National Toxicology Program

The basic parts of the study design are:

1. Dose range finding study . A Dose-Range Finding Study (DRF) is conducted to determine suitable doses for the cohabitation and breeding phases of the study. A Limited DRF involves dosing for 14 days and monitoring food and water consumption and body weights. For an Extended DRF, the test chemical is administered for 7 days to male and female animals prior to cohabitation; then, with continued dosing, animals are housed as breeding pairs for 18 days, then separated, and maintained for an additional 10 days. Based on body weight, food and water consumption, and fertility data (pup, litter, and pregnancy data), the maximum tolerated dose is then estimated. In some instances, the Extend DRF may also included dosing of pups from PND 4-28 to further refine dose level selection for the main study. This DRF study seeks to assess a maximally tolerated dose level (defined as that dose which will cause no more than a 10% reduction in terminal body weight ) to be employed on the main study.
2. Main Study. The Main Study, or continuous breeding phase, is designed to determine the effect of the MTD and 2 lower dose levels on fertility and reproduction in experimental animals. In the main study, treatment of F₀ animals (usually 20 males and 20 females in each dose group) is continued for up to 36 weeks (2 wk. prior to cohabitation, production of 3 litters each consisting of 2 weeks of cohabitation, 3-4 weeks gestation, 4 weeks lactation and 1 week post-natal evaluation totaling ~33 weeks for 3 litters, followed by a 3 wk. separated holding period).
3. Crossover mating Phase . If the fertility of F₀ animals is adversely affected, a crossover mating phase is conducted to determine the affected sex(es). This involves the mating of F₀ males and females from the high dose and control groups with untreated näive females and males. If the crossover mating indicates a substantial male contribution to the adverse reproductive effect, a dominant lethal (DL) test may be then performed. The DL design uses näive females which are mated to treated males and sacrificed in late gestation for analysis of uterine contents for total implantation sites, live fetuses, resorption moles, and dead embryos and fetuses. If the crossover mating indicates a substantial female contribution to the adverse reproductive effect, the Project Officer may request that induction of pseudopregnancy and ability to superovulate may be assessed in the females.
4. F₁ Offspring mating and Assessment. From the third litter of each F₀ pair, 2 F₁ males and 2 F₁ females are kept after weaning for mating at 10 weeks of age to produce an F₂ generation for assessment. The post-weaning F₁ offspring from all dose groups and controls are treated at the same dose levels used for their F₀ parents. During the maturation of the second-generation animals, the day of first vaginal opening, the onset of estrous cycling, testicular descent, and prepuce separation are noted. At sexual maturity (74 + 10 d for mice; 91 + 10 d for rats), 20 animals for each tested group are randomized and cohabited with a further 20 nonsiblings within the same dose group for 7 days. Chemical exposure continues until termination; after mating, the animals are separated, and the females retained for 22 days. After delivery and data collection, F₂ pups are humanely sacrificed and discarded. After a sufficient delay to permit the resumption of cycling, vaginal cytology data are collected for 16 days from adult female F₁ animals. Then, 20 males and 20 females from both the control and treated groups of mating pairs of F₁ animals are necropsied and organ weight and sperm data are collected and tissues saved for possible histologic analysis.

Necropsies

After weaning of F₁ offspring, completion of any crossover or dominant lethal studies, and vaginal cytology assessment of F 0 females, all of the F₀ animals are necropsied. The endpoints for this necropsy are weights of whole body, liver, kidneys, right testis, right epididymis, right cauda epididymis, prostate, seminal vesicles (with coagulating glands attached), and ovaries, as well as any other known target organs for the compound on test. The ovaries are fixed in Bouin's and retained in 70% ethanol for possible study. The vagina, cervix, and uterus are fixed in formalin, but not weighed. Testes are fixed in 4% buffered paraformaldehyde for <= 48 hrs, then transferred to PBS. Testes and epididymides are embedded in glycol methacrylate and stained with hematoxylin and periodic acid - Schiff's stain. Prior to necropsy, vaginal smears are recorded for 12 days. For males, additional endpoints include cauda epididymal sperm number and density, morphology, and motility, and testicular spermatid head count. After production of 3 F₂ litters, all F₁ animals are necropsied and weights of whole body, liver, kidneys, right and left testis, right and left epididymis, right and left cauda epididymis, prostate, seminal vesicles (with coagulating glands attached), and ovaries obtained. The ovaries are fixed in Bouin's and retained for possible study. Data are collected as for the F₀ males and sperm motility data are gathered from all males undergoing necropsy using a computer-assisted semen analyzer.