Fourteen-Day and Thirteen-Week Studies:
In the 14-day studies (at 94-1,500 mg/kg in rats and 62-1,000 mg/kg in mice), no compound-related deaths or body weight changes were observed and no adverse effects were observed at necropsy.
In the 13-week studies (at 94-1,500 mg/kg in rats and 31-2,000 mg/kg in mice), compound-related kidney lesions were observed in rats and mice of each sex. These lesions included variable degrees of degeneration and necrosis of epithelial cells in the proximal convoluted tubules, regeneration of tubular epithelium, and granular casts in the tubules. In a few female rats of the highest dose group, necrosis of the renal papillae and suppurative inflammation were also observed. Mean body weights were generally comparable among groups of rats and mice. Mice receiving 2,000 mg/kg had body weights 11%-12% lower than those of vehicle controls. Five of 10 female rats that received the highest dose of 1,500 mg/kg died before the end of the study, but no compound-related deaths occurred in male rats or mice of either sex.
Based on these results, 2-year studies of C.I. Acid Orange 3 were conducted by administering the dye by gavage in corn oil at 0, 375, or 750 mg/kg to groups of 50 F344/N rats of each sex, 5 days per week for 103 weeks. Groups of 50 male B6C3F1 mice were administered 0, 125, or 250 mg/kg C.I. Acid Orange 3 on the same schedule, and groups of 50 female B6C3F1 mice were administered 0, 250, or 500 mg/kg. These doses were selected on the basis of the nature and severity of the renal lesions in both species.
Body Weights and Survival in the Two-Year Studies:
Mean body weights of high dose rats were generally more than 10% lower than those of vehicle controls after week 52 for males and week 70 for females. Mean body weights forlow dose groups were comparable to those of vehicle controls. The survival of high dose male (after week 33) and female (after week 14) rats was lower (P<0.05) than that of vehicle controls and was attributed to nephrotoxicity (final survival-- male: vehicle control, 36/50; low dose, 30/50; high dose, 0/50; female: 43/50; 34/50; 7/50). Mean body weights of dosed male and female mice were lower than those of vehicle controls (high dose, 5%-11% after week 74; low dose, 7%-17% after week 48). Survival of both the low dose (after week 102) and high dose (after week 100) groups of male mice was lower than that of the vehicle controls (final survival: 38/50; 25/50; 26/50). Although survival was lower than usual, no notable differences in survival were observed between groups of female mice (final survival: 23/50; 23/50; 24/50).
Nonneoplastic and Neoplastic Lesions in the Two-Year Studies:
For both species, the kidney was the major target organ for C.I. Acid Orange 3. These findings are summarized in the accompanying table. The incidences of renal pelvic epithelial hyperplasia were increased in dosed rats of each sex. No renal neoplasms were observed in dosed male rats, but a tubular cell adenocarcinoma was observed in a vehicle control male rat. Six transitional cell carcinomas of the kidney were observed in high dose female rats; kidney transitional cell neoplasms have not been observed in 1,697 corn oil vehicle control female F344/N rats.
Nonneoplastic lesions characteristic of secondary renal hyperparathyroidism or secondary to uremia also occurred in dosed rats. The lesions included parathyroid hyperplasia, fibrous dysplasia of bone, erosion and ulcers of the glandular stomach, and mineralization of the aorta and glandular stomach.
Epithelial hyperplasia of the urinary bladder was observed in one low dose and three high dose female mice. A squamous cell carcinoma was seen in the urinary bladder of one low dose female mouse. Even though no squamous cell urinary bladder neoplasms have been observed in 1,665 corn oil vehicle control female B6C3F1 mice, this single neoplasm in a low dose animal was not considered to be related to the administration of C.I. Acid Orange 3.
C.I. Acid Orange 3 was mutagenic with and without exogenous metabolic activation in Salmonella typhimurium strains TA97; TA98; and TA100; no mutagenicity was observed for strain TA1535.
The data, documents, and pathology materials from the 2-year studies of C.I. Acid Orange 3 have been audited. The audit findings show that the conduct of the studies is documented adequately and support the data and results given in this Technical Report.
Under the conditions of these 2-year gavage studies, there was no evidence of carcinogenic activity of C.I. Acid Orange 3 for male F344/N rats administered 375 mg/kg; because of a marked reduction in survival and no indication of carcinogenicity, the 750 mg/kg group was considered to be inadequate for assessment of carcinogenic activity. There was clear evidence of carcinogenic activity of C.I. Acid Orange 3 for female F344/N rats as shown by the occurrence of transitional cell carcinomas of the kidney in the 750 mg/kg group; this group had reduced survival and chemically related nonneoplastic lesions of the kidney. There was no evidence of carcinogenic activity of C.I. Acid Orange 3 for male B6C3F1 mice administered 125 or 250 mg/kg or for female B6C3F1 mice administered 250 or 500 mg/kg. Nonneoplastic lesions of the kidney were observed in both dose groups of both sexes of rats and mice.