title: 13-week subchronic toxicity studies of direct blue 6, direct black 38, and direct brown 95 dyes.
abstract: Thirteen-week subchronic toxicity studies of direct blue 6, direct black 38, and direct brown 95 dyes were conducted by administering the test chemicals in feed to Fischer 344 rats and B6C3F1 mice. Groups of 10 rats and 10 mice of each sex were administered one of the three dyes at one of five concentrations for 13 weeks and then necropsied, beginning the second day after the end of the dosing period. The concentrations used for the rats were 190, 375, 750, 1,500, and 3,000 ppm. The concentrations used for the mice were 750, 1,500, 3,000, 6,000, and 12,500 ppm, except for the female mice administered direct brown 95 dye, which were given concentrations of 375, 750, 1,500, 3,000, and 6,000 ppm. Matched controls consisted of groups of 10 untreated rats and 10 untreated mice of each sex. Mean body weights of the male and female rats administered the two or three highest doses of any one of the test dyes were lower than mean body weights of the corresponding controls, and the depressions in mean body weight were dose related. Mean body weights of the male and female mice administered the highest dose of any one of the test dyes were slightly lower than mean body weights of the corresponding controls; mean body weights of mice administered lower doses were generally unaffected. All male and female rats administered 3,000 ppm of any one of the dyes or 1,500 ppm of direct brown 95 dye died before the end of the studies. One male administered 1,500 ppm direct blue 6 dye, six males administered 1,500 ppm direct black 38 dye, and two males administered 750 ppm direct brown 95 dye also died by the end of the studies. No deaths occurred in any other dosed group or in any control group of rats. All male and female mice administered the test dyes survived to the end of the studies, except for one male whose death was attributed to bacterial infection. Benzidine and monoacetyl benzidine were detected in the urine of male and female rats and mice administered the test dyes, but neither compound was detected in the urine of control rats and mice. Determinations of methemoglobin in control and dosed rats showed no differences. In rats, neoplastic lesions occurred only in dosed groups and consisted of hepatocellular carcinomas and neoplastic nodules of the liver. The incidences of hepatocellular carcinomas in female rats administered 3,000 ppm direct blue 6 dye (4/9) and male rats administered 1,500 ppm direct black 38 dye (4/9) were significant (P=0.033) when related to the incidences of the tumors in the corresponding controls (0/10); hepatocellular carcinomas were also observed in two male rats administered 1,500 ppm direct blue 6 dye and in one female rat administered 1,500 ppm direct brown 95 dye. No control rats from any of the three studies developed hepatocellular carcinomas. When incidences of neoplastic nodules were combined with those of hepatocellular carcinomas, the significance increased to P<0.001 for male rats administered 1,500 ppm direct blue 6 dye, P=0.001 for females administered 3,000 ppm direct blue 6 dye, P<0. 001 for males administered 1, 500 ppm direct black 38 dye, and P=0.007 for females administered 1,500 ppm direct brown 95 dye. No controls developed neoplastic nodules. Female rats administered direct black 38 dye developed no hepatocellular carcinomas, but had an incidence of neoplastic nodules of 5/10, with a significance of P=0.016. Male rats administered direct brown 95 dye developed neither hepatocellular carcinomas nor neoplastic nodules, but as indicated below, had significant incidences of preneoplastic lesions. The failure of groups of rats administered 3,000 ppm dye to develop tumors when other groups administered 1,500 ppm did develop tumors may be due to earlier deaths at the higher dose. Preneoplastic hepatic lesions (basophilic foci) occurred only in dosed rats and did not occur in the controls. The incidences of the basophilic foci were significant (P<0.033) in male (4/9) and female (7/9) rats administered 3,000 ppm direct blue 6 dye and in male rats (7/8) administered 1, 500 ppm direct brown 95 dye. Basophilic foci also occurred, at lower incidences, in males (1/10) administered 1,500 ppm direct blue 6 dye, in males (3/9) administered 1,500 ppm direct black 38 dye, in females (1/8) administered 3,000 ppm direct black 38 dye, in males administered 750 ppm (3/10) or 3,000 ppm (2/9) direct brown 95 dye, and in females administered 1,500 ppm (3/8) or 3,000 ppm (3/8) direct brown 95 dye. When incidences of foci of cellular alteration, a possible preneoplastic lesion, were added to those of basophilic foci, significance occurred in additional dosed groups. In mice, no neoplastic lesions occurred in the liver or other tissues of groups administered the different dyes. However, three mice administered 12,500 ppm direct black 38 dye and one mouse administered 12,500 ppm direct brown 95 dye had foci of cellular alteration, in which the cells were basophilic when compared with surrounding normal cells. It is concluded that under the conditions of these 13-week subchronic toxicity studies, direct blue 6 and direct black 38 dyes were carcinogenic in male and female Fischer 344 rats and direct brown 95 was carcinogenic in female rats; all three dyes induced hepatocellular carcinomas and neoplastic nodules in the liver. The test dyes were not carcinogenic for B6C3F1 mice in the 13-week subchronic toxicity studies.