[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.
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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
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\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.
(1) Cornacoff, J.B., Graham, C.S., and LaBrie, T.K. Eds. Burleson,
G.R., Dean, J.H., and Munson, A.E. Phenotypic identification of
peripheral blood mononuclear leukocytes by flow cytometry as an adjunct
to immunotoxicity evaluation. Vol. 1. Methods in Immunotoxicology
(Wiley-Liss, Inc., New York, 1995) pp. 211-226.
(2) Cunningham, A.J. A method of increased sensitivity for
detecting single antibody-forming cells. Nature. 207:1106-1107 (1965).
(3) Djeu, Julie Y. Eds. Burleson, G.R., Dean, J.H., and Munson,
A.E. Natural Killer Activity. Methods in Immunotoxicology. pp. 437-449
(1995).
(4) Holsapple, M.P. Eds. Burleson, G.R., Dean, J.H., and Munson,
A.E. The plaque-forming cell (PFC) response in Immunotoxicology: An
approach to monitoring the primary effector function of B lymphocytes.
Vol. 1. Methods in Immunotoxicology (Wiley-Liss, Inc., New York, 1995)
pp. 71-108.
(5) Ladics, G.S. and Loveless, S.E. Cell surface marker analysis of
splenic lymphocyte populations of the CD rat for use in
immunotoxicological studies. Toxicology Methods. 4: 77-91 (1994).
(6) Ladics, G.S., Smith, C., Heaps, K., and Loveless, S.E.
Evaluation of the humoral immune response of CD rats following a 2-week
exposure to the pesticide carbaryl by the oral, dermal, or inhalation
routes. Journal of Toxicology Environmental Health. 42:143-156 (1994).
(7) Ladics., G.S., Smith, C., Heaps, K., Elliot, G.S., Slone, T.W.,
and Loveless, S.E. Possible incorporation of an immunotoxicological
functional assay for assessing humoral immunity for hazard
identification purposes in rats on standard toxicology study.
Toxicology. 96:225-238 (1995).
(8) Luster, M.I., Portier, C., Pait, D.G., White, K.L., Jr.,
Gennings, C., Munson, A.E., and Rosenthal, G.J. Risk assessment in
immunotoxicology I. Sensitivity and predictability of immune tests.
Fundamental Applied Toxicology. 18:200-210 (1992).
(9) Luster, M.I., Portier, C., Pait, D.G., Rosenthal, G.J.
Germolec. D.R., Corsini, E., Blaylock, B.L., Pollock, P., Kouchi, Y.,
Craig, W., White, D.L., Munson, A.E., and Comment, C.E. Risk Assessment
in Immunotoxicology II. Relationships Between Immune and Host
Resistance Tests. Fundamental Applied Toxicology. 21:71-82 (1993).
(10) Temple, L., Kawabata, T. T., Munson, A. E., and White, Jr., K.
L. Comparison of ELISA and plaque-forming cell assays for measuring the
humoral immune response to SRBC in rats and mice treated with
benzo[a]pyrene or cyclophosphamide. Fundamental Applied Toxicology.
21:412-419 (1993).
(11) Temple, L., Butterworth, L., Kawabata, T.T., Munson, A.E., and
White, Jr., K.L. Eds. Burleson, G.R., Dean, J.H., and Munson, A.E.
ELISA to Measure SRBC Specific Serum IgM: Method and Data Evaluation.
Vol. 1. Methods in Immunotoxicology (Wiley-Liss, Inc., New York, 1995)
pp. 137-157.
[FR Doc. 97-21413 Filed 8-14-97; 8:45 am]
BILLING CODE 6560-50-F