“All animals were checked at least once daily to determine their general health status. Animals were observed by cage at regular intervals during exposure. Animals were given a frequent and detailed individual clinical examination at intervals appropriate to the study design….

Body weight and food consumption. Individual body weights were recorded at least weekly at the same time as clinical examinations were conducted. Food consumption was recorded weekly for each cage of rats throughout the 90-day study. Food consumption for the 2-year study was recorded weekly by cage of rats for the first 14 weeks, at a 2-week interval for the following 2 weeks, and every 4 weeks thereafter.

Ophthalmoscopy. The eyes of all animals were examined before the start of the 90-day and 2-year studies. All animals designated for termination at the end of the 90-day study were examined similarly. The eyes of all surviving controls and animals exposed to 50,000 ppm HFC 134a were examined at Weeks 52 and 104 in the 2-year study. Examinations were carried out using a Fisons binocular indirect ophthalmoscope (Keelcr, London, UK) after instillation of 0.5% v/v tropicamide (Mydriacil, Alcon Laboratories Ltd., Watford, Herts., UK).

Hematology and clinical chemistry. Blood was collected from the tail vein from predesignated animals, different animals being designated for samples for hematology and clinical chemistry. In the 90-day study it was collected from 5 animals per sex per group in Weeks 5, 10, and 14 (the latter applied only to those animals allocated to the recovery period). In the 2-year study it was collected from 10 animals per sex per group in Weeks 14, 27, 53, and 79. Blood was taken by cardiac puncture under terminal anesthesia from all animals at the end of the 90-day study, all animals designated for the interim kill at 52 weeks in the 2-year study, and those predesignated animals at the end of the 2-year study. When a predesignated animal died before termination, it was replaced by an alternative animal. For hematology approximately 1 ml of blood was collected in tubes containing EDTA as anticoagulant. The following parameters were determined for each sample: hemoglobin, red cell count, hematocrit, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, total white cell count, and platelet count Peripheral blood films were prepared for Romanowsky-staining and were examined for all animals in the 90-day study and control and those exposed to 50,000 ppm HFC 134a in the 2- year study. This examination included a differential white cell count as well as an assessment of red cell morphology. Reticulocyte counts were performed on animal bloods showing anemia. In addition, approximately 2 ml of blood taken by cardiac puncture was collected in tubes containing trisodium citrate and prothrombin and kaolin-cephalin times were measured on a "Coag-a-mate" (Organon Technika). For clinical chemistry studies approximately 1 ml of blood was collected in tubes containing lithium heparin as anticoagulant and the following parameters were measured in the plasma: urea, creatinine, glucose, albumin, total protein, cholesterol, triglycerides and total bilirubin concentrations, alkaline phosphatase, creatine kinase, alanine aminotransferase, aspartate transaminase, ƴ-glutamyltransferase, and lactate dehydrogenase activities. At interim and terminal kills the following parameters were measured in plasma: sodium, potassium, chloride, calcium, magnesium, and phosphorus (as phosphate).

Urine analysis. Urine analysis was conducted on the same animals as those used for blood chemistry analysis during Weeks 4, 9 and 12 in the 90-day study and Weeks 13, 26,52,78, and 104 for the 2-year study. Urine samples were collected overnight. The following measurements were made on each sample: volume, pH, specific gravity, protein, creatinine, and fluoride. Semiquantitative analysis of glucose, ketones, urobilinogen, and blood were made using BM-Test-7 Strips BCL.

Pathology. Any rats considered moribund or terminated on humane grounds during the studies were killed by exsanguination using cardiac puncture, under terminal anesthesia induced by exposure to halothane Ph Eur (Fluothane, Zeneca Pharmaceuticals, Macclesfield, Cheshire, UK) vapor and subjected to a full postmortem examination as soon after death as possible. Rats found dead were also subjected to a postmortem examination as soon as practical and always within 24 hr of death. At interim and terminal kills, all designated rats were deeply anesthetized as described above and exsanguinated by cardiac puncture. This provided blood samples for clinical chemistry and hematology and also gave uniform conditions for organ weights. The animals were then subjected to a full postmortem examination and the lungs, testes, adrenal glands, kidneys, liver, heart, spleen, ovaries, and brain were weighed. Adrenal gland, aorta, bladder, bone (femur), bone marrow, brain, cecum, cervix, colon, duodenum, heart, ileum, jejunum, kidney, larynx, liver, lung, cervical and mesenteric lymph nodes, nasal cavity, sciatic nerve, esophagus, ovary, pancreas, pituitary gland, prostate gland, rectum, salivary gland, seminal vesicle, spinal cord, spleen, stomach, thymus, thyroid gland, trachea, uterus, voluntary muscle, and abnormal tissues from the 90-day study were preserved in 10% neutral-buffered formol saline. In addition, oral cavity, parathyroid gland, sternum, thymic lymph node, tongue, and vagina were preserved from the 2-year study in the same preservative. From both studies, testis, epididymis, skin, and mammary gland were fixed in Bouin's solution and eyes and Harderian gland in Davidson's fixative. All tissues from the control and 50,000 ppm HFC 134a-exposed groups, together with all lungs and any abnormal tissues, but excepting Harderian gland, mammary gland, skin, spinal cord, and voluntary muscle, were submitted for histology from the 90-day study. All tissues excepting those from the oral cavity were submitted for histology from the 2-year study. Tissues for histology were routinely processed, embedded in paraffin wax, and 5-µm-thick sections were cut and stained with hematoxylin and eosin. All sections were examined by light microscopy. Pathology findings from the 2-year study were subject to an independent peer review.

Statistical Methods

Inhalation toxicity studies. Initial body weights were considered by analysis of variance, and body weights for subsequent weeks by analysis of covariance, on initial body weight, separately for males and females. Weekly food consumption, food utilization, hematology, clinical blood, and urine chemistry were considered by analysis of variance. Organ weights were considered by analysis of variance and covariance on final body weight. All analyses were carried out using the GLM procedure in SAS (1985) and unbiased estimates of treatment group means were provided by the least-square means. Differences from control were tested statistically by comparing each treatment group least square mean using a two-sided Student's t test based on the error mean square in the analysis. Kaplan-Meier survival estimates (Kaplan and Meier, 1958) were calculated separately for each sex and treatment group. Intergroup comparisons of mortality comparing each treated group with the control group were performed using the log-rank test (Peto and Pike, 1973). The incidence of selected neoplastic findings were analyzed using Fischer's exact test, the incidence in each treated group being compared with the control incidence. In addition, the incidence of these selected findings were tested for dose-related trends using the log-rank test (Peto and Pike, 1973). Both analyses were one-sided, looking for a positive relationship with dose.”

Male Wistar-derived Rats