53

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

H2N NH2

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

NH2 H . 2HCl 2N

C12H10N2.2HCl 

Relative molecular mass: 257.18

Description: Crystalline solid (O’Neil, 

2006)

Solubility: Soluble in water and alcohol 

(O’Neil, 2006)

1.2 Uses

Benzidine has been used since the 1850s as the 

reagent base for the production of a large number 

of dyes, particularly azo dyes for wool, cotton, and 

leather (IARC, 2010). In the past, benzidine also 

has been used in clinical laboratories for detection of blood, as a rubber compounding agent, 

in the manufacture of plastic films, for detection 

of hydrogen peroxide in milk, and for quantitative determination of nicotine. Most of these uses 

have been discontinued because of toxicological 

concerns. Some dyes used as stains for microscopy and similar laboratory applications may 

contain benzidine as an impurity (ATSDR, 2001; 

NTP, 2005).

Manufacture of benzidine is prohibited in 

several individual countries (e.g. Japan, Republic 

of Korea, Canada, and Switzerland) and in 

Europe through European Union (EU) legislation. It has not been manufactured on a large 

scale for commercial purposes in the United 

BENZIDINE

Benzidine was considered by previous IARC Working Groups in 1987 and 2008 (IARC, 1987, 

2010). Since that time new data have become available, which have been incorporated in 

this Monograph, and taken into consideration in the present evaluation.
IARC MONOGRAPHS – 100F

States of America (USA) since 1976, although 

small quantities remain available for diagnostic 

testing. It is reportedly produced and/or supplied 

in research quantities in Germany, Hong Kong 

Special Administrative Region, India, the 

People’s Republic of China (China), Switzerland, 

and the USA (IARC, 2010). Production and use 

of benzidine in dye production has been reported 

in some developing countries, as has the transfer 

of benzidine production from other European 

countries to the former Serbia and Montenegro 

and to the Republic of Korea (Carreón et al.,

2006a).

In 1994, the German Government prohibited the use of certain azo dyes in consumer 

goods that come in direct, prolonged contact 

with human skin (e.g. clothing, bedding, footwear, gloves, etc.). The dyes affected are those 

that, after reduction of one or more azo groups, 

may release one or more of 20 specific aromatic 

amines (including benzidine) in detectable 

concentrations (i.e. > 30 parts per million (ppm)). 

In 2002, a EU Directive (76/769/EEC) expanded 

coverage to articles that come in contact with 

the oral cavity and added two amines to the list 

(Ahlström et al., 2005; ETAD, 2008). The US 

Food and Drug Administration limits benzidine content in food colourants to 1 part per 

billion (ppb). While exposure via ingestion is 

considered highly unlikely, other impurities in 

synthetic colouring agents may be metabolized 

to benzidine after ingestion (ATSDR, 2001).

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).

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 

54

Table 1.1 Estimated numbers of workers 

exposed to benzidine in the European Union

Industry, occupational activity

Manufacture of textiles 160

Education services 3700

Research and scientific institutes 1900

Medical, dental, other health & 

veterinary services

1300

TOTAL 6900

From: CAREX (1999)
Benzidine

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.

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 

55
IARC MONOGRAPHS – 100F

56 Table 3.1 Carcinogenicity studies of benzidine in experimental animals

Species, strain (sex)DurationReference

RouteDosing regimen,

Animals/group at start

Incidence of tumours Significance Comments

Mouse, B6C3F1

(M)90 wkVesselinovitch et al. (1975)

FeedFour groups of 50 M mice were fed 150 ppm benzidine as the dihydrochloride (certified ACS grade) in the diet for 45 wk. Groups of 50 mice were sacrificed at 45, 60, 75, and 90 wk of age. A group of 100 male mice that were sacrificed at 90 wk of age served as untreated controls

Hepatocellular carcinomas: 1/98, 2/50, 5/50$, 

14/50*, 24/50*Hepatocellular adenomas: 1/98, 6/50$, 15/50*, 

17/50*, 11/50*

*[

P < 0.0001], $

[P < 0.05]

Mouse, B6C3F1

(M)90 wkVesselinovitch et al. (1975)

FeedThree groups of 50 M mice were fed 150 ppm benzidine as the dihydrochloride salt (certified ACS grade) in the diet for 39, 54, or 84 wk. All animals were sacrificed at 90 wk of age. A group of 100 M mice that were sacrificed at 90 wk of age served as untreated controls.

Hepatocellular tumours [benign and malignant]:1/98, 35/50*, 25/50*, 44/50*

*[

P < 0.0001]

Mouse, B6C3F1

(M, F)90 wkVesselinovitch et al. (1979)

FeedGroups of 43–65 M and F offspring mice were fed and/or exposed to a diet containing 150 ppm benzidine as the dihydrochloride (1) through fed mothers from the 12th d of gestation (prenatal) to delivery; (2) through fed mothers with litters from delivery to weaning; (3) to offspring from weaning to 90 wk of age; (4) during the prenatal and pre-weaning period; or (5) prenatally, during pre-weaning and in adulthood. Groups 98 M and 100 F mice served as untreated controls.

Hepatocellular tumours [benign and malignant]:Group 1–17/55* (M), 2/62 (F)Group 2–62/65*, 2/43Group 3–25/44*, 48/50*

Group 4–49/49*, 12/48*

Group 5–50/50*, 47/50*Controls–1/98, 0/100

*[

P < 0.0001] Age NR

Purity NR

Mouse, B6C3F1

(M, F)90 wkVesselinovitch (1983)

FeedGroups of pregnant F or weanling M + F B6C3F1

mice were fed diets containing 150 ppm of benzidine as the dihydrochloride:group 1–prenatal exposure (12th d of gestation to delivery) group 2–pre-weaning exposure (delivery to weaning)group 3–from weaning to 90 wkGroups of 98 M + 96 F mice were sacrificed at 90 wk and served as controls.

Hepatocellular adenomas and carcinomas:Group 1: 8/36* (M), 2/56 (F)Group 2: 35/52$, 9/43$

Group 3: 22/26$, 16/25$Controls: 1/98, 0/96Hepatocellular carcinomas:Group 1: 3/36* (M), 1/56 (F)Group 2: 26/52$, 5/43*

Group 3: 17/26$, 16/25$

Controls: 0/98, 0/96

*[

P < 0.05], $

[P < 0.0001)

Liver was the only tissue examined.Purity NR
Benzidine

57

Species, strain (sex)DurationReference

RouteDosing regimen,

Animals/group at start

Incidence of tumours Significance Comments

Mouse , F1

(C57BL/6Jf C3Hf/Nctr females × BALB/cStCrLfC3Hf/Nctr males) and mono-hybrid (F1

females and F1

males) up to 80 wkNelson et al. (1982)

Drinking-waterGroups of M + F mice (F1) and MC mice 

were given drinking-water containing 0, 30, 60, 120, 200, or 400 ppm of benzidine as the dihydrochloride. Groups of mice were sacrificed after 40, 60, or 80 wk of treatment.

Hepatocellular adenomas and carcinomas:F1 M, 40 wk–0/49, 0/98, 0/72, 0/51, 3/50, 1/28

F1 F, 40 wk–0/48, 2/98, 1/72, 0/49, 5/50, 13/29F1 M, 60 wk–1/48, 0/73, 4/49, 9/48, 9/47, 12/23

F1 F, 60 wk–1/48, 3/74, 4/52, 24/58, 54/61, 41/41F1 M, 80 wk–0/46, 5/44, 6/47, 13/45, 8/21, 16/20F1 F, 80 wk–0/47, 9/43, 23/43, 34/37, 9/9, 0/1

MC M, 40 wk–0/50, 1/101, 0/71, 1/48, 0/52, 1/27MC F, 40 wk–0/48, 0/97, 0/72, 3/51, 6/50, 10/26MC M, 60 wk–0/48, 3/69, 3/46, 8/50, 8/43, 7/26MC F, 60 wk–1/48, 7/72, 12/54, 26/56, 47/60, 33/38MC M, 80 wk–2/45, 2/41, 7/43, 14/44, 7/19, 11/17 MC F, 80 wk–0/48, 12/43, 20/42, 31/32, 7/8, 5/6

No statistics provided[significant for many groups]

Age NRPurity NRInitial number of animals unclear

Mouse, F1

(C57BL/6Jf C3Hf/Nctr females × BALB/cStCrLfC3Hf/Nctr males) and mono-hybrid (F1

females and F1

males) 33 mo (Lifetime)Littlefield et al. 

(1983, 1984)

Drinking-waterGroups of 72–120 M + F mice (F1) and MC mice 

were given drinking-water containing 0, 30, 40, 60, 80, 120, or 160 ppm (M) and 0, 20, 30, 40, 60, 80, or 120 ppm (F) of benzidine as the dihydrochloride.

Hepatocellular carcinomas:F1 M: 14/125, 24/119, 30/96, 23/71, 35/71, 51/71, 

49/71F1 F: 3/124, 51/120, 52/95, 45/72, 55/71, 60/69, 64/72MC M: 17/123, 20/118, 20/95, 23/72, 24/71, 37/71, 32/71MC F: 10/125, 54/119, 43/95, 31/71, 37/72, 51/69, 56/72The authors also noted that a dose effect was observed for Harderian gland adenomas in F1

and MC males (

P = 0.02) and females (

P = 0.02) 

and for angiomas of the uterus in F1 and MC 

females (

P = 0.07).

Authors indicate that for all four strain/sex combinations, there was a significant doserelated trend for fatal liver tumours, incidental liver tumours, and the pooled estimate using Peto’s test, however details on statistics were not provided.

Purity NR

Mouse, B6C3F1

(M, F)90 wkVesselinovitch et al. (1975)

GavageGroups of 75 M and 75 F mice were given 0.5 or 1.0 mg benzidine as the dihydrochloride salt twice weekly. All animals were sacrificed at 90 wk of age. Groups of 100 M and 100 F mice served as untreated controls, killed at 90 wk.

Hepatocellular tumours [benign and malignant]: M–1/98, 3/75, 12/75*F–0/100, 4/75$, 17/75*

No statistics provided*[

P < 0.0005], $

[P < 0.05]

Purity NRVehicle NR

Table 3.1 (continued)
IARC MONOGRAPHS – 100F

58

Species, strain (sex)DurationReference

RouteDosing regimen,

Animals/group at start

Incidence of tumours Significance Comments

Mouse, ICR (F)63 wkMiyakawa & Yoshida (1980)

FeedGroups of F mice were implanted with a 45-mg glass bead in the urinary bladder and divided into three groups: 30 were fed basal diet; 60 were fed diet containing 0.2% benzidine; and 60 were fed diet containing a mixture of 0.2% benzidine and 2% dl-tryptophan. Treated groups received diets starting at six wk of age for 20 wk and were then fed the control diet for 40–43 wk.

Hepatomas [adenomas]: 0/30, 34/41*, 24/51No bladder tumours were found in any of the animals.

*P < 0.01, compared with control and tryptophan treated groups

Purity NR

Mouse, Strain NR (M, F)LifetimeBonser et al. (1956)

Subcutaneous InjectionGroups of 30 M and 30 F mice received 0.1 ml of a freshly made 3% solution of benzidine (purity NR) in arachis oil by subcutaneous injection, twice/wk for 50 wk, and were then observed for life. Groups of 30 M and 30 F mice served as vehicle controls. 

Hepatomas:M–0/30, 4/30;F–0/30, 3/30 

No statistics provided

Age NR

Rat, SpragueDawley (F)9 moGriswold et al. 

(1968)

GavageFour groups of 10–20 F rats were given benzidine at doses of 12, 25, 35 or 50 mg/rat in sesame oil by stomach tube, daily for 30 d, and sacrificed after a 9-mo observation period. A group of 140 F rats served as vehicle controls.

Mammary carcinomas:3/132, 5/10*, 7/9*, -, 4/5*

No statistics provided*[

P < 0.0001]

Small number of animals per group, high mortality in top 2 dose groups.Purity NR

Rat, Sherman (M, F)LifetimeSpitz et al. 

(1950)

Subcutaneous injection

Groups of 45 to 155 M and F rats were injected with 15 mg/rat of “technical” benzidine, “pure” benzidine, or benzidine sulfate, dissolved in 1 mL of olive oil once/wk throughout life. A group of 50 rats served as vehicle controls.

Hepatomas:Technical–8/78 (M), 0/155 (F); Pure–5/45 (M), 1/107 (F); Sulfate–not tested (M), 1/153 (F)External auditory canal carcinomas (M+F): Technical −54/233*; Pure −32/152*; Sulfate −16/153*None of the above tumours were reported in the 50 control animals.

No statistics provided*[significant]

Survival at 300 d was 56% for controls and 15% to 3% for treated rats. Poor survival due to heat (no air conditioning in animal rooms).Sex of control animals NR

Table 3.1 (continued)
Benzidine

59

Species, strain (sex)DurationReference

RouteDosing regimen,

Animals/group at start

Incidence of tumours Significance Comments

Rat, CD (F)46 wkMorton et al. (1981)

Intraperitoneal injectionThree groups of 30 F rats were given injections of 0, 10, or 30 μmol/kg bw benzidine in trioctanoin suspensions, twice/wk for 4 wk.

Mammary gland tumours (all): 3/30, 7/30, 12/29* Mammary gland adenocarcinomas: 1/30, 2/30, 7/29**Zymbal gland tumours (benign and malignant): 1/30, 1/30, 7/29** No tumours of the liver were found.

*P < 0.01 **P < 0.05 Purity NR

Frog, Rana temporaria

38 wkKhudoley (1977)

Subcutaneous injectionA group of 37 frogs (1–1.5 yr old) received a subcutaneous injection, once/wk, of 0.2–0.5 mL of a 0.5% solution of benzidine in mineral oil, for up to 38 wk (total dose, 45–114 mg/animal). A group of 67 frogs given subcutaneous injections of 0.2–0.5 mL mineral oil once weekly for 42 wk served as vehicle controls.

Liver tumours: 0/67, 3/14*Tumours of the haematopoietic system: 0/67, 4/14*

*[P < 0.01] Fourteen animals 

in the treated group were still alive when the first tumour appeared at 16 wk. The high mortality was the result of exceeding the maximum tolerated dose. Sex NR. Purity NR. Histopathology not further specified

Table 3.1 (continued)

bw, body weight; d, day or days; F, female; M, male; MC, monohybrid cross; mo, month or months; NR, not reported; d, day; wk, week or weeks
IARC MONOGRAPHS – 100F

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′-(deoxyguanosin8-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)-Nacetylbenzidine 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 

60
Benzidine

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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