§ 79.63 Fertility assessment/teratology.
(a) Purpose. Fertility assessment/teratology is an in vivo study designed to provide information on potential health hazards to the fetus arising from the mother's repeated inhalation exposure to vehicle/engine emissions before and during her pregnancy. By including a mating of test animals, the study provides preliminary data on the effects of repeated vehicle/engine emissions exposure on gonadal function, conception, and fertility. Since this is a one-generation test that ends with examination of full-term fetuses, but not of live pups, it is not capable of determining effects on reproductive development which would only be detected in viable offspring of treated parents.
(b) Definitions. For the purposes of this section, the following definitions apply:
Developmental toxicity means the ability of an agent to induce in utero death, structural or functional abnormalities, or growth retardation after contact with the pregnant animal.
Estrous cycle means the periodic recurrence of the biological phases of the female reproductive system which prepare the animal for conception and the development of offspring. The phases of the estrous cycle for a particular animal can be characterized by the general condition of the cells present in the vagina and the presence or absence of various cell types.
Vaginal cytology evaluation means the use of wet vaginal cell smears to determine the phase of a test animal's estrous cycle and the potential for adverse exposure effects on the regularity of the animal's cycle. In the rat, common cell types found in the smears correlate well with the various stages of the estrous cycle and to changes occurring in the reproductive tract.
(c) Principle of the test method. (1) For a two week period before exposures start, daily vaginal cell smears are examined from a surplus of female test animals to identify and cull those females which are acyclic. After culling, testers shall randomly assign at each exposure concentration (including unexposed) a minimum of twenty-five females for breeding and fifteen non-bred females for later histologic evaluation. Test animals shall be exposed by inhalation to graduated concentrations of the test atmosphere for a minimum of six hours per day over the next 13 weeks. Males and females in both test and control groups are mated after nine weeks of exposure. Exposures for pregnant females continue through gestation day 15, while exposures for males and all non-pregnant females shall continue for the full exposure period.
(2) Beginning two weeks before the start of the mating period, daily vaginal smears resume for all to-be-bred females to characterize their estrous cycles. This will continue for four weeks or until a rat's pregnancy is confirmed, i.e., day 0, by the presence of sperm in the cell smear. On pregnancy day 20, shortly before the expected date of delivery, each pregnant female is sacrificed, her uterus removed, and the contents examined for embryonic or fetal deaths, and live fetuses. At the end of the exposure period, males and all non-pregnant females shall be weighed, and various organs and tissues, as appropriate, shall be removed and weighed, fixed with stain, and sectioned for viewing under a light microscope.
(3) This assay may be done separately or in combination with the subchronic toxicity study, pursuant to the provisions in § 79.62.
(d) Limit test. If a test at one dose level of the highest concentration that can be achieved while maintaining a particle size distribution with a mass median aerodynamic diameter (MMAD) of 4 micrometers (µm) or less, using the procedures described in section 79.60 of this part produces no observable toxic effects and if toxicity would not be expected based upon data of structurally related compounds, then a full study using three dose levels might not be necessary. Expected human exposure though may indicate the need for a higher dose level.