"Male Sprague-Dawley rats (CD-strain), 250-260 g body weight, nine weeks old, were purchased from Charles River Breeding Laboratory, Wilmington, MA, and were housed four per cage in light cycle (12 hr light, 12 hr dark), humidity, and temperature-controlled rooms, and were allowed to acclimitize for a week prior to inhalation exposure. Food (NIH #31 diet) and water were given ad libiturn, except during inhalation exposure to test chemical.

To assess the subchronic toxicity of monochlorodifluoromethane (Freon 22), animals were randomly divided into two groups, each consisting of 16 male rats. One group was exposed to 50 000 ppm Freon 22, five hrs per day for eight weeks, in a 4.0 L stainless steel exposure chamber, described previously (Hinners et al., 1969), at a flow rate of 100 L/min. The control group was exposed to filtered air at an identical rate. The chamber Freon 22 concentration was constantly monitored at 4.5 micron (peak) with an infrared gas analyzer (Wilks Miran l-A: Infrared Gas Analyzer). Control and experimental animals were weighed weekly and physically examined. At the end of the eight-week period, six rats from each experimental group were sacrificed by cervical dislocation to determine organ weights, histopathology, clinical chemistry, and hematology. Hematologic and clinical indices were determined from blood collection via the right jugular vein. Plasma glucose, blood urea nitrogen (BUN), creatine, Na*, K', CI-, CO2,

uric acid, cholesterol, triglycerides, albumin, total protein, Ca'*, phosphorus, total bilirubin, creatine phosphokinase (CPK), alkaline phosphatase (ALP), lactic dehydrogenase (LDH), serum glutamic oxalate transaminase (SCOT), and serum glutamic pyruvate transaminase (SGPT) were determined with an Automated Technicon SMAC System (Westgard eta/., 1976). Red blood cells (RBC), white blood cells (WBC) (segmented neutrophile, Iymphocyte, monocyte, and eosinophiles), hemoglobin, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hematocrit (MCHC) were also quantified with a Technicon Hemalog 8 (Brown, 1979).

At sacrifice, all major tissues and organs were examined both grossly and microscopically. The liver, pancreas, brain, lung, heart, muscle, kidney, thymus, spleen, pituitary glands, thyroid glands, adrenal glands, testis, epididymis, seminal vesicle, ventral prostate gland, and coagulating glands were weighed at sacrifice, and the weights of all organs were expressed relative to that of the brain. The liver, pancreas, brain, lung, heart, muscle, kidney, thymus, and spleen were fixed with 10% phosphate buffered formulin (pH 7.4), while the pituitary, thyroid, adrenal, prostate, coagulating glands, testis, epididymis, and seminal vesicle were fixed in Bouins Solution and stained with hematoxylin eosin for subsequent microscopic examination."

"Both ventral and dorsal prostate glands were immediately dissected into an ice cold 15 mL centrifuge tube and weighed. The prostatic glands were homogenized in 5 vol of 10-3M Tris buffer (pH 7,0); an aliquot of homogenate was used for determination of fructose, as described previously (Ashwell, 1957). The homogenate was then centrifuged at 10(90 g for 30 min, and the filtrate was used to assay for acid phosphatase activity (Lee et al., 1978). All enzymes substrates, D-fructose, and other chemicals were obtained from Sigma Chemical Company, St. Louis, MO 63178."

"Protein concentration enzyme preparations were determined by the procedure of Lowry et al. (1951 )."

"Immediately after the final exposure, heparinized whole blood was collected from the right jugular vein of each of the remaining 10 rats which were under ether anesthesia. After the suture, each rat was returned to the cage for fertility studies. After blood centrifugation, the plasma was collected and radioimmunoassayed for FSH and LH. Rat FSH-RP-1 and LH-RP-1 were used as the reference preparations. Assay sensitivity is 20 ng/mL for FSH and 2 ng/mL for LH, Intra-assay variation is less than 10% (Kreuger et al., 1974). Rat FSH-RP-1 and LH-RP-1 were supplied by the National Institute of Arthritis and Metabolic Disease, Hormone Distribution Program, Bethesda, MD 20014."

"At the end of eight weeks of Freon 22 exposure, the remaining 10 rats from each group were selected for serial mating studies to assess the time of onset of infertility (Lee et al., 1 978). Each rat was maintained in an individual cage for 10 weeks and placed on a NIH #31 diet, with free access to drinking water. Each male rat was housed singly with a virgin female for a period of seven days. Females were examined daily for vaginal plugs to assess copulation. After the seven day period, the female rats were removed from the males and replaced by other virgin females. Breeding studies were terminated at the end of 10 weeks. Nine days following removal of females from males, females were sacrificed and the number of corpora lutea (CL) and implants were scored and examined to determine the number of live implants, reabsorption sites, and the number of dead implants."

"The Fisher's nonparametric test (Siegel, 1956) was used to analyze fertility data. The statistical differences between control and experimental groups in other studies were calculated using the student t test (Snedecor and Cochran, 1967). "

Male Sprague-Dawley CD Rats