United States Prevention, Pesticides EPA712-C-96-225
Environmental Protection and Toxic Substances June 1996
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.5385
In Vivo Mammalian
Cytogenetics Tests:
Bone Marrow
Chromosomal Analysis
"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 870.5385 In vivo mammalian cytogenetics tests: Bone mar-
row chromosomal analysis.
(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 material used in developing this har-
monized OPPTS test guideline is OPPT 40 CFR 798.5385 In Vivo Mam-
malian Bone Marrow Cytogenetics Tests: Chromosomal Analysis; OPP
84-2 Mutagenicity Testing (Pesticide Assessment Guidelines, Subdivision
F—Hazard Evaluation; Human and Domestic Animals) EPA report 5407
09-82-025, 1982; and OECD guideline 475 Genetic Toxicology: In Vivo
Mammalian Bone Marrow Cytogenetics Tests—Chromosomal Analysis.
(b) Purpose. The in vivo bone marrow cytogenetic test is a muta-
genicity test for the detection of structural chromosomal aberrations. Chro-
mosomal aberrations are generally evaluated in first posttreatment mitoses.
With the majority of chemical mutagens, induced aberrations are of the
chromatid type but chromosome type aberrations also occur.
(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 aberrations are damage expressed as breakage of sin-
gle chromatids or breakage and/or reunion between chromatids.
Chromosome-type aberrations are changes which result from damage
expressed in both sister chromatids at the same time.
(d) Test method—(1) Principle. Animals are exposed to test chemi-
cals by appropriate routes and are sacrificed at sequential intervals. Chro-
mosome preparations are made from bone marrow cells. The stained prep-
arations are examined and metaphase cells are scored for chromosomal
aberrations.
(2) Description. The method employs bone marrow of laboratory ro-
dents which have been exposed to test chemicals. Prior to sacrifice, ani-
mals are further treated with a spindle inhibitor, (e.g., colchicine or
Colcemid ®) to arrest the cells in ometaphase. Chromosome preparations
from the cells are stained and scored for chromosomal aberrations.
(3) Animal selection—(i) Species and strain. Any appropriate mam-
malian species may be used. Examples of commonly used rodent species
are rats, mice, and hamsters.
(ii) Age. Healthy young adult animals should be used.
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(iii) Number and sex. At least five female and five male animals
per experimental and control group should be used. Thus, 10 animals
would be sacrificed per time per group treated with the test compound
if several test times after treatment are included in the experimental sched-
ule. The use of a single sex or smaller number of animals should be justi-
fied.
(iv) Assignment to groups. Animals should be randomized and as-
signed to treatment and control groups.
(4) Control groups—(1) Concurrent controls, (i) Concurrent posi-
tive and negative (vehicle) controls should be included in the assay.
(ii) Positive controls. A single dose positive control showing a sig-
nificant response at any one time point is adequate. A compound known
to produce chromosomal aberrations in vivo should be employed as the
positive control.
(5) Test chemicals—(i) Vehicle. When possible, test chemicals
should be dissolved in isotonic saline or distilled water. Water insoluble
chemicals may be dissolved or suspended in appropriate vehicles. The ve-
hicles used should neither interfere with the test chemical nor produce
toxic effects. Fresh preparations of the test compound should be employed.
(ii) Dose levels. For an initial assessment, one dose of the test sub-
stance may be used, the dose being the maximum tolerated dose (to a
maximum of 5,000 mg/kg) or that producing some indication of
cytotoxicity (e.g., partial inhibition of mitosis) or should be the highest
dose attainable (to a maximum of 5,000 mg/kg). Additional dose levels
may be used. For determination of dose-response, at least three dose levels
should be used.
(iii) Route of administration. The usual routes are oral or by
intraperitoneal injection. Other routes may be appropriate.
(iv) Treatment schedule. In general, test substances should be ad-
ministered once only. However, based on toxicological information a re-
peated treatment schedule may be employed.
(e) Test performance—(1) Generally the test may be performed
in two assays, (i) Animals should be treated with the test substance once
at the selected doses. Samples should be taken at three times after treat-
ment. For rodents, the central sampling interval is 24 h. Since cell cycle
kinetics can be influenced by the test substance, one earlier and one later
sampling interval adequately spaced within the range of 6 to 48 h should
be applied. Where the additional dose levels are tested in a subsequent
experiment, samples should be taken at the predetermined most sensitive
interval or, if this is not established, at the central sampling time. If the
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most sensitive interval is known and documented with data, only this one
time point should be sampled.
(ii) If a repeated treatment schedule is used at the selected doses,
samples should be taken 6 and 24 h after the last treatment; other sampling
times may be used if justified. Where the additional dose levels are tested
in a subsequent experiment, samples should be taken at the predetermined
most sensitive interval or, if this is not established, at 6 h after the last
treatment.
(2) Administration of spindle inhibitor. Prior to sacrifice, animals
should be injected IP with an appropriate dose of a spindle inhibitor (e.g.,
colchicine or Colcemid®) to arrest cells in ometaphase.
(3) Preparation of slides. Immediately after sacrifice, the bone mar-
row should be obtained, exposed to hypotonic solution, and fixed. The
cells should then be spread on slides and stained. Chromosome prepara-
tions should be made following standard procedures.
(4) Analysis. The number of cells to be analyzed per animal should
be based upon the number of animals used, the negative control frequency,
the predetermined sensitivity, and the power chosen for the test. Slides
should be coded before microscopic analysis.
(f) Data and report—(1) Treatment of results. Data should be pre-
sented in tabular form for both cells and animals. Different types of struc-
tural chromosomal aberrations should be listed with their numbers and a
mean frequency per cell for each animal in all treated and control groups.
Gaps (achromatic lesions) should be recorded separately and not included
in the total abberration frequency. Differences among animals within each
group should be considered before making comparisons between treated
and control groups.
(2) Statistical evaluation. Data should be evaluated by appropriate
statistical methods.
(3) Interpretation of results, (i) There are several criteria for deter-
mining a positive result, one of which is a statistically significant dose-
related increase in the number of structual chromosomal aberrations or
abnormal metaphase figures. Another criterion may be based upon detec-
tion of a reproducible and statistically significant positive response for a
least one of the test points.
(ii) A test substance which does not produce either a statistically sig-
nificant dose-related increase in the number of chromosomal aberrations
or abnormal metaphase figures or a statistically significant and reproduc-
ible positive response at any one of the test points is considered
nonmutagenic in this system.
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(iii) Both biological and statistical significance should be considered
together in the evaluation.
(4) Test evaluation, (i) Positive results in the in vivo bone marrow
cytogenetics assay indicate that under the test conditions the test substance
induces chromosomal aberrations in the bone marrow of the test species.
(ii) Negative results indicate that under the test conditions, the test
substance does not induce chromosomal aberrations in the bone marrow
of the test species.
(5) Test report. In addition to the reporting recommendations as
specified under 40 CFR part 792, subpart J, the following specific informa-
tion should be reported:
(i) Species, strain, age, weight, number and sex of animals in each
treatment and control group.
(ii) Test chemical vehicle, dose levels used, rationale for dose selec-
tion.
(iii) Route of administration, treatment and sampling schedules, tox-
icity data, negative and positive controls.
(iv) Identity of spindle-inhibitor, its concentration and duration of
treatment.
(v) Details of the protocol used for chromosome preparation, number
of cells scored per animal, type and number of aberrations given separately
for each treated and control animal.
(vi) Mitotic index, where applicable.
(vii) Criteria for scoring aberrations.
(viii) Number and frequency of aberrant cells per animal in each treat-
ment and control groups.
(ix) Total number of aberrations per group.
(x) Number of cells with aberrations per group.
(xi) Dose-response relationship, if applicable.
(g) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) Adler, I.D. et al., In vivo cytogenetic effects of trimethyl phos-
phate and of TEPA on bone marrow cells of male rats, Mutation Research
13:263-273 (1971).
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(2) Evans, H.J. Cytological methods for detecting chemical mutagens,
Chemical Mutagens: Principles and Methods for Their Detection, Vol. 4.
Ed. A. Hollaender. Plenum, New York and London. (1976) pp. 1-29.
(3) Kilian, J.D. et al. A collaborative study to measure intralaboratory
variation with the in vivo bone morrow metaphase procedure. Handbook
of mutagenicity test procedures. Eds. Kilby, B.J., Legator, M. Nichols,
C., Ramel, D. Elsevier/North Holland Biomedical Press, Amsterdam
(1977) 243-260.
(4) Preston, J.R. et al., Mammalian in vivo and vitro cytogenetics
assays: Report of the Gene-Tox Program. Mutation Research 87:143-188
(1981).
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