3,3'-Dimethylbenzidine dihydrochloride 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.
3,3'-Dimethylbenzidine dihydrochloride 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
3,3'-dimethylbenzidine dihydrochloride (approximately 99% pure) in
drinking water to groups of F344/N rats of each sex for 14 days, 13 weeks, or 9 or 14 months. The 14-month exposures were planned as 24-month exposures but were terminated early because of rapidly declining animal survival, due primarily to
neoplasia. These studies were performed only in rats because similar studies were being performed in mice at the National Center for Toxicological Research (NCTR). Hematologic and serum chemical analyses and
thyroid hormone determinations were conducted in conjunction with the 13-week and 9-month studies. Genetic toxicology studies were conducted in Salmonella typhimurium, Chinese hamster ovary (CHO) cells, and Drosophila melanogaster. 14-Day Studies: Rats were exposed to
3,3'-dimethylbenzidine dihydrochloride in
drinking water at doses ranging from 600 to 7,500 ppm. All five males and one female in the 7,500 ppm group and 1/5 males in the 5,000 ppm group died. Final mean
body weights were decreased in males receiving 1,250 ppm or more and in all exposed females, and final mean
body weights of animals receiving 2,500 ppm or more were lower than initial weights. Water consumption decreased with increasing chemical concentration. Compound-related effects observed in rats receiving 5,000 ppm or more included minimal to slight hepatocellular
necrosis, accumulation of brown pigment (presumably bile) in individual hepatocytes, increased severity of nephropathy relative to controls, and severe lymphocytic
atrophy of the thymus. Treated animals also showed an increased severity of
atrophy of the bone marrow relative to controls, varying degrees of lymphocytic
atrophy of the mandibular and mesenteric lymph nodes and spleen, increased vacuolization and
necrosis of cells of the adrenal cortex, focal acinar cell degeneration in the pancreas, and, in males, increased immature sperm forms in the testis and epididymis. 13-Week Studies:
3,3'-Dimethylbenzidine dihydrochloride was administered in
drinking water at doses of 300, 500, 1,000, 2,000, and 4,000 ppm. All rats receiving 4,000 ppm and 4/10 males and 1/10 females receiving 2,000 ppm died before the end of the studies. Depressions in final mean
body weight relative to controls ranged from 12% to 48% for males and from 9% to 42% for females. Water consumption decreased with increasing dose. At compound concentrations of 300 to 2,000 ppm, mean water consumption was 29% to 83% of control values. Compound-related effects included an increase in the severity of nephropathy relative to controls; hepatocellular
necrosis and accumulation of brown pigment (presumably bile) in sinusoidal lining cells; lymphocytic
atrophy of the thymus, spleen, and mandibular and mesenteric lymph nodes;
atrophy of the bone marrow in the higher-dose groups; degeneration of pancreatic acinar cells; and, in males, immature sperm forms in the testis and epididymis. Decreases in serum
triiodothyronine (T3) values were observed in exposed females, and decreases in mean
thyroxin (T4) concentrations in exposed males and females; no significant changes were observed in
thyroid stimulating hormone (TSH) levels in exposed rats. Based on the decreased survival, reductions in water consumption and
body weight gain, and chemical-induced hepatocellular and renal lesions observed in the 13-week studies, the doses selected for the 9- and 14-month
drinking water studies of
3,3'-dimethylbenzidine dihydrochloride were 0, 3
3,3'-dimethylbenzidine dihydrochloride were 0, 30, 70, and 150 ppm. Seventy rats of each sex were used in the control group, 45 in the low-dose group, 75 in the mid-dose group, and 70 in the high-dose group. 9-Month Studies: Ten rats of each sex in the control and 150 ppm dose groups were evaluated after 9 months. Chemical-related effects observed in exposed animals included alveolar/
bronchiolar carcinoma in one male,
basal cell carcinoma of the skin in one male, a
squamous cell carcinoma of the oral cavity in one female, preputial gland
carcinoma in two males, clitoral gland
carcinoma in three females,
adenocarcinoma of the small intestine in two males, Zymbal's gland
carcinoma in two males and three females,
hepatocellular carcinoma in two males, and
adenomatous polyps of the large intestine in three males. Other effects seen in dosed rats included focal cellular alteration in the liver, lymphoid
atrophy in the spleen, and increased severity of nephropathy relative to controls. An increase in serum T3 values was observed in exposed males, and a decrease in mean T4 concentrations in exposed males and females. TSH concentrations were increased in exposed male and female rats.
Body Weights and Survival in the 14-Month Studies: The average amount of
3,3'-dimethylbenzidine dihydrochloride consumed per day was approximately 1.8, 4.0, or 11.2, mg/kg for low-, mid-, or high-dose male rats and 3.0, 6.9, or 12.9 mg/kg for low-, mid-, or high-dose female rats. The mean
body weight of high-dose males was about 85% of the control value by week 28. By the end of the study, mean
body weights of low-, mid-, and high-dose males were 97%, 92%, and 70% of the control values, respectively. Mean
body weights of high- and mid-dose females were about 85% of the control values at week 32 and week 44, respectively. At the end of the study, mean
body weights of exposed females were about 94%, 81%, and 74% of the control values for low-, mid-, and high-dose groups, respectively. Because of extensive
neoplasia, many exposed males and females were dying or were sacrificed moribund in the first year, and all high-dose males died by week 55. The studies were terminated at weeks 60 to 61, at which time the group survivals were male: control, 60/60, low dose, 41/45; mid dose, 50/75; high dose, 0/60; female: 59/60; 39/45; 32/75; 10/60. Nonneoplastic Effects in the 14-Month Studies: Increases in nonneoplastic lesions in dosed rats included cystic degeneration and foci of cellular alteration in the liver; exacerbation of nephropathy; and focal or multifocal
hyperplasia of the Zymbal's gland, preputial and clitoral glands, and alveolar epithelium. Neoplastic Effects in the 14-Month Studies:
Neoplasms were observed in exposed rats at many sites: skin, Zymbal's gland, preputial and clitoral glands, liver, oral cavity, small and large intestine, mammary gland, lung, brain, and mesothelium. The incidence of these neoplastic effects in male and female rats is summarized in the table at the end of this section (see page 8 of the Technical Report). Genetic Toxicology:
3,3'-Dimethylbenzidine dihydrochloride was mutagenic in Salmonella typhimurium strain TA98 with exogenous metabolic activation; it was not mutagenic in strains TA100, TA1535, or TA97 with or without activation.
3,3'-Dimethylbenzidine dihydrochloride induced sister-chromatid exchanges (CHO) and
chromosomal aberrations in CHO cells in the absence of exogenous metabolic activation; these effects were not evident in test with S9 activation. Sex-linked recessive lethal mutations were induced in germ cells of adult male Drosophila melanogaster administered
3,3'-dimethylbenzidine dihydrochloride in feed or by injection. No reciprocal translocations occurred in D. melanogaster germ cells following exposure to
3,3'-dimethylbenzidine dihydrochloride. Conclusions: Under the conditions of these 14-month
drinking water studies, there was clear evidence of carcinogenic activity of
3,3'-dimethylbenzidine dihydrochloride for male F344/N rats, as indicated by benign and
malignant neoplasms of the skin, Zymbal's gland, preputial gland, liver, oral cavity, small and large intestine, lung, and mesothelium. Increased incidences of
neoplasms of the brain 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, small and large intestine, mammary gland, and lung. Increased incidences of
neoplasms of the brain and mononuclear cell
leukemia may have been related to chemical administration. Synonyms:
o-tolidine dihydrochloride; 3,3'-dimethylbiphenyl-4,4'-diamine dihydrochloride; 3,3'-dimethylbiphenyl-4,4'-biphenyldiamine dihydrochloride; 4,4'-diamino-3,3'-dimethylbiphenyl dihydrochloride