title: NTP Technical Report on the Toxicology and Carcinogenesis Studies of 2,4-Diaminophenol Dihydrochloride (CAS NO. 137-09-7) in F344/N Rats and B6C3F1 Mice (Gavage Studies).
abstract: 2,4-Diaminophenol dihydrochloride is used in the manufacture of dyes and as a color accelerator in photographic developers. Toxicology and carcinogenesis studies were conducted by administering 2,4-diaminophenol dihydrochloride (greater than 97% pure) in corn oil by gavage to F344/N rats and B6C3F1 mice for 16 days, 13 weeks, and 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, mouse lymphoma cells, Drosophila melanogaster, and Chinese hamster ovary cells. 16-Day Studies: Groups of five rats of each sex received doses of 0, 6, 13, 25, 50, or 100 mg/kg and groups of five mice of each sex received doses of 0, 13, 25, 50, 100, or 200 mg/kg of 2,4-diaminophenol dihydrochloride in corn oil by gavage. There were no deaths among rats during the 16-day studies and chemical exposure had no effect on final mean body weights. Organ weight differences were unrelated to chemical exposure. Renal tubule necrosis was present in male and female rats in the 25, 50, and 100 mg/kg dose groups. All male and four female mice that received 200 mg/kg, all males and three females that received 100 mg/kg, and two female mice that received 50 mg/kg died before the end of the studies. Final mean body weights of surviving dosed mice were similar to controls. Absolute liver weights were increased in female mice that received 50 and 100 mg/kg and relative liver weights were increased in all female dose groups. Renal tubule necrosis was present in the 100 mg/kg male and female dose groups. 13-Week Studies: Groups of ten male and female rats received doses of 0, 12, 25, 50, 100, or 200 mg/kg and groups of ten male and female mice received doses of 0, 5, 9, 19, 38, or 75 mg/kg of 2,4-diaminophenol dihydrochloride in corn oil by gavage. All female and nine male rats that received 200 mg/kg and four males and one female in the 100 mg/kg rat groups died before the end of the studies. Final mean body weights of male rats that received 50 or 100 mg/kg were significantly lower than controls. Relative kidney weights were significantly increased in all male dose groups; absolute and relative kidney and liver weights were significantly increased in females that received 50 and 100 mg/kg. Lesions in rats associated with chemical exposure included renal tubule necrosis in males that received 25 mg/kg and above and in females that received 100 or 200 mg/kg. Forestomach ulcers with acanthosis and hyperkeratosis were present at 50 mg/kg and above in both sexes. Pigment, presumably 2,4-diaminophenol dihydrochloride or a metabolite, was present in the duodenum and within the renal tubule epithelium of all dose groups. Splenic lymphoid depletion was present in both sexes and bone marrow hyperplasia was present in groups of males that received 100 or 200 mg/kg. Hemosiderin was present in Kupffer cells of males that received 100 or 200 mg/kg. The severity of splenic extramedullary hematopoiesis increased with dose in males. There were no deaths among mice attributed to chemical exposure. Final mean body weight of male mice that received 75 mg/kg was 8% lower than for the controls. Absolute and relative liver weights were increased in males that received 9 mg/kg or above and in all female dose groups. Absolute and relative kidney weights of 38 and 75 mg/kg male mice and all dosed female mice were significantly increased. Absolute heart weights were increased in the 19, 38, and 75 mg/kg female groups. Lesions in 75 mg/kg mice included increased incidences of renal tubule regeneration; forestomach acanthosis and hyperkeratosis occurred in 38 and 75 mg/kg groups. Hemosiderin was present in the spleen of mice from all dose groups and in hepatic Kupffer cells of female mice that received 38 or 75 mg/kg. Pigment was present in the duodenum of males and females that received 9 mg/kg and above and in the renal tubule epithelium of both sexes that received 75 mg/kg. 2-Year Studies: The 2-year studies were conducted by administering 2,4-diaminophenol dihydrochloride in corn oil by gavage to groups of 60 male and 60 female F344/N rats and B6C3F1 mice. Tence. Ten animals from each dose group were evaluated after 15 months. Rats received doses of 0, 12.5, or 25 mg/kg; mice received doses of 0, 19, or 38 mg/kg. Body Weight and Survival in the 2-Year Studies: Mean body weights of high-dose male and female rats and of high-dose male mice were lower than those of the respective controls. Survival of dosed rats and mice was similar to controls throughout the 2-year studies. Neoplastic and Nonneoplastic Effects in the 2-Year Studies: The severity of nephropathy increased in high-dose male rats. The incidence of renal tubule cell hyperplasia was increased in rats that received 2,4-diaminophenol dihydrochloride; however, there were no renal tubule cell neoplasms attributed to chemical exposure. Renal tubule cell hyperplasia was present in three high-dose male mice and a renal tubule adenoma was present in one of the high-dose males with hyperplasia; two additional high-dose males also had renal tubule adenomas. These are uncommon neoplasms in male mice and were considered related to chemical exposure. In a supplemental review of step-sectioned kidneys, six additional hyperplasia were seen in high-dose male mice and adenomas in three other high-dose males. A tubule cell carcinoma was present in one low-dose female mouse. The incidences of several nonneoplastic kidney lesions, including necrosis and renal tubule regeneration, were also increased in male and female mice. Pigment was observed in the lamina propria of the duodenum, the submucosa of the forestomach, and in pancreatic and mesenteric lymph nodes in dosed rats. Hemosiderin was present in the renal tubule epithelial cells of low- and high-dose rats. The incidence of forestomach acanthosis was increased in dosed male mice and pigment was present in liver Kupffer cells, in the lamina propria of the duodenum, and in the mesenteric lymph nodes of male and female mice. Pigment and hemosiderin were also present in the renal tubule epithelium of dosed mice. Genetic Toxicology: 2,4-Diaminophenol dihydrochloride was mutagenic in the Salmonella typhimurium strain TA98 in the presence of exogenous metabolic activation (S9); it was not mutagenic in strain TA98 in the absence of S9, nor was it mutagenic in strains TA100, TA1535, or TA1537 with or without S9. 2,4-Diaminophenol dihydrochloride was positive in the mouse lymphoma assay for induction of trifluorothymidine resistance in L5178Y cells without S9; it was not tested with S9. No induction of sister chromatid exchanges or chromosomal aberrations was observed in Chinese hamster ovary cells treated with 2,4-diaminophenol dihydrochloride with and without S9. In the Drosophila melanogaster sex-linked recessive lethal assay, 2,4-diaminophenol dihydrochloride gave equivocal results when administered in the feed and negative results when administered by injection. Conclusions: Under the conditions of these 2-year gavage studies, there was no evidence of carcinogenic activity of 2,4-diaminophenol dihydrochloride in male or female F344/N rats that received 12.5 or 25 mg/kg. There was some evidence of carcinogenic activity of 2,4-diaminophenol dihydrochloride in male B6C3F1 mice based on increased incidences of renal tubule adenomas; there was no evidence of carcinogenic activity of 2,4-diaminophenol dihydrochloride in female B6C3F1 mice that received 19 or 38 mg/kg. Administration of 2,4-diaminophenol dihydrochloride to rats was associated with increased severity of nephropathy in males and females, increased incidence of nephropathy in females, and focal renal tubule hyperplasia in males and females. In mice, chemical exposure was associated with renal tubule necrosis and regeneration in males and females and forestomach acanthosis in males. Synonyms: Acrol, amidol, dianol