“Developmental Toxicity Study

Rabbits were exposed in rotary stainless steel and glass chambers having an internal volume of 3.4 m3 (Doe and Tinston, 1981). Each chamber was supplied with air (conditioned to a nominal temperature of 23 ± 3*C and a nominal relative humidity of 40-60%) at a flow rate of 700 liters/min. Temperature and humidity were measured frequently during each exposure using wet and dry bulb hygrometers (Casella, London UK) and ranged between 20.5 and 23.5'C and 46 and 68%, respectively. Groups of 28 rabbits were used and exposure concentrations were nominally 0 (control), 2500, 10,000, and 40,000 ppm HFC 134a. Animals were assigned randomly to each group. Pregnancy was induced through artificial insemination using sperm collected from males of proven fertility. After insemination, each female was given an intravenous injection of 25 IU chorionic gonadotrophin to promote ovulation. Females were in the weight range of 2.8-4.0 kg on the day of insemination (Day 1 of gestation).

Experimental observations. The clinical condition of each animal was assessed daily. The body weight of each animal was recorded on Days 1 and 4 and 7-19 (inclusive) and on Days 22, 26, and 30 of gestation. The amount of food consumed by each animal was measured by giving a weighed quantity of food every 3 or 4 days and calculating consumption from the residues remaining. Food wastage was not measured. Animals that aborted were removed from the study.

Terminal investigations. On Day 30 of gestation, animals were killed by iv injection of Euthatal (200 mg/ml, May and Baker Ltd., Dagenham, UK). A postmortem examination was performed and the intact gravid uterus (minus ovaries) was removed and weighed. The ovaries and uterus were then examined and the number of corpora lutea in each ovary, the number and position of implantations (subdivided into live fetuses, early intrauterine deaths [showing decidual or placenta! tissue only], and late intrauterine deaths [showing embryonic or fetal tissue in addition to placental tissue]) and fetal weight were recorded. Group mean values for maternal body weight gain, maternal food consumption, gravid uterus weight, litter weight, fetal weight, number of corpora lutea, number of implantations, percentage of preimplantation loss, number of live fetuses, number of early and late intrauterine deaths, and postimplantation losses were calculated. Each fetus was examined for external abnormalities and deft palate. All fetuses were also examined internally for visceral abnormalities, sexed, eviscerated, and fixed in methanol. After approximately 24 hr the head of each fetus was cut along the frontal-parietal suture line and the brain was examined for macroscopic abnormalities. The carcasses were then returned to methanol for subsequent processing and staining with alizarin red S. The stained fetal skeletons were examined for abnormalities and the degree of ossification was assessed. The individual bones of the manus and pes were assessed. Abnormalities were classified as major (rare or possibly lethal or both) or minor (deviations from normal that are not uncommon at external visceral or skeletal examination) defects. Variations were also recorded and classified as minor defects or variants depending on the frequency of occurrence in relation to background data.

Statistical Methods

Developmental toxicity study. Data relating to animals which were nonpregnant or had aborted their litters were excluded from the statistical analysis. Maternal body weight gain and food consumption, number of implantations and live fetuses per female, percentage pre- and postimplantation loss, percentage of implantations which were early or late intrauterine deaths, gravid uterus weight, litter weight, mean fetal weight, mean manus and pes scores per fetus, and percentage of fetuses with minor external/ visceral defects only and minor skeletal defects only were considered by analysis of variance. Analyses were carried out using the GLM procedure in SAS (1985). Each treatment group mean was compared with the control group mean using Student's T test based on the error mean square in the analysis. Fetal data were converted to the litter level before calculating group means or carrying out analyses of variance. Percentages were transformed before analysis using the double arcsine transformation of Freeman and Tukey (1950). The proportion of females with pre- and postimplantation loss, early and late intrauterine deaths, the proportion of male fetuses, the proportion of fetuses with major or minor (only) external/ visceral defects, major or minor (only) skeletal defects, skeletal variants, and individual variants were analyzed by Fischer's exact test, comparing each group with the control group. All statistical tests were one-sided, except maternal body weight gain, maternal food consumption, and the proportion of fetuses which were male, which were two-sided.”

F1 Male and Female White New Zealand Rabbits