[Code of Federal Regulations]
[Title 40, Volume 28]
[Revised as of July 1, 2002]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR799.9538]
[Page 416-421]
TITLE 40--PROTECTION OF ENVIRONMENT
CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
PART 799--IDENTIFICATION OF SPECIFIC CHEMICAL SUBSTANCE AND MIXTURE TESTING REQUIREMENTS--Table of Contents
Subpart H--Health Effects Test Guidelines
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
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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 [plusmn] 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 [plusmn] 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 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
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a detectable increase over background. Positive control doses should be
chosen so that the effects are clear but do not immediately reveal the
identity of the coded slides to the reader. It is acceptable that the
positive control be administered by a route different from the test
substance and sampled at only a single time. The use of chemical class
related positive control chemicals may be considered, when available.
Examples of positive control substances include:
------------------------------------------------------------------------
Chemical CAS No.
------------------------------------------------------------------------
Triethylenemelamine....................... [CAS no. 51-18-3]
Ethyl methanesulphonate................... [CAS no. 62-50-0]
Ethyl nitrosourea......................... [CAS no. 759-73-9]
Mitomycin C............................... [CAS no. 50-07-7]
Cyclophosphamide (monohydrate)............ [CAS no. 50-18-0]
[CAS no. 6055-19-2]
------------------------------------------------------------------------
(3) Negative controls, treated with solvent or vehicle alone, and
otherwise treated in the same way as the treatment groups, shall be
included for every sampling time, unless acceptable inter-animal
variability and frequencies of cells with chromosome aberrations are
available from historical control data. If single sampling is applied
for negative controls, the most appropriate time is the first sampling
time. In the absence of historical or published control data
demonstrating that no deleterious or mutagenic effects are induced by
the chosen solvent/vehicle, untreated animals should be used.
(3) Procedure--(i) Number and sex of animals. Each treated and
control group shall include at least 5 analyzable animals per sex. If at
the time of the study there are data available from studies in the same
species and using the same route of exposure that demonstrate that there
are no substantial differences in toxicity between sexes, then testing
in a single sex will be sufficient. Where human exposure to chemicals
may be sex-specific, as for example with some pharmaceutical agents, the
test should be performed with animals of the appropriate sex.
(ii) Treatment schedule. (A) Test substances are preferably
administered as a single treatment. Test substances may also be
administered as a split dose, i.e. two treatments on the same day
separated by no more than a few hrs, to facilitate administering a large
volume of material. Other dose regimens should be scientifically
justified.
(B) Samples shall be taken at two separate times following treatment
on one day. For rodents, the first sampling interval is 1.5 normal cell
cycle length (the latter being normally 12-18 hr) following treatment.
Since the time required for uptake and metabolism of the test substance
as well as its 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 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
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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 [plusmn] 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.
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(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.
(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.
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(2) Preston, R.J., Dean, B.J., Galloway, S., Holden, H., McFee,
A.F., and Shelby, M. Mammalian In Vivo Cytogenetic Assays: Analysis of
Chromosome Aberrations in Bone Marrow Cells. Mutation Research. 189,
157-165 (1987).
(3) Richold, M., Chandley, A., Ashby, J., Gatehouse, D.G., Bootman,
J., and Henderson, L. Ed. D.J. Kirkland. In Vivo Cytogenetic Assays.
Basic Mutagenicity Tests, UKEMS Recommended Procedures. UKEMS
Subcommittee on Guidelines for Mutagenicity Testing. Report. Part I
revised. (Cambridge University Press, Cambridge, NY, Port Chester,
Melbourne, Sydney, 1990) pp. 115-141.
(4) Tice, R.R., Hayashi, M., MacGregor, J.T., Anderson, D., Blakey,
D.H., Holden, H.E., Kirsch-Volders, M., Oleson Jr., F.B., Pacchierotti,
F., Preston, R.J., Romagna, F., Shimada, H., Sutou, S., and Vannier, B.
Report from the Working Group on the In Vivo Mammalian Bone Marrow
Chromosomal Aberration Test. Mutation Research. 312, 305-312 (1994).
(5) Fielder, R.J., Allen, J.A., Boobis, A.R., Botham, P.A., Doe, J.,
Esdaile, D.J., Gatehouse, D.G., Hodson-Walker, G., Morton, D.B.,
Kirkland, D. J., and Richold, M. Report of British Toxicology Society/UK
Environmental Mutagen Society Working Group: Dose Setting in In Vivo
Mutagenicity Assays. Mutagenesis. 7, 313-319 (1992).
(6) Lovell, D.P., Anderson, D., Albanese, R., Amphlett, G.E., Clare,
G., Ferguson, R., Richold, M., Papworth, D.G., and Savage, J.R.K. Ed.
Kirkland,D. J. Statistical Analysis of In Vivo Cytogenetic Assays. UKEMS
Sub-Committee on Guidelines for Mutagenicity Testing. Report Part III.
Statistical Evaluation of Mutagenicity Test Data (Cambridge University
Press, Cambridge, 1989) pp. 184-232.
(7) Locke-Huhle, C. Endoreduplication in Chinese Hamster Cells
During Alpha-Radiation Induced G2 Arrest. Mutation Research. 119, 403-
413 (1983).
(8) Huang, Y., Change, C., and Trosko, J. E. Aphidicolin-Induced
Endoreduplication in Chinese Hamster Cells. Cancer Research. 43, 1362-
1364 (1983).
[62 FR 43824, Aug. 15, 1997, as amended at 64 FR 35079, June 30, 1999]