[Code of Federal Regulations]

[Title 40, Volume 31]

[Revised as of July 1, 2006]

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.



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    (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]