C.I. Acid Red 114 is one of five chemicals being evaluated in 2-year carcinogenicity and toxicity studies as part of the NTP's Benzidine Dye Initiative. This Initiative was designed to evaluate representative benzidine congeners, benzidine congener-derived dyes, and benzidine-derived dyes. C.I. Acid Red 114 was nominated for study because of the potential for human exposure during production of bisazobiphenyl dyes and because benzidine, a structurally related chemical, is a known human carcinogen.
Toxicology and carcinogenesis studies were conducted by administering desalted, industrial grade C.I. Acid Red 114 in drinking water to groups of F344/N rats of each sex for 13 days, 13 weeks, 9 or 15 months, or 2 years. These studies were performed only in rats because studies of benzidine congeners were being performed in mice at the National Center for Toxicological Research (NCTR). Genetic toxicology studies were conducted in Salmonella typhimurium, Chinese hamster ovary cells, and Drosophila melanogaster.
Rats were exposed to C.I. Acid Red 114 in drinking water at doses of 0, 10,000, 20,000, or 30,000 ppm. All control and dosed rats survived except one male rat in the 20,000 ppm dose group. Final mean body weights in the three dosed groups were 94%, 83%, or 77% of controls for males and 92%, 88%, or 80% of controls for females. Water consumption declined with increased dose. Clinical findings included red stained fur, ears, and tail in all test animals. On gross necropsy, organs and tissues were also stained red.
C.I. Acid Red 114 was administered in drinking water at doses of 0, 600, 1,200, 2,500, 5,000, or 10,000 ppm. All control and dosed animals survived until the end of the study. Final mean body weights in the five dosed groups were 97%, 89%, 87%, 87%, or 85% of controls for males and 97%, 94%, 94%, 92%, or 89% of controls for females. Water consumption was decreased in dosed animals. As was seen in the 13-day studies, major organs and tissues from treated animals were stained red. Kidney toxicity characterized by regeneration and karyomegaly of tubule epithelial cells with chronic inflammation was observed in female rats at doses of 1,200 ppm or above. Treatment-related increases in relative liver weights and elevated liver enzyme levels were seen in males and females, centrilobular pallor in the liver was seen in all male dose groups. Because of these body weight differences, decreases in water consumption, and organ toxicity, the doses chosen for the 2-year studies were 70,150, and 300 ppm for males and 150, 300, and 600 for females.
Male rats received doses of 0, 70, 150, or 300 ppm of C.I. Acid Red 114, and female rats received 0, 150, 300, or 600 ppm. Seventy animals were in the control and high-dose groups, 45 in the low-dose groups, and 75 in the mid-dose groups. Ten animals were evaluated from the control and high-dose groups at 9 months, and ten animals from all dose groups were evaluated at 15 months. The average amount of compound consumed per day was 4, 8, or 20 mg/kg for males and 9, 20, or 70 mg/kg for females.
Survival and Body Weights: Survival at 105 weeks for male rats receiving 0, 70, 150, or 300 ppm was 24/50, 15/35, 26/65, and 1/50; for females receiving 0, 150, or 300 ppm, survival was 36/50, 13/35, and 6/64. All female rats receiving 600 ppm died by week 89. The decreased survival in treated groups was due primarily to the development of chemical-related neoplasms. Of the surviving animals, the final mean body weights for males receiving 70 or 150 ppm were 94% and 90% of control and for females receiving 150 or 300 ppm, 99% and 84% of control. These weight differences began in the second year of the studies and were attributed in part to the development of neoplasms in the dosed groups.
At 9 and 15 months, a few neoplasms were seen in the liver, lung, clitoral gland, skin, Zymbal's gland, oral cavity epithelium, and small and large intestine, and the number of neoplasms at these sites increased as the studies progressed. At 2 years, there was a clear carcinogenic response in the skin, Zymbal's gland, and liver of male and female rats, and in the clitoral gland, oral cavity epithelium, small and large intestine, and lung in female rats. Treatment-related increases were also seen in the incidence in neoplasms of the oral cavity epithelium, adrenal gland, and lung of male rats, and in mononuclear cell leukemia and in neoplasms of the mammary gland and adrenal gland in female rats. The incidence of these neoplasms was generally lower, but was significant and considered to be marginally related to chemical treatment. The same neoplastic effects have been previously observed in some or all of the NTP studies with dimethoxybenzidine, dimethylbenzidine, or C.I. Direct Blue 15.
In a standard preincubation protocol, C.I. Acid Red 114 was mutagenic in Salmonella typhimurium strain TA98 in the presence of induced hamster liver S9, and an equivocal response was noted in strain TA100 with hamster liver S9. However, no significant mutagenic activity was noted in strains TA1535 or TA1537 with or without S9 activation. In a modified S. typhimurium gene mutation test which employed reductive metabolism followed by oxidative metabolism with S9 liver enzymes, C.I. Acid Red 114 was strongly mutagenic in strain TA1538. C.I. Acid Red 114 did not induce sister chromatid exchanges or chromosomal aberrations in Chinese hamster ovary cells with or without S9 activation; reductive metabolism was not used in these cytogenetic tests. No increase in sex-linked recessive lethal mutations was observed in germ cells of male Drosophila melanogaster administered C.I. Acid Red 114 by feeding or injection.
Under the conditions of these 2-year drinking water studies, there was clear evidence of carcinogenic activity of C.I. Acid Red 114 for male F344/N rats, as indicated by benign and malignant neoplasms of the skin, Zymbal's gland, and liver. Increased incidences of neoplasms of the oral cavity epithelium, adrenal gland, and lung may have been related to chemical administration. There was clear evidence of carcinogenic activity for female F344/N rats, as indicated by benign and malignant neoplasms of the skin, Zymbal's gland, clitoral gland, liver, oral cavity epithelium, small and large intestines, and lung. Increased incidences of mononuclear cell leukemia, mammary gland adenocarcinoma, and adrenal gland pheochromocytomas may have been related to chemical administration.