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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