United States Prevention, Pesticides EPA712-C-96-224
Environmental Protection and Toxic Substances June 1996
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.5380
In Vivo Mammalian
Cytogenetics Tests:
Spermatogonial
Chromosomal
Aberrations
"Public Draft'
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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.).
Public Draft Access Information: This draft guideline is part of a
series of related harmonized guidelines that need to be considered as a
unit. For copies: These guidelines are available electronically from the
EPA Public Access Gopher (gopher.epa.gov) under the heading "Environ-
mental Test Methods and Guidelines" or in paper by contacting the OPP
Public Docket at (703) 305-5805 or by e-mail:
guidelines@epamail.epa.gov.
To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section, Office
of Pesticide Programs, Field Operations Division (7506C), Environmental
Protection Agency, 401 M St. SW., Washington, DC 20460. In person:
bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Ar-
lington, VA. Comments may also be submitted electronically by sending
electronic mail (e-mail) to: guidelines@epamail.epa.gov.
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202-512-0132 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from the EPA Public Access Gopher
(gopher.epa.gov) under the heading "Environmental Test Methods and
Guidelines."
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OPPTS 870.5380 In vivo mammalian cytogenetics tests:
Spermatogonial chromosomal aberrations.
(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 are the OPPT 40 CFR 798.5380 In Vivo
Mammalian Cytogenetics Tests: Spermatogonial Chromosomal Aberra-
tions and OPP 84-2 Mutagenicity Testing (Pesticide Assessment Guide-
lines, Subdivision F—Hazard Evaluation; Human and Domestic Animals)
EPA report 540/09-82-025, 1982.
(b) Purpose. The purpose of the in vivo mammalian Spermatogonial
cell assay for chromosomal aberrations is to identify those agents that
reach the mammalian gonad and induce structural chromosomal aberra-
tions in Spermatogonial mitoses. It is recognized that other assays and
endpoints are useful for addressing the questions of gonadal exposure and
induced genetic effects. This guideline, however, is restricted to the detec-
tion of chromosomal aberrations in Spermatogonial 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.
Spermatogonia are the mitotically dividing cells which are the
premeiotic progenitors of haploid gametes in the male gonad.
Structural chromosomal aberrations are changes in the morphology
of the chromosome which are detectable during the metaphase stage of
cell division. Aberration types can be observed as deletions, intrachanges
or interchanges. These include breakage and reunion events which are ex-
pressed as chromatid-type aberrations, which involve single chromatids,
or chromosome-type aberrations, which involve both sister chromatids.
(d) Test method—(1) Principle. The method relies upon testicular
cell preparations from animals which have been exposed to the test sub-
stance by an appropriate route of administration and sacrificed at selected
intervals after treatment. Prior to sacrifice, animals are treated with a spin-
dle inhibitor such as colchicine in order to accumulate metaphase cells.
In general, cells are examined at the first mitosis after exposure to the
test agent. Chromosome preparations are made, stained and microscopi-
cally analyzed.
(2) Animal selection—(i) Species and strain. Although other mam-
malian species are useful for Spermatogonial analyses, mice are the most
widely used. This guideline emphasizes the use of the mouse as the test
animal. At this time, there is no basis for recommending a specific strain.
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(ii) Age. Young, sexually mature animals should be used.
(iii) Number and sex. At least eight male animals per treatment and
control group are recommended. For those agents that may cause mortality,
sufficient animals should be treated to ensure that there is a minimum
of five survivors per group.
(iv) Assignment to groups. Animals should be randomized and as-
signed to treatment and control groups.
(3) Control groups—(i) Concurrent controls. Concurrent positive
and negative (vehicle) controls should be included in each assay.
(ii) Positive controls. A compound known to produce chromosomal
aberrations in spermatogonia should be employed as the positive control.
When feasible, the positive control should be administered in the same
vehicle and by the same route as the test chemical. In view of the animal-
to-animal variability in response that may occur in this assay, it is sug-
gested that the positive control be run at more than one dose level.
(4) Test chemicals—(i) Vehicle. When appropriate, solid and liquid
substances should be dissolved or suspended in distilled water or isotonic
saline. Water-insoluble chemicals may be dissolved or suspended in other
appropriate vehicles. The vehicle used should not interfere with the test
compound nor produce toxic effects. DMSO has been observed to induce
adverse effects with certain substances, and is not recommended as a sol-
vent. However, if it is used, it should not be used at toxic concentrations.
Fresh preparations of the test compound should be employed.
(ii) Dose levels. The use of three dose levels is recommended. Dose
selection should be made on the basis of range-finding experiment(s) that
establish the maximum tolerated dose (MTD), which should be the highest
dose tested. The MTD may be defined as the lowest dose that produces
signs of overt toxicity or a significant suppression of mitoses. Alter-
natively, some high proportion (e.g. 50 to 80 percent) of the LD50 may
be used as indicative that an acceptable MTD was achieved. Other doses
should be selected to cover reasonable intervals on a dose response curve.
For example, the second dose may be approximately 50 percent of the
high dose and the third dose no less than 25 percent of the high dose.
The highest dose for freely soluble nontoxic chemicals should be
5,000 mg/kg. Chemicals in suspension should be tested to the maximum
amount feasible. Range-finding data should be based upon the species,
strain, route of administration and treatment schedule to be used in the
mutagenicity assay proper.
(iii) Route of administration. The usual routes of administration are
intraperitoneal injection or oral gavage. Other routes may be acceptable;
if other routes are used, they should be justified.
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(iv) Treatment schedule. It has been routine practice in chromosomal
aberration studies to administer the test chemical once only. However,
based on toxicological information, a repeated treatment schedule may be
employed. Under a multiple-treatment schedule, the spermatogonial chro-
mosomal aberration assay may be run as part of subchronic studies. In
at least one treatment group the treatment should induce some sign of
toxicity in the animals. Other multiple treatment protocols, e.g. two to five
treatments separated by 24 h, may be employed.
(e) Test performance—(1) Treatment and sampling times, (i) Two
harvest times are recommended after single dose administration. Since the
cell cycle time for mouse spermatogonial cells is estimated to be between
24 to 30 h, it is recommended that the first harvest be at 24 to 30 h
after treatment and the second approximately 24 h after the first.
(ii) These harvest times should allow the detection of chromosomal
aberrations induced by test agents which either do not affect or which
delay the cell cycle. The 24- to 30-h harvest time would be expected
to sample cells which, in the absence of delay or in the presence of slight
delay, had completed at least one cell cycle. The second harvest time
would be expected to detect metaphase cells which under nondelayed con-
ditions had progressed through approximately two cell cycles. If there is
cell cycle delay caused by the test agent, the second harvest time would
be expected to yield variable ratios of first and second generation meta-
phase cells.
(iii) A single harvest is recommended after multiple dose treatment.
This harvest should be approximately one cell cycle (24 to 30 h) after
administration of the last dose. It is sufficient to harvest both negative
and positive controls at only one time following treatment.
(2) Administration of spindle inhibitor. Approximately 3 to 5 h be-
fore they are sacrificed, animals should be injected intraperitoneally with
an appropriate dose of a spindle inhibitor (e.g. colchicine) to arrest cells
in metaphase.
(3) Preparation of slides. Immediately after sacrifice, testicular sam-
ples should be obtained. Spermatogonial cells are exposed to hypotonic
solution and fixed. Some methods use acid or enzyme steps to increase
the yield of spermatogonial cells for analysis. The cells should then be
spread on slides and stained. Chromosome preparations should be made
following standard procedures.
(4) Confirmatory experiments. Good scientific practice suggests that
every experiment should be verified in an independent repeat assay. How-
ever, assuming that the procedures described above have been properly
conducted, there is no absolute requirement for a repeat assay. In the case
of a marginal or equivocal response, or of a positive response at a single
dose, a repeat assay should be required. In the repeat assay, modification
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of study parameters (e.g. dose level, number of doses, and sacrifice time)
should be considered.
(5) Analysis. Slides should be coded so that the persons evaluating
the assay are unaware of the identity of treatment and control groups. Cells
from all animals surviving treatment should be scored. Where possible,
at least 50 spermatogonial cells per animal should be scored. When it is
not feasible to score 50 cells from an animal, the maximum practical num-
ber of cells should be scored. Scoring less than 50 cells per animal must
be justified.
(f) Data and report—(1) Treatment of results. Individual animal
data should be presented in tabular form for each treatment and control
group. For each animal, the number of cells scored, the numbers and types
of structural aberrations, the aberrations per cell and the percent of cells
with aberrations should be listed. In addition to the individual animal data,
group means and standard deviations should be presented. Group data
should be based upon the mean frequencies per animal. Gaps (achromatic
lesions) should be recorded separately and should not be included in the
calculation of aberration frequencies.
(2) Statistical evaluation. Data should be evaluated by appropriate
statistical methods.
(3) Interpretation of results. There are several degrees of response
in a mutagenicity assay. Although most assays will give clearly positive
or negative results, in some tests the data set will preclude making a defini-
tive judgement about the activity of the test agent. These equivocal or
questionable responses will occur regardless of protocol employed or the
number of times the assay is repeated. For these chemicals, it is essential
that all available information on the test agent be evaluated when making
judgments about mutagenic activity or lack thereof in a specific assay.
(i) There are several criteria for determining a positive response. The
two most common criteria will be discussed here, although it is recognized
that there are others which also define a valid positive response. One cri-
terion is a statistically significant dose-related increase in the number of
spermatogonia with chromosomal aberrations. Another criterion is based
upon detection of a reproducible and statistically significant positive re-
sponse for at least one of the test substance concentrations.
(ii) If these or other acceptable criteria for a positive response are
not met, and assuming that a definitive judgement can be made about the
lack of genetic activity, the test agent is considered to be nonmutagenic
in this assay.
(iii) In all instances, scientific judgement should enter into the evalua-
tion of test results.
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(4) Test evaluation, (i) A positive spermatogonial aberration assay
indicates that, under the test conditions, the substance induces a significant
increase in the frequency of chromosomal aberrations in the gonad of the
test species.
(ii) Negative results indicate that under the test conditions the sub-
stance does not produce a significant increase in the frequency of chromo-
somal aberrations in the gonad of the test species.
(5) Test report. In addition to the reporting recommendations as
specified under 40 CFR part 792, subpart J, and under paragraph (g)(l)
of this guideline, the following specific information should be reported:
(i) Species, strain, source, age, weight, number, and sex of animals
in each treatment and control group.
(ii) Identity and purity of the test chemical, vehicle, dose levels used,
rationale for dose selection, and route of administration.
(iii) Identity of spindle inhibitor, its concentration, and duration of
treatment.
(iv) Rationale for and description of treatment and sampling sched-
ules, toxicity data, negative and positive controls.
(v) Details of the protocol used for slide preparation and staining of
cells.
(vi) Criteria for scoring aberrations.
(vii) The statistical methods used.
(viii) Dose-response relationship, if applicable.
(ix) Historical control data, if available, for both positive and negative
controls.
(x) Results obtained and statistical analysis.
(g) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) Ashby, J. et al., Overview of the study in relation to protocol
design of the rodent bone-marrow micronucleus assay. Mutation Research
234:223-248 (1990).
(2) Adler, I.D. and Brewen, J.G., Effect of chemicals on chromosome
aberration production in male and female germ cells. In Chemical
Mutagens, Principles and Methods in their Detection. Eds. F.J. de Serres,
A. Hollaender. Plenum, New York (1982) pp. 1-35.
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(3) Adler, I.D., Cytogenetic tests in mammals, Mutagenicity Testing,
A Practical Approach. Eds. S. Venitt, J.M. Parry, (Oxford: IRL Press,
1984) pp. 275-306.
(4) Albanese, R. et al. Mammalian germ cell cytogenetics. Report
of the UKEMS subcommittee of guidelines for mutagenicity studies, Part
II, 1984. pp. 145-172.
(5) Brewen, J.G. and Preston, R.J. Analysis of chromosome aberra-
tions in mammalian germ cells, Chemical Mutagens: Principles and Meth-
ods for Their Detection, Vol 5. Eds. A. Hollaender, F.J. de Serres, (New
York: Plenum Press, 1978) pp. 127-150.
(6) Preston, R.J. et al., Mammalian in vivo and in vitro cytogenetics
assays: Report of the Gene-Tox Program. Mutation Research 87:143-188
(1981).
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