title: NTP Toxicology and Carcinogenesis Studies of HC Yellow 4 (CAS No. 59820-43-8) in F344 Rats and B6C3F1 Mice (Feed Studies).
abstract: HC Yellow 4 is used in semipermanent hair dyes. Toxicology and carcinogenesis studies were conducted by administering HC Yellow 4 (greater than 93% pure) in feed to groups of F344/N rats and B6C3F1 mice of each sex for 14 days, 13 weeks, and 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, cultured Chinese hamster ovary cells, and Drosophila melanogaster. 14-Day Studies: Groups of five rats of each sex were given 0, 5,000, 10,000, 20,000, 40,000, or 80,000 ppm and groups of five mice of each sex were given 0, 1,250, 2,500, 5,000, 10,000, or 20,000 ppm HC Yellow 4 in feed for 14 days. All animals survived to the end of the studies. Final mean body weights of male rats that received 20,000 ppm or more, female rats that received 10,000 ppm or more, and female mice that received 20,000 ppm were significantly lower than those of the controls. The mean body weights of exposed and control groups of male mice were similar. No chemical-related decrease in feed consumption was observed. No chemical-related clinical findings or changes in absolute or relative organ weights occurred in rats or mice. No gross or microscopic changes were related to HC Yellow 4 administration in rats or mice. 13-Week Studies: Groups of 10 rats of each sex were fed diets containing 0, 2,500, 5,000, 10,000, 20,000, or 40,000 ppm and groups of 10 mice of each sex were fed diets containing 0, 5,000, 10,000, 20,000, 40,000, or 80,000 ppm HC Yellow 4 for 13 weeks. All rats survived to study termination. Chemical-related deaths occurred at the two highest dose levels in male and female mice. Final mean body weights of male rats that received 10,000 ppm or greater, female rats that received 20,000 or 40,000 ppm, and mice that received 10,000 ppm or greater were significantly lower than those of the controls. There were no biologically significant changes in absolute or relative organ weights. Mineralization of the renal papilla occurred in all male rats in the 40,000 ppm group. Thyroid pigmentation occurred in rats receiving 40,000 ppm and in mice at all dose levels. Uterine atrophy occurred in female rats in the 20,000 and 40,000 ppm groups and female mice in the 40,000 and 80,000 ppm groups. Lymphoid depletion and atrophy of the spleen occurred in male mice that received 40,000 or 80,000 ppm and female mice that received 80,000 ppm. Atrophy of the thymus occurred in male and female mice that received 40,000 or 80,000 ppm. 2-Year Studies: Groups of 70 male rats were fed diets containing 0, 2,500, or 5,000 ppm and groups of 70 female rats and 70 mice of each sex were fed diets containing 0, 5,000, or 10,000 ppm HC Yellow 4 for up to 2 years. Interim evaluations were performed on 10 rats and 10 mice from each dose group at 6 and 15 months. No biologically significant changes in absolute or relative organ weight or hematology or clinical chemistry values were found in these rats or mice. No compound-related lesions were seen in exposed rats. In exposed mice, pigmentation of the thyroid gland was observed at the 6-month interim evaluations; pigmentation and hyperplasia of the thyroid gland were seen at the 15-month interim evaluations. Body Weight, Survival, and Feed Consumption in the 2-Year Studies: The mean body weight of female rats that received 10,000 ppm was significantly lower than that of the controls. The mean body weights of mice receiving 10,000 ppm were 20% to 30% lower than those of the controls during the second year of the studies. The survival of exposed rats and mice was similar to that of the controls. Neoplasms and Nonneoplastic Lesions in the 2-Year Studies: Pituitary gland pars distalis adenomas were marginally increased in exposed male rats (0 ppm, 17/45; 2,500 ppm, 20/49; 5,000 ppm, 28/49), and there was a concomitant dose-related increase in the incidence of hyperplasia (8/45, 13/49, 18/49). There was no increase in the incidence of pituitary gland adenomas or carcinomas in female rats (34/49, 35/48, 30/49). In mice, no neoplasms were considered related to chemical administration. However, a dose-related incr, a dose-related increased incidence of thyroid gland pigmentation and follicular cell hyperplasia occurred in both sexes of mice. Genetic Toxicology: HC Yellow 4 was mutagenic in Salmonella typhimurium strains TA100, TA1537, and TA98 with and without exogenous metabolic activation (S9); the response in strain TA1535 without S9 was equivocal. HC Yellow 4 induced sister chromatid exchanges in Chinese hamster ovary cells in the absence but not the presence of S9 activation; no induction of chromosomal aberrations occurred in Chinese hamster ovary cells, with or without S9. HC Yellow 4 induced sex-linked recessive lethal mutations in germ cells of adult male Drosophila melanogaster when administered by injection; results of a reciprocal translocation test in D. melanogaster were negative. Conclusions: Under the conditions of these 2-year feed studies, there was equivocal evidence of carcinogenic activity of HC Yellow 4 in male F344/N rats based on the increased incidence of pituitary gland adenomas and hyperplasia. The male rats may have been able to tolerate a slightly higher dose of the chemical. There was no evidence of carcinogenic activity of HC Yellow 4 in female F344/N rats given 5,000 or 10,000 ppm. There was no evidence of carcinogenic activity of HC Yellow 4 in male or female B6C3F1 mice given 5,000 or 10,000 ppm. There was a chemical-related increase in the incidence of thyroid gland pigmentation and follicular cell hyperplasia in mice. Synonym: N,O-di(2-hydroxyethyl)-2-amino-5-nitrophenol