United States Prevention, Pesticides EPA712-C-98-227
Environmental Protection and Toxic Substances August 1998
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.5450
Rodent Dominant Lethal
Assay
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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 870.5450 Rodent dominant lethal assay.
(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 798.5450 Rodent domi-
nant Lethal assay and OECD 478 Genetic Toxicology: Rodent Dominant
Lethal Assay.
(b) Purpose. Dominant lethal (DL) effects cause embryonic or fetal
death. Induction of a dominant lethal event after exposure to a chemical
substance indicates that the substance has affected germinal tissue of the
test species. Dominant lethals are generally accepted to be the result of
chromosomal damage (structural and numerical anomalies) but gene
mutations and toxic effects cannot be excluded.
(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 definition also applies to this test guideline.
Dominant lethal mutation is one occurring in a germ cell which does
not cause dysfunction of the gamete, but which is lethal to the fertilized
egg or developing embryo.
(d) Reference substances. These may include, but need not be lim-
ited to, triethylenemelamine, cyclophosphamide, or ethyl
methanesulfonate.
(e) Test method—(1) Principle. Generally, male animals are exposed
to the test substance and mated to untreated virgin females. The various
germ cell stages can be tested separately by the use of sequential mating
intervals. The females are sacrificed after an appropriate period of time
and the contents of the uteri are examined to determine the numbers of
implants and live and dead embryos. The calculation of the dominant lethal
effect is based on comparison of the live implants per female in the treated
group to the live implants per female in the control group. The increase
of dead implants per female in the treated group over the dead implants
per female in the control group reflects the post-implantation loss. The
post-implantation loss is calculated by determining the ratio of dead to
total implants from the treated group compared to the ratio of dead to
total implants from the control group. Pre-implantation loss can be esti-
mated on the basis of corpora lutea counts or by comparing the total im-
plants per female in treated and control groups.
(2) Description, (i) Several treatment schedules are available. The
most widely used requires single administration of the test substance.
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Other treatment schedules, such as treatment on five consecutive days, may
be used if justified by the investigator.
(ii) Individual males are mated sequentially to virgin females at ap-
propriate intervals. The number of matings following treatment is governed
by the treatment schedule and should ensure that germ cell maturation
is adequately covered. Females are sacrificed in the second half of preg-
nancy and the uterine contents examined to determine the total number
of implants and the number of live and dead embryos.
(3) Animal selection—(i) Species. Rats or mice are generally used
as the test species. Strains with low background dominant lethality, high
pregnancy frequency, and high implant numbers are recommended.
(ii) Age. Healthy, sexually mature animals should be used.
(iii) Number. An adequate number of animals should be used taking
into account the spontaneous variation of the biological characteristics
being evaluated. The number chosen should be based on the predetermined
sensitivity of detection and power of significance. For example, in a typical
experiment, the number of males in each group should be sufficient to
provide between 30 and 50 pregnant females per mating interval.
(iv) Assignment to groups. Animals should be randomized and as-
signed to treatment and control groups.
(4) Control groups—(i) Concurrent controls. Generally concurrent
positive and negative (vehicle) controls should be included in each experi-
ment. When acceptable positive control results are available from experi-
ments conducted recently (within the last 12 months) in the same labora-
tory, these results can be used instead of a concurrent positive control.
(ii) Positive controls. Positive control substances should be used at
a dose which demonstrates the test sensitivity.
(5) Test chemicals—(i) Vehicle. When possible, test substances
should be dissolved or suspended in isotonic saline or distilled water.
Water-insoluble chemicals may be dissolved or suspended in appropriate
vehicles. The vehicle used should neither interfere with the test chemical
nor produce toxic effects. Fresh preparations of the test chemical should
be employed.
(ii) Dose levels. Normally, three dose levels should be used. The
highest dose should produce signs of toxicity (e.g., slightly reduced fertil-
ity and slightly reduced body weight). However, in an initial assessment
of dominant lethality a single high dose may be sufficient. Nontoxic sub-
stances should be tested at 5 g/kg or, if this is not practicable, then as
the highest dose attainable.
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(iii) Route of administration. The usual routes of administration are
oral or by IP injection. Other routes may be appropriate.
(f) Test performance. (1) Individual males are mated sequentially
at appropriate predetermined intervals to one or two virgin females. Fe-
males should be left with the males for at least the duration of one estrus
cycle or alternatively until mating has occurred as determined by the pres-
ence of sperm in the vagina or by the presence of a vaginal plug.
(2) The number of matings following treatment should be governed
by the treatment schedule and should ensure that germ cell maturation
is adequately covered.
(3) Females should be sacrificed in the second half of pregnancy and
uterine contents examined to determine the number of implants and live
and dead embryos. The ovaries may be examined to determine the number
of corpora lutea.
(g) Data and report—(1) Treatment of results. Data should be tab-
ulated to show the number of males, the number of pregnant females, and
the number of nonpregnant females. Results of each mating, including the
identity of each male and female, should be reported individually. For each
female, the dose level and week of mating and the frequencies of live
implants and of dead implants should be enumerated. If the data are re-
corded as early and late deaths, the tables should make that clear. If pre-
plantation loss is estimated, it should be reported. Pre-implantation loss
can be calculated as the difference between the number of corpora lutea
and the number of implants or as a reduction in the average number of
implants per female in comparison with control matings.
(2) Statistical evaluation. Data should be evaluated by appropriate
statistical methods. Differences among animals within the control and
treatment groups should be considered before making comparisons be-
tween treated and control groups.
(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 dominant lethals. Another criterion may
be based upon detection of a reproducible and statistically significant posi-
tive 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 dominant lethals or a statis-
tically 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.
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(4) Test evaluation, (i) A positive DL assay suggests that under the
test conditions the test substance may be genotoxic in the germ cells of
the treated sex of the test species.
(ii) A negative result suggests that under the conditions of the test
the test substance may not be genotoxic in the germ cells of the treated
sex 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, and weights of animals used, number of ani-
mals of each sex in experimental and control groups.
(ii) Test substance, vehicle used, dose levels and rationale for dosage
selection, negative (vehicle) and positive controls, and experimental obser-
vations, including signs of toxicity.
(iii) Route and duration of exposure.
(iv) Mating schedule.
(v) Methods used to determine that mating has occurred (where appli-
cable).
(vi) Criteria for scoring dominant lethals including the number of
early and late embryonic deaths.
(vii) Dose-response relationship, if applicable.
(h) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) Brewen, J.G. et al. Studies on chemically induced dominant
lethality. I. The cytogenetic basis of MMS-induced dominant lethality in
post-meiotic germ cells. Mutation Research 33:239-250 (1975).
(2) Ehling, U.H. et al. Standard protocol for the dominant lethal test
on male mice. Set up by the Work Group Dominant lethal mutations of
the ad hoc Committee Chemogenetics. Archives of Toxicology 39:173-185
(1978).
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