"Experimental procedures In the chronic toxicity and oncogenicity study ten rats of each sex were killed after 1 yr and the survivors after 2 yr. Exposures for the dominant lethal test were continued for 15 wk followed by eight weekly matings of each male with two untreated virgin females. These females were obtained as 5-wk-old animals from the same supplier approximately 12 wk before the start of mating. All husbandry and environmental conditions were as described for exposed rats. The females were killed 17 days after the start of cohabitation. At autopsy, corpora lutea were counted and the uterine contents were examined for implantation sites, early and late resorption sites, and viable foetuses. Triethylene melamine (0.5 mg, kg body weight) was administered ip to a group of ten unexposed male rats maintained under the same conditions as the other groups, to provide a positive control.

For the cytogenetics study exposure was continued for 13 wk and male rats were killed 2 to 4 hr after ip colchicine administration (4 mg/kg body weight). Femoral bone-marrow obtained at autopsy was suspended in Hanks buffered salt solution. After washing and centrifugation, the 140 g pellet was suspended in hypotonic potassium chloride fixed in 3:1 (v/v) acetic acid-methanol and dropped onto cold, clean microscope slides. Metaphase spreads were stained with slightly alkaline orcein and 50 spreads were scored per animal. The mitotic index, chromosome numbers, chromatid and chromosome gaps, breaks, exchanges and rearrangements were recorded.

All rats were weighed weekly during wk 1-14 and 81-104 and biweekly during wk 16-80. Weekly clinical observations were made and palpations carried out throughout the exposure period. Ophthalmoscopic examinations were carried out before starting and after 12 and 24 months of exposure.

Haematological and clinical chemistry determinations on whole blood (EDTA anticoagulant) or serum from the retro-orbital plexus of ten rats/sex/group were carried out after 12, 18, and 24 months of exposure. The determinations included erythrocyte numbers, haematocrit, haemoglobin concentration, leucocyte counts, bilirubin, urea nitrogen, glucose, glutamyl transpeptidase, creatinine phosphokinase, alanine and aspartate aminotransferases, and alkaline phosphatase. Analyses of 16-hr overnight urine samples collected at the same time intervals included appearance, osmolality, specific gravity, pH, protein, glucose, ketones, occult blood, bile pigments, and microscopic evaluation of sediment.

All rats whether found dead, killed in extremis or killed at a scheduled interval, were given detailed autopsies and selected tissues were preserved. Organ weights were determined for all rats killed after 12 months or at the end of the study for the following organs: brain, pituitary, heart, lungs, liver, kidneys. spleen, adrenals, gonads, and thymus. Morphological evaluation of approximately 45 tissues, gross lesions, and tissue masses from all rats in the control and high exposure groups were carried out using haematoxylin and eosin stain; brain sections stained with luxol fast blue and lung sections stained with haematoxylin/eosin/trichrome/silver were also prepared and examined. In addition, adrenal glands from all rats in the low and intermediate exposure groups were examined after haematoxylin and eosin staining.

Analysis of variance, using the F distribution and Dunnett's test (Dunnett, 1955), was used for statistical analysis of parametric data. Kruskal-Wallis and Dunn summed rank tests were used for non-parametric data (Hollander & Wolfe, 1973). Bartlett's test (Snedecor & Cochran, 1967) for equality of variance was conducted at the 1% two-sided risk level. All other tests were conducted at the 5% and 1% two-sided risk levels. Neoplastic lesions and survival data were analysed using National Cancer Institute statistical programs (Thomas, Breslow & Gart, 1977). Bone-marrow cytogenetic data was analysed using Student's t-test and one-way analysis of variance (Snedecor & Cochran, 1967)."

Female Sprague-Dawley-derived CD Rats