[Code of Federal Regulations]
[Title 40, Volume 31]
[Revised as of July 1, 2007]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR798.5450]

[Page 202-204]
 
                   TITLE 40--PROTECTION OF ENVIRONMENT
 
         CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
 
PART 798_HEALTH EFFECTS TESTING GUIDELINES--Table of Contents
 
                       Subpart F_Genetic Toxicity
 
Sec.  798.5450  Rodent dominant lethal assay.

    (a) 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.
    (b) Definition. A 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.
    (c) Reference substances. These may include, but need not be limited 
to, triethylenemelamine, cyclophosphamide or ethyl methanesulfonate.
    (d) 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 estimated on the basis of corpora lutea 
counts or by comparing the total implants per female in treated and 
control groups.

[[Page 203]]

    (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 
appropriate 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 pregnancy 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 shall be used.
    (iii) Number. An adequate number of animals shall 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 shall be 
sufficient to provide between 30 and 50 pregnant females per mating 
interval.
    (iv) Assignment to groups. Animals shall be randomized and assigned 
to treatment and control groups.
    (4) Control groups--(i) Concurrent controls. Generally concurrent 
positive and negative (vehicle) controls shall be included in each 
experiment. When acceptable positive control results are available from 
experiments conducted recently (within the last 12 months) in the same 
laboratory these results can be used instead of a concurrent positive 
control.
    (ii) Positive controls. Positive control substances shall be used at 
a dose which demonstrates the test sensitivity.
    (5) Test chemicals--(i) Vehicle. When possible, test substances 
shall be dissolved or suspended in isotonic saline or distilled water. 
Water-insoluble chemicals may be dissolved or suspended in appropriate 
vehicles. The vehicle used shall 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 shall be used. The 
highest dose shall produce signs of toxicity (e.g., slightly reduced 
fertility and slightly reduced body weight). However, in an initial 
assessment of dominant lethality a single high dose may be sufficient. 
Nontoxic substances shall be tested at 5g/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.
    (e) Test performance. (1) Individual males are mated sequentially at 
appropriate predetermined intervals to one or two virgin females. 
Females 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 presence 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.
    (f) Data and report--(1) Treatment of results. Data shall be 
tabulated 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, shall be reported individually. 
For each female, the dose level and week of mating and the frequencies 
of live implants and of dead implants shall be enumerated. If the data 
are recorded as early and late deaths, the tables shall make that clear. 
If preimplantation loss is estimated, it shall be reported.

[[Page 204]]

Preimplantation 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 shall be evaluated by appropriate 
statistical methods. Differences among animals within the control and 
treatment groups shall be considered before making comparisons between 
treated and control groups.
    (3) Interpretation of results. (i) There are several criteria for 
determining 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 positive response for at least one of the test 
points.
    (ii) A test substance which does not produce either a statistically 
significant dose-related increase in the number of dominant lethals 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) 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 
information shall be reported:
    (i) Species, strain, age and weights of animals used, number of 
animals 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, experimental 
observations, including signs of toxicity.
    (iii) Route and duration of exposure.
    (iv) Mating schedule.
    (v) Methods used to determine that mating has occurred (where 
applicable).
    (vi) Criteria for scoring dominant lethals including the number of 
early and late embryonic deaths.
    (vii) Dose-response relationship, if applicable.
    (g) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) Brewen, J.G., Payne, H.S., Jones, K.P., Preston, R.J. ``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., Machemer, L., Buselmaier, E., Dycka, D., Frohberg, 
H., Kratochvilova, J., Lang, R., Lorke, D., Muller, D., Pheh, J., 
Rohrborn, G., Roll, R., Schulze-Schencking, M., Wiemann, H. ``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).

[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19081, May 20, 1987]