Toxicology and Carcinogenesis Studies of3,3'-Dimethoxybenzidine Dihydrochloride (CAS No. 20325-40-0) in F344/NRats(Drinking Water Studies)
Chemical Formula: C14H16N2O2 · 2HCl
3,3'-Dimethoxybenzidine dihydrochloride is an off-white powder with amelting point of 274 degrees C. 3,3'-Dimethoxybenzidine is used principally as anintermediate in the production of commercial bisazobiphenyl dyes for coloringtextiles, paper, plastic, rubber, and leather. In the synthesis of thebisazobiphenyl dyes, the amine groups of 3,3'-dimethoxybenzidine arechemically linked with other aromatic amines. A small quantity of3,3'-dimethoxybenzidine is also used as an intermediate in the production of o-dianisidinediisocyanate, which is used in isocyanate-based adhesive systems and as acomponent of polyurethane elastomers.
3,3'-Dimethoxybenzidine dihydrochloride was evaluated in toxicity andcarcinogenicity studies as part of the National Toxicology Program'sBenzidine Dye Initiative. This Initiative was designed to evaluate the representativebenzidine congeners and benzidine congener-derived and benzidine-deriveddyes. 3,3'-Dimethoxybenzidine dihydrochloride was nominated for study becauseof the potential for human exposure during production of bisazobiphenyl dyes andbecause benzidine, a structurally related chemical, is a known humancarcinogen.
Toxicology and carcinogenesis studies were conducted by administering3,3'-dimethoxybenzidine dihydrochloride (greater than 97.5% pure) indrinking water to groups of F344/N rats of each sex for 14 days, 13 weeks, 9 months,or 21-months. The 21-month studies were intended to last 24 months but wereterminated early because of rapidly declining survival due to neoplasia.Studies were performed only in rats because similar studies are beingperformed in mice at the National Center for Toxicology Research. Genetic toxicologystudies were conducted with Salmonella typhimurium, Chinesehamster over (CHO) cells, and Drosophila melanogaster.
All rats receiving drinking water concentrations up to4,500 ppm lived to the end of the studies. Rats that received water containing4,500 ppm 3,3'-dimethoxybenzidine dihydrochloride lost weight. Waterconsumption decreased with increasing concentration of chemical and at4,500 ppm was less than one-fourth that by the controls. Lymphoid depletion of thethymus in males and hypocellularity of the bone marrow in males and femaleswere seen at the 4,500-ppm concentration, but not at the next lowerconcentration or in controls.
All rats receiving concentrations up to 2,500 ppmlived to the end of the studies. Final mean body weights of rats given drinking watercontaining 1,250 or 2,500 ppm 3,3'-dimethoxybenzidine dihydrochloridewere 5%-20% lower than those of controls. Water consumption at theseconcentrations was 40%-60% that consumed by controls. Compound-related effects in ratsgiven water containing 2,500 ppm 3,3'-dimethoxybenzidine dihydrochlorideincluded a mind exacerbation of naturally occurring nephropathy and the presence of ayellow-brown pigment (lipofuscin) in the cytoplasm of thyroid follicular cells.Serum triiodothyronine (T3) and thyroxin (T4) concentrations in femalesreceiving 330 ppm or more and T4 concentrations in males receiving 170 ppmor more were significantly lower than in controls. Thyrotropin (TSH)concentrations were comparable in controls and exposed rats.
Based on the chemical-related nephropathy and reductions in waterconsumption and body weight gain observed in the 13-week studies, doses for thelong-term studies in male and female rats were 0 or 330 ppm 3,3'-dimethoxybenzidinedihydrochloride in drinking water administered for 9 months and 0, 80, 170, or330 ppm administered for 21 months.
Ten rats of each sex in control and 330-ppm groupswere evaluated after 9 months. Significant decreases in T3 and T4 concentrationswere seen in exposed male and female rats. Other lesions seen in exposedrats included foci of alteration in the liver, a carcinoma of the preputial gland inone male, a carcinoma of the clitoral gland in one female, and carcinoma ofthe Zymbal gland in two males.
Body Weights and Survival in the Twenty-One-Month Studies:
The averageamount of 3,3'-dimethoxybenzidine dihydrochloride consumed per day wasapproximately 6, 12, or 21 mg/kg for low, mid, or high dose male rats and 7, 14, or 23 mg/kgfor low, mid, or high dose female rats. Mean body weights of male and female rats began to decrease relative to those of controls after about 1 year ofexposure at 170 or 330 ppm and were 6%-22% lower for males and 7%-17%lower for females. Survival of rats exposed to 3,3'-dimethoxybenzidinedihydrochloride was reduced because animals were dying with neoplasms or being killed in amoribund condition (survival at 21 months--male: control, 44/60, 73%; lowdose, 8/45, 18%; mid dose, 0/75; high dose, 0/60; female: 45/60, 75%; 15/45, 33%;6/75, 8%; 0/60). Because of these early compound-related deaths, thestudies were terminated at 21 months.
Nonneoplastic and Neoplastic Effects in the Twenty-One-Month Studies:
Increased incidences of several nonneoplastic lesions were observed inexposed rats, including hematopoietic cell proliferation in the spleen and cystic andcentrilobular degeneration and necrosis of the liver. Neoplasms attributed to3,3'-dimethoxybenzidine dihydrochloride exposure were observed in rats atmany tissue sites, including the skin, Zymbal gland, preputial and clitoral glands,oral cavity, small and large intestines, liver, brain, mesothelium, mammarygland, and uterus/cervix. The incidences of these neoplasms in male andfemale rats are given in the abstract summary table (see page 5 of the TechnicalReport).
3,3'-Dimethoxybenzidine was mutagenic in S.typhimurium strain TA100 with exogenous metabolic activation and instrain TA98 without activation; a weakly positive response was observed in strainTA1535 with metabolic activation. 3,3'-Dimethoxybenzidine induced sisterchromatid exchanges and chromosomal aberrations in CHO cells with andwithout exogenous metabolic activation. 3,3'-Dimethoxybenzidine did not inducesex-linked recessive lethal mutations in adult male D. melanogasterexposed via feeding or injection.
Under the conditions of these 21-month drinking water studies,there was clear evidence of carcinogenic activity of3,3'-dimethoxybenzidine dihydrochloride for male F344/N rats, as indicatedby benign and malignant neoplasms of the skin, Zymbal gland, preputial gland,oral cavity, intestine, liver, and mesothelium. Increased incidences ofastrocytomas of the brain may have been related to chemical administration.There was clear evidence of carcinogenic activity of3,3'-dimethoxybenzidine dihydrochloride for female F344/N rats, as indicatedby benign and malignant neoplasms of the Zymbal gland, clitoral gland, andmammary gland. Increases in neoplasms of the skin, oral cavity, large intestine, liver,and uterus/cervix were also considered to be related to chemicaladministration of 3,3'-dimethoxybenzidine dihydrochloride.
Synonyms: o-dianisidine dihydrochloride;3,3'-dimethoxy-1,1-biphenyl)-4,4'-diamine dihydrochloride;3,3'-dimethoxy-4,4'-diaminobiphenyl dihydrochloride
Report Date: January 1990