Toxicology and carcinogenesis studies of HC Red No. 3 (97% pure), a semipermanent hair dye, were conducted by administering the chemical in corn oil by gavage for 105 weeks to groups of 50 male and 50 female F344/N rats and for 104 weeks to groups of 50 male and 50 female B6C3F1 mice. The dosage regimen used for rats was 0, 250, or 500 mg/kg per day and for mice, 0, 125. or 250 mg/kg per day. Doses were administered 5 days per week. In prior 13-week studies, these doses produced no signs of toxicity when administered 5 days per week.
In the 2-year studies, the administration of HC Red No. 3 did not affect body weight gains of male or female rats or mice. Body weight gains by all groups of female mice were reduced because of a reproductive tract infection. Survival of male and female rats and mice was not reduced by administration of HC Red No. 3. The survival of female mice, including vehicle controls, was reduced relative to historical survival rates due to a reproductive tract infection. The infection, accompanied by weight loss, high mortality, and suppurative inflammation of multiple organs, was found in 36/50 vehicle control, 32/50 low dose, and 29/50 high dose female mice. Klebsiella pneumoniae was isolated from infected tissues.
Pigmentation of various tissues in both rats and mice was a common observation in both the 13-week and the 2-year studies. The pigment was not identified but was presumed to be a derivative of HC Red No. 3. Very minimal nephropathy was found in dosed female rats, but its relationship to HC Red No. 3 is equivocal. Mild nephrosis was found in dosed female mice, but this effect may have been secondary to the infection of the genital tract.
There was an increase in the incidence of mammary gland fibroadenomas or cystadenomas in low dose female rats. The incidence of this lesion in high dose female rats was not increased (vehicle control, 14/50, 28%; low dose, 25/50, 50%; high dose, 11/50, 22%). Largely because of the lack of a dose response, the increased incidence in the low dose females was not considered to be due to HC Red No. 3. No increased incidences of neoplasms were seen in male rats.
Transitional cell papillomas of the urinary bladder were detected in one high dose male rat, two low dose female rats, and one high dose female rat; none was observed in the vehicle controls. These uncommon neoplasms were found in animals that survived to the termination of the study and were not accompanied by other proliferative lesions.
The incidence of hepatocellular adenomas or carcinomas (combined) was increased in high dose male mice, whereas the incidence of these neoplasms in low dose male mice was significantly lower than that in the vehicle controls (25/50; 15/50; 35/50). Hepatocellular carcinomas in three vehicle control, one low dose, and five high dose male mice metastasized to the lung. The incidences of liver neoplasms in dosed female mice were not significantly different from those in the vehicle control group.
HC Red No. 3 was mutagenic in Salmonella typhimurium strains TA97, TA98, and TA100, but not in TA1535, in the presence or absence of Aroclor 1254-induced male Sprague-Dawley rat or male Syrian hamster liver S9 when tested by the preincubational protocol.
An audit of the experimental data was conducted for these 2-year toxicology and carcinogenesis studies on HC Red No. 3. No data discrepancies were found that influenced the final interpretations.
Under the conditions of these 2-year gavage studies of HC Red No. 3, there was no evidence of carcinogenicity for male or female F344/N rats given 250 or 500 mg/kg per day. There was equivocal evidence of carcinogenicity for male B6C3F1 mice as indicated by an increased incidence of hepatocellular adenomas or carcinomas (combined) in the 250 mg/kg dose group. Poor survival coupled with lack of significant findings rendered the study in female B6C3F1 mice an inadequate study of carcinogenicity. Both sexes of both species may have been able to tolerate higher doses of HC Red No. 3. Therefore, the sensitivity of these studies for detecting carcinogenesis may have been limited.