1. Exposure Data

1.1. Identification of the agents

1.1.1. Benzidine

• Chem. Abstr. Serv. Reg. No.: 92-87-5
• Chem. Abstr. Serv. Name: [1,1′-Biphenyl]-4,4′-diamine

• C₁₂H₁₂N₂
• Relative molecular mass: 184.24
• Description: White to slightly reddish, crystalline powder; darkens on exposure to air and light (O’Neil, 2006).
• Solubility: Slightly soluble in water, diethyl ether, and dimethyl sulfoxide (DMSO); soluble in ethanol (Lide, 2008)

1.1.2. Benzidine dihydrochloride

• Chem. Abstr. Serv. Reg. No.: 531-85-1
• Chem. Abstr. Serv. Name: [1,1′-Biphenyl]-4,4′-diamine, dihydrochloride

• C₁₂H₁₀N₂.2HCl
• Relative molecular mass: 257.18
• Description: Crystalline solid (O’Neil, 2006)
• Solubility: Soluble in water and alcohol (O’Neil, 2006)

1.3. Human exposure

1.3.1. Occupational exposure

Estimates of numbers of workers potentially exposed to benzidine have been published by CAREX (CARcinogen EXposure) in Europe. CAREX is an international information system that provides selected exposure data and documented estimates of the number of exposed workers by country, carcinogen, and industry (Kauppinen et al., 2000). Based on data on occupational exposure to known and suspected carcinogens collected from 1990 to 1993, it is estimated from the CAREX database that 6900 workers were exposed to benzidine in the European Union. Table 1.1 presents the results for benzidine by industry in the EU (CAREX, 1999).

Table 1.1

Estimated numbers of workers exposed to benzidine in the European Union.

NIOSH (1984) estimated from the US National Occupational Exposure Survey (1981–83) that 1554 workers (including 426 women) were potentially exposed to benzidine and 2987 workers (2464 women) to benzidine dihydrochloride.

Studies that reported airborne or urinary concentrations and results of dermal wipes of benzidine or benzidine derivatives in the benzidine-based dye industry have been reviewed (IARC, 2010).

1.3.2. Non-occupational exposure

Because benzidine may be produced only for captive consumption (i.e. in-house by the producer only, in closed systems with stringent controls), its direct release into the environment is expected to be low.

Benzidine-based dyes can contain various amounts of benzidine as a contaminant. The general population can be exposed to benzidine when in contact with consumer goods containing benzidine or benzidine based-dyes such as leather products (Ahlström et al., 2005), clothes and toys (Garrigós et al., 2002). Some food colourants such as tartrazine and sunset yellow FCF have been reported to contain trace amounts of benzidine (< 5 to 270 ng/g) (Lancaster & Lawrence, 1999).

2. Cancer in Humans

In a previous IARC Monograph (IARC, 2010) it was concluded that there is sufficient evidence in humans for the carcinogenicity of benzidine in the human bladder. Numerous case reports from different countries have been published (IARC, 1972, 1982, 1987, 2010). In one extreme instance, all five of a group of workers permanently employed in the manufacture of benzidine for 15 years or more developed bladder cancer (IARC, 1982). Vigliani & Barsotti (1962) reported on 20 workers with tumours of the urinary bladder between 1931 and 1948 among 83 Italian dyestuff workers involved in benzidine production and use. Case et al. (1954) found 10 bladder-cancer deaths among dyestuff workers exposed only to benzidine (standardized mortality ratio (SMR) 13.9 [95%CI: 6.7–25.5]). In benzidine-exposed workers in the chemical dye industry in China, the morbidity from bladder cancer increased with increasing duration of exposure (p for trend < 0.01) (Sun & Deng, 1980), while in a cohort of benzidine manufacturers in the USA, risks were significantly elevated for those with ≥2 years exposure to benzidine (SMR 13.0 [95%CI: 4.8–28.4]) (Meigs et al., 1986). In a cohort of dyestuff workers in Torino, Italy, the SMR was 100.8 [95%CI: 60.8–167.2] during exposure and 14.8 [95%CI: 71–31.0] at 20 or more years after exposure ceased (Piolatto et al., 1991). In a study of workers from a chemical manufacturing plant in Shanghai, China, an interaction was found between benzidine exposure and cigarette smoking in the development of bladder cancer (Wu, 1988). Relative to those who did not smoke and had no exposure to benzidine, the risks (RR) for bladder cancer were 6.2 (P = 0.05) for smokers who were not exposed to benzidine; 63.4 (P < 0.05) for non-smokers exposed to benzidine; and 152.3 (P < 0.01) for smokers exposed to benzidine. In another study of Chinese workers in benzidine production and use facilities, the odds ratios (OR) for bladder cancer were 1.0, 2.7 (1.1–6.3) and 4.4 (1.8–10.8) for low, medium and high cumulative exposure to benzidine, respectively, after adjustment for life-time cigarette smoking (Carreón et al., 2006b). In a case–control study in Canada, excesses of renal cell cancer in relation to duration of exposure to benzidine (P < 0.004) were noted, but other consistently supporting data were not found (Hu et al., 2002).

Overall, case reports and epidemiological investigations from several countries show strong and consistent associations between benzidine exposure and risk for bladder cancer.

3. Cancer in Experimental Animals

Studies on the carcinogenicity of benzidine in the mouse, rat, hamster, rabbit, dog, and frog by oral, subcutaneous injection, intraperitoneal injection, or inhalation routes of exposure have been reviewed by previous IARC Working Groups (IARC, 1972, 1982, 1987, 2010). There have been no additional carcinogenicity studies in animals reported since the most recent evaluation (IARC, 2010). Results of adequately conducted carcinogenicity studies are summarized in Table 3.1.

Table 3.1

Carcinogenicity studies of benzidine in experimental animals.

Benzidine and its dihydrochloride were adequately tested for carcinogenicity by oral administration (feed, drinking-water or gavage) in eight experiments in mice and one experiment in rats; by subcutaneous injection in one experiment in rats and one experiment in frogs; and in rats in one experiment by intraperitoneal injection.

Following oral administration to male and female mice, newborn and adult, of different strains, benzidine significantly increased the incidence of hepatocellular tumours (benign and/or malignant) in both sexes (Vesselinovitch et al., 1975, 1979; Miyakawa & Yoshida, 1980; Vesselinovitch, 1983; Nelson et al., 1982; Littlefield et al., 1983, 1984). Oral administration of benzidine caused a markedly increased incidence of mammary carcinomas in female rats (Griswold et al., 1968). Subcutaneous administration of benzidine or its sulfate to male and female rats produced a high incidence of external auditory canal carcinomas (Spitz et al., 1950), while subcutaneous administration of benzidine to frogs caused an increase in tumours of the liver and haematopoietic system (Khudoley, 1977). The intraperitoneal administration of benzidine to female rats resulted in an increase in the incidence of mammary gland adenocarcinomas and combined benign and malignant Zymbal gland tumours (Morton et al., 1981). Other studies were found to be inadequate for evaluation.

4. Other Relevant Data

A general section on “Aromatic amines: metabolism, genotoxicity, and cancer susceptibility” appears as Section 4.1 in the Monograph on 4-aminobiphenyl in this Volume.

Benzidine, N-acetylbenzidine, and N,N′-diacetylbenzidine have been detected in the urine of workers exposed to benzidine. The predominant DNA adduct identified in exfoliated bladder cells was N′-(deoxyguanosin-8-yl)-N-acetylbenzidine (Rothman et al., 1996a). Thus, N-monoacetylation of benzidine does not interfere with the formation of a DNA-reactive intermediate, and may occur before the cytochrome P450-catalysed formation of N′-hydroxy-N-acetylbenzidine. In contrast, N,N′-diacetylation may be a detoxification pathway. N-Acetylbenzidine may be N-glucuronidated or N-hydroxylated in the liver. In the bladder, the N′-hydroxyl-N-acetylbenzidine or N′-acetoxy-N-acetylbenzidine formed by NAT-mediated O-acetylation may react with DNA to form covalent adducts. NAT1 is more efficient than NAT2 in catalysing the N-acetylation of benzidine or of N-acetylbenzidine (Zenser et al., 1996). At low exposure levels, N-acetylation of benzidine is favoured over that of N-acetylbenzidine. Human neutrophils can also form N′-(deoxyguanosin-8-yl)-N-acetylbenzidine from N-acetylbenzidine by a reaction catalysed by myeloperoxidase (Lakshmi et al., 2000). N′-hydroxy-N-acetylbenzidine may also be formed through peroxidative activation by prostaglandin H synthase (Zenser et al., 1999a, b). Levels of benzidine-related DNA adducts in exfoliated urothelial cells among exposed workers were not affected by acetylator phenotype (Rothman et al., 1996a), or GSTM1 genotype (Rothman et al., 1996b).

For several benzidine-based azo dyes, both metabolism and molecular changes identical to those of benzidine have been observed. Metabolic conversion of Direct Black 38, Direct Blue 6, and Direct Brown 95 to benzidine has been observed in the Rhesus monkey (Rinde & Troll, 1975). Azoreductase activity in intestinal bacteria and in the liver catalyses the formation of benzidine from benzidine-based dyes (Cerniglia et al., 1982; Bos et al., 1986).

Benzidine is a multiorgan carcinogen in experimental animals; it induces bladder tumours in dogs, liver tumours in mice and hamsters, and mammary gland tumours in rats. In the presence of a liver-derived metabolic activation system – which in some cases leads to reductive metabolism followed by oxidative metabolism – benzidine and benzidine-based dyes (e.g. Direct Black 38, CI Acid Red 114, CI Direct Blue 15, and CI Pigment Red) were mutagenic in several strains of S. typhimurium. Benzidine, N-acetylbenzidine, and N,N′-diacetylbenzidine have been measured in the urine of workers exposed to Direct Black 38, and benzidine- or 4-ABP-related haemoglobin adducts have been measured in blood (Dewan et al., 1988; Beyerbach et al., 2006). Significant increases in the incidence of chromosomal aberrations in peripheral lymphocytes have been observed in workers exposed to benzidine or benzidine-based dyes (Mirkova & Lalchev, 1990). In workers exposed to benzidine, the accumulation of mutant p53 protein increased with increasing exposures (Xiang et al., 2007). Similarly, benzidine induced DNA lesions in TP53 in the bladder, liver, and lung of exposed rats (Wu & Heng, 2006), increased the frequency of micronucleated bone-marrow cells and induced unscheduled DNA synthesis in mice, and increased DNA strand-breaks in the liver of exposed rats.

Based on bio-monitoring studies in workers, animal carcinogenicity data and genotoxicity data, it is reasonable to use the same carcinogenic hazard classification for benzidine-based dyes that are metabolized to benzidine as for benzidine.

5. Evaluation

There is sufficient evidence in humans for the carcinogenicity of benzidine. Benzidine causes cancer of the urinary bladder.
There is sufficient evidence in experimental animals for the carcinogenicity of benzidine.

There is strong mechanistic evidence indicating that the carcinogenicity of benzidine in humans operates by a genotoxic mechanism of action that involves metabolic activation, formation of DNA adducts, and induction of mutagenic and clastogenic effects. Metabolic activation to DNA-reactive intermediates occurs by multiple pathways including N-oxidation in the liver, O-acetylation in the bladder, and peroxidative activation in the mammary gland and other organs.

Benzidine is carcinogenic to humans (Group 1).

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