United States       Prevention, Pesticides     EPA712-C-98-227
          Environmental Protection    and Toxic Substances     August 1998
          Agency         (7101)
&EPA    Health Effects Test
          OPPTS 870.5450
          Rodent Dominant Lethal

     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

     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

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

     (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.

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.

     (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.

     (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-

     (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