United States Prevention, Pesticides EPA712-C-98-224
Environmental Protection and Toxic Substances August 1998
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.5380
Mammalian
Spermatogonial
Chromosome Aberration
Test
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INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on disks or paper
copies: call (202) 512-0132. This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http://www.epa.gov/epahome/research.htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelines/OPPTS Harmonized Test
Guidelines."
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OPPTS 870.5380 Mammalian spermatogonial chromosome aberration
test.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.} and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).
(2) Background. The source materials used in developing this har-
monized OPPTS test guideline is OECD 483, Mammalian Spermatogonial
Chromosome Aberration Test.
(b) Purpose. (1) The purpose of the in vivo mammalian
spermatogonial chromosome aberration test is to identify those substances
that cause structural aberrations in mammalian spermatogonial cells (see
paragraphs (i)(l), (i)(2), (i)(3), (i)(4), and (i)(5) of this guideline). Struc-
tural aberrations may be of two types, chromosome or chromatid. With
the majority of chemical mutagens, induced aberrations are of the chro-
matid type, but chromosome-type aberrations also occur. This guideline
is not designed to measure numerical aberrations and is not routinely used
for this purpose. Chromosome mutations and related events are the cause
of many human genetic diseases.
(2) This test measures chromosome events in spermatogonial germ
cells and is, therefore, expected to be predictive of induction of inheritable
mutations in germ cells.
(c) Definitions. The definitions in section 3 of TSCA and in 40 CFR
Part 792—Good Laboratory Practice Standards (GLP) apply to this test
guideline. The following definitions also apply to this test guideline.
Chromatid-type aberration is structural chromosome damage ex-
pressed as breakage of single chromatids or breakage and reunion between
chromatids.
Chromosome-type aberration is structural chromosome damage ex-
pressed as breakage, or breakage and reunion, of both chromatids at an
identical site.
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, ob-
served as deletions, intrachanges or interchanges.
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(d) Initial considerations. (1) Rodents are routinely used in this test.
This in vivo cytogenetic test detects chromosome aberrations in
spermatogonial mitoses. Other target cells are not the subject of this guide-
line.
(2) To detect chromatid-type aberrations in spermatogonial cells, the
first mitotic cell division following treatment should be examined before
these lesions are lost in subsequent cell divisions. Additional information
from treated spermatogonial stem cells can be obtained by meiotic chro-
mosome analysis for chromosome-type aberrations at diakinesis-metaphase
I when the treated cells become spermatocytes.
(3) This in vivo test is designed to investigate whether somatic cell
mutagens are also active in germ cells. In addition, the spermatogonial
test is relevant to assessing mutagenicity hazard in that it allows consider-
ation of factors of in vivo metabolism, pharmacokinetics, and DNA-repair
processes.
(4) A number of generations of spermatogonia are present in the testis
with a spectrum of sensitivity to chemical treatment. Thus, the aberrations
detected represent an aggregate response of treated spermatogonial cell
populations, with the more numerous differentiated spermatogonial cells
predominating. Depending on their position within the testis, different gen-
erations of spermatogonia may or may not be exposed to the general cir-
culation, because of the physical and physiological Sertoli cell barrier and
the blood-testis barrier.
(5) 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) Principle of the test method. Animals are exposed to the test
substance by an appropriate route of exposure and are sacrificed at appro-
priate 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 germ cells and stained, and metaphase
cells are analyzed for chromosome aberrations.
(f) Description of the method—(1) Preparations—(i) Selection of
animal species. Male Chinese hamsters and mice are commonly used.
However, males of other appropriate mammalian species may be used.
Commonly used laboratory strains of healthy young-adult animals should
be employed. At the commencement of the study the weight variation of
animals should be minimal and not exceed +20 percent of the mean
weight.
(ii) Housing and feeding conditions. The temperature in the experi-
mental animal room should be 22 °C ( ±3 °C). Although the relative hu-
midity should be at least 30 percent and preferably not exceed 70 percent
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other than during room cleaning, the aim should be 50-60 percent. Light-
ing should be artificial, the sequence being 12 hours light, 12 hours dark.
For feeding, conventional laboratory diets may be used with an unlimited
supply of drinking water. The choice of diet may be influenced by the
need to ensure a suitable admixture of a test substance when administered
by this method. Animals may be housed individually, or be caged in small
groups.
(iii) Preparation of the animals. Healthy young-adult males should
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 accli-
mated to the laboratory conditions for at least 5 days prior to the start
of the study.
(iv) Preparation of doses. Solid test substances should 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 di-
rectly or diluted prior to dosing. Fresh preparations of the test substance
should be employed unless stability data demonstrate the acceptability of
storage.
(2) Test conditions—(i) Solvent/vehicle. The solvent/vehicle should
not produce toxic effects at the dose levels used and should not be sus-
pected of chemical reaction with the test substance. If other than well-
known solvents/vehicles are used, their inclusion should be supported with
reference data indicating their compatibility. It is recommended that wher-
ever possible, the use of an aqueous solvent/vehicle should be considered
first.
(ii) Controls. (A) Concurrent positive and negative (solvent/vehicle)
controls should be included in each test. Except for treatment with the
test substance, animals in the control groups should be handled in an iden-
tical manner to animals in the treated groups.
(B) Positive controls should produce structural chromosome aberra-
tions in vivo in spermatogonial cells when administered 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 sub-
stance 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:
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Chemical
Cyclophosphamide (monohydrate)
Cyclohexylamine
Mitomycin C
Monomeric acrylamide
Triethvlenemelamine
50-18-0
(6055-19-2)]
108-91-8]
50-07-7]
79-06-1]
51-18-31
CAS number
(C) Negative controls, treated with solvent or vehicle alone, and oth-
erwise treated in the same way as the treatment groups, should be included
for every sampling time, unless acceptable inter-animal variability and fre-
quency of cells with chromosome aberrations are demonstrated by histori-
cal control data. In addition, untreated controls should also be used unless
there are historical or published control data demonstrating that no delete-
rious or mutagenic effects are induced by the chosen solvent/vehicle.
(g) Procedure—(1) Number of animals. Each treated and control
group should include at least five analyzable males.
(2) Treatment schedule, (i) Test substances are preferably adminis-
tered once or twice (i.e. as a single treatment or as two treatments). 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 hours, to facilitate ad-
ministering a large volume of material. Other dose regimens should be
scientifically justified.
(ii) In the highest dose group, two sampling times after treatment
should be used. Since cell cycle kinetics can be influenced by the test
substance, one early and one late sampling time are used around 24 and
48 hours after treatment. For doses other than the highest dose, a sampling
time of 24 hours or 1.5 cell cycle length after treatment should be taken,
unless another sampling time is known to be more appropriate for detec-
tion of effects (see paragraph (i)(6) of this guideline).
(iii) In addition, other sampling times may be used. For example, in
the case of chemicals which may induce chromosome lagging, or may
exert S-independent effects, earlier sampling times may be appropriate (see
paragraph (i)(l) of this guideline).
(iv) The appropriateness of a repeated treatment schedule needs to
be identified on a case-by-case basis. Following a repeated treatment
schedule the animals should then be sacrificed 24 hours (1.5 cell-cycle
length) after the last treatment. Additional sampling times may be used
where appropriate.
(v) Prior to sacrifice, animals are injected intraperitoneally with an
appropriate dose of a metaphase arresting substance (e.g., Colcemid® or
colchicine). Animals are sampled at an appropriate interval thereafter. For
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mice this interval is approximately 3-5 hours, for Chinese hamsters this
interval is approximately 4-5 hours.
(3) Dose levels. If a range finding study is performed because there
are no suitable data available, it should be performed in the same labora-
tory, using the same species, strain, and treatment regimen to be used in
the main study (see paragraph (i)(7) of this guideline). If there is toxicity,
three-dose levels are used for the first sampling time. These dose levels
should 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 spermatogonial cells (e.g., a reduction in the ratio of
spermatogonial mitoses to first and second meiotic metaphases; this reduc-
tion should not exceed 50 percent).
(4) Limit test. If a test at one dose level of at least 2,000 mg/kg
body weight/day 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. Ex-
pected human exposure may indicate the need for a higher dose level to
be used in the limit test.
(5) Administration of doses. The test substance is usually adminis-
tered 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/lOOg body weight.
The use of volumes higher than these must be justified. Except for irritat-
ing 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.
(6) Chromosome preparation. Immediately after sacrifice, cell sus-
pensions should be obtained from one or both testes, exposed to hypotonic
solution and fixed. The cells should be then spread on slides and stained.
(7) Analysis. For each animal at least 100 well-spread metaphases
should be analyzed (i.e. a minimum of 500 metaphases per group). This
number could be reduced when high numbers of aberrations are observed.
All slides, including those of positive and negative controls, should be
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independently coded before microscopic analysis. Since fixation proce-
dures often result in the breakage of a proportion of metaphases with loss
of chromosomes, the cells scored should contain a number of centromeres
equal to the number 2n±2.
(h) Data and reporting—(1) Treatment of results, (i) Individual
animal data should be presented in tabular form. The experimental unit
is the animal. For each animal the number of cells with structural chro-
mosome aberration(s) and the number of chromosome aberrations per cell
should be evaluated. Different types of structural chromosome aberrations
should be listed with their numbers and frequencies for treated and control
groups. Gaps are recorded separately and reported but generally not in-
cluded in the total aberration frequency.
(ii) If mitosis as well as meiosis is observed, the ratio of
spermatogonial mitoses to first and second meiotic metaphases should be
determined as a measure of cytotoxicity for all treated and negative control
animals in a total sample of 100 dividing cells per animal to establish
a possible cytotoxic effect. If only mitosis is observed, the mitosis index
should be determined in at least 1,000 cells for each animal.
(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 (see paragraph (i)(8) of this guideline). Statistical signifi-
cance should not be the only determining factor for a positive response.
Equivocal results should be clarified by further testing preferably using
a modification of experimental conditions.
(ii) A test substance for which the results do not meet the criteria
in paragraph (h)(2)(i) of this guideline is considered nonmutagenic 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 in vivo spermatogonial chromosome ab-
erration test indicate that a substance induces chromosome aberrations in
the germ cells of the species tested. Negative results indicate that, under
the test conditions, the test substance does not induce chromosome aberra-
tions in the germ cells of the species tested.
(v) The likelihood that the test substance or its metabolites reach the
target tissue should be discussed.
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(3) Test report. The test report should include the following informa-
tion:
(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 and age 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) Data from range finding study, if conducted.
(B) Rationale for dose level selection.
(C) Rationale for route of administration.
(D) Details of test substance preparation.
(E) Details of the administration of the test substance.
(F) Rationale for sacrifice times.
(G) Conversion from diet/drinking water test substance concentration
(ppm) to the actual dose (mg/kg body weight/day), if applicable.
(H) Details of food and water quality.
(I) Detailed description of treatment and sampling schedules.
(J) Methods for measurement of toxicity.
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(K) Identity of metaphase arresting substance, its concentration and
duration of treatment.
(L) Methods of slide preparation.
(M) Criteria for scoring aberrations.
(N) Number of cells analyzed per animal.
(O) Criteria for considering studies as positive, negative, or equivocal.
(v) Results.
(A) Signs of toxicity.
(B) Mitotic index.
(C) Ratio of spermatogonial mitoses cells to first and second meiotic
metaphases.
(D) Type and number of aberrations, given separately for each animal.
(E) Total number of aberrations per group.
(F) Number of cells with aberrations per group.
(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.
(L) Changes in ploidy, if seen.
(vi) Discussion of the results.
(vii) Conclusion.
(i) References. The following references should be consulted for ad-
ditional background information on this test guideline.
(1) Adler, I.D. Clastogenic Potential in Mouse Spermatogonia of
Chemical Mutagens Related to Their Cell-Cycle Specifications. Genetic
Toxicology of Environmental Chemicals, Part B: Genetic Effects and Ap-
plied Mutagenesis, Ramel, C., Lambert, B. and Magnusson, J. (Eds.) Liss,
New York, pp. 477-484 (1986).
8
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(2) Adler, I.D. Cytogenetic Tests in Mammals. Mutagenicity Testing:
a Practical Approach. Ed. S. Venitt and J. M. Parry. IRL Press, Oxford,
Washington DC, pp. 275-306 (1984).
(3) Evans, E.P., Breckon, G. and Ford, C.E. An Air-Drying Method
for Meiotic Preparations from Mammalian Testes. Cytogenetics and Cell
Genetics 3, 289-294 (1964).
(4) Richold, M. In Vivo Cytogenetics Assays. D.J.Kirkland (Ed.)
Basic Mutagenicity Tests, UKEMS Recommended Procedures. UKEMS
Subcommittee on Guidelines for Mutagenicity Testing. Report. Part I re-
vised. Cambridge University Press, Cambridge, New York, Port Chester,
Melbourne, Sydney, pp. 115-141 (1990).
(5) Yamamoto, K. and Kikuchi, Y. A New Method for Preparation
of Mammalian Spermatogonial Chromosomes. Mutation Research 52,
207-209 (1978).
(6) Adler I.D. et al. International Workshop on Standardisation of
Genotoxicity Test Procedures. Summary Report of the Working Group on
Mammalian Germ Cell Tests. Mutation Research 312, 313-318 (1994).
(7) Fielder, R. J. et al. Report of British Toxicology Society/UK Envi-
ronmental Mutagen Society Working Group: Dose setting in In Vivo Muta-
genicity Assays. Mutagenesis 7, 313-319 (1992).
(8) Lovell, D.P. et al. Statistical Analysis of In Vivo Cytogenetic As-
says. D. J. Kirkland (Ed.) 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, pp. 184-232 (1989).
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