"Parent F0 Animals

One-hundred seventy-four 4-week-old (±1 day) SPF Sprague-Dawley male and female rats [Crl: CD (SD) B R VAF/Plus strain] within a 40-g weight range were obtained from Charles River Ltd. The mean weight for males was 145 g and that for females was 125 g. Males and females were obtained from separate litters. After an acclimatization period of 6 days they were weighed again and assigned to five groups.

Experimental Design

Treatment of the F0 males and females by inhalation began when they were approximately 6 weeks of age and continued up to weaning of the F1 pups, at which point most F0 animals were euthanized. Twelve males and 12 females per group were retained for a total exposure of 39 weeks. These animals were used for additional clinical studies. Direct treatment of the males and females was started when the animals were approximately 4 weeks of age and continued up to the weaning of the F2 pups.

Observations

Observations and measurements of body weight and food consumption were similar to procedures previously described (Rusch, 1995). Mating procedures and pregnancy indicators were also described previously. However, F0 animals in this study were only mated once. Histological procedures were also the same as in the prior study.

Litter Data

As soon as possible after parturition, the young were counted, individually identified, sexed, weighed, and examined for external abnormalities. All litters were examined daily, and the pups were weighed on Days 2, 4, 7, 14, 18, and 21 postpartum. On Day 4 postpartum, the pups were weighed and the litter was standardized to a total litter size of eight pups (ideally four male and four female pups) by random selection for each sex. No pups were culled from litters of eight or less, regardless of sex ratio. On Day 21 postpartum, one male and one female pup in each of 28 litters per group from F1 litters were retained for further study. The 28 litters were those weaned as close to the median weaning data as possible for the study or group, paying attention to retaining as wide a genetic pool as possible. Selection of pups was made using computer-generated random number tables. Where less than 28 litters in a group weaned or were within a reasonable range of weaning dates, or where all pups in a litter were of one sex, the complement was restored by taking a second pup from an appropriate litter. Shortly after Day 21, all excess pups were killed and examined externally and internally for abnormalities. Sex of the pups was confirmed by gonadal inspection. Tissues showing macroscopic abnormality were preserved in 10% neutral buffered formalin.

Sexual Maturation (F1 Litters)

The onset of vaginal opening was monitored in females from 28 days postpartum. The occurrence of cleavage of the balanopreputial skinfold was monitored in males from Day 35 postpartum.

Individual Litter Values

In assessing litter parameters for each litter reared to weaning, implantation loss, pup loss at birth, cumulative pup loss to Day 4 (prior to cull), and cumulative pup loss from Day 4 (as percentage survival from Day 4) were calculated. The following indices on the growth of pups were calculated: viability index (number of surviving pups on Day 4/number of pups born) and viability to weaning index (number of pups alive at weaning/ number of live pups at Day 4 postcull).

Biochemical/Hormonal Assays

Blood samples were obtained from F0 and F1 animals. In view of blood volumes required to perform clinical investigations, 12 animals/sex/group (F0 main group and F1 animals) were bled for biochemical/hormonal assays. Blood samples were withdrawn under light ether anaesthesia from the orbital sinus into heparin anticoagulant tubes. Animals were fasted overnight prior to the laboratory investigations.

For the F0 and F1 generations, blood samples were obtained at 14 and again at 15 weeks of age due to volumes needed to complete the biochemical/hormonal assays (Study Weeks 9, 10 for F0; Weeks 15, 16 for F1). For F0 animals, except For the estradiol and progesterone assays, the biochemical and hormonal assays were performed on samples taken at 14 weeks of age. For F1 animals, the hormonal assays were performed on samples taken at 14 weeks of age and the biochemical assays on samples taken at 15 weeks of age. For the F0 retained group, blood samples were obtained at 44 weeks of age for the biochemical and hormonal assays and at 45 weeks of age for the cholecystokinin (CCK) assay (Study Weeks 39, 40). The following assays were performed on males and females in all groups: Total cholesterol (chol.), esterified cholesterol (by subtraction: total chol. — free chol.); free cholesterol (Hitachi 737 clinical chemistry analyzer); high-density lipoprotein cholesterol (HDL chol., Hitachi 737 clinical chemistry analyzer); low-density lipoprotein cholesterol (LDL chol., Hitachi 737 clinical chemistry analyzer); very-low-density lipoprotein and chylomicron cholesterol (VLDL, by subtraction); and total cholesterol (HDL + LDL chol.). In addition, serum triglyceride levels were measured on the retained group and in the F1 generation (Roche Cobas centrifugal analyzer and a BM test kit). The following hormonal assays were conducted on males of the F0 and F1 generations: Follicle-stimulating hormone (radioimmunoassay using Amersham rat FSH kit); luteinizing hormone (radioimmunoassay using Amersham rat LH kit); estradiol (radioimmunoassay using Diagnostic Products Corporation test kit); progesterone (radioimmunoassay using Diagnostic Products Corporation test kit); and testosterone (radioimmunoassay using Farmes Diagnostic Products Corporation test kit).

Statistical Analysis

Group mean values from individual litter values are presented to include valid data from any animals with viable young at termination. The basic sample unit was the litter. Statistical analyses were routinely performed on litter data. Significance tests were normally two-tailed. These procedures have been described previously (Rusch, 1995)."

F1 Male Parental Rats