"Body Weights

All rats were weighed twice per week throughout the study.

K. Food Consumption and Food Efficiency

The amount of food consumed by each test group was determined once per week throughout the study. Group food consumption was determined by weighing the amount of chow in the food bucket at the beginning of the interval and subtracting the diet remaining and the amount of spillage from feeders at the end of the interval. From these determinations and body weight data, mean individual daily food consumption and food efficiency were calculated.

L. Clinical Observations and Mortality

Cage-site examinations to detect moribund or dead rats and abnormal behavior and appearance among rats were conducted twice daily throughout the study. At every weighing, each rat was individually handled and carefully examined for abnormal behavior or appearance, and palpable masses.

H. Clinical Laboratory Evaluation

A clinical laboratory evaluation was conducted the same day the rats were sacrificed (test days 30 and 31 for males and females, respectively).

Two days prior to collection of blood samples for clinical evaluation, all surviving rats were placed in metabolism cages and allowed to acclimate to the new cages. The day prior to blood collection, the rats were returned to metabolism cages following the exposure period. After at least two hours of access to food and water, the food was removed and urine collection initiated. At the end of the urine collection period (approximately 15 hours), blood samples were collected. Blood samples for hematological and clinical chemical measurements were collected from the orbital sinus of each rat while the rat was under light carbon dioxide anesthesia.

Hematology: The hematological parameters examined at each sampling time consisted of erythrocyte, leukocyte, differential leukocyte, and platelet counts, hemoglobin, hematocrit, mean corpuscular hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin concentration. Blood smears for reticulocyte counts were prepared from each rat, but evaluation was not required to clarify any findings.

Clinical Chemistry: In conjunction with the hematological examination, blood serum was evaluated for gamma glutamyl transferase, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, and creatine kinase activities, and concentrations of blood urea nitrogen, total serum protein, albumin, globulin (calculated), creatinine, total bilirubin, cholesterol, triglycerides, glucose, calcium, sodium, potassium, phosphorus, and chloride. Blood samples for measurement of plasma fluoride concentration were collected from surviving rats at the time of necropsy.

Urine Analyses: Urine collected during the fasting period and used for clinical chemical and hematological examinations was subjected to quantitative measures of volume, osmolality, and fluoride ion, and semi-quantitative measures of pH, glucose, protein, bilirubin, urobilinogen, ketone, and occult blood. Urine color and transparency were recorded, and sediment from each urine sample was microscopically examined.

N. Pathological Evaluation

At the end of the study (test days 30 and 31 for males and females, respectively), all surviving rats were sacrificed and necropsied. The order of sacrifice for the pathological evaluation was random among all treatment groups within a sex.

Bone marrow smears were prepared from all rats-sacrificed by design, but evaluation was not required to clarify any findings.

The lungs. brain, heart, liver, spleen, kidneys, ovaries, adrenals, and testes were weighed wet at necropsy. Organ weight/final body weight ratios were calculated.

A representative sample of the liver from each rat surviving to terminal sacrifice was collected and frozen. The frozen liver samples were stored in a freezer for determination of peroxisome proliferation…

Collected tissues were preserved in 10% buffered neutral formalin (except testes, eyes, skin, mammary tissue, and epididymides, which were preserved in Bouin's solution) until processed. All tissues collected from rats in the high-concentration and control groups were further processed to slides, stained with hematoxylin and eosin, and examined microscopically. Nose, lungs, liver, kidneys, and all gross lesions from rats in the low- and intermediate-concentration groups were processed to slides and examined microscopically. The remaining tissues collected from rats in the low- and intermediate-concentration groups were processed to paraffin blocks.

O. Determination of Peroxisome Proliferation

Liver samples were collected from each rat at the time of necropsy, frozen, and maintained at -85°F until analyzed for peroxisomal Beta-oxidation activity of fatty acids by the method of Lazarow.(6) Samples from control and 50,000 ppm exposed rats were thawed, homogenized separately in a Tris and sucrose buffer solution, and centrifuged to remove connective tissue, nuclei, and intact cells. The remaining supernatant from each sample was centrifuged and the pellet containing the peroxisomes retained. Each pellet was resuspended in a buffer and incubated with the reaction mixture containing [14Cjpalmitoyl-CoA. The reaction was suspended by transferring the reaction vessel to an ice bath and adding perchloric acid. Following centrifugation, the radioactivity of the supernatant (formation of acid soluble [14Cjacetyl-CoA) was measured by liquid scintillation spectrometry.

P. Statistical Analyses

Body weights, body weight gains, organ weights, and clinical laboratory measurements were analyzed by a one-way analysis of variance. When the corresponding F-test for differences among test group means was significant, pairwise comparisons between test and control groups were made with the Dunnett's test. Incidence of clinical observations was evaluated by the Fisher's Exact test with a Bonferroni correction and the Cochran-Armitage test for trend.

Bartlett's test for homogeneity of variances was performed on the clinical laboratory data. When the results of the Bartlett's test were significant at alpha = 0.005, the Kruskal-Wallis test was employed and the Mann-Whitney U test was used to compare means from the control groups and each of the groups exposed to HCFC-124.

Hepatic peroxisomal Beta-oxidation activity data were evaluated by a one-way analysis of variance. Pairwise comparisons between test and control groups were made with the least significant differences test.

Except for Bartlett's test, all other significance was judged at alpha = 0.05."

Histiocytosis, Alveolar

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