United States Prevention, Pesticides EPA712-C-96-228
Environmental Protection and Toxic Substances June 1996
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.5460
Rodent Heritable
Translocation Assays
'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 870.5460 Rodent heritable translocation assays.
(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 OPPT 40 CFR 798.5460 Rodent Heri-
table Translocation Assays and OECD guideline 485 Genetic Toxicology:
Mouse Heritable Translocation Assay.
(b) Purpose. This test detects transmitted chromosomal damage
which manifests as balanced reciprocal translocations in progeny de-
scended from parental males treated with chemical mutagens.
(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.
Diakinesis and metaphase I are stages of meiotic prophase scored
cytologically for the presence of multivalent chromosome association char-
acteristic of translocation carriers.
A heritable translocation is one in which distal segments of
nonhomologous chromosomes are involved in a reciprocal exchange.
(d) Test method—(1) Principle. When a balanced reciprocal
translocation is induced in a parental male germ cell, the resulting progeny
is translocation heterozygote.
(i) Basis for fertility screening. Male translocation heterozygotes
may be completely sterile. This class consists of two types of
translocations:
(A) Translocations between nonhomologous chromosomes in which
at least one of the breaks occurs close to one end of a chromosome.
(B) Those that carry multiple translocations. The majority of male
translocation heterozygotes are semisterile—they carry one or (rarely) two
translocations. The degree of semisterility is dependent upon the propor-
tions of balanced and unbalanced (duplication-deficiency) gametes pro-
duced in the ejaculate as a function of meiotic segregation. Balanced and
unbalanced sperm are equally capable of fertilizing an egg. Balanced
sperm lead to viable progeny. Unbalanced sperm result in early embryonic
lethality.
(ii) Basis for cytological screening. The great majority of male
translocation heterozygotes can be identified cytologically through analysis
of diakinesis metaphase I spermatocytes. Translocation heterozygotes are
characterized by the presence of multivalent chromosome association such
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as a ring or chain of four chromosomes held together by chiasmata in
paired homologous regions. Some translocation carriers can be identified
by the presence of extra long and/or extra short chromosomes in
spermatogonial and somatic cell metaphase preparations.
(2) Description. Essentially, two methods have been used to screen
for translocation heterozygosity—one method uses a mating sequence to
identify sterile and semisterile males followed by cytological examination
of suspect male individuals; the other method deletes the mating sequence
altogether and all FI male progeny are examined cytologically for presence
of translocation. In the former approach, the mating sequence serves as
a screen which eliminates most fully fertile animals for cytological con-
firmation as translocation heterozygotes.
(3) Animal selection—(i) Species. The mouse is the species generally
used, and is recommended.
(ii) Age. Healthy sexually mature animals shall be used.
(iii) Number. The number of male animals necessary is determined
by the following factors:
(A) The use of either historical or concurrent controls.
(B) The power of the test.
(C) The minimal rate of induction required.
(D) Whether positive controls are used.
(E) The level of significance desired.
(iv) Assignment to groups. Animals shall be randomized and as-
signed to treatment and control groups.
(4) Control groups—(i) Concurrent controls. No concurrent posi-
tive or negative (vehicle) controls are recommended as routine parts of
the heritable translocation assay. However, investigators not experienced
in performing translocation testing shall include a substance known to
produce translocations in the assay as a positive control reference chemi-
cal.
(ii) Historical controls. At the present time, historical control data
must be used in tests for significance. When statistically reliable historical
controls are not available, negative (vehicle) controls shall be used.
(5) Test chemicals—(i) Vehicle. When appropriate for the route of
administration, solid and liquid test substances should be dissolved or sus-
pended in distilled water or isotonic saline. Water-insoluble chemicals may
be dissolved or suspended in appropriate vehicles. The vehicle used shall
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neither interfere with the test chemical nor produce toxic effects. Fresh
preparations of the test chemical should be employed.
(ii) Dose levels. At least two dose levels shall be used. The highest
dose level shall result in toxic effects (which shall not produce an inci-
dence of fatalities which would prevent a meaningful evaluation) or shall
be the highest dose attainable or 5 g/kg body weight.
(iii) Route of administration. Acceptable routes of administration
include oral, inhalation, admixture with food or water, and IP or IV injec-
tion.
(e) Test performance—(1) Treatment and mating. The animals
shall be dosed with the test substances 7 days per week over a period
of 35 days. After treatment, each male shall be caged with two untreated
females for a period of 1 week. At the end of 1 week, females shall be
separated from males and caged individually. When females give birth,
the day of birth, litter size, and sex of progeny shall be recorded. All
male progeny should be weaned, and all female progeny should be dis-
carded.
(2) Testing for translocation heterozygosity. When males are sexu-
ally mature, testing for translocation heterozygosity shall begin. One of
two methods shall be used; the first method involves mating, determining
those FI progeny which are sterile or semisterile and subsequent
cytological analysis of suspect progeny; the other method does not involve
mating and determining sterility or semisterility; all progeny are examined
cytologically.
(i) Determination of sterility or semisterility—(A) Conventional
method. Females are mated, usually three females for each male, and each
female is killed at midpregnancy. Living and dead implantations are count-
ed. Criteria for determining normal and semisterile males are usually estab-
lished for each new strain because the number of dead implantations varies
considerably among strains.
(B) Sequential method. Males to be tested are caged individually
with females and the majority of the presumably normal males are identi-
fied on the basis of a predetermined size of one or two litters. Breeding
pens are examined daily on weekdays beginning 18 days after pairing.
Young are discarded immediately after they are scored. Males that sire
a litter whose size is the same as or greater than the minimum set for
a translocation-free condition are discarded with their litter. If the litter
size is smaller than the predetermined number, a second litter is produced
with the same rule applying. Males that cannot be classified as normal
after production of a second litter are tested further by the conventional
method or by cytological confirmation of translocation.
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(ii) Cytological analysis. For cytological analysis of suspected
semisteriles, the air-drying technique is used. Observation of at least two
diakinesis-metaphase 1 cells with mutivalent association constitutes the re-
quired evidence for the presence of a translocation. Sterile males are exam-
ined by one of two methods, those with testes of normal size and sperm
in the epididymis are examined by the same techniques used for
semisteriles. Animals with small testes are examined by squash prepara-
tions or, alternatively, by examination of mitotic metaphase preparations.
If squash preparations do not yield diakinesis-metaphase 1 cells, analysis
of spermatogonia or bone marrow for the presence of unusually long or
short chromosomes should be performed.
(f) Data and report—(1) Treatment of results, (i) Data shall be
presented in tabular form and shall include the number of animals at risk,
the germ cell stage treated, the number of partial steriles and semisteriles
(if the fertility test is used), the number of cytogenetically confirmed
translocation heterozygotes (if the fertility test is used, report the number
of confirmed steriles and confirmed partial steriles), the translocation rate,
and either the standard error of the rate or the upper 95 percent confidence
limit on the rate.
(ii) These data shall be presented for both treated and control groups.
Historical or concurrent controls shall be specified, as well as the
randomization procedure used for concurrent controls.
(2) Statistical evaluation. Data shall be evaluated by appropriate sta-
tistical 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 heritable translocations. Another criterion
may be based upon detection of a reproducible and statistically significant
positive response for at 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 heritable translocations or
a statistically significant and reproducible positive response at any one of
the test points is considered nonmutagenic in this system.
(iii) Both biological and statistical significance should be considered
together in the evaluation.
(4) Test evaluation, (i) Positive results in the heritable translocation
assay indicate that under the test conditions the test substance causes heri-
table chromosomal damage in the test species.
(ii) Negative results indicate that under the test conditions the test
substance does not cause heritable chromosomal damage in the test spe-
cies.
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(5) Test report. In addition to the reporting recommendations as
specified under 40 CFR part 792, subpart J, the following specific informa-
tion shall be reported:
(i) Species, strain, age, weight, and number of animals of each sex
in each group.
(ii) Test chemical vehicle, route and schedule of administration, and
toxicity data.
(iii) Dosing regimen, doses tested, and rationale for dosage selection.
(iv) Mating schedule and number of females mated to each male.
(v) The use of historical or concurrent controls.
(vi) Screening procedure including the decision criteria used and the
method by which they were determined.
(vii) Dose-response relationship, if applicable.
(g) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) Generoso, W.M. et al. Heritable translocation test in mice. Muta-
tion Research 76:191-215 (1980).
(2) [Reserved]
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