Rats "were weighed once per week throughout the study. The amount of food consumed by each sex per group was determined once per week throughout the study. From these determinations and body weight data, mean individual daily food consumption, and food efficiency were calculated.

Cage-site examinations to detect moribund or dead animals and abnormal behavior and appearance were conducted twice daily throughout the study. At every weighing, each animal was individually handled and examined for abnormal behavior and appearance, and palpable masses. Ophthalmological examinations were conducted by a veterinary ophthalmologist prior to the first exposure, and prior to the 90-day sacrifice. At least 1 hr before each examination, one or two drops of 1 % atropine sulfate solution were placed in each eye of every animal. Both eyes were examined by focal illumination and indirect ophthalmoscopy. The examinations were conducted under subdued lighting.

A functional observational battery (FOB) (Irwin, 1968) was conducted at 0, 4, 8, 13, and 16 weeks on 10 rats per group designated for recovery (see below). The FOB conducted on weeks 4, 8, and 13 occurred on nonexposure days (such as weekends), beginning approximately 18 hr after the preceding exposure period. Rats were counterbalanced by sex and by dose group over time to minimize the influence of uncontrolled factors. The experimenter conducting the FOB was blind with respect to treatment group of each rat. While in the home cage, each rat was observed and scored for posture, palpebral closure, writhing, circling, biting, and vocalization. Upon removal from the cage, the following were recorded: ease of removal, ease of handling, piloerection, bite marks on tail and/or paws, palpebral closure, fur appearance, lacrimation, and salivation. In a free roaming space, presence or absence of the following were recorded: convulsions, grooming, gait abnormalities, degree of locomotion, arousal, and responses to approach, touch, finger snap, and tail pinch. In addition, pupillary response, grip strength, hind limb foot splay, and presence or absence of diarrhea and urination were assessed."

Clinical laboratory evaluations were conducted on 10 rats per sex per concentration at approximately 45 and 90 days after initiation of the study, and on surviving animals at the conclusion of the recovery period. "A complete blood cell count (CBC), serum biochemical panel (20 analytes), urinalysis, and plasma and urine fluoride were evaluated for each rat."

At the conclusion of the 90-day period, 10 rats per sex per concentration were allowed to recover for approximately 1 month. During the recovery period, exposures were not conducted, but in-life observations and measurements (clinical observations, body weight, and food consumption) were continued. After approximately 90 days on test, the 10 rats per sex per concentration designated for the 90-day portion of the study were sacrificed and necropsied. Approximately 1 month later, all surviving rats which were designated for recovery were sacrificed and necropsied. "The lungs, brain, heart, liver, spleen, kidneys, ovaries, adrenals, and testes were weighed, and organ weight/final body weight ratios were calculated. The following tissues were collected from all rats: skin, bone marrow, lymph nodes, spleen, thymus, aorta, heart, nose, trachea, lungs, larynx, pharynx, salivary glands, esophagus, stomach, liver, pancreas, small intestine, large intestine, tongue, kidneys, bladder, pituitary, thyroid-parathyroid, adrenals, prostate, mammary gland, testes, ovaries, epididymides, uterus, seminal vesicles, vagina, brain, spinal cord, peripheral nerve, bone, muscle, eyes, exorbital lacrimal glands, harderian glands, and all gross lesions. Collected tissues were preserved in an appropriate fixative until processed." All tissues collected from rats in the 50,000 ppm group and control group were further processed to slides, stained with hematoxylin and eosin, and examined microscopically. Nose, lungs, liver, kidneys, and all gross lesions from animals in the intermediate groups were processed to slides and examined microscopically.

A representative sample of liver was collected and frozen (-85°C) from five rats per sex per concentration sacrificed at the 90-day and recovery sacrifices (recovery samples for rats were not analyzed). "Each sample was analyzed for peroxisomal beta-oxidation activity of fatty acids by the method of Lazarow (1981). The peroxisomal fraction was incubated with a reaction mixture containing [14C]-palmitoyl-CoA. Following centrifugation, the radioactivity from the formation of acid soluble [14C]acetyl-CoA was measured by liquid scintillation spectrometry."

"Body weight, body weight gain, food consumption, organ weights, hepatic peroxisomal beta-oxidation activity data, and clinical chemistry parameters 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. Bartlett's test for homogeneity of variances was performed on the organ weight and clinical laboratory data from the subchronic studies. 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 or Dunn's test was used to compare means from the control groups and each of the groups exposed to HCFC-124."

The incidence of clinical observations was evaluated by the Cochran-Armitage test for trend and Fisher's exact test (with a Bonferroni correction for clinical signs data).

Male Crl:CD-1(ICR)BR Mice