C.I. Basic Red 9 monohydrochloride is a triphenylmethane dye used for coloring textiles, leather, and paper and as a biological stain. Toxicology and carcinogenesis studies were conducted by administering the test chemical in feed to groups of 50 male and 50 female F344/N rats and B6C3F1 mice for 103 weeks at concentrations of 0, 1,000, or 2,000 ppm for male rats and 0, 500, or 1,000 ppm for female rats and mice of each sex. The average daily doses of C.I. Basic Red 9 monohydrochloride were estimated to be 49 and 103 mg/kg for male rats, 28 and 59 for female rats, 196 and 379 mg/kg for male mice, and 149 and 407 mg/kg for female mice. Two lots of the test chemical were used in the 2-year studies with purities of 93% (water content approximately 9%) and 99%.
In rats, the thyroid gland and pituitary gland were identified as target sites in the 13-week studies. Therefore, 10 additional rats of each sex were added to the control and high dose groups in the 2-year studies to examine the effects on these organs after 1 year of exposure.
In the 1-year studies in rats, final mean body weights were slightly decreased in both sexes.The thyroid gland weight to body ratio of dosed males was 1.7 times that of the controls, and the concentration of serum thyroxin in male and female rats was significantly lower than that of the controls at week 52. Compound-related histopathologic effects included thyroid gland cysts in both sexes (1/10; 1/10)and thyroid gland follicular cell hyperplasia (1/10), adenomas (1/10), and carcinomas (1/10) and fatty metamorphosis of the liver (4/10, two of these with focal necrosis) in males; no effect was seen in the controls.
The doses selected for the 2-year studies were based on the results of the 13-week studies. The absence of toxicologic signs, histopathologic changes,significant body weight depressions, or mortality after 13 weeks of exposure to C.I. Basic Red 9 monohydrochloride suggested that these concentrations would not shorten survival. However, throughout the 2-year studies, mean body weights of high dose rats and dosed mice were lower than those of the controls, and significantly reduced survival relative to controls was observed for high dose rats of each sex (P<0.001), low dose male mice(P<0.03), and low dose and high dose female mice (P<0.001).
In the 2-year studies, several types of neoplastic lesions occurred with significantly increased incidences in dosed animals (see table page 12 of the Technical Report). High dose male rats had increased incidences of squamous cell carcinomas, trichoepitheliomas, and sebaceous adenomas of the skin. Greater incidences of follicular cell carcinomas and of follicular cell adenomas were found in the thyroid glands of high dose male rats than in controls, whereas in high dose female rats, the combined incidence of follicular cell adenomas or carcinomas was greater than that in controls. Dosed rats of each sex had increased incidences of subcutaneous fibromas, and high dose rats had increased incidences of Zymbal gland carcinomas. Hepatocellular carcinomas were the compound-related neoplasms common to both species; the incidences were increased in both high dose male rats and in dosed mice of each sex. Dosed female mice had an increased incidence of pheochromocytomas or malignant pheochromocytomas. In addition, marginally increased incidences of mammary gland tumors (23/50;32/50; 32/50) in female rats, and malignant lymphomas (17/50; 24/50; 25/50)in female mice were observed.
C.I. Basic Red 9 monohydrochloride was mutagenic in strains TA98 and TA100 of Salmonella typhimurium by the preincubational protocol with or without metabolic activation. It was not mutagenic in strains TA1535 and TA1537 in this system with or without metabolic activation. It was mutagenic in the L5178Y/TK+/-mouse lymphoma assay with or without metabolic activation. C.I. Basic Red 9monohydrochloride did not induce chromosomal aberrations in Chinese hamster ovary cells; it did induce sister-chromatid exchanges in the presence of Aroclor 1254-induced male Sprague-Dawley rat liver S9. C.I. Basic Red 9 monohydrochloride also induced unscheduled DNA synthesis in F344 male rat hepatocytes in vitro.
An audit of the experimental data was conducted for these 2-year studies of C.I. Basic Red 9 monohydrochloride. No data discrepancies were found that caused squamous cell influenced the final interpretations.
Under the conditions of these 2-year feed studies, there was clear evidence of carcinogenicity of C.I. Basic Red 9monohydrochloride for male and female F344/N rats and for male and female B6C3F1 mice. In male rats, C.I. Basic Red 9 monohydrochloride caused squamous cell carcinomas, trichoepitheliomas and sebaceous adenomas of the skin,subcutaneous fibromas, thyroid gland follicular cell adenomas and follicular cell carcinomas, Zymbal gland carcinomas, and hepatocellular carcinomas. In female rats, C.I. Basic Red 9 monohydrochloride caused subcutaneous fibromas, thyroid gland follicular cell adenomas or carcinomas(combined), and Zymbal gland carcinomas. In male mice, C.I. Basic Red 9monohydrochloride caused hepatocellular carcinomas. In female mice, C.I.Basic Red 9 monohydrochloride caused hepatocellular carcinomas and adrenal gland pheochromocytomas or malignant pheochromocytomas (combined). Exposure to C.I.Basic Red 9 monohydrochloride also may have been related to increased incidences of mammary gland tumors in female rats and hematopoietic system tumors in female mice.