 12-18 hr) following 
treatment. Since the time required for uptake and metabolism of the 
test substance as well as its effect on cell cycle kinetics can affect 
the optimum time for chromosome aberration detection, a later sample 
collection 24 hr after the first sample time is recommended. If dose 
regimens of more than one day are used, one sampling time at 1.5 normal 
cell cycle lengths after the final treatment should be used.
    (C) Prior to sacrifice, animals shall be injected intraperitoneally 
with an appropriate dose of a metaphase arresting agent (e.g. 
Colcemid or colchicine). Animals are sampled at an 
appropriate interval thereafter. For mice this interval is 
approximately 3-5 hrs; for Chinese hamsters this interval is 
approximately 4-5 hrs. Cells shall be harvested from the bone marrow 
and analyzed from chromosome aberrations.
    (iii) Dose levels. If a range finding study is performed because 
there are no suitable data available, it shall be performed in the same 
laboratory, using the same species, strain, sex, and treatment regimen 
to be used in the main study (an approach to dose selection is 
presented in the reference under paragraph (g)(5) of this section). If 
there is toxicity, three dose levels shall be used for the first 
sampling time. These dose levels shall cover a range from the maximum 
to little or no toxicity. At the later sampling time only the highest 
dose needs to be used. The highest dose is defined as the dose 
producing signs of toxicity such that higher dose levels, based on the 
same dosing regimen, would be expected to produce lethality. Substances 
with specific biological activities at low non-toxic doses (such as 
hormones and mitogens) may be exceptions to the

[[Page 43852]]

dose-setting criteria and should be evaluated on a case-by-case basis. 
The highest dose may also be defined as a dose that produces some 
indication of toxicity in the bone marrow (e.g. greater than 50% 
reduction in mitotic index).
    (iv) Limit test. If a test at one dose level of at least 2,000 mg/
kg body weight using a single treatment, or as two treatments on the 
same day, produces no observable toxic effects, and if genotoxicity 
would not be expected based on data from structurally related 
compounds, then a full study using three dose levels may not be 
considered necessary. For studies of a longer duration, the limit dose 
is 2,000 mg/kg/body weight/day for treatment up to 14 days, and 1,000 
mg/kg/body weight/day for treatment longer than 14 days. Expected human 
exposure may indicate the need for a higher dose level to be used in 
the limit test.
    (v) Administration of doses. The test substance is usually 
administered by gavage using a stomach tube or a suitable intubation 
cannula, or by intraperitoneal injection. Other routes of exposure may 
be acceptable where they can be justified. The maximum volume of liquid 
that can be administered by gavage or injection at one time depends on 
the size of the test animal. The volume should not exceed 2 ml/100g 
body weight. The use of volumes higher than these must be justified. 
Except for irritating or corrosive substances which will normally 
reveal exacerbated effects with higher concentrations, variability in 
test volume should be minimized by adjusting the concentration to 
ensure a constant volume at all dose levels.
    (vi) Chromosome preparation. Immediately after sacrifice, bone 
marrow shall be obtained, exposed to hypotonic solution and fixed. The 
cells shall be then spread on slides and stained.
    (vii) Analysis. (A) The mitotic index should be determined as a 
measure of cytotoxicity in at least 1,000 cells per animal for all 
treated animals (including positive controls) and untreated negative 
control animals.
    (B) At least 100 cells should be analyzed for each animal. This 
number could be reduced when high numbers of aberrations are observed. 
All slides, including those of positive and negative controls, shall be 
independently coded before microscopic analysis. Since slide 
preparation procedures often result in the breakage of a proportion of 
metaphases with loss of chromosomes, the cells scored should therefore 
contain a number of centromeres equal to the number 2n  2.
    (f) Data and reporting--(1) Treatment of results. Individual animal 
data shall be presented in tabular form. The experimental unit is the 
animal. For each animal the number of cells scored, the number of 
aberrations per cell and the percentage of cells with structural 
chromosome aberration(s) shall be evaluated. Different types of 
structural chromosome aberrations shall be listed with their numbers 
and frequencies for treated and control groups. Gaps shall be recorded 
separately and reported but generally not included in the total 
aberration frequency. If there is no evidence for a difference in 
response between the sexes, the data may be combined for statistical 
analysis.
    (2) Evaluation and interpretation of results. (i) There are several 
criteria for determining a positive result, such as a dose-related 
increase in the relative number of cells with chromosome aberrations or 
a clear increase in the number of cells with aberrations in a single 
dose group at a single sampling time. Biological relevance of the 
results should be considered first. Statistical methods may be used as 
an aid in evaluating the test results (some statistical methods are 
described in the reference under paragraph (g)(6) of this section). 
Statistical significance should not be the only determining factor for 
a positive response. Equivocal results should be clarified by further 
testing preferably using a modification of experimental conditions.
    (ii) An increase in polyploidy may indicate that the test substance 
has the potential to induce numerical chromosome aberrations. An 
increase in endoreduplication may indicate that the test substance has 
the potential to inhibit cell cycle progression. This phenomenon is 
described in the references under paragraphs (g)(7) and (g)(8) of this 
section.
    (iii) A test substance for which the results do not meet the 
criteria described in paragraph (f)(2)(i) of this section is considered 
non-mutagenic in this test.
    (iv) Although most experiments will give clearly positive or 
negative results, in rare cases the data set will preclude making a 
definite judgment about the activity of the test substance. Results may 
remain equivocal or questionable regardless of the number of 
experiments performed.
    (v) Positive results from the in vivo chromosome aberration test 
indicate that a substance induces chromosome aberrations in the bone 
marrow of the species tested. Negative results indicate that, under the 
test conditions, the test substance does not induce chromosome 
aberrations in the bone marrow of the species tested.
    (vi) The likelihood that the test substance or its metabolites 
reach the general circulation or specifically the target tissue (e.g., 
systemic toxicity) should be discussed.
    (3) Test report. The test report shall include the following 
information:
    (i) Test substance:
    (A) Identification data and CAS No., if known.
    (B) Physical nature and purity.
    (C) Physicochemical properties relevant to the conduct of the 
study.
    (D) Stability of the test substance, if known.
    (ii) Solvent/vehicle:
    (A) Justification for choice of vehicle.
    (B) Solubility and stability of the test substance in solvent/
vehicle, if known.
    (iii) Test animals:
    (A) Species/strain used.
    (B) Number, age and sex of animals.
    (C) Source, housing conditions, diet, etc.
    (D) Individual weight of the animals at the start of the test, 
including body weight range, mean and standard deviation for each 
group.
    (iv) Test conditions:
    (A) Positive and negative (vehicle/solvent) controls.
    (B) Data from range-finding study, if conducted.
    (C) Rationale for dose level selection.
    (D) Details of test substance preparation.
    (E) Details of the administration of the test substance.
    (F) Rationale for route of administration.
    (G) Methods for verifying that the test substance reached the 
general circulation or target tissue, if applicable.
    (H) Conversion from diet/drinking water test substance 
concentration parts per million (ppm) to the actual dose (mg/kg body 
weight/day), if applicable.
    (I) Details of food and water quality.
    (J) Detailed description of treatment and sampling schedules.
    (K) Methods for measurement of toxicity.
    (L) Identity of metaphase arresting substance, its concentration 
and duration of treatment.
    (M) Methods of slide preparation.
    (N) Criteria for scoring aberrations.
    (O) Number of cells analyzed per animal.
    (P) Criteria for considering studies as positive, negative or 
equivocal.
    (v) Results:
    (A) Signs of toxicity.
    (B) Mitotic index.
    (C) Type and number of aberrations, given separately for each 
animal.
    (D) Total number of aberrations per group with means and standard 
deviations.

[[Page 43853]]

    (E) Number of cells with aberrations per group with means and 
standard deviations.
    (F) Changes in ploidy, if seen.
    (G) Dose-response relationship, where possible.
    (H) Statistical analyses, if any.
    (I) Concurrent negative control data.
    (J) Historical negative control data with ranges, means and 
standard deviations.
    (K) Concurrent positive control data.
    (vi) Discussion of the results.
    (vii) Conclusion.
    (g) References. For additional background information on this test 
guideline, the following references should be consulted. These 
references are available for inspection at the TSCA Nonconfidential 
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M 
St., SW., Washington, DC, 12 noon to 4 p.m., Monday through Friday, 
except legal holidays.
    (1) Adler, I.D. Eds. S. Venitt and J.M. Parry. Cytogenetic Tests in 
Mammals. Mutagenicity Testing: A Practical Approach. (IRL Press, 
Oxford, Washington DC, 1984) pp. 275-306.
    (2) Preston, R.J., Dean, B.J., Galloway, S., Holden, H., McFee, 
A.F., and Shelby, M. Mammalian In Vivo Cytogenetic Assays: Analysis of 
Chromosome Aberrations in Bone Marrow Cells. Mutation Research. 189, 
157-165 (1987).
    (3) Richold, M., Chandley, A., Ashby, J., Gatehouse, D.G., Bootman, 
J., and Henderson, L. Ed. D.J. Kirkland. In Vivo Cytogenetic Assays. 
Basic Mutagenicity Tests, UKEMS Recommended Procedures. UKEMS 
Subcommittee on Guidelines for Mutagenicity Testing. Report. Part I 
revised. (Cambridge University Press, Cambridge, NY, Port Chester, 
Melbourne, Sydney, 1990) pp. 115-141.
    (4) Tice, R.R., Hayashi, M., MacGregor, J.T., Anderson, D., Blakey, 
D.H., Holden, H.E., Kirsch-Volders, M., Oleson Jr., F.B., Pacchierotti, 
F., Preston, R.J., Romagna, F., Shimada, H., Sutou, S., and Vannier, B. 
Report from the Working Group on the In Vivo Mammalian Bone Marrow 
Chromosomal Aberration Test. Mutation Research. 312, 305-312 (1994).
    (5) Fielder, R.J., Allen, J.A., Boobis, A.R., Botham, P.A., Doe, 
J., Esdaile, D.J., Gatehouse, D.G., Hodson-Walker, G., Morton, D.B., 
Kirkland, D. J., and Richold, M. Report of British Toxicology Society/
UK Environmental Mutagen Society Working Group: Dose Setting in In Vivo 
Mutagenicity Assays. Mutagenesis. 7, 313-319 (1992).
    (6) Lovell, D.P., Anderson, D., Albanese, R., Amphlett, G.E., 
Clare, G., Ferguson, R., Richold, M., Papworth, D.G., and Savage, 
J.R.K. Ed. Kirkland,D. J. Statistical Analysis of In Vivo Cytogenetic 
Assays. UKEMS Sub-Committee on Guidelines for Mutagenicity Testing. 
Report Part III. Statistical Evaluation of Mutagenicity Test Data 
(Cambridge University Press, Cambridge, 1989) pp. 184-232.
    (7) Locke-Huhle, C. Endoreduplication in Chinese Hamster Cells 
During Alpha-Radiation Induced G2 Arrest. Mutation Research. 119, 403-
413 (1983).
    (8) Huang, Y., Change, C., and Trosko, J. E. Aphidicolin-Induced 
Endoreduplication in Chinese Hamster Cells. Cancer Research. 43, 1362-
1364 (1983).


Sec. 799.9539  TSCA mammalian erythrocyte micronucleus test.

    (a) Scope. This section is intended to meet the testing 
requirements under section 4 of TSCA.
    (1) The mammalian erythrocyte micronucleus test is used for the 
detection of damage induced by the test substance to the chromosomes or 
the mitotic apparatus of erythroblasts by analysis of erythrocytes as 
sampled in bone marrow and/or peripheral blood cells of animals, 
usually rodents.
    (2) The purpose of the micronucleus test is to identify substances 
that cause cytogenetic damage which results in the formation of 
micronuclei containing lagging chromosome fragments or whole 
chromosomes.
    (3) When a bone marrow erythroblast develops into a polychromatic 
erythrocyte, the main nucleus is extruded; any micronucleus that has 
been formed may remain behind in the otherwise anucleated cytoplasm. 
Visualization of micronuclei is facilitated in these cells because they 
lack a main nucleus. An increase in the frequency of micronucleated 
polychromatic erythrocytes in treated animals is an indication of 
induced chromosome damage.
    (b) Source. The source material used in developing this TSCA test 
guideline is the OECD guideline 474 (February 1997). This source is 
available at the address in paragraph (g) of this section.
    (c) Definitions. The following definitions apply to this section:
    Centromere (kinetochore) is a region of a chromosome with which 
spindle fibers are associated during cell division, allowing orderly 
movement of daughter chromosomes to the poles of the daughter cells.
    Micronuclei are small nuclei, separate from and additional to the 
main nuclei of cells, produced during telophase of mitosis (meiosis) by 
lagging chromosome fragments or whole chromosomes.
    Normochromatic erythrocyte is a mature erythrocyte that lacks 
ribosomes and can be distinguished from immature, polychromatic 
erythrocytes by stains selective for ribosomes.
    Polychromatic erythrocyte is a immature erythrocyte, in an 
intermediate stage of development, that still contains ribosomes and 
therefore can be distinguished from mature, normochromatic erythrocytes 
by stains selective for ribosomes.
    (d) Initial considerations. (1) The bone marrow of rodents is 
routinely used in this test since polychromatic erythrocytes are 
produced in that tissue. The measurement of micronucleated immature 
(polychromatic) erythrocytes in peripheral blood is equally acceptable 
in any species in which the inability of the spleen to remove 
micronucleated erythrocytes has been demonstrated, or which has shown 
an adequate sensitivity to detect agents that cause structural or 
numerical chromosome aberrations. Micronuclei can be distinguished by a 
number of criteria. These include identification of the presence or 
absence of a kinetochore or centromeric DNA in the micronuclei. The 
frequency of micronucleated immature (polychromatic) erythrocytes is 
the principal endpoint. The number of mature (normochromatic) 
erythrocytes in the peripheral blood that contain micronuclei among a 
given number of mature erythrocytes can also be used as the endpoint of 
the assay when animals are treated continuously for 4 weeks or more. 
This mammalian in vivo micronucleus test is especially relevant to 
assessing mutagenic hazard in that it allows consideration of factors 
of in vivo metabolism, pharmacokinetics and DNA-repair processes 
although these may vary among species, among tissues and among genetic 
endpoints. An in vivo assay is also useful for further investigation of 
a mutagenic effect detected by an in vitro system.
    (2) If there is evidence that the test substance, or a reactive 
metabolite, will not reach the target tissue, it is not appropriate to 
use this test.
    (e) Test method--(1) Principle. Animals are exposed to the test 
substance by an appropriate route. If bone marrow is used, the animals 
are sacrificed at appropriate times after treatment, the bone marrow 
extracted, and preparations made and stained (test techniques described 
in the references under paragraphs (g)(1), (g)(2), and (g)(3) of this 
section may be used). When peripheral blood is used, the blood is 
collected at appropriate times after treatment and smear preparations 
are

[[Page 43854]]

made and stained (the test techniques described in the references under 
paragraphs (g)(3), (g)(4), (g)(5), and (g)(6) of this section may be 
used). For studies with peripheral blood, as little time as possible 
should elapse between the last exposure and cell harvest. Preparations 
are analyzed for the presence of micronuclei.
    (2) Description--(i) Preparations--(A) Selection of animal species. 
Mice or rats are recommended if bone marrow is used, although any 
appropriate mammalian species may be used. When peripheral blood is 
used, mice are recommended. However, any appropriate mammalian species 
may be used provided it is a species in which the spleen does not 
remove micronucleated erythrocytes or a species which has shown an 
adequate sensitivity to detect agents that cause structural or 
numerical chromosome aberrations. Commonly used laboratory strains of 
young healthy animals should be employed. At the commencement of the 
study, the weight variation of animals should be minimal and not exceed 
 20% of the mean weight of each sex.
    (B) Housing and feeding conditions. The temperature in the 
experimental animal room should be 22 deg.C ( 3 deg.C). 
Although the relative humidity should be at least 30% and preferably 
not exceed 70% other than during room cleaning, the aim should be 50-
60%. Lighting should be artificial, the sequence being 12 hrs light, 12 
hrs dark. For feeding, conventional laboratory diets may be used with 
an unlimited supply of drinking water. The choice of diet may be 
influenced by the need to ensure a suitable admixture of a test 
substance when administered by this route. Animals may be housed 
individually, or caged in small groups of the same sex.
    (C) Preparation of the animals. Healthy young adult animals shall 
be randomly assigned to the control and treatment groups. The animals 
are identified uniquely. The animals are acclimated to the laboratory 
conditions for at least 5 days. Cages should be arranged in such a way 
that possible effects due to cage placement are minimized.
    (D) Preparation of doses. Solid test substances shall be dissolved 
or suspended in appropriate solvents or vehicles and diluted, if 
appropriate, prior to dosing of the animals. Liquid test substances may 
be dosed directly or diluted prior to dosing. Fresh preparations of the 
test substance should be employed unless stability data demonstrate the 
acceptability of storage.
    (ii) Test conditions--(A) Solvent/vehicle. The solvent/vehicle 
should not produce toxic effects at the dose levels used, and should 
not be suspected of chemical reaction with the test substance. If other 
than well-known solvents/vehicles are used, their inclusion shall be 
supported with reference data indicating their compatibility. It is 
recommended that wherever possible, the use of an aqueous solvent/
vehicle should be considered first.
    (B) Controls. (1) Concurrent positive and negative (solvent/
vehicle) controls shall be included for each sex in each test. Except 
for treatment with the test substance, animals in the control groups 
should be handled in an identical manner to animals of the treatment 
groups.
    (2) Positive controls shall produce micronuclei in vivo at exposure 
levels expected to give a detectable increase over background. Positive 
control doses should be chosen so that the effects are clear but do not 
immediately reveal the identity of the coded slides to the reader. It 
is acceptable that the positive control be administered by a route 
different from the test substance and sampled at only a single time. In 
addition, the use of chemical class-related positive control chemicals 
may be considered, when available. Examples of positive control 
substances include:

                                                                        
------------------------------------------------------------------------
                 Chemical                              CAS No.          
------------------------------------------------------------------------
Ethyl methanesulphonate...................  [CAS no. 62-50-0]           
Ethyl nitrosourea.........................  [CAS no. 759-73-9]          
Mitomycin C...............................  [CAS no. 50-07-7]           
Cyclophosphamide (monohydrate)............  [CAS no. 50-18-0]           
                                            [CAS no. 6055-19-2]         
Triethylenemelamine.......................  [CAS no. 51-18-3]           
------------------------------------------------------------------------

    (3) Negative controls, treated with solvent or vehicle alone, and 
otherwise treated in the same way as the treatment groups shall be 
included for every sampling time, unless acceptable inter-animal 
variability and frequencies of cells with micronuclei are demonstrated 
by historical control data. If single sampling is applied for negative 
controls, the most appropriate time is the first sampling time. In 
addition, untreated controls should also be used unless there are 
historical or published control data demonstrating that no deleterious 
or mutagenic effects are induced by the chosen solvent/vehicle.
    (4) If peripheral blood is used, a pre-treatment sample may also be 
acceptable as a concurrent negative control, but only in the short 
peripheral blood studies (e.g., one to three treatment(s)) when the 
resulting data are in the expected range for the historical control.
    (3) Procedure--(i) Number and sex of animals. Each treated and 
control group shall include at least 5 analyzable animals per sex 
(techniques described in the reference under paragraph (g)(7) of this 
section may be used). If at the time of the study there are data 
available from studies in the same species and using the same route of 
exposure that demonstrate that there are no substantial differences 
between sexes in toxicity, then testing in a single sex will be 
sufficient. Where human exposure to chemicals may be sex-specific, as 
for example with some pharmaceutical agents, the test should be 
performed with animals of the appropriate sex.
    (ii) Treatment schedule. (A) No standard treatment schedule (i.e. 
one, two, or more treatments at 24 h intervals) can be recommended. The 
samples from extended dose regimens are acceptable as long as a 
positive effect has been demonstrated for this study or, for a negative 
study, as long as toxicity has been demonstrated or the limit dose has 
been used, and dosing continued until the time of sampling. Test 
substances may also be administered as a split dose, i.e., two 
treatments on the same day separated by no more than a few hrs, to 
facilitate administering a large volume of material.

[[Page 43855]]

    (B) The test may be performed in two ways:
    (1) Animals shall be treated with the test substance once. Samples 
of bone marrow shall be taken at least twice, starting not earlier than 
24 hrs after treatment, but not extending beyond 48 hrs after treatment 
with appropriate interval(s) between samples. The use of sampling times 
earlier than 24 hrs after treatment should be justified. Samples of 
peripheral blood shall be taken at least twice, starting not earlier 
than 36 hrs after treatment, with appropriate intervals following the 
first sample, but not extending beyond 72 hrs. When a positive response 
is recognized at one sampling time, additional sampling is not 
required.
    (2) If two or more daily treatments are used (e.g. two or more 
treatments at 24 hr intervals), samples shall be collected once between 
18 and 24 hrs following the final treatment for the bone marrow and 
once between 36 and 48 hrs following the final treatment for the 
peripheral blood (techniques described in the reference under paragraph 
(g)(8) of this section may be used).
    (C) Other sampling times may be used in addition, when relevant.
    (iii) Dose levels. If a range finding study is performed because 
there are no suitable data available, it should be performed in the 
same laboratory, using the same species, strain, sex, and treatment 
regimen to be used in the main study (guidance on dose setting is 
provided in the reference in paragraph (g)(9) of this section). If 
there is toxicity, three dose levels shall be used for the first 
sampling time. These dose levels shall cover a range from the maximum 
to little or no toxicity. At the later sampling time only the highest 
dose needs to be used. The highest dose is defined as the dose 
producing signs of toxicity such that higher dose levels, based on the 
same dosing regimen, would be expected to produce lethality. Substances 
with specific biological activities at low non-toxic doses (such as 
hormones and mitogens) may be exceptions to the dose-setting criteria 
and should be evaluated on a case-by-case basis. The highest dose may 
also be defined as a dose that produces some indication of toxicity in 
the bone marrow (e.g. a reduction in the proportion of immature 
erythrocytes among total erythrocytes in the bone marrow or peripheral 
blood).
    (iv) Limit test. If a test at one dose level of at least 2,000 mg/
kg body weight using a single treatment, or as two treatments on the 
same day, produces no observable toxic effects, and if genotoxicity 
would not be expected based upon data from structurally related 
substances, then a full study using three dose levels may not be 
considered necessary. For studies of a longer duration, the limit dose 
is 2,000 mg/kg/body weight/day for treatment up to 14 days, and 1,000 
mg/kg/body weight/day for treatment longer than 14 days. Expected human 
exposure may indicate the need for a higher dose level to be used in 
the limit test.
    (v) Administration of doses. The test substance is usually 
administered by gavage using a stomach tube or a suitable intubation 
cannula, or by intraperitoneal injection. Other routes of exposure may 
be acceptable where they can be justified. The maximum volume of liquid 
that can be administered by gavage or injection at one time depends on 
the size of the test animal. The volume should not exceed 2 ml/100g 
body weight. The use of volumes higher than these must be justified. 
Except for irritating or corrosive substances which will normally 
reveal exacerbated effects with higher concentrations, variability in 
test volume should be minimized by adjusting the concentration to 
ensure a constant volume at all dose levels.
    (vi) Bone marrow/blood preparation. Bone marrow cells shall be 
obtained from the femurs or tibias immediately following sacrifice. 
Cells shall be removed from femurs or tibias, prepared and stained 
using established methods. Peripheral blood is obtained from the tail 
vein or other appropriate blood vessel. Blood cells are immediately 
stained supravitally (the test techniques described in the references 
under paragraphs (g)(4), (g)(5), and (g)(6) of this section may be 
used) or smear preparations are made and then stained. The use of a DNA 
specific stain (e.g. acridine orange (techniques described in the 
reference under paragraph (g)(10) of this section may be used) or 
Hoechst 33258 plus pyronin-Y) can eliminate some of the artifacts 
associated with using a non-DNA specific stain. This advantage does not 
preclude the use of conventional stains (e.g., Giemsa). Additional 
systems (e.g. cellulose columns to remove nucleated cells (the test 
techniques described in the references under paragraph (g)(12) of this 
section may be used)) can also be used provided that these systems have 
been shown to adequately work for micronucleus preparation in the 
laboratory.
    (vii) Analysis. The proportion of immature among total (immature + 
mature) erythrocytes is determined for each animal by counting a total 
of at least 200 erythrocytes for bone marrow and 1,000 erythrocytes for 
peripheral blood (techniques described in the reference under paragraph 
(g)(13) of this section maybe used). All slides, including those of 
positive and negative controls, shall be independently coded before 
microscopic analysis. At least 2,000 immature erythrocytes per animal 
shall be scored for the incidence of micronucleated immature 
erythrocytes. Additional information may be obtained by scoring mature 
erythrocytes for micronuclei. When analyzing slides, the proportion of 
immature erythrocytes among total erythrocytes should not be less than 
20% of the control value. When animals are treated continuously for 4 
weeks or more, at least 2,000 mature erythrocytes per animal can also 
be scored for the incidence of micronuclei. Systems for automated 
analysis (image analysis) and cell suspensions (flow cytometry) are 
acceptable alternatives to manual evaluation if appropriately justified 
and validated.
    (f) Data and reporting--(1) Treatment of results. Individual animal 
data shall be presented in tabular form. The experimental unit is the 
animal. The number of immature erythrocytes scored, the number of 
micronucleated immature erythrocytes, and the number of immature among 
total erythrocytes shall be listed separately for each animal analyzed. 
When animals are treated continuously for 4 weeks or more, the data on 
mature erythrocytes should also be given if it is collected. The 
proportion of immature among total erythrocytes and, if considered 
applicable, the percentage of micronucleated erythrocytes shall be 
given for each animal. If there is no evidence for a difference in 
response between the sexes, the data from both sexes may be combined 
for statistical analysis.
    (2) Evaluation and interpretation of results. (i) There are several 
criteria for determining a positive result, such as a dose-related 
increase in the number of micronucleated cells or a clear increase in 
the number of micronucleated cells in a single dose group at a single 
sampling time. Biological relevance of the results should be considered 
first. Statistical methods may be used as an aid in evaluating the test 
results (the test techniques described in the references paragraphs 
(g)(14) and (g)(15) of this section may be used). Statistical 
significance should not be the only determining factor for a positive 
response. Equivocal results should be clarified by further testing 
preferably using a modification of experimental conditions.
    (ii) A test substance for which the results do not meet the 
criteria described is considered non-mutagenic in this test.

[[Page 43856]]

    (iii) Although most experiments will give clearly positive or 
negative results, in rare cases the data set will preclude making a 
definite judgement about the activity of the test substance. Results, 
may remain equivocal or questionable regardless of the number of times 
the experiment is repeated. Positive results in the micronucleus test 
indicate that a substance induces micronuclei which are the result of 
chromosomal damage or damage to the mitotic apparatus in the 
erythroblasts of the test species. Negative results indicate that, 
under the test conditions, the test substance does not produce 
micronuclei in the immature erythrocytes of the test species.
    (iv) The likelihood that the test substance or its metabolites 
reach the general circulation or specifically the target tissue (e.g. 
systemic toxicity) should be discussed.
    (3) Test report. In addition to the reporting requirements as 
specified under 40 CFR part 792, subpart J, the following specific 
information shall be reported. Both individual and summary data should 
be presented.
    (i) Test substance:
    (A) Identification data and CAS no., if known.
    (B) Physical nature and purity.
    (C) Physiochemical properties relevant to the conduct of the study.
    (D) Stability of the test substance, if known.
    (ii) Solvent/vehicle:
    (A) Justification for choice of vehicle.
    (B) Solubility and stability of the test substance in the solvent/
vehicle, if known.
    (iii) Test animals:
    (A) Species/strain used.
    (B) Number, age, and sex of animals.
    (C) Source, housing conditions, diet, etc.
    (D) Individual weight of the animals at the start of the test, 
including body weight range, mean and standard deviation for each 
group.
    (iv) Test conditions:
    (A) Positive and negative (vehicle/solvent) control data.
    (B) Data from range-finding study, if conducted.
    (C) Rationale for dose level selection.
    (D) Details of test substance preparation.
    (E) Details of the administration of the test substance.
    (F) Rationale for route of administration.
    (G) Methods for verifying that the test substance reached the 
general circulation or target tissue, if applicable.
    (H) Conversion from diet/drinking water test substance 
concentration parts per million (ppm) to the actual dose (mg/kg body 
weight/day), if applicable.
    (I) Details of food and water quality.
    (J) Detailed description of treatment and sampling schedules.
    (K) Methods of slide preparation.
    (L) Methods for measurement of toxicity.
    (M) Criteria for scoring micronucleated immature erythrocytes.
    (N) Number of cells analyzed per animal.
    (O) Criteria for considering studies as positive, negative or 
equivocal.
    (v) Results:
    (A) Signs of toxicity.
    (B) Proportion of immature erythrocytes among total erythrocytes.
    (C) Number of micronucleated immature erythrocytes, given 
separately for each animal.
    (D) Mean  standard deviation of micronucleated immature 
erythrocytes per group.
    (E) Dose-response relationship, where possible.
    (F) Statistical analyses and method applied.
    (G) Concurrent and historical negative control data.
    (H) Concurrent positive control data.
    (vi) Discussion of the results.
    (vii) Conclusion.
    (g) References. For additional background information on this test 
guideline, the following references should be consulted. These 
references are available for inspection at the TSCA Nonconfidential 
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M 
St., SW., Washington, DC, 12 noon to 4 p.m., Monday through Friday, 
except legal holidays.
    (1) Heddle, J.A. A Rapid In Vivo Test for Chromosomal Damage. 
Mutation Research. 18, 187-190 (1973).
    (2) Schmid, W. The Micronucleus Test. Mutation Research. 31, 9-15 
(1975).
    (3) Mavournin, K.H., Blakey, D.H., Cimino, M.C., Salamone, M.F., 
and Heddle, J.A. The In Vivo Micronucleus Assay in Mammalian Bone 
Marrow and Peripheral Blood. A report of the U.S. Environmental 
Protection Agency Gene-Tox Program. Mutation Research. 239, 29-80 
(1990).
    (4) Hayashi, M., Morita, T., Kodama, Y., Sofuni, T., and Ishidate, 
Jr., M. The Micronucleus Assay with Mouse Peripheral Blood 
Reticulocytes Using Acridine Orange-Coated Slides. Mutation Research. 
245, 245-249 (1990).
    (5) The Collaborative Study Group for the Micronucleus Test (1992). 
Micronucleus Test with Mouse Peripheral Blood Erythrocytes by Acridine 
Orange Supravital Staining: The Summary Report of the 5th Collaborative 
Study by CSGMT/JEMS. MMS. Mutation Research. 278, 83-98.
    (6) The Collaborative Study Group for the Micronucleus Test (CSGMT/
JEMMS.MMS, The Mammalian Mutagenesis Study Group of the Environmental 
Mutagen Society of Japan) Protocol recommended for the short-term mouse 
peripheral blood micronucleus test. Mutagenesis. 10, 153-159 (1995).
    (7) Hayashi, M., Tice, R.R., MacGregor, J.T., Anderson, D., Blakey, 
D.H., Kirsch-Volders, M., Oleson, Jr. F.B., Pacchierotti, F., Romagna, 
F., Shimada, H., Sutou, S., and Vannier, B. In Vivo Rodent Erythrocyte 
Micronucleus Assay. Mutation Research. 312, 293-304 (1994).
    (8) Higashikuni, N. and Sutou, S. An optimal, generalized sampling 
time of 30 +/- 6 h after double dosing in the mouse 
peripheral blood micronucleus test. Mutagenesis. 10, 313-319 (1995).
    (9) Fielder, R.J., Allen, J.A., Boobis, A.R., Botham, P.A., Doe, 
J., Esdaile, D.J., Gatehouse, D.G., Hodson-Walker, G., Morton, D.B., 
Kirkland, D. J., and Richold, M. Report of British Toxicology Society/
UK Environmental Mutagen Society Working Group: Dose Setting in In Vivo 
Mutagenicity Assays. Mutagenesis. 7, 313-319 (1992).
    (10) Hayashi, M., Sofuni, T., and Ishidate, Jr., M. An Application 
of Acridine Orange Fluorescent Staining to the Micronucleus Test. 
Mutation Research. 120, 241-247 (1983).
    (11) MacGregor, J.T., Wehr, C.M., and Langlois, R.G. A Simple 
Fluorescent Staining Procedure for Micronuclei and RNA in Erythrocytes 
Using Hoechst 33258 and Pyronin Y. Mutation Research. 120, 269-275 
(1983).
    (12) Romagna, F. and Staniforth, C.D. The automated bone marrow 
micronucleus test. Mutation Research. 213, 91-104 (1989).
    (13) Gollapudi, B. and McFadden, L.G. Sample size for the 
estimation of polychromatic to normochromatic eruthrocyte ratio in the 
bone marrow micronucleus test. Mutation Research. 347, 97-99 (1995).
    (14) Richold, M., Ashby, J., Bootman, J., Chandley, A., Gatehouse, 
D.G., and Henderson, L. Ed. Kirkland, D.J. In Vivo Cytogenetics Assays. 
Basic Mutagenicity Tests, UKEMS Recommended Procedures. UKEMS 
Subcommittee on Guidelines for Mutagenicity Testing. Report. Part I 
revised (Cambridge University Press, Cambridge, New York, Port Chester, 
Melbourne, Sydney, 1990) pp. 115-141.
    (15) Lovell, D.P., Anderson, D., Albanese, R., Amphlett, G.E., 
Clare, G., Ferguson, R., Richold, M., Papworth, D.G., and Savage, 
J.R.K. Ed. D.J.

[[Page 43857]]

Kirkland. Statistical Analysis of In Vivo Cytogenetic Assays. 
Statistical Evaluation of Mutagenicity Test Data. UKEMS Sub-Committee 
on Guidelines for Mutagenicity Testing, Report, Part III. (Cambridge 
University Press, Cambridge, New York, Port Chester, Melbourne, Sydney, 
1989) pp. 184-232.
    (16) Heddle, J.A., Salamone, M.F., Hite, M., Kirkhart, B., 
Mavournin, K., MacGregor, J.G., and Newell, G.W. The Induction of 
Micronuclei as a Measure of Genotoxicity. Mutation Research. 123: 61-
118 (1983).
    (17) MacGregor, J.T., Heddle, J.A., Hite, M., Margolin, G.H., Ramel 
C., Salamone, M.F., Tice, R.R., and Wild, D. Guidelines for the Conduct 
of Micronucleus Assays in Mammalian Bone Marrow Erythrocytes. Mutation 
Research. 189: 103-112 (1987).
    (18) MacGregor, J.T., Wehr, C.M., Henika, P.R., and Shelby, M.E. 
(1990). The In Vivo Erythrocyte Micronucleus Test: Measurement at 
Steady State Increases Assay Efficiency and Permits Integration with 
Toxicity Studies. Fundamental Applied Toxicology. 14: 513-522.
    (19) MacGregor, J.T., Schlegel, R. Choy, W.N., and Wehr, C.M. Eds. 
Hayes, A.W., Schnell, R.C., and Miya, T.S. Micronuclei in Circulating 
Erythrocytes: A Rapid Screen for Chromosomal Damage During Routine 
Toxicity Testing in Mice. Developments in Science and Practice of 
Toxicology (Elsevier, Amsterdam, 1983) pp. 555-558.


Sec. 799.9620  TSCA neurotoxicity screening battery.

    (a) Scope. This section is intended to meet the testing 
requirements under section 4 of TSCA. This neurotoxicity screening 
battery consists of a functional observational battery, motor activity, 
and neuropathology. The functional observational battery consists of 
noninvasive procedures designed to detect gross functional deficits in 
animals and to better quantify behavioral or neurological effects 
detected in other studies. The motor activity test uses an automated 
device that measures the level of activity of an individual animal. The 
neuropathological techniques are designed to provide data to detect and 
characterize histopathological changes in the central and peripheral 
nervous system. This battery is designed to be used in conjunction with 
general toxicity studies and changes should be evaluated in the context 
of both the concordance between functional neurological and 
neuropatholgical effects, and with respect to any other toxicological 
effects seen. This test battery is not intended to provide a complete 
evaluation of neurotoxicity, and additional functional and 
morphological evaluation may be necessary to assess completely the 
neurotoxic potential of a chemical.
    (b) Source. The source material used in developing this TSCA test 
guideline is the OPPTS harmonized test guideline 870.6200 (June 1996 
Public Draft). This source is available at the address in paragraph (g) 
of this section.
    (c) Definitions. The following definitions apply to this section.
    ED is effective dose.
    Motor activity is any movement of the experimental animal.
    Neurotoxicity is any adverse effect on the structure or function of 
the nervous system related to exposure to a chemical substance.
    Toxic effect is an adverse change in the structure or function of 
an experimental animal as a result of exposure to a chemical substance.
    (d) Principle of the test method. The test substance is 
administered to several groups of experimental animals, one dose being 
used per group. The animals are observed under carefully standardized 
conditions with sufficient frequency to ensure the detection and 
quantification of behavioral and/or neurologic abnormalities, if 
present. Various functions that could be affected by neurotoxicants are 
assessed during each observation period. Measurements of motor activity 
of individual animals are made in an automated device. The animals are 
perfused and tissue samples from the nervous system are prepared for 
microscopic examination. The exposure levels at which significant 
neurotoxic effects are produced are compared to one another and to 
those levels that produce other toxic effects.
    (e) Test procedures--(1) Animal selection--(i) Species. In general, 
the laboratory rat should be used. Under some circumstances, other 
species, such as the mouse or the dog, may be more appropriate, 
although not all of the battery may be adaptable to other species.
    (ii) Age. Young adults (at least 42 days old for rats) shall be 
used.
    (iii) Sex. Both males and females shall be used. Females shall be 
nulliparous and nonpregnant.
    (2) Number of animals. At least 10 males and 10 females should be 
used in each dose and control group for behavioral testing. At least 
five males and five females should be used in each dose and control 
group for terminal neuropathology. If interim neuropathological 
evaluations are planned, the number should be increased by the number 
of animals scheduled to be perfused before the end of the study. 
Animals shall be randomly assigned to treatment and control groups.
    (3) Control groups. (i) A concurrent (vehicle) control group is 
required. Subjects shall be treated in the same way as for an exposure 
group except that administration of the test substance is omitted. If 
the vehicle used has known or potential toxic properties, both 
untreated or saline treated and vehicle control groups are required.
    (ii) Positive control data from the laboratory performing the 
testing shall provide evidence of the ability of the observational 
methods used to detect major neurotoxic endpoints including limb 
weakness or paralysis, tremor, and autonomic signs. Positive control 
data are also required to demonstrate the sensitivity and reliability 
of the activity-measuring device and testing procedures. These data 
should demonstrate the ability to detect chemically induced increases 
and decreases in activity. Positive control groups exhibiting central 
nervous system pathology and peripheral nervous system pathology are 
also required. Separate groups for peripheral and central 
neuropathology are acceptable (e.g. acrylamide and trimethyl tin). 
Positive control data shall be collected at the time of the test study 
unless the laboratory can demonstrate the adequacy of historical data 
for this purpose, i.e. by the approach outlined in this section.
    (4) Dose level and dose selection. At least three doses shall be 
used in addition to the vehicle control group. The data should be 
sufficient to produce a dose-effect curve. The Agency strongly 
encourage the use of equally spaced doses and a rationale for dose 
selection that will maximally support detection of dose-effect 
relations. For acute studies, dose selection may be made relative to 
the establishment of a benchmark dose (BD). That is, doses may be 
specified as successive fractions, e.g. 0.5, 0.25, ...n of the BD. The 
BD itself may be estimated as the highest nonlethal dose as determined 
in a preliminary range-finding lethality study. A variety of test 
methodologies may be used for this purpose, and the method chosen may 
influence subsequent dose selection. The goal is to use a dose level 
that is sufficient to be judged a limit dose, or clearly toxic.
    (i) Acute studies. The high dose need not be greater than 2 g/kg. 
Otherwise, the high dose should result in significant neurotoxic 
effects or other clearly toxic effects, but not result in an incidence 
of fatalities that would preclude a meaningful evaluation of the

[[Page 43858]]

data. This dose may be estimated by a BD procedure as described under 
paragraph (e)(4) of this section, with the middle and low dose levels 
chosen as fractions of the BD dose. The lowest dose should produce 
minimal effect, e.g. an ED10, or alternatively, no effects.
    (ii) Subchronic and chronic studies. The high dose need not be 
greater than 1 g/kg. Otherwise, the high dose level should result in 
significant neurotoxic effects or other clearly toxic effects, but not 
produce an incidence of fatalities that would prevent a meaningful 
evaluation of the data. The middle and low doses should be fractions of 
the high dose. The lowest dose should produce minimal effects, e.g. an 
ED10, or alternatively, no effects.
    (5) Route of exposure. Selection of route may be based on several 
criteria including, the most likely route of human exposure, 
bioavailability, the likelihood of observing effects, practical 
difficulties, and the likelihood of producing nonspecific effects. For 
many materials, it should be recognized that more than one route of 
exposure may be important and that these criteria may conflict with one 
another. Initially only one route is required for screening for 
neurotoxicity. The route that best meets these criteria should be 
selected. Dietary feeding will generally be acceptable for repeated 
exposures studies.
    (6) Combined protocol. The tests described in this screening 
battery may be combined with any other toxicity study, as long as none 
of the requirements of either are violated by the combination.
    (7) Study conduct--(i) Time of testing. All animals shall be 
weighed on each test day and at least weekly during the exposure 
period.
    (A) Acute studies. At a minimum, for acute studies observations and 
activity testing shall be made before the initiation of exposure, at 
the estimated time of peak effect within 8 hrs of dosing, and at 7 and 
14 days after dosing. Estimation of times of peak effect may be made by 
dosing pairs of rats across a range of doses and making regular 
observations of gait and arousal.
    (B) Subchronic and chronic studies. In a subchronic study, at a 
minimum, observations and activity measurements shall be made before 
the initiation of exposure and before the daily exposure, or for 
feeding studies at the same time of day, during the 4th, 8th, and 13th 
weeks of exposure. In chronic studies, at a minimum, observations and 
activity measurements shall be made before the initiation of exposure 
and before the daily exposure, or for feeding studies at the same time 
of day, every 3 months.
    (ii) Functional observational battery--(A) General conduct. All 
animals in a given study shall be observed carefully by trained 
observers who are unaware of the animals' treatment, using standardized 
procedures to minimize observer variability. Where possible, it is 
advisable that the same observer be used to evaluate the animals in a 
given study. If this is not possible, some demonstration of 
interobserver reliability is required. The animals shall be removed 
from the home cage to a standard arena for observation. Effort should 
be made to ensure that variations in the test conditions are minimal 
and are not systematically related to treatment. Among the variables 
that can affect behavior are sound level, temperature, humidity, 
lighting, odors, time of day, and environmental distractions. Explicit, 
operationally defined scales for each measure of the battery are to be 
used. The development of objective quantitative measures of the 
observational end-points specified is encouraged. Examples of 
observational procedures using defined protocols may be found in the 
references under paragraphs (g)(5), (g)(6), and (g)(9) of this section. 
The functional observational battery shall include a thorough 
description of the subject's appearance, behavior, and functional 
integrity. This shall be assessed through observations in the home cage 
and while the rat is moving freely in an open field, and through 
manipulative tests. Testing should proceed from the least to the most 
interactive with the subject. Scoring criteria, or explicitly defined 
scales, should be developed for those measures which involve subjective 
ranking.
    (B) List of measures. The functional observational battery shall 
include the following list of measures:
    (1) Assessment of signs of autonomic function, including but not 
limited to:
    (i) Ranking of the degree of lacrimation and salivation, with a 
range of severity scores from none to severe.
    (ii) Presence or absence of piloerection and exophthalmus.
    (iii) Ranking or count of urination and defecation, including 
polyuria and diarrhea. This is most easily conducted during the open 
field assessment.
    (iv) Pupillary function such as constriction of the pupil in 
response to light or a measure of pupil size.
    (v) Degree of palpebral closure, e.g., ptosis.
    (2) Description, incidence, and severity of any convulsions, 
tremors, or abnormal motor movements, both in the home cage and the 
open field.
    (3) Ranking of the subject's reactivity to general stimuli such as 
removal from the cage or handling, with a range of severity scores from 
no reaction to hyperreactivity.
    (4) Ranking of the subject's general level of activity during 
observations of the unperturbed subject in the open field, with a range 
of severity scores from unresponsive to hyperactive.
    (5) Descriptions and incidence of posture and gait abnormalities 
observed in the home cage and open field.
    (6) Ranking of any gait abnormalities, with a range of severity 
scores from none to severe.
    (7) Forelimb and hindlimb grip strength measured using an objective 
procedure (the procedure described in the reference under paragraph 
(g)(8) of this section may be used).
    (8) Quantitative measure of landing foot splay (the procedure 
described in the reference under paragraph (g)(3) of this section may 
be used).
    (9) Sensorimotor responses to stimuli of different modalities will 
be used to detect gross sensory deficits. Pain perception may be 
assessed by a ranking or measure of the reaction to a tail-pinch, tail-
flick, or hot-plate. The response to a sudden sound, e.g., click or 
snap, may be used to assess audition.
    (10) Body weight.
    (11) Description and incidence of any unusual or abnormal 
behaviors, excessive or repetitive actions (stereotypies), emaciation, 
dehydration, hypotonia or hypertonia, altered fur appearance, red or 
crusty deposits around the eyes, nose, or mouth, and any other 
observations that may facilitate interpretation of the data.
    (C) Additional measures. Other measures may also be included and 
the development and validation of new tests is encouraged. Further 
information on the neurobehavioral integrity of the subject may be 
provided by:
    (1) Count of rearing activity on the open field.
    (2) Ranking of righting ability.
    (3) Body temperature.
    (4) Excessive or spontaneous vocalizations.
    (5) Alterations in rate and ease of respiration, e.g., rales or 
dyspnea.
    (6) Sensorimotor responses to visual or proprioceptive stimuli.
    (iii) Motor activity. Motor activity shall be monitored by an 
automated activity recording apparatus. The device used must be capable 
of detecting both increases and decreases in activity, i.e., baseline 
activity as measured by the device must not be so low as to preclude 
detection of decreases nor so high as to preclude detection of 
increases in activity. Each device shall be tested by standard 
procedures to ensure, to the extent possible, reliability of operation

[[Page 43859]]

across devices and across days for any one device. In addition, 
treatment groups must be balanced across devices. Each animal shall be 
tested individually. The test session shall be long enough for motor 
activity to approach asymptotic levels by the last 20% of the session 
for nontreated control animals. All sessions shall have the same 
duration. Treatment groups shall be counterbalanced across test times. 
Effort should be made to ensure that variations in the test conditions 
are minimal and are not systematically related to treatment. Among the 
variables which can affect motor activity are sound level, size and 
shape of the test cage, temperature, relative humidity, lighting 
conditions, odors, use of the home cage or a novel test cage, and 
environmental distractions.
    (iv) Neuropathology: Collection, processing and examination of 
tissue samples. To provide for adequate sampling as well as optimal 
preservation of cellular integrity for the detection of 
neuropathological alterations, tissue shall be prepared for 
histological analysis using in situ perfusion and paraffin and/or 
plastic embedding procedures. Paraffin embedding is acceptable for 
tissue samples from the central nervous system. Plastic embedding of 
tissue samples from the central nervous system is encouraged, when 
feasible. Plastic embedding is required for tissue samples from the 
peripheral nervous system. Subject to professional judgment and the 
type of neuropathological alterations observed, it is recommended that 
additional methods, such as glial fibrillary acidic protein (GFAP) 
immunohistochemistry and/or methods known as Bodian's or Bielchowsky's 
silver methods be used in conjunction with more standard stains to 
determine the lowest dose level at which neuropathological alterations 
are observed. When new or existing data provide evidence of structural 
alterations it is recommended that the GFAP immunoassay also be 
considered. A description of this technique can be found in the 
reference under paragraph (g)(10) of this section.
    (A) Fixation and processing of tissue. The nervous system shall be 
fixed by in situ perfusion with an appropriate aldehyde fixative. Any 
gross abnormalities should be noted. Tissue samples taken should 
adequately represent all major regions of the nervous system. The 
tissue samples should be postfixed and processed according to 
standardized published histological protocols (protocols described in 
the references under paragraphs (g)(1), (g)(2), or (g)(11) of this 
section may be used). Tissue blocks and slides should be appropriately 
identified when stored. Histological sections should be stained for 
hematoxylin and eosin (H&E), or a comparable stain according to 
standard published protocols (some of these protocols are described in 
the references under paragraphs (g)(1) and (g)(11) of this section).
    (B) Qualitative examination. Representative histological sections 
from the tissue samples should be examined microscopically by an 
appropriately trained pathologist for evidence of neuropathological 
alterations. The nervous system shall be thoroughly examined for 
evidence of any treatment-related neuropathological alterations. 
Particular attention should be paid to regions known to be sensitive to 
neurotoxic insult or those regions likely to be affected based on the 
results of functional tests. Such treatment-related neuropathological 
alterations should be clearly distinguished from artifacts resulting 
from influences other than exposure to the test substance. A stepwise 
examination of tissue samples is recommended. In such a stepwise 
examination, sections from the high dose group are first compared with 
those of the control group. If no neuropathological alterations are 
observed in samples from the high dose group, subsequent analysis is 
not required. If neuropathological alterations are observed in samples 
from the high dose group, samples from the intermediate and low dose 
groups are then examined sequentially.
    (C) Subjective diagnosis. If any evidence of neuropathological 
alterations is found in the qualitative examination, then a subjective 
diagnosis shall be performed for the purpose of evaluating dose-
response relationships. All regions of the nervous system exhibiting 
any evidence of neuropathological changes should be included in this 
analysis. Sections from all dose groups from each region will be coded 
and examined in randomized order without knowledge of the code. The 
frequency of each type and severity of each lesion will be recorded. 
After all samples from all dose groups including all regions have been 
rated, the code will be broken and statistical analysis performed to 
evaluate dose-response relationships. For each type of dose-related 
lesion observed, examples of different degrees of severity should be 
described. Photomicrographs of typical examples of treatment-related 
regions are recommended to augment these descriptions. These examples 
will also serve to illustrate a rating scale, such as 1+, 2+, and 3+ 
for the degree of severity ranging from very slight to very extensive.
    (f) Data reporting and evaluation. The final test report shall 
include the following information:
    (1) Description of equipment and test methods. A description of the 
general design of the experiment and any equipment used shall be 
provided. This shall include a short justification explaining any 
decisions involving professional judgment.
    (i) A detailed description of the procedures used to standardize 
observations, including the arena and scoring criteria.
    (ii) Positive control data from the laboratory performing the test 
that demonstrate the sensitivity of the procedures being used. 
Historical data may be used if all essential aspects of the 
experimental protocol are the same. Historical control data can be 
critical in the interpretation of study findings. The Agency encourages 
submission of such data to facilitate the rapid and complete review of 
the significance of effects seen.
    (2) Results. The following information shall be arranged by test 
group dose level.
    (i) In tabular form, data for each animal shall be provided 
showing:
    (A) Its identification number.
    (B) Its body weight and score on each sign at each observation 
time, the time and cause of death (if appropriate), total session 
activity counts, and intrasession subtotals for each day measured.
    (ii) Summary data for each group must include:
    (A) The number of animals at the start of the test.
    (B) The number of animals showing each observation score at each 
observation time.
    (C) The mean and standard deviation for each continuous endpoint at 
each observation time.
    (D) Results of statistical analyses for each measure, where 
appropriate.
    (iii) All neuropathological observations shall be recorded and 
arranged by test groups. This data may be presented in the following 
recommended format:
    (A) Description of lesions for each animal. For each animal, data 
must be submitted showing its identification (animal number, sex, 
treatment, dose, and duration), a list of structures examined as well 
as the locations, nature, frequency, and severity of lesions. Inclusion 
of photomicrographs is strongly recommended for demonstrating typical 
examples of the type and severity of the neuropathological alterations 
observed. Any diagnoses derived from

[[Page 43860]]

neurological signs and lesions including naturally occurring diseases 
or conditions, should be recorded.
    (B) Counts and incidence of neuropathological alterations by test 
group. Data should be tabulated to show:
    (1) The number of animals used in each group and the number of 
animals in which any lesion was found.
    (2) The number of animals affected by each different type of 
lesion, the locations, frequency, and average grade of each type of 
lesion.
    (3) Evaluation of data. The findings from the screening battery 
should be evaluated in the context of preceding and/or concurrent 
toxicity studies and any correlated functional and histopathological 
findings. The evaluation shall include the relationship between the 
doses of the test substance and the presence or absence, incidence and 
severity, of any neurotoxic effects. The evaluation shall include 
appropriate statistical analyses, for example, parametric tests for 
continuous data and nonparametric tests for the remainder. Choice of 
analyses should consider tests appropriate to the experimental design, 
including repeated measures. There may be many acceptable ways to 
analyze data.
    (g) References. For additional background information on this test 
guideline, the following references should be consulted. These 
references are available for inspection at the TSCA Nonconfidential 
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M 
St., SW., Washington, DC, 12 noon to 4 p.m., Monday through Friday, 
except legal holidays.
    (1) Bennet, H.S. et al. Science and art in the preparing tissues 
embedded in plastic for light microscopy, with special reference to 
glycol methacrylate, glass knives and simple stains. Stain Technology. 
51:71-97 (1976).
    (2) Di Sant Agnese, P.A. and De Mesy Jensen, K. Dibasic staining of 
large epoxy sections and application to surgical pathology. American 
Journal of Clinical Pathology. 81:25-29 (1984).
    (3) Edwards, P.M. and Parker, V.H. A simple, sensitive and 
objective method for early assessment of acrylamide neuropathy in rats. 
Toxicology and Applied Pharmacology. 40:589-591 (1977).
    (4) Finger, F.W. Ed. Myers, R.D. Measuring Behavioral Activity. 
Vol. 2. Methods in Psychobiology (Academic, NY, 1972) pp.1-19.
    (5) Gad, S. A neuromuscular screen for use in industrial 
toxicology. Journal of Toxicology and Environmental Health. 9:691-704 
(1982).
    (6) Irwin, S. Comprehensive observational assessment: Ia. A 
systematic quantitative procedure for assessing the behavioral 
physiological state of the mouse. Psychopharmacologia. 13:222-257 
(1968).
    (7) Kinnard, E.J. and Watzman, N. Techniques utilized in the 
evaluation of psychotropic drugs on animals activity. Journal of 
Pharmaceutical Sciences. 55:995-1012 (1966).
    (8) Meyer, O.A. et al. A method for the routine assessment of fore- 
and hindlimb grip strength of rats and mice. Neurobehavioral 
Toxicology. 1:233-236 (1979).
    (9) Moser V.C. et al. Comparison of chlordimeform and carbaryl 
using a functional observational battery. Fundamental and Applied 
Toxicology. 11:189-206 (1988).
    (10) O'Callaghan, J.P. Quantification of glial fibrillary acidic 
protein: Comparison of slot-immunobinding assays with a novel sandwich 
ELISA. Neurotoxicology and Teratology. 13:275-281 (1991).
    (11) Pender, M.P. A simple method for high resolution light 
microscopy of nervous tissue. Journal of Neuroscience Methods. 15:213-
218 (1985).
    (12) Reiter, L.W. Use of activity measures in behavioral 
toxicology. Environmental Health Perspectives. 26:9-20 (1978).
    (13) Reiter, L.W. and MacPhail, R.C. Motor activity: A survey of 
methods with potential use in toxicity testing. Neurobehavorial 
Toxicology. 1--Supplement. 1:53-66 (1979).
    (14) Robbins, T.W. Eds. Iversen, L.L., Iverson, D.S., and Snyder, 
S.H. A critique of the methods available for the measurement of 
spontaneous motor activity. Vol 7. Handbook of Psychopharmacology 
(Plenum, NY, 1977) pp. 37-82.


Sec. 799.9780  TSCA immunotoxicity.

    (a) Scope. This section is intended to meet the testing 
requirements under section 4 of TSCA. This section is intended to 
provide information on suppression of the immune system which might 
occur as a result of repeated exposure to a test chemical. While some 
information on potential immunotoxic effects may be obtained from 
hematology, lymphoid organ weights and histopathology (usually done as 
part of routine toxicity testing), there are data which demonstrate 
that these endpoints alone are not sufficient to predict immunotoxicity 
(Luster et al., 1992, 1993 see paragraphs (j)(8) and (j)(9) of this 
section). Therefore, the tests described in this section are intended 
to be used along with data from routine toxicity testing, to provide 
more accurate information on risk to the immune system. The tests in 
this section do not represent a comprehensive assessment of immune 
function.
    (b) Source. The source material used in developing this TSCA test 
guideline is the OPPTS harmonized test guideline 870.7800 (June 1996 
Public Draft). This source is available at the address in paragraph (j) 
of this section.
    (c) Definitions. The following definitions apply to this section.
    Antibodies or immunoglobulins (Ig) are part of a large family of 
glycoprotein molecules. They are produced by B cells in response to 
antigens, and bind specifically to the eliciting antigen. The different 
classes of immunoglobulins involved in immunity are IgG, IgA, IgM, IgD, 
and IgE. Antibodies are found in extracellular fluids, such as serum, 
saliva, milk, and lymph. Most antibody responses are T cell-dependent, 
that is, functional T and B lymphocytes, as well as antigen-presenting 
cells (usually macrophages), are required for the production of 
antibodies.
    Cluster of differentiation (CD) refers to molecules expressed on 
the cell surface. These molecules are useful as distinct CD molecules 
are found on different populations of cells of the immune system. 
Antibodies against these cell surface markers (e.g., CD4, CD8) are used 
to identify and quantitate different cell populations.
    Immunotoxicity refers to the ability of a test substance to 
suppress immune responses that could enhance the risk of infectious or 
neoplastic disease, or to induce inappropriate stimulation of the 
immune system, thus contributing to allergic or autoimmune disease. 
This section only addresses potential immune suppression.
    Natural Killer (NK) cells are large granular lymphocytes which 
nonspecifically lyse cells bearing tumor or viral antigens. NK cells 
are up-regulated soon after infection by certain microorganisms, and 
are thought to represent the first line of defense against viruses and 
tumors.
    T and B cells are lymphocytes which are activated in response to 
specific antigens (foreign substances, usually proteins). B cells 
produce antigen-specific antibodies (see the definition for 
``antibodies or immunoglobulins''), and subpopulations of T cells are 
frequently needed to provide help for the antibody response. Other 
types of T cell participate in the direct destruction of cells 
expressing specific foreign

[[Page 43861]]

(tumor or infectious agent) antigens on the cell surface.
    (d) Principles of the test methods. (1) In order to obtain data on 
the functional responsiveness of major components of the immune system 
to a T cell dependent antigen, sheep red blood cells (SRBC), rats and/
or mice1 shall be exposed to the test and control substances 
for at least 28 days.2 The animals shall be immunized by 
intravenous or intraperitoneal injection of SRBCs approximately 4 days 
(depending on the strain of animal) prior to the end of the exposure. 
At the end of the exposure period, either the plaque forming cell (PFC) 
assay or an enzyme linked immunosorbent assay (ELISA) shall be 
performed to determine the effects of the test substance on the splenic 
anti-SRBC (IgM) response or serum anti-SRBC IgM levels, respectively.
---------------------------------------------------------------------------

    1 If absorption/distribution/metabolism/excretion (ADME) data 
are similar between species, then either rats or mice may be used 
for the test compound in question. If such data are lacking, both 
species should be used.
    2 Because there is a fairly rapid turnover of many of the cells 
in the immune system, 28 days is considered sufficient for the 
purposes of the anti-SRBC tests.
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    (2) In the event the test substance produces significant 
suppression of the anti-SRBC response, expression of phenotypic markers 
for major lymphocyte populations (total T and total B), and T cell 
subpopulations (T helpers (CD4) and T cytotoxic/suppressors 
(CD8)), as assessed by flow cytometry, may be performed to 
determine the effects of the test substance on either splenic or 
peripheral-blood lymphocyte populations and T cell subpopulations. When 
this study is performed, the appropriate monoclonal antibodies for the 
species being tested should be used. If the test substance has no 
significant effect on the anti-SRBC assay, a functional test for NK 
cells may be performed to test for a chemical's effect on non-specific 
immunity.3 For tests performed using cells or sera from 
blood (ELISA or flow cytometry), it is not necessary to destroy the 
animals, since immunization with SRBCs at 28 days is not expected to 
markedly affect the results of other assays included in subchronic or 
longer-term studies (these tests are discussed in the reference under 
paragraph (j)(7) of this section). The necessity to perform either a 
quantitative analysis of the effects of a chemical on the numbers of 
cells in major lymphocyte populations and T Cell subpopulations by flow 
cytometry, or a splenic NK cell activity assay to assess the effects of 
the test compound on non-specific immunity shall be determined on a 
case-by-case basis, depending upon the outcome of the anti-SRBC assay.
---------------------------------------------------------------------------

    3 When these optional tests are included, the phenotypic or NK 
cell analyses may be performed at 28 days of exposure, or at a later 
timepoint if ADME data suggest that a longer exposure is more 
appropriate.
---------------------------------------------------------------------------

    (e) Limit test. If a test at one dose level of at least 1,000 mg/kg 
body weight (or 2 mg/L for inhalation route of exposure) using the 
procedures described for this study produces no observable toxic 
effects or if toxic effects would not be expected based upon data of 
structurally related compounds, then a full study using three dose 
levels might not be necessary. Expected human exposure may indicate the 
need for a higher dose level.
    (f) Test procedures--(1) Animal selection--(i) Species and strain. 
These tests are intended for use in rats and/or mice. Commonly used 
laboratory strains shall be employed.4 All test animals 
shall be free of pathogens, internal and external parasites. Females 
shall be nulliparous and nonpregnant. The species, strain, and source 
of the animals shall be identified.
---------------------------------------------------------------------------

    4 The study director shall be aware of strain differences in 
response to SRBC. For example, if the 
B6C3F1 hybrid mouse is used in the 
PFC assay, a response of 800-1,000 PFC/106 spleen cells 
in control mice should be the minimally acceptable PFC response.
---------------------------------------------------------------------------

    (ii) Age/weight. (A) Young, healthy animals shall be employed. At 
the commencement of the study, the weight variation of the animals used 
shall not exceed  20% of the mean weight for each sex.
    (B) Dosing shall begin when the test animals are between 6 and 8 
weeks old.
    (iii) Sex. Either sex may be used in the study; if one sex is known 
or believed to be more sensitive to the test compound, then that sex 
shall be used.
    (iv) Numbers. (A) At least eight animals shall be included in each 
dose and control group. The number of animals tested shall yield 
sufficient statistical power to detect a 20% change based upon the 
interanimal variation which may be encountered in these assays.
    (B) To avoid bias, the use of adequate randomization procedures for 
the proper allocation of animals to test and control groups is 
required.
    (C) Each animal shall be assigned a unique identification number. 
Dead animals, their preserved organs and tissues, and microscopic 
slides shall be identified by reference to the animal's unique number.
    (v) Husbandry. (A) Animals may be group-caged by sex, but the 
number of animals per cage shall not interfere with clear observation 
of each animal. The biological properties of the test substance or 
toxic effects (e.g., morbidity, excitability) may indicate a need for 
individual caging.
    (B) The temperature of the experimental animal rooms shall be at 22 
 3 deg.C.
    (C) The relative humidity of the experimental animal rooms shall be 
between 30 and 70%.
    (D) Where lighting is artificial, the sequence shall be 12 hrs 
light, 12 hrs dark.
    (E) Control and test animals shall be maintained on the same type 
of bedding and receive feed from the same lot. The feed shall be 
analyzed to assure adequacy of nutritional requirements of the species 
tested and for impurities that might influence the outcome of the test. 
Rodents shall be fed and watered ad libitum with food replaced at least 
weekly.
    (F) The study shall not be initiated until the animals have been 
allowed an adequate period of acclimatization or quarantine to 
environmental conditions. The period of acclimatization shall be at 
least 1 week in duration.
    (2) Control and test substances. (i) The test substance shall be 
dissolved or suspended in a suitable vehicle. Ideally, if a vehicle or 
diluent is needed, it shall not elicit toxic effects or substantially 
alter the chemical or toxicological properties of the test substance. 
It is recommended that an aqueous solution should be used. If 
solubility is a problem a solution in oil may be used. Other vehicles 
may be considered, but only as a last resort.
    (ii) One lot of the test substance shall be used, if possible, 
throughout the duration of the study, and the research sample shall be 
stored under conditions that maintain its purity and stability. Prior 
to the initiation of the study, there shall be a characterization of 
the test substance, including the purity of the test compound and if 
technically feasible, the name and quantities of any known contaminants 
and impurities.
    (iii) If the test or positive control substance is to be 
incorporated into feed or another vehicle, the period during which the 
test substance is stable in such a mixture shall be determined prior to 
the initiation of the study. Its homogeneity and concentration shall 
also be determined prior to the initiation of the study and 
periodically during the study. Statistically randomized samples of the 
mixture shall be analyzed to ensure that proper mixing, formulation, 
and storage procedures are being followed, and that the appropriate 
concentration of the test

[[Page 43862]]

or control substance is contained in the mixture.
    (3) Control groups. (i) A concurrent, vehicle-treated control group 
is required.
    (ii) A separate untreated control group is required if the toxicity 
of the vehicle is unknown.
    (iii) A positive control group with a known immunosuppressant 
(e.g., cyclophosphamide) shall be included in the study. A group of at 
least eight animals shall be given the immunosuppressive chemical.
    (4) Dose levels. (i) In repeated-dose toxicity tests, it is 
desirable to have a dose-response relationship and a no observed 
immunotoxic effect level. Therefore, at least three dose levels and a 
negative control shall be used, unless a limit test is performed as 
specified under paragraph (e) of this section.
    (ii) The highest dose level shall not produce significant stress, 
malnutrition, or fatalities, but ideally should produce some measurable 
sign of general toxicity (e.g., a 10% loss of body weight).
    (iii) The lowest dose level ideally shall not produce any evidence 
of immunotoxicity.
    (5) Administration of the test substance. (i) The test substance, 
vehicle, or positive control substance shall be administered for at 
least 28 days for the anti-SRBC assay. The route of administration of 
the test material will usually be oral; however, this shall be 
determined by the likely route of occupational or indoor exposure. 
Therefore, under certain conditions, the dermal or inhalation route of 
exposure may be more relevant for the study. All animals shall be dosed 
by the same method during the entire experimental period.
    (ii) If the test substance is administered by gavage, the animals 
are dosed with the test substance ideally on a 7-days-per-week basis. 
However, based primarily on practical considerations, dosing by gavage 
on a 5-days-per-week basis shall be acceptable. If the test substance 
is administered in the drinking water, or mixed directly into the diet, 
then exposure shall be on a 7-days-per-week basis.
    (A) For substances of low toxicity, it is important to ensure that 
when administered in the diet, the quantities of the test substance 
involved do not interfere with normal nutrition. When the test 
substance is administered in the diet, either a constant dietary 
concentration in parts per million (ppm) or a constant dose level in 
terms of the animal's body weight shall be used; the alternative used 
should be specified.
    (B) For a substance administered by gavage, the dose shall be given 
at approximately the same time each day, and adjusted at intervals 
(weekly for mice, twice per week for rats) to maintain a constant dose 
level in terms of the animal's body weight.
    (iii) If the test substance is administered dermally, use 
paragraphs (f)(5)(iii)(A) through (f)(5)(iii)(D) of this section.
    (A) Dose levels and dose selection. (1) In this test, it is 
desirable to determine a dose-response relationship as well as a NOEL. 
Therefore, at least three dose levels plus a control and, where 
appropriate, a vehicle control (corresponding to the concentration of 
vehicle at the highest dose level) group should be used. Doses should 
be spaced appropriately to produce test groups with a range of toxic 
effects. The data should be sufficient to produce a dose-response 
curve.
    (2) The highest dose level should elicit signs of toxicity but not 
produce severe skin irritation or an incidence of fatality which would 
prevent a meaningful evaluation. If application of the test substance 
produces severe skin irritation, the concentration may be reduced, 
although this may result in a reduction in, or absence of, other toxic 
effects at the high dose level. If the skin has been badly damaged 
early in the study, it may be necessary to terminate the study and 
undertake a new one at lower concentrations.
    (3) The intermediate dose levels should be spaced to produce a 
gradation of toxic effects.
    (4) The lowest dose level should not produce any evidence of toxic 
effects.
    (B) Preparation of animal skin. Shortly before testing, fur should 
be clipped from not less than 10% of the body surface area for 
application of the test substance. In order to dose approximately 10% 
of the body surface, the area starting at the scapulae (shoulders) to 
the wing of the ileum (hipbone) and half-way down the flank on each 
side of the animal should be shaved. Shaving should be carried out 
approximately 24 hrs before dosing. Repeated clipping or shaving is 
usually needed at approximately weekly intervals. When clipping or 
shaving the fur, care should be taken to avoid abrading the skin which 
could alter its permeability.
    (C) Preparation of test substance. (1) Liquid test substances are 
generally used undiluted, except as indicated in paragraph 
(f)(5)(iii)(A)(2) of this section.
    (2) Solids should be pulverized when possible. The substance should 
be moistened sufficiently with water or, when necessary, a suitable 
vehicle to ensure good contact with the skin. When a vehicle is used, 
the influence of the vehicle on toxicity of, and penetration of the 
skin by, the test substance should be taken into account.
    (3) The volume of application should be kept constant, e.g. less 
than 300 L for the rat; different concentrations of 
test solution should be prepared for different dose levels.
    (D) Administration of test substance. (1) The duration of exposure 
should be at least for 90 days.
    (2) The animals should be treated with test substance for at least 
6 hrs/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.
    (3) The test substance should be applied uniformly over the 
treatment site.
    (4) 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.
    (5) During the exposure period, the test substance should be held 
in contact with the skin with a porous gauze dressing. The test site 
should 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.
    (iv) If the test substance is administered by the inhalation route, 
use the procedures under paragraphs (e)(2), (e)(3), (e)(6), (e)(8), 
(e)(9), and (e)(10) of 40 CFR 799.9346. The exposure time for the anti-
SRBC test shall be at least 28 days.
    (6) Observation period. Duration of the observation period shall be 
at least 28 days.
    (7) Observation of animals. (i) Observations shall be made at least 
once each day for morbidity and mortality. Appropriate actions shall be 
taken to minimize loss of animals to the study (e.g., necropsy of those 
animals found dead and isolation or euthanasia of weak or moribund 
animals).
    (ii) A careful clinical examination shall be made at least once a 
week. Observations shall be detailed and carefully recorded, preferably 
using explicitly defined scales. Observations shall 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

[[Page 43863]]

motor activity, gait and posture, reactivity to handling or sensory 
stimuli, grip strength, and stereotypes or bizarre behavior (e.g., 
self-mutilation, walking backwards).
    (iii) Signs of toxicity shall be recorded as they are observed, 
including the time of onset, degree and duration.
    (iv) Food and water consumption shall be determined weekly.
    (v) Animals shall be weighed immediately prior to dosing, weekly 
(twice per week for rats) thereafter, and just prior to euthanasia.
    (vi) Any moribund animals shall be removed and euthanized when 
first noticed. Necropsies shall be conducted on all moribund animals, 
and on all animals that die during the study.
    (vii) The spleen and thymus shall be weighed in all animals at the 
end of the study.
    (g) Immunotoxicity tests--(1) Functional tests. Either a splenic 
PFC assay or an ELISA shall be used to determine the response to 
antigen administration.
    (i) Antibody plaque-forming cell (PFC) assay. If the antibody PFC 
assay is performed, the criteria listed under paragraphs (g)(1)(i)(A) 
through (g)(1)(i)(F) of this section shall be adhered to. Assays 
described in the references under paragraphs (j)(2) and (j)(4) of this 
section may be used.
    (A) The T cell-dependent antigen, SRBC, shall be injected 
intravenously or intraperitoneally, usually at 24 days after the first 
dosing with the test substance.5 Although the optimum 
response time is usually 4 days after immunization, some strains of 
test animal may deviate from this time point. The strain to be used 
shall be evaluated for the optimum day for PFC formation after 
immunization.
---------------------------------------------------------------------------

    5 If the SRBCs are administered by the intraperitoneal route, 
the study director should be aware that a low percentage of animals 
may not respond because the antigen was accidentally injected into 
the intestinal tract.
---------------------------------------------------------------------------

    (B) The activity of each new batch of complement shall be 
determined. For any given study, the SRBCs shall be from a single 
sheep, or pool of sheep, for which the shelf life and dose for optimum 
response has been determined.
    (C) Modifications of the PFC assay described in paragraph (g)(1)(i) 
of this section exist and may prove useful; however, the complete 
citation shall be made for the method used, any modifications to the 
method shall be reported, and the source and, where appropriate, the 
activity or purity of important reagents shall be given. Justification 
or rationale shall be provided for each protocol modification. 
Discussions of modifications of the PFC assay are available in the 
references under paragraphs (j)(5),(j)(6), and (j)(10) of this section
    (D) Samples shall be randomized and shall be coded for PFC 
analysis, so that the analyst is unaware of the treatment group of each 
sample examined.
    (E) Spleen cell viability shall be determined.
    (F) The numbers of IgM PFC per spleen, and the number of IgM PFC 
per 106 spleen cells shall be reported.
    (ii) Immunoglobulin quantification. As an alternative to a PFC 
assay, the effects of the test substance on the antibody response to 
antigen may be determined by an Enzyme-Linked Immunosorbent Assay 
(ELISA). Comparison between the PFC and ELISA assays for immunotoxicity 
assessment are discussed in the references under paragraphs (j)(5), 
(j)(6), and (j)(10) of this section. Test animals shall be immunized 
with SRBCs as for the PFC assay. IgM titers in the serum of each test 
animal shall be determined (usually 4 days after immunization). As with 
the PFC assay, the optimum dose of SRBCs and optimum time for 
collection of the sera shall be determined for the species and strain 
of animal to be tested. Several methods are described in the reference 
under paragraph (j)(11) of this section).
    (iii) Natural killer (NK) cell activity. The methods described in 
the reference under paragraph (j)(3) of this section may be used to 
demonstrate the effects of at least 28 days of exposure to a test 
substance on spontaneous cytotoxic activity. In this assay, splenocytes 
from treated and untreated test animals are incubated with 
51Cr-labeled YAC-1 lymphoma cells. The amount of radiolabel 
released from the target cells after incubation with the effector cells 
for four hrs is used as a measure of NK cytolysis. The following points 
shall be adhered to when using the NK cell assay:
    (A) Assay controls shall be included to account for spontaneous 
release of radiolabel from target cells in the absence of effector 
cells, and also for the determination of total release of radiolabel.
    (B) Target cells other than YAC-1 lymphoma cells may be appropriate 
for use in the assay. In all cases, target cell viability shall be 
determined.
    (C) Modifications of the protocol exist that may prove useful. 
However, complete citation shall be made to the method used. 
Modifications shall be reported, and where appropriate, the source, 
activity, and/or purity of the reagents should be given. Justification 
or rationale shall be provided for each protocol modification.
    (2) Enumeration of splenic or peripheral blood total B cells, total 
T cells, and T cell subpopulations. The phenotypic analysis of total B 
cell, total T cell, and T cell subpopulations from the spleen or 
peripheral blood by flow cytometry should be performed after at least 
28 days of dosing; this may be performed at a later timepoint, if ADME 
data suggest that a longer exposure is more appropriate. If an exposure 
period longer than 28 days is used, then these tests may be performed 
in conjunction with subchronic (ninety day oral, dermal, or inhalation) 
toxicity studies, when these studies are required. Methods described in 
the references under paragraphs (j)(1) and (j)(5) of this section may 
be used.
    (h) Data and reporting--(1) Treatment of results--(i) Data shall be 
summarized in tabular form, showing for each test group the number of 
animals at the start of the test, the number of animals showing 
effects, the types of effects and the percentage of animals displaying 
each type of effect.
    (ii) All observed results, quantitative and incidental, shall be 
evaluated by an appropriate statistical method. Any generally accepted 
statistical methods may be used; the statistical methods including 
significance criteria shall be selected during the design of the study.
    (2) Evaluation of study results. The findings of an immunotoxicity 
study shall be evaluated in conjunction with the findings of preceding 
studies and considered in terms of other toxic effects. The evaluation 
shall include the relationship between the dose of the test substance 
and the presence or absence, and 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 test shall provide a satisfactory estimation of a no-
observed-effect level. It may indicate the need for an additional study 
and provide information on the selection of dose levels.
    (3) Test report. In addition to the reporting requirements as 
specified under 40 CFR part 792, subpart J, the following specific 
information shall be reported. Both individual and summary data should 
be presented.
    (i) The test substance characterization shall include:
    (A) Chemical identification.
    (B) Lot or batch number.
    (C) Physical properties.
    (D) Purity/impurities.
    (E) Identification and composition of any vehicle used.

[[Page 43864]]

    (ii) The test system shall contain data on:
    (A) Species, strain, and rationale for selection of animal species, 
if other than that recommended.
    (B) Age, body weight data, and sex.
    (C) Test environment including cage conditions, ambient 
temperature, humidity, and light/dark periods.
    (D) When inhalation is the route of exposure, a description of the 
exposure equipment and data shall be included as follows:
    (1) Description of test conditions; the following exposure 
conditions shall be reported:
    (i) Description of exposure apparatus including design, type, 
volume, source of air, system for generating aerosols, method of 
conditioning air, treatment of exhaust air and the method of housing 
the animals in a test chamber.
    (ii) The equipment for measuring temperature, humidity, and 
particulate aerosol concentrations and size should be described.
    (2) Exposure data shall be tabulated and presented with mean values 
and a measure of variability (e.g., standard deviation) and include:
    (i) Airflow rates through the inhalation equipment.
    (ii) Temperature and humidity of air.
    (iii) Actual (analytical or gravimetric) concentration in the 
breathing zone.
    (iv) Nominal concentration (total amount of test substance fed into 
the inhalation equipment divided by volume of air).
    (v) Particle size distribution, calculated mass median aerodynamic 
diameter (MMAD) and geometric standard deviation (GSD).
    (vi) Explanation as to why the desired chamber concentration and/or 
particle size could not be achieved (if applicable) and the efforts 
taken to comply with this aspect of the section.
    (E) Identification of animal diet.
    (iii) The test procedure shall 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.
    (iv) Test results should include the following data:
    (A) Group animal toxic response data shall be tabulated 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 shall be presented, as well as summary 
(group mean data).
    (C) Date of death during the study or whether animals survived to 
termination.
    (D) Date of observation of each abnormal sign and its subsequent 
course.
    (E) Absolute and relative spleen and thymus weight data.
    (F) Feed and water consumption data, when collected.
    (G) Results of immunotoxicity tests.
    (H) Necropsy findings of animals that were found moribund and 
euthanized or died during the study.
    (I) Statistical treatment of results, where appropriate.
    (i) Quality control. A system shall be developed and maintained to 
assure and document adequate performance of laboratory staff and 
equipment. The study shall be conducted in compliance with the 40 CFR 
Part 792--Good Laboratory Practice.
    (j) 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.
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