| Use | MFR AZO DYES [R1]
CURING AGENT FOR ISOCYANATE-CONTAINING POLYMERS [R6]
RUBBER & PLASTIC COMPOUNDING INGREDIENT [R7]
3,3'-DICHLOROBENZIDINE IS USED ALONE AND IN BLENDS WITH
4,4'-METHYLENEBIS(2-CHLOROANILINE) AS A CURING AGENT FOR
LIQUID-CASTABLE POLYURETHANE ELASTOMERS; IN COLOR TEST FOR
/DETECTION/ OF GOLD [R8]
3,3'-Dichlorobenzidine base and salts are used as chemical intermediates to produce pigments
that are produced commercially in the USA (Pigment Yellows 12, 13, 14, 17, 34, & 55). [R9]
The yellow pigments derived from 3,3'-dichlorobenzidine and salts can be used as substitutes for
the lead chromate pigments. [R9]| Apparent Color | Gray
or purple crystalline solid
|
|---|
| Odor | N/A
|
|---|
| Boiling Point | 402 DEG C
|
|---|
| Melting Point | 132-133 DEG C
|
|---|
| Molecular Weight | 253.13
|
|---|
| Misc | READILY SOL IN BENZENE, DIETHYL ETHER, ETHANOL,
AND GLACIAL ACETIC ACID; SLIGHTLY SOL IN DILUTE HYDROCHLORIC ACID;
Almost insoluble in water
SPEC: The absorption maximum for 3,3'-dichlorobenzidine in aqueous ethanol is 290 nm with
significant absorption in the ultraviolet region extending to 340 nm. In dilute aqueous solution the
absorption maxima have been observed at 282 nm and 211 nm.; Intense mass spectral peaks: 252
m/z (100%), 254 m/z (66%), 253 m/z (16%), 126 m/z (16%); MASS: 1771 (National Bureau of
Standards EPA-NIH Mass Spectra Data Base, NSRDS-NBS-63)
OCPP: Undergoes usual reactions of benzidine derivatives, eg, formation of diazonium salts &
alkyl deriv; conversion factor: ppm= 0.0966 times mg/cu m
When combined with ferric chloride or bleaching powder, a green color is produced.
HAS GENERAL CHARACTERISTICS OF PRIMARY AROMATIC AMINES
MP: 165 deg C
TXHR: The Human Health Assessment Group in EPA's Office of Health and Environmental
Assessment has evaluated 3'3-dichlorobenzidine for carcinogenicity. According to their analysis,
the weight-of-evidence for 3'3-dichlorobenzidine is group B2, which is based on suffieicnt
evidence in animals. No data on humans is available. As a group B2 chemical,
3'3-dichlorobenzidine is considered to be probably carcinogenic to humans. [R26]
MEDS: Urine cytology surveillance has proved useful in evaluating tumors in dyestuff plant
workers. The system consists of two-stage tests: periodic urine cytology, followed by, in
suspicious cases, urological examinations. [R27]
PRECAUTIONS FOR "CARCINOGENS": Whenever medical surveillance is indicated, in
particular when exposure to a carcinogen has occurred, ad hoc decisions should be taken
concerning ... /cytogenetic and/or other/ tests that might become useful or mandatory. /Chemical
Carcinogens/ [R18, 23]
HTOX: Dermal exposure to 3,3'-dichlorobenzidine base has caused dermatitis in dye workers.
[R28]
... PRONOUNCED STERNUTATORY /SNEEZING/ EFFECT.
/3,3'-DICHLOROBENZIDINE DIHYDROCHLORIDE/ [R2]
3,3'-Dichlorobenzidine is mutagenic in unscheduled DNA synthesis human fibroblasts without
activation by the liquid scintillation counting method. [R29]
The health records of 59 workers at a dyestuff plant in Great Britain who were exposed from
1953 through 1973 to 3,3'-dichlorobenzidine only /were examined/ and compared them to those
working with both benzidine and 3,3'-dichlorobenzidine, and to unexposed populations ... . It was
calculated that the 3,3'-dichlorobenzidine process worker was actually exposed to
3,3'-dichlorobenzine for a maximum of 10 hours per work week. Men whose total
3,3'-dichlorobenzidine exposure was less than 245 hours (six months' full-time work) were
excluded from the study, leaving 35 segregated 3'3-dichlorobenzidine workers. These 35 workers,
representing a total of 68,505 hours of 3,3'-dichlorobenzidine exposure, had no urinary tract
tumors, no other tumors, and two deaths from other causes (coronary thrombosis, cerebral
hemorrhage). In contrast, among 14 mixed benzidine and 3,3'-dichlorobenzidine workers with
16,200 hours exposure (approximately 60% worked with benzidine, 40% worked
3,3'-dichlorobenzidine), three men developed tumors of the bladder, and one man developed
carcinoma of the bronchus. One death from coronary thrombosis /was noted/. [R30]
The authors reported the results of an epidemiological study of workers exposed to
3,3'-dichlorobenzidine in manufacture and utilization in a plant in the United States. A survey of
the number of 3,3'-dichlorobenzidine exposed workers who developed neoplasms and the type of
neoplasm was presented. These included lung cancer (2 workers), leukemia bone marrow (1),
lipoma (6), rectum papilloma (3), sigmoid colon carcinoma (2), prostate carcinoma (1), breast
muscle myoblastoma (1), and skin basal cell epithelioma (1). A total of 17 workers of the total of
207 workers surveyed had developed neoplasms. [R31]
3,3'-DICHLOROBENZIDINE (10-7 TO 10-4 MOLAR) INDUCED UNSCHEDULED DNA
SYNTHESIS IN HELA CELLS IN THE PRESENCE OF A PHENOBARBITAL-INDUCED
RAT LIVER ACTIVATION SYSTEM. [R28]
The following 5 countries have designated, by regulation or guidelines, 3,3'-dichlorobenzidine as
a skin irritant & as a carcinogen: Australia, Belgium, the Federal Republic of Germany,
Switzerland & the USA. In addition, Finland, Italy & Sweden have designated it as a carcinogen.
In UK, 3,3'-dichlorobenzidine & its salts are "controlled substances", subject to special preventive
measures, including prescribed medical exam. [R8]
No case report is known in which 3,3'-dichlorobenzidine has been associated with the occurrence
of cancer in man. 3,3'-Dichlorobenzidine may, however, have contributed to cases of bladder
cancer attributed to benzidine, as both substances may be prepared in the same plant. [R32]
Urine cytology surveillance of occupational bladder tumor incidence was evaluated by reviewing
clinical history of 9 bladder tumor cases found in dyestuff plant workers. A bladder tumor
surveillance system was organized for workers (179 active and 65 retired) who were exposed to
benzidine or beta-naphthylamine in the plant. The urine cytology surveillance was found to be
useful for two reasons: first, it detected tumors in 5 out of 6 cases surveyed by the system, and
second, four cases screened by cytology had tumors curable by transurethral operation, while
other cases underwent cystectomy. Calculated average latent periods from the first and last
exposure was 26.4 years (SD= 6.0) and 14.0 years (SD= 6.2). [R27]
NTOX: Twenty-six male ICR/JCL mice (age at start not specified) were fed diet containing
0.1% 3,3'-dichlorobenzidine (purity unspecified) for ... 12 months. Animals were killed after 6 or
12 months of treatment. All (100%) had hepatomas, with mean numbers of 8 & 18
hepatomas/mouse, respectively. Of 39 control mice maintained on normal diet & killed at 6, 12, &
18 months, 0, 9.5 & 38.5% had hepatomas, with mean numbers of 0, 2 & 5 hepatomas/mouse,
respectively. (The Working Group noted the absence of data on the survival of treated & control
animals). [R33]
Two groups of outbred Rappolovo rats (110-130 g) 15 female & 35 male were fed diets
containing 3,3'-dichlorobenzidine in form of paste (45.3% 3,3'-dichlorobenzidine, 50% water &
4.7% unspecified impurities) at dose of 10 to 20 mg/day, which was admin 6 days a wk for 12
months (total dose, 4.5 g/rat). ... Animals that survived were: 34 at 6 mo, 27 at 12 mo, & 29 ... at
time of appearance of the 1st tumor (11 mo). ... /They/ were observed for life. Twenty-three rats
developed tumors, incl 7 tumors of the Zymbal gland, 3 skin tumors, 7 mammary gland tumors, 2
adenocarcinomas of the ileum, 3 bladder tumors, 3 tumors of hematopoietic system, 2 tumors of
connecutive tissue ... /&/ 2 of salivary gland, 1 tumor of liver & 1 of thyroid. In 130 controls
injected with octadecylamine & methylstearylamine, no tumors were found within 23 months.
(The Working Group noted the absence of adequate controls). [R33]
Two groups of rats (50 male & 50 female, 5-wk-old ChR-CD) were fed a diet containing 1000
mg/kg (ppm) 3,3'-dichlorobenzidine (purity & impurities unspecified) for 349-353 days. Equal
numbers of animals were maintained on a control diet for period of 12 mo. Six rats per group &
per sex were killed at 12 mo. Of remaining treated rats, 6 survived ... 15 mo, at which time they
were killed; controls were maintained under observation ... to approx 2 yr. A statistically
significant (p< 0.05) incr in incidence of tumors was observed in treated compared with control
animals, for the following target sites: In male treated rats, 9/44 granulocytic leukemias, 7/44
mammary adenocarcinomas, & 8/44 Zymbal gland carcinomas; The corresponding incidences in
control rats were 2/44, 0/44 & 0/44. In female rats ... 26/44 treated animals developed mammary
adenocarcinomas versus 3/44 in control rats. [R33]
In lifetime studies ... /using Syrian golden hamsters/ dietary levels of 0.1% 3,3'-dichlorobenzidine
(purity & impurities unspecified) did not induce tumors in 30 /females/ ... when compared to
similar number of untreated animals. However, in later studies in similar groups of animals, 0.3%
3,3'-dichlorobenzidine in diet produced 4 transitional-cell carcinomas of bladder & some liver-cell
& cholangiomatous tumors. These tumors were not found in control animals. [R34]
Six female 1-yr-old beagle dogs were each given 100 mg 3,3'-dichlorobenzidine (reported to be
100% pure) in gelatin capsules 3 times per wk for 6 wk, then 5 times/wk continuously for periods
... /up/ to 7.1 yr. Intake of 3,3'-dichlorobenzidine was between 9.1 & 12.8 mg/kg body wt/dose.
One dog was sacrificed after 3.5 yr /and/ ... had no tumors. Another dog was sacrificed after 6.6
yr ... (total intake, 164 g) had undifferentiated carcinoma of urinary bladder. Of the 4 dogs killed
at 7.1 yr (total intake, 176 g/dog), 4/4 had papillary transitional-cell carcinomas of urinary bladder
& 3/4 had hepatocellular carcinomas. None of 6 control dogs had these tumors. However, 4/6
control animals killed at 8-9 yr of age had major tumors of mammary gland (adenocarcinomas &
carcinosarcoma). [R34]
Groups of outbred rats (25 females & 36 males) were given weekly sc injections of a 8.8%
suspension of dichlorobenzidine paste (45.3% 3,3'-dichlorobenzidine, 50% water & 4.7%
unspecified impurities) in glycerol, at dose of 120 mg/rat. Because of toxic effects, beginning at 6
mo, 3'3-dichlorobenzidine dose was reduced to 20 mg/rat. Total dose was 1.62 to 3 g/rat over
10-11 mo. The animals were observed for life. The numbers of rats that survived were: 40 at 6
mo, 23 at 12 mo, & 35 at time (7 mo) of appearance of 1st tumor. Twenty-six (74.3%) had
tumors at different sites: 10 had tumors of Zymbal gland; 5, skin tumors; 6, tumors of mammary
gland; 7, local subcutaneous sarcomas; 2, remote tumors of connective tissue; 2, tumors of
hematopoietic system; & 1, a tumor of salivary gland. No tumor occurred within 23 mo in 130
controls injected with octadecylaime or methylstearylamine. (The Working Group noted the
absence of adequate controls.) [R34]
... RATS /WERE INJECTED/ SC WITH 15-60 MG 3'3-DICHLOROBENZIDINE/RAT IN
SUNFLOWER SEED OIL OR GLYCEROL AND WATER AT UNSPECIFIED INTERVALS
FOR 10-13 MONTHS. TOTAL PERCENTAGE OF TUMOR-BEARING ANIMALS WAS
74%. SKIN, SEBACEOUS AND MAMMARY GLAND TUMORS WERE OBSERVED MOST
FREQUENTLY, THERE WERE ALSO INTESTINAL, URINARY BLADDER AND BONE
TUMORS. AMONG 50 CONTROL RATS INJECTED WITH VEHICLE ALONE, OR LEFT
UNTREATED, 1 TUMOR WAS REPORTED. (THE WORKING GROUP NOTED
INADEQUATE REPORTING OF EXPERIMENT) [R34]
A group of 20 female Sprague-Dawley rats (40 days of age) was given 10 doses of
3,3'-dichlorobenzidine dihydrochloride (purity & impurities unspecified) every 3 days by gastric
intubation (total dose 300 mg/rat, which was max tolerated dose) and observed for 9 months,
when the surviving animals (14) were killed. No mammary tumors were observed in 15 rats
autopsied, while 100% of positive control group (treated with 7,12-dimethylbenz(a)anthracene) &
3% of animals treated with sesame oil only had mammary tumors. /3,3'-Dichlorobenzidine
dihydrochloride/ [R33]
During last wk of pregnancy, a group of BALB/c mice (number & age not specified) were
treated with 5 sc injections of 2 mg/injection 3,3'-dichlorobenzidine (total dose, 10 mg/mouse).
Another group was treated with 0.1 ml sesame oil. Of offspring that lived 12-20 mo, 13/24 had
tumors, compared with 6/30 of control progeny. A significant increase in incidence of lymphoid
leukemias (7/24 in treated & 0/30 in control animals (sex unspecified)) was observed in offspring.
Tumors were also observed at other sites, but there was no statistically significant increase over
incidence in controls (5/24 lung adenomas versus 3/30; 4/24 mammary tumors versus 3/30).
[R35]
Analogs of benzidine were assayed for mutagenicity using Salmonella typhimurium TA-98 &
TA-100 in the presence and absence of a mouse liver enzyme preparation /SRP: S-9 fraction/.
Only 4-aminobiphenyl produced frameshift & base pair substitution mutations &
3,3'-dichlorobenzidine was the only compound (cmpd) which was mutagenic without the liver
enzyme factor. When hydrochloride salts of the parent cmpd were tested, the mutagenicity for
Salmonella tester strains was reduced except for 3,3'-dichlorobenzidine. [R36]
... ONE HR EXPOSURE OF SPRAGUE-DAWLEY RATS TO A CONCENTRATED DUST
ATMOSPHERE /OF 3,3'-DICHLOROBENZIDINE/ (ABOUT 50% AT OR LESS THAN 5
UM) CAUSED SOME IRRITATION & MODERATE PULMONARY CONGESTION, BUT
NO DEATHS IN EITHER OF THE SEXES. TEN MALE RATS EXPOSED TO
CONCENTRATED DUST ... 2 HR/DAY FOR 7 DAYS CONTINUED TO GAIN WEIGHT &
SHOWED NO OUTWARD SIGN OF ADVERSE EFFECTS. ... FOUR OF FIVE RABBITS
DIED FOLLOWING /SKIN/ APPLICATION OF 1 G/KG OF /3'3-DICHLOROBENZIDINE/
BASE FOR 24 HR. [R2]
ALL ANIMAL TESTS OF ACUTE & CHRONIC TOXICITY /OF
3,3'-DICHLOROBENZIDINE/ INDICATE BOTH /3'3-DICHLOROBENZIDINE/ BASE &
SALT TO /HAVE/ LOW /TOXICITY/; /UPON EXAM AFTER DEATH ARISING FROM
ORAL LD50 STUDIES/ ... GI CONGESTION & HEMORRHAGING WERE CHIEF
AUTOPSY FINDINGS. NINE OF 14 RATS SURVIVED MORE THAN 288 DAYS /AFTER/
INGESTING 700 MG/KG OF BASE IN OLIVE OIL, 5 DAYS/WK. LIVER DAMAGE & ONE
TUMOR WERE ... /FOUND/. [R2]
FEMALE BEAGLE DOGS GIVEN /3,3'-DICHLOROBENZIDINE/ (100 MG/DAY ORALLY,
3 TIMES/WK, FOR 6 WK, THEN 5 TIMES/WK CONTINUOUSLY FOR 7.1 YR), SHOWED
ELEVATED PLASMA GLUTAMIC-PYRUVIC TRANSAMINASE ACTIVITY, LIVER
CARCINOMAS WITH METASTASES, HEPATOCELLULAR, & PAPILLARY
TRANSITIONAL CELL CARCINOMAS OF URINARY BLADDER.
3,3'-DICHLOROBENZIDINE WAS FOUND TO BE CARCINOGENIC FOR THE LIVER &
URINARY BLADDER IN DOGS UNDER THE CONDITIONS EMPLOYED (P< 0.025,
FISHER'S EXACT TEST, 1 TAIL). [R37]
PURIFIED & TECHNICAL GRADE 3,3'-DICHLOROBENZIDINE HAD SOME DIRECT
MUTAGENIC ACTIVITY TOWARD SALMONELLA TYPHIMURIUM TA1538, BUT THIS
WAS INCREASED OVER 50-FOLD BY ADDITION OF RAT LIVER MIXED FUNCTION
OXIDASE PREPARATION (PREPN). IN PRESENCE OF LIVER PREPN IT WAS APPROX
10 TIMES MORE ACTIVE THAN BENZIDINE. [R38]
3,3'-DICHLOROBENZIDINE WAS BOUND COVALENTLY TO DNA &
POLYRIBONUCLEOTIDES VIA DIRECT INTERACTION OF PARENT CMPD &
REACTIVE METABOLITES GENERATED IN PRESENCE OF RAT LIVER S-9
FRACTION. BINDING IN PRESENCE OF S-9 WAS CONSIDERABLY GREATER THAN
THE NONENZYMIC BINDING. A GOOD CORRELATION BETWEEN THE AMT OF IN
VITRO BINDING OF 3,3'-DICHLOROBENZIDINE TO DNA & THE MUTAGENICITY IN
THAT BOTH MUTAGENICITY & COVALENT BINDING WERE HIGH IN PRESENCE OF
RAT LIVER S-9 FRACTION & LOW IN ITS ABSENCE. [R39]
At concn of 5 ug/ml ... /3,3'-DCB/ enhanced transformation of high-passage cells containing
Rauscher leukemia virus in the Fischer rat embryo cell system. ... It induced cell transformation in
the baby hamster kidney test. ... [R28]
... 3,3'-Dichlorobenzidine /injected ip into mice/ at low concn increased, and at high concn,
decreased the sister chromatid exchange frequency /in bone marrow cells/. [R40]
The ability of the hepatocarcinogen 3,3'-dichlorobenzidine to induce cytochrome p448 mediated
monooxygenase in the liver was studied in rats. 3,3'-Dichlorobenzidine was given intraperitoneally
at 40 mg/kg. Animals were killed after 48 hrs and microsomes were prepared. The difference
absorption spectrum of reduced hepatic microsomal cytochrome p450 was studied. Pretreatment
with 40 mg/kg 3,3'-dichlorobenzidine for 2 consecutive days, 40 mg/kg phenobarbital for 3 days
was administered to compare inductive effects. Inductions of microsomal
ethoxycoumarin-O-deethylase, p-nitrophenetole O-deethylase, aniline hydroxylase, and
aminopyrine-N-demethylase were measured. Aryl hydrocarbon hydroxylase activity was also
determined. Enzymatically mediated covalent binding of benzo(a)pyrene to microsomal protein
was determined using radiolabeled benzo(a)pyrene. The effects of alpha-naphthoflavone, an
inhibitor of 3-methylcholanthrene microsomal monooxygenase activity, and SKF-525A, an
inhibitor of phenobarbital pretreated microsomes, were studied in DCB induced activities.
3,3'-dichlorobenzidine caused a shift in the absorption maximum of the reduced form of
cytochrome p450. 3,3'-Dichlorobenzidine caused induction of ethoxycoumarin-O-deethylase and
p-nitrophenetole O-deethylase in quantities comparable to those induced by
3-methylcholanthrene. Aryl hydrocarbon hydroxylase activity was stimulated by
3,3'-dichlorobenzidine, but much less than 3-methylcholanthrene. Covalent binding of
benzo(a)pyrene to microsomal protein was greater in rats pretreated with 3,3'-dichlorobenzidine.
All 3'3-dichlorobenzidine induced enzymic activities were inhibited by alpha-naphthoflavone and
not SKF-525A. Microsomes from rats pretreated with 3,3'-dichlorobenzidine had a higher
capacity for metabolizing 3,3'-dichlorobenzidine than untreated animals. /It was/ concluded that
3,3'-dichlorobenzidine is a potent inducer of cytochrome p448 though it is only a halogenated
biphenyl derivative, not a polychlorinated biphenyl. [R41]
3,3'-Dichlorobenzidine is mutagenic in Salmonella typhimurium TA100 and TA1535 with
metabolic activation with Arochlor 1254 induced rat liver S-9 by the standard plate method.
[R42]
For 12 months, 6 times weekly, 0.5 to 1.0 ml of a 4.4% suspension of 3,3'-dichlorobenzidine
/was given/ to a strain of rats of both sexes assumed to have a low spontaneous tumor rate. Each
rat received a total dose of 4.53 g. Neoplasms were detected in 22 of 29 (75.8%) surviving
animals. Tumors, primarily carcinomas, were observed in a broad spectrum of organs including
mammary gland, Zymbal's gland (sebaceous gland of the external auditory meatus), bladder, skin,
small intestine, liver, thyroid gland, kidney, hematopoietic (lymphatic) system, and salivary glands.
[R43]
An assay of 3'3-dichlorobenzidine carcinogenicity was also done with mice. Mice received 0.1 ml
of a 1.1% 3,3'-dichlorobenzidine suspension in their food for 10 months, receiving a total dose of
127.5 to 135 mg 3,3'-dichlorobenzidine. Hepatic tumors were found in 4 of 18 mice surviving
after 18.5 months (22.2%). A sebaceous gland carcinoma and a lung adenoma were also seen.
[R44]
An oral LD50 was reported for albino rats of 7.07 g/kg using the free amine and 3.82 g/kg in
male and female Sprague-Dawley rats with the hydrochloride salt. When the compound was
applied topically to the skin of male and female rats, the LD50 was 8 g/kg. Exposure of rats to a
concentrated atmosphere of dichlorobenzidine hydrochloride dust for 14 days, or to 355 mg free
base 2 hours daily for 7 days resulted in no mortalities. [R45]
PURIFIED & TECHNICAL GRADE 3,3'-DICHLOROBENZIDINE HAD SOME DIRECT
MUTAGENIC ACTIVITY TOWARD SALMONELLA TYPHIMURIUM TA1538, BUT THIS
WAS INCREASED OVER 50-FOLD BY ADDITION OF RAT LIVER MIXED FUNCTION
OXIDASE PREPARATION. IN PRESENCE OF LIVER PREPN IT WAS APPROX 10 TIMES
MORE ACTIVE THAN BENZIDINE. [R46]
The Ames Salmonella/microsome test was used to compare the mutagenic response of
Salmonella typhimurium TA100, TA98, TA1538, and TA1535 to 12 benzidine derivatives, ie,
benzidine, 3,3'-dimethoxybenzidine, 3,3'-dimethylbenzidine, 3,3'-dichlorobenzidine, and the
corresponding N- and N,N'-diacetylated derivatives. With a few exceptions, the mutagenic
response to this series of compounds varied in the order TA98 greater than TA1538 greater than
TA100 greater than TA1535 = 0, and the N-monoacetylated derivatives were more mutagenic
than either the parent diamines or the N,N'-diacetyl derivatives. The relative mutagenicities of the
parent amines for TA98, were 3,3'-dichlorobenzidine much greater than 3,3'-dimethoxybenzidine
greater than benzidine greater than 3,3'-dimethylbenzidine. [R47]
Covalent binding of benzidine and some congeners to hemoglobin was studied in female Wistar
rats after oral administration. Hemoglobin adducts were hydrolyzed under alkaline conditions, and
the arylamines analyzed by high performance liquid chromatography. With benzidine, three
cleavage products were observed, the major component being monoacetylbenzidine. This
indicates that 4-nitroso-4'-N- acetylaminobiphenyl is the major reactive metabolite in erythrocytes.
In addition benzidine and 4-aminobiphenyl were identified. With 3,3'-dichlorobenzidine
dihydrochloride, 3,3'-dimethoxybenzidine and 3,3'-dimethylbenzidine two cleavage products were
observed, the parent diamines being present in excess to or in amounts comparable to the
monoacetyl derivative. With 3,3',5,5'-tetramethylbenzidine a hemoglobin adduct could not be
found. When the azo dye direct red 28 was administered to the animals, the three cleavage
products typical for benzidine were found, indicating that benzidine became bioavailable after
reductive cleavage of the azo compound. In this case the fraction of 4-aminobiphenyl was greater
than after benzidine. It is proposed to use the analysis of hemoglobin adducts in human blood to
control the exposure of individuals to these carcinogenic chemicals in the course of biochemical
effect monitoring. [R48]
The binding of benzidine, 3,3'-dichlorobenzidine, and the asymmetrically substituted chlorinated
benzidines 3,5-dichlorobenzidine and 3,5,3'-trichlorobenzidine to the rat hepatic cytosolic
aromatic hydrocarbon receptor was measured, to assess the mechanism of p450I induction by
3,3'-dichlorobenzidine. Binding affinity to the hydrocarbon receptor protein was determined by
displacement of labelled 2,3,7,8-tetrachlorodibenzo-p-dioxin from the receptor. The rank order of
affinities were 3,5,3'-trichlorobenzidine > 3,5-dichlorobenzidine > 3,3'-dichlorobenzidine.
Benzidine did not displace 2,3,7,8-tetrachlorodibenzo-p-dioxin from the recptor protein.
4-Aminobiphenyl a structural link between the benzidine and biphenyl series competed weakly
with 2,3,7,8-tetrachlorodibenzo-p-dioxin. The results are consistent with the induction of p450
enzymes by 3,3'-dichlorobenzidine in vivo is mediated by the hydrocarbon receptor. [R49]
Single oral dose of benzidine (300 mg/kg) and 3,3'-dichlorobenzidine (1000 mg/kg) to male ICR
mice elicited positive response in the bone marrow micronucleus test. In the transplacental
micronucleus test, the compounds were administered to pregnant females in the same manner. A
significant increase in the frequency of micronuclei occurred in the fetal liver, but not in the bone
marrow of mothers. [R50]
The relationship between the structure and mutagenicity of benzidine analogues was examined.
The mutagenic activities of benzidine, benzidine dihydrochloride, and their analogues,
3,3'-diaminobenzidine, 3,3'-dichlorobenzidine, 3,3'-dimethoxybenzidine, 3,3'-dimethoxybenzidine
dihydrochloride, and N,N,N',N'-tetramethylbenzidine, were evaluated in the Ames assay.
Salmonella strains (TA-100) and (TA-98) were used, with or without metabolic activation by liver
S9 mix from male Sprague-Dawley rats induced wit aroclor-1254. In the absence of S9 mix only
3,3'-dichlorobenzidine in strain TA-98 showed significant mutagenic activity. In the presence of
S9 mix all compounds except N,N,N',N'-tetramethylbenzidine were mutagenic in strain TA-100.
Bridged diphenyl compounds showed mutagenicity equal to or greater than that of the parent
compound, benzidine. In strain TA-98 biphenyl compounds were more mutagenic than the
bridged diphenyl compounds. No significant differences in mutagenicity were seen between the
free base and dihydrochloride salt forms of benzidine or its dimethoxy analogue. Compounds
capable of inducing frame shift mutations must have a planar aromatic moiety and an activated
electrophilic side chain, mutagenicity in this series of compounds increases with decreasing
basicity of the aniline moiety. [R51]
The kinetics of total deoxyribonucleic acid adducts were compared in the liver, bladder
epithelium and small intestinal epithelium of rats and mice followng a single oral dose (100
mg/kg) of 3,3'-dichlorobenzidine (14)C-3,3'-dichlorobenzidine). Peak deoxyribonucleic acid
binding (expressed as pmol 3,3'-dichlorobenzidine bound/mg deoxyribonucleic acid) in rat tissues
was 153.5, 144.8 and 36.9 in the intestine, bladder and liver, respectively, whereas in mouse
tissues, the binding was 72.5, 58.2 and 55.8, respectively. In either species, the half-life of the
deoxyribonucleic acid adducts in the liver (13.5 and 13.8 days in rats and mice, respectively) was
comparable to that in the bladder epithelium (14.8 and 12.7 days in rats and mice, respectively)
but longer than that in the intestinal epithelium (5.9 and 4.7 days in rats and mice, respectively).
Peak total 3,3'-dichlorobenzidine binding in hepatic but not intestinal or bladder epithelial
deoxyribonucleic acid correlated positively with total urinary 3,3'-dichlorobenzidine metabolites.
In vitro, mouse hepatic S9 was 57% more active in catalyzing the formation of deoxyribonucleic
acid binding derivatives of 3,3'-dichlorobenzidine, in parallel with the higher in vivo maximum
hepatic deoxyribonucleic acid binding in mice than in rats. Thus, a single oral dose of
3,3'-dichlorobenzidine in rats and mice leads to extensive binding of the chemical to tissue
deoxyribonucleic acid, with the rate of removal of the adducts not differing between target and
non target tissues. [R52]
The effect of pretreatment with 3,3'-dichlorobenzidine on k(i) enzymic and nonenzymic lipid
peroxidation in hepatic microsomes as measured by malondialdehyde formation, and (ii) hepatic
antioxidant status as measured by the contents of vitamin E and reduced glutathione and the
activity of glutathione peroxidase, was examined in the rat. 3,3'-Dichlorobenzidine pretreatment
(20 mg/kg/day, ip, for 2 days) caused an increase in NDAPH-dependent (enzymic) lipid
peroxidation, but had no effect on thelinoleic acid hydroperoxide dependent (nonenzymic) lipid
peroxidation in microsomes. 3,3'-Dichlorobenzidine pretreatment also caused a 44% decrease in
the content of glutathione or the activities of glutathione peroxidases in the liver. The
3,3'-dichlorobenzidine induced increase in in vitro microsomal lipid peroxidation is interpreted as
resulting from the diminution of vitamin E induced by 3,3'-dichlorobenzidine in vivo. [R53]
Administration of a single oral dose (20 mg/kg) of U-14(C) 3,3'-dichlorobenzidine to rats
resulted in the in vivo covalent binding of the compound to hepatic lipids. More than 70% of the
lipid 3,3'-dichlorobenzidine adducts were accounted for in microsomes. Loss of the lipid bound
3,3'-dichlorobenzidine residues from either total liver or endoplasmic reticulum occurred in at
least two phases an initial fast phase and a terminal slow phase. In vitro studies with hepatic
microsomes in the presence of antibodies to specific p450 isozymes and chemical inhibitors to
determine the enzymes that activate 3,3'-dichlorobenzidine to the lipid binding derivative(s)
implicated cytochrome p450 dependent The 3,3'-dichlorobenzidine bound microsomal lipids were
not mutagenic to Salmonella TA98 in the Ames test. The results suggest that adduct formation
between 3,3'-dichlorobenzidine and membrane lipids may provide a measure of
3,3'-dichlorobenzidine activation. [R54]
In a modified Ames assay, mutagenicity is observed in Salmonella typhimurium strain TA98
following the incubation of dichlorobenzidine, bacteria, and hydrogen peroxide.
14(C)-3,3'-dichlorobenzidine becomes covalently bound to Salmonella typhimurium
macromolecules, including deoxyribonucleic acid, when exogenous hydrogen peroxide is
supplied. Binding was approximately twofold higher in TA4124 (oxyR1) than in TA4123
(oxyR+). Bacterial hydroperoxidases may catalyze the activation of dichlorobenzidine to
mutagenic and deoxyribonucleic acid binding species in this system. [R55]
NTXV: LD50 Mouse (female) oral 352 mg/kg/day (7 consecutive days) [R56]
LD50 Mouse (male) oral 368 mg/kg/day (7 consecutive days) [R56]
LD50 Mouse (female) oral 488 mg/kg/day (7 consecutive days) [R56]
LD50 Rat (Sprague-Dawley) oral 3.82 g/kg. /3,3'-Dichlorobenzidine dihydrochloride/ [R56]
IARC: +Inadequate evidence of carcinogenicity in humans. Sufficient evidence of carcinogenicity
in animals. OVERALL EVALUATION: Group 2B: The agent is possibly carcinogenic to
humans. [R57]
ADE: No data were avail on extent of intestinal or dermal absorption of 3,3'-dichlorobenzidine
in experimental animals; however, the appearance of systemic toxicity following oral admin or
dermal application indicates some degree of absorption by way of these routes. [R35]
Constituents that co-chromatographed with 3,3'-dichlorobenzidine were isolated from urine of
workers occupationally exposed to 3,3'-dichlorobenzidine. In one worker drenched with a slurry
of this cmpd in water, urinary excretion was incr approx 10-fold over the rate observed in other
workers. [R28]
3,3'-Dichlorobenzidine crosses the placenta of mice as demonstrated by growth changes in
explanted kidneys of embryos from BALB/c mice treated during pregnancy ... & by incr tumor
incidences in offspring /exposed in utero to 3,3'-dichlorobenzidine/. ... [R35]
FOUR HR AFTER IV ADMIN OF 14(C)-LABELED BENZIDINE OR
3,3'-DICHLOROBENZIDINE TO DOGS, RADIOACTIVITY LEVELS IN URINE &
BLADDER WERE 30 & 42% HIGHER, RESPECTIVELY, FOR BENZIDINE THAN FOR
3,3'-DICHLOROBENZIDINE. IN GENERAL, THE EXCRETION RATES WERE SIMILAR,
WITH EXCRETION PRACTICALLY CEASING SIMULTANEOUSLY 1 WK AFTER
TREATMENT. [R58]
The disposition of the carcinogen, 3,3'-dichlorobenzidine, was studied in the male rat following
oral administration. (14)C-3,3'-dichlorobenzidine was absorbed by the rat with the maximum
plasma radioactivity levels being found within 8 hr after dosing. The radioactivity was distributed
throughout the body 24 hr after admin with the highest levels found in the liver, followed by
kidney, lung, and spleen. Repeated admin (6 doses) of (14)C-3,3'-dichlorobenzidine to animals did
not result in a substantial accumulation of (14)C in the tissues. The elimination of radioactivity
from the plasma, liver, kidney and lung was biphasic, showing an initial rapid decline (half-lives
1.68, 5.78, 7.14, and 3.85 hr, respectively) followed by a slower disappearance phase (half-lives
33.0, 77.0, 138.6, and 43.3 hr, respectively). Approximately half of the total (14)C in the liver and
kidney was covalently bound to cellular macromolecules 72 hr after dosing. Excretion of
(14)C-3,3'-dichlorobenzidine-derived radioactivity was mainly via the feces. Approximately
23-33% of the admin dose was recovered in the urine and 58-72% in the feces of rats within 96
hr. More than 65% of the admin (14)C was eliminated in the bile of bile duct-cannulated rats
within 24 hr of dosing. The radioactivity excreted in the urine and bile was primarily in the form
of free (urine 71.2%, bile 25.5%) and conjugated (urine 19.6%, bile 57.9%) metabolites of
3,3'-dichlorobenzidine. [R59]
Fecal excretion was the predominant route of elimination in rats, dogs, and possibly in monkeys.
[R60]
Male Wistar rats & male beagles given 0.2 mg/kg body wt (14)C-3,3'-dichlorobenzidine IV
(dissolved in 0.5% Tween 20R in water) displayed multiphasic blood clearances. ... Fecal
excretion was major route of elimination in rats, dogs & rhesus monkey, accounting for 30-85%
of admin dose within 7 days; 10-40% was eliminated in urine. Urinary excretion was delayed 3-5
hr after treatment, & most of urinary products were ... /3,3'-dichlorobenzidine/ metabolites. ...
Some parent cmpd was eliminated in urine during 1st few hours. ... The majority of ... dose could
be recovered from bile, intestine & liver within 14 hr of treatment, indicating the importance of
hepato-biliary excretion in elimination. ... By 7 or 14 days after treatment, residual radioactivity
was recovered primarily from excretory organs (kidney, bladder, liver, bile) or their products, but
also from adrenals (rat) & lung (dog). [R28]
Dichlorobenzidine could be detected in the urine of monkeys fed 100 mcg/kg. When
(14)C-dichlorobenzidine, at 0.2 mg/kg body weight, was injected iv into monkeys, 7% of the total
dose was excreted unchanged in urine and 26 to 46% in feces ... [R61]
When dichlorobenzidine was administered ip to rats, it cleared rapidly from the peritoneal cavity
and accumulated in the liver, intestines, and peritoneal fat. After 24 hr, only liver and body carcass
contained about 4% dichlorobenzidine. [R62]
Five rabbits were administered single oral doses of 50 mg/kg of Pigment Yellow 13.
Contamination of the pigment with dichlorobenzidine was less than 2 ppm. Analysis was
performed on urine samples collected 48 and 72 hr after dosing. No dichlorobenzidine was found
in any of the urine samples. [R63]
... When a 10 kg mongrel dog was injected ip with 1 g of a tragacanth suspension of
dichlorobenzidine, a total of 0.13% of the compound was excreted in urine unchanged. In 24
hours, the urinary excretion was 0.047% (0.472 mg). However, studies in the feces revealed that
10 times more dichlorobenzidine (1.84%) was excreted by this route, with a 24 hour excretion
value of 0.054% (0.54 mg). Excretion in urine was negligible by the 9th day and in the feces by
the 15th day. [R62]
... About 8% of administered radioactivity was excreted in the urine of dogs and 84% in feces in
7 days when dogs were injected iv with radioactive material. It cleared from the blood with a
half-life of 0.3 to 1.7 hours during the first few hours; after 24 hours clearance is much slower
(half-life 88 hours). Most of the radioactivity was distributed in bile, intestine, liver, and urine 4
hours after administration. [R64]
REMOVAL FROM THE BLOOD WAS DIVIDED INTO FOUR PHASES. PHASE I WAS
IDENTICAL FOR BOTH CMPD OVER FIVE MIN. PHASE II WAS CHARACTERIZED BY
A RAPID DECREASE IN 3,3'-DICHLOROBENZIDINE. IN PHASE III, HALF-LIVES OF
3,3'-DICHLOROBENZIDINE IN DOGS AND RATS WERE 1.7 AND 1.4 HR COMPARED
TO 4.1 AND 2.7 HR FOR BENZIDINE. PHASE IV WAS PARALLEL FOR BOTH CMPD
WITH HALF-LIVES OF 86 AND 88 HR, RESPECTIVELY. BENZIDINE CONCN WERE
ABOUT 50 PERCENT HIGHER IN THE DOG AND DOUBLE IN THE RAT. EXCRETION
CURVES IN RATS AND DOGS SHOWED A MULTIPHASIC COURSE SIMILAR TO
BLOOD. ABOUT 16 PERCENT OF THE EXCRETED RADIOACTIVITY WAS IN THE
URINE OF RATS FOR BOTH CMPD. ABOUT 66 PERCENT OF THE RADIOACTIVITY OF
BENZIDINE WAS RENALLY EXCRETED IN DOGS; LESS THAN 16 PERCENT OF
3,3'-DICHLOROBENZIDINE WAS COLLECTED IN URINE. IN MONKEYS, 7 PERCENT
OF THE 3,3'-DICHLOROBENZIDINE WAS EXCRETED UNCHANGED IN URINE,
COMPARED TO 0.1 PERCENT BENZIDINE. [R65]
The dermal absorption of benzidine derivatives was studied in rats. (14)C labeled benzidine,
3,3'-dichlorobenzidine, or 3,3'-dimethoxybenzidine were applied in doses of 1 mg/kg. The heart,
spleen, kidney, fat, urinary bladder, ear, bone marrow, brain, muscle, and stomach had less than
0.1 percent of the radioactivity of the compounds at all time intervals. Blood levels of (14)C
activity were 0.1 to 0.5% of the dose and were relatively consistent among the compounds. (14)C
activity in the urine and feces was found in trace quantities at 1 or 8 hours, but it increased to 10
to 20% of the dose at the 24 hour assay. Radioactivity from benzidine was consistently higher in
the urine and feces than from the derivatives. The amount of (14)C activity disappearing from the
site of application was the same for benzidine and 3,3'-dichlorobenzidine, reaching about 50% in
24 hours. 3,3'-Dimethoxybenzidine was absorbed less rapidly; only 29% had disappeared from the
application site in 24 hours. All (14)C activity that disappeared from the site of application was
found in the tissues and excreta. Benzidine and its derivatives are readily absorbed through intact
mammalian skin. [R66]
METB: Four hours after the iv administration of (14)C-labeled benzidine or
3,3'-dichlorobenzidine to dogs, the radioactivity levels in the urine and bladder were 30 and 42%
higher, resp, for 14(C)-labeled benzidine than for 3,3'-dichlorobenzidine. In general, the excretion
rates were similar for (14)C-labeled benzidine and 3,3'-dichlorobenzidine, with excretion
practically ceasing one week after treatment. During the first few hours after administration of
(14)C-labeled benzidine to monkeys, its metabolites were primarily detected in the urine, whereas
3,3'-dichlorobenzidine was excreted almost entirely as the unchanged base. The urinary
(14)C-labeled benzidine metabolites appeared to be N-acetylated cmpds. [R58]
Mongrel dogs (sex unspecified) given 1 g 3,3'-dichlorobenzidine ip as suspension in gum
tragacanth excreted less than 2% in feces & less than 0.2% in urine as parent cmpd. Thus,
3'3-dichlorobenzidine is probably degraded rapidly in vivo. ... One metabolite obtained from
monkey urine /following iv admin/ co-chromatographed with monoacetyl benzidine, a urinary
metabolite of benzidine in monkeys. [R35]
Dichlorobenzidine (250 mg) was administered orally to four men and determined the urinary
excretion of metabolites over a 24-hour period. An average of 1.07% (range, 0.32 to 1.55) of the
administered dichlorobenzidine was excreted in the form of N-hydroxyacetyl derivatives and
0.23% (range, 0.11 to 0.45) was excreted as a glucuronide conjugate of an N-hydroxyacetyl
derivative. ... [R67]
(14)C Benzidine is rapidly oxidized by a peroxidase/hydrogen peroxide system to products
which bind irreversibly to deoxyribonucleic acid. The peroxidase/hydrogen peroxide system also
catalyzed the binding of dichlorobenzidine, o-tolidine and o-dianisidine to deoxyribonucleic acid
bind. The binding could be prevented by various biological hydrogen donors, thiols, or phenolic
antioxidants. [R68]
The peroxidatic oxidation of 3,3'-dichlorobenzidine by horseradish peroxidase in the presence of
hydrogen peroxide was examined. The horseradish peroxidase-catalyzed oxidation of
3,3'-dichlorobenzidine yielded two transient and one stable spectral species whereas that of
benzidine yielded three stable spectral species. One species from benzidine, but not a similar
species from 3,3'-dichlorobenzidine was scavenged by butylated hydroxyanisole, glutathione,
N-acetylcysteine or 2-deoxyguanosine. The major product from the horseradish peroxidase
catalyzed oxidation of 3,3'-dichlorobenzidine is azo-3,3'-dichlorobenzidine. None of the products
from enzymic or chemical oxidation of either 3,3'-dichlorobenzidine or benzidine was directly
mutagenic to salmonella typhimurium TA98 in the Ames test; however the chemically oxidized
and enzymic products from 3,3'-dichlorobenzidine were mutagenic in the presence of hydrogen
peroxide. [R69]
The differential mutagen activation of 3,3'-dichlorobenzidine, benzidine, o-tolidine, and
o-dianisidine was studied using Salmonella typhimurium (TA-98). Male Sprague-Dawley rats
were pretreated with 3,3'-dichlorobenzidine, 3-methylcholanthrene, or phenobarital; S9 and
microsomal suspensions were prepared. Three rat liver enzyme systems were used: S9, S9 plus
acetylcoenzyme-A, and microsomes. Activation of the benzidines to mutagens was performed by
preincubating S9 or microsomes containing test amines at optimal mutagenic concentrations.
3,3'-Dichlorobenzidine activation. Activation of o-tolidine was inhibited by 3,3'-dichlorobenzidine
inhibited almost completely benzidine pretreatement and increased by phenobarbital pretreatment.
The response of o-dianisidine was similar to that of 3,3'-dichlorobenzidine. The addition of
acetylcoenzyme-A did not significantly alter the activation of 3,3'-dichlorobenzidine, but increased
activation of benzidine after pretreatment with phenobarbital or 3-methylcholanthrene. The
microsomes from 3,3'-dichlorobenzidine pretreated rats were the most active in mutagenic
activation. Microsome catalyzed activation of o-dianisidine and o-tolidine was inhibited by the
pretreatment with all inducers. Benzidine activation was increased only by 3-methylcholanthrene
pretreatment. 3,3'-Dichlorobenzidine stimulated its own microsome catalyzed activation ninefold
on the basis of cytochrome p450. Dithiothreitol had no effect on the microsome catalyzed
activation of 3,3'-dichlorobenzidine and glutathione depletion did not alter the S9 catalyzed
activation of 3,3'-dichlorobenzidine. After the addition of reduced NAPDH, only
3,3'-dichlorobenzidine elicited the formation of a species absorbing at 440 nanometers.
3,3'-Dichlorobenzidine is the most mutagenic of the four benzidines under conditions of
cytochrome p450 catalyzed activation. [R70]
Studies were carried out, using antibodies to specific cytochrome p450 isozymes, to identify the
isozymes involved in the NADPH-dependent activation of 3,3'-dichlorobenzidine by rat hepatic
microsomes to mutagens in the Ames test. 3,3'-Dichlorobenzidine activation was not affected by a
monoclonal antibody specific for p450c or by a monoclonal antibody specific for p450b, but was
inhibited 69% by a polyclonal antibody made against p450 dependent. 3,3'-Dichlorobenzidine
activation was also inhibited 46% by antibody specific for NADPH-cytochrome p450 reductase.
Further, addition of methimazole, a high affinity substrate for the flavin containing
monooxygenase, reduced the residual mutagenicity in the systems containing antibody to p450
dependent and cytochrome reductase to 9% dn 19%, respectively, of the appropriate control
values. P450 dependent contributes to a majority of the p450 dependent activation of
3,3'-dichlorobenzidine in hepatic microsomes. The flavin-containing monooxygenase may
contribute to the microsomal activation of 3,3'-dichlorobenzidine. [R71]
The direct and hydrogen peroxide dependent mutagenicity of 3,3'-dichlorobenzidine were
compared in Salmonella tester strains TA98, TA98/1,8-DNP6, TA100 and TA102 using the
Ames test. 3,3'-Dichlorobenzidine exhibited both direct and hydrogen peroxide dependent
mutagenicity to both tester strains TA98, TA98/1,8-DNP6. This hydrogen peroxide dependent
mutagenicity of 3,3'-dichlorobenzidine was prevented by horseradish peroxidase.
3,3'-Dichlorobenzidine, in contrast to its effects in tester strains TA98, was not mutagenic to
TA100 and TA102 either directly or in the presence of hydrogen peroxide. These results suggest
that mechanisms, perhaps enzymes endogenous to tester strains TA98, may play a role in the
activation of 3,3'-dichlorobenzidine. [R72]
Dichlorobenzidine can be peroxidatively activated in Salmonella typhimurium Ames tester
strains. Mutagenicity is observed when an Salmonella typhimurium strain which is sensitive to
frame shift mutagens is incubated with dichlorobenzidine and hydrogen peroxide. The bacterial
enzyme, hydroperoxidase I, is responsible for much of this activation. A tester strain lacking
hydroperoxidase I activity was much less sensitive than was the parent strain. When
hydroperoxidase I activity was restored in this strain sensitivity to peroxide dependent
dichlorobenzidine mutagenicity was enhanced. [R73]
The thioether level of urine samples was assessed to determine the validity of thioether excretion
as a parameter for occupational chemical exposure. Male but not female smokers showed
significantly higher urinary thioether levels than nonsmokers. Significantly increased thioether
concentrations were detected in urine samples from patients undergoing chemotherapy and in
persons exposed to 3,3'-dichlorobenzidine. /It was/ concluded that the analytical technique used is
valid for biological monitoring but that thioether in urine is a nonspecific parameter; urine levels
of thioether can be regarded as an indicator of exposure to electrophilic chemicals, but normal
thioether concentrations do not exclude exposure. [R74]
BHL: Male Wistar rats & male beagles given (14)C-3,3'-dichlorobenzidine (0.2 mg/kg body wt),
IV dissolved in 0.5% Tween 20R in water displayed multiphasic blood clearances. The final
phase, predominated by 24 hr after treatment, had half-life of 68 hr in rats & 86 hr in dogs. [R28]
The disposition of the carcinogen, 3,3'-dichlorobenzidine, was studied in the male rat following
oral admin. (14)C-3'3-dichlorobenzidine was absorbed by the rat with the maximum plasma
radioactivity levels being found within 8 hr after dosing. ... The elimination of radioactivity from
the plasma, liver, kidney and lung was biphasic, showing an initial rapid decline (half-lives 1.68,
5.78, 7.14, and 3.85 hr, respectively) followed by a slower disappearance phase (half-lives 33.0,
77.0, 138.6, and 43.3 hr, respectively). [R59]
... About 8% of administered radioactivity was excreted in the urine of dogs and 84% in feces in
7 days when dogs were injected iv with radioactive material. It cleared from the blood with a
half-life of 0.3 to 1.7 hours during the first few hours; after 24 hours clearance is much slower
(half-life 88 hours). ... [R75]
Male Wistar rats (110 to 300 g) were given 0.2 mg/kg (14)C-dichlorobenzidine by injection into
the tail vein, the label disappeared from the blood with a half-life of 0.2 to 1.4 hours during the
early phase and 65 hours after 24 hours. ... [R64]
INTC: Admin in conjunction with other chem: 9 groups of 22 (& 1 of 96) male Wistar rats were
given the following cmpd alone or in sequence for a period of 4 wk per cmpd:
Ortho-N-butyl-N-(4-hydroxybutyl)nitrosamine (0.01% in drinking-water),
N-(4-(5-nitro-2-furyl)-2-thiazolyl)formamide (0.15% in diet), N-fluorenylacetamide (0.025% in
diet), & 3,3'-dichlorobenzidine (0.3% in diet). An untreated control group consisted of 12 rats.
The animals were killed when 40 wk old. 3,3'-Dichlorobenzidine when given in sequence with 1
or more of the other cmpd induced histological changes of liver (cystic change of bile ducts &
oval cell proliferation) in 44-60% of animals (p< 0.05 when compared with ... /control group/).
No change was seen in liver when 3'3-dichlorobenzidine was given alone. Incidence of urinary
bladder tumors was not significantly incr when 3,3'-dichlorobenzidine was added to diet. [R34]
ENVS: 3,3'-Dichlorobenzidine (DCB) may be released as emissions or in wastewater during its
production or use as an intermediate in the manufacture of pigments. Strict regulations requiring
its use in closed systems should limit its release. If released into water, it will rapidly adsorb to
sediment and particulate matter where it is very tightly bound, possibly chemically bound, and not
be readily dislodged. It will undergo very rapid photooxidation in surface layers of water (half-life
90 sec) forming 3-chlorobenzidine and benzidine. Redox reactions and reactions involving free
radicals may also be important. It will bioconcentrate in fish. When released on land, it will tightly
bind to the soil and possibly undergo chemical reactions with soil components. Very slow
mineralization occurs (2% in 32 weeks). If released to the atmosphere it will most likely be
adsorbed to particulate matter and rapidly photodegrade. Exposure to DCB will be primarily
occupational. (SRC)
NATS: 3,3'-Dichlorobenzidine has not been reported to occur naturally(1). [R76]
ARTS: 3,3'-Dichlorobenzidine (DCB) may be released as emissions and in wastewater during its
production, use as an intermediate in the manufacture of pigments, and use as curing agents in
polyurethane elastomers(1). Regulations in the US requiring that DCB and its salts be used in
isolated or closed systems would reduce its release(1). [R76]
FATE: TERRESTRIAL FATE: When released on land, 3,3'-dichlorobenzidine will adsorb
tightly to soil and possibly react chemically with soil components. Very slow mineralization (2%
in 32 weeks(1)) can occur under aerobic conditions. Oxidation by certain naturally occurring soil
cations may be important(SRC). [R77]
AQUATIC FATE: When released in water, 3,3'-dichlorobenzidine (DCB) will rapidly sorb to
sediment and particulate matter in the water column. It will rapidly photodegrade in the surface
layers of water (half-life 90 sec(1)) forming 3-chlorobenzidine and benzidine. DCB is relatively
resistant to biodegradation and this would be a minor loss process. Chemical oxidative processes
involving metal cations occur for the parent molecule, benzidine, and may also occur for DCB.
[R78]
ATMOSPHERIC FATE: Should 3,3'-dichlorobenzidine be released into the atmosphere, it
would most likely be associated with particulate matter or aerosols and be subject to gravitational
settling. While its degradation in the atmosphere has not been studied, it would probably rapidly
photodegrade. (SRC)
BIOD: In laboratory biodegradability tests using sewage seed, 9-99% of 3,3'-dichlorobenzidine
(DCB) degraded in 28 days when yeast extract was present at concentrations of 50 to 400 mg/l.
However, no degradation occurred without this additional nutrient. Nonetheless, DCB was
considered inherently biodegradable although the precise role of the yeast extract was
unexplained(1). When incubated with natural aquatic communities from eutrophic and
mesotrophic lakes, 25% of the DCB degraded in a month(2). When incubated in soil under
aerobic conditions, only 2% mineralization occurred in 32 weeks and no degradation
intermediates were detected(3). Under anaerobic conditions no mineralization occurred in a
year(3). [R79]
Dichlorobenzidine is not readily degradeable by microorganisms obtained from freshwater lakes
and activated sludge. [R45]
ABIO: 3,3'-Dichlorobenzidine (DCB) has a strong absorption band at 282 nm(1) and degrades
rapidly in dilute aqueous solutions (half-life 90 sec) when exposed to noon-day summer
sunlight(1). The photodegradation products are 3-chlorobenzidine, benzidine and other water
insoluble colored materials. Short-lived intermediates are also observed when chlorine-water is
added to a dilute aqueous solution of DCB(1). Half-lives of 3-4 minutes were determined in
laboratory irradiation experiments(1) which also revealed that the photodegradation was acid
catalyzed. The photolability of the compound is much lower in hydrocarbon solvents which may
lead to enhanced stability in water contaminated with hydrocarbon(1). Hydrolysis is not expected
to be an important process for DCB(2). While no data could be found for DCB, unsubstituted
benzidine is very rapidly oxidized by Fe(III) and other naturally occurring cations. While the
chlorosubstituted benzidine would have less of a tendency to oxidize, this type of chemical
reaction could be very important environmentally both in soil and in water(2). [R80]
BIOC: The equilibrium bioconcentration factors using (14)-C-dichlorobenzidine in whole
bluegill sunfish is 495-507, the biconcentration factors is 114-175 in edible portion and 814-856 in
non-edible parts(1). The bioconcentration factors in Golden ide and in algae are 610 and 940
respectively(2). [R81]
KOC: The distribution coefficient of 3,3'-dichlorobenzidine (DCB) to natural sediments at pH 7
ranged from 26.7 to 128(1). The adsorption was initially very rapid(1). Adsorption at pH 9 was
reduced by 30-50% and desorption was low(1). Attempts to extract the DCB from the sediment
revealed that is was very tightly bound(1). It is strongly adsorbed to Brookston clay loam and
Rubicon sand with the Freundlich KF(1/n) values of 1100(0.85) and 273(0.74), respectively(2).
Aromatic amines are known to form covalent bonds to humic materials(3) but no data are
available specific to DCB. [R82]
VWS: No 3,3'-dichlorobenzidine was lost from soil due to volatilization during persistance
studies over 32 and 52 weeks under aerobic and anaerobic conditions, respectively(1). The rate of
evaporation from water is unknown but based on the reported boiling point of unsubstituted
benzidine of 402 deg C(2), evaporation from water should not be a significant transport
process(2). [R83]
EFFL: Wastewater from metal finishing 10 ppb max; Nonferrous metals manufacture 2.0 ppb
max, 0.3 ppb avg; Paint and ink formulation 10 ppb max; Coal mining 3 ppb max(1). Nationwide
urban runoff program (51 catchments in 19 cities-86 samples) not detected(2). [R84]
Analysis of purge wells and seepage water near a waste disposal lagoon receiving
DCB-manufacture wastes showed levels of DCB ranging from 0.13 to 0.27 mg/l. [R85]
The author analyzed the water of the Sumida River in Tokyo during 1964. ... Calorimetric
analysis revealed that total aromatic amine content of the water (including dichlorobenzidine)
reached levels up to 0.562 mg/l, although levels of DCB itself were not quantified. [R86]
SEDS: The computurized water quality database called STORET maintained by EPA shows that
no DCB was detected in 347 sediment samples at a detection limit of 1 mg/kg (dry weight)(1).
DCB was qualitatively identified in sediment/soil/water mixture obtained from contaminated areas
of Love Canal, Niagara Falls(2). [R87]
FISH: STORET data base of EPA indicates that no DCB was detected in 83 biota samples at
detection limit of 2.5 mg/kg (wet weight)(1). [R88]
RTEX: Exposure to 3,3'-dichlorobenzidine is primarily occupational in workers connected with
its manufacture, conversion to derived pigments, and in curing polyurethane elastomers(1).
Exposure is most likely to be through inhalation of dust or mist or by dermal adsorption(1,2).
[R89]
PBEX: Occupational exposure to 3,3'-dichlorobenzidine has and probably still does occur during
its manufacture and conversion to derived pigments. Rubber workers were formerly and may still
be exposed to 3,3'-dichlorobenzidine used for curing polyurethane elastomers. [R32]
In 1973, 18 US companies had been confirmed to be using 3,3'-dichlorobenzidine (DCB) and
166 employees were potentially exposed(1). Although no experimental is available, NIOSH
estimated that approximately 1000 people were exposed to DCB in 1974(2). In pigment
manufacturing plants in Japan, the exposure levels are 2 ppb within 10 min of charging reaction
vessels, dropping to 0.2 ppb within 20 min(1). [R90]
The author surveyed the workplace and factory workers involved in manufactuing
dichlorobenzidine-based organic pigments in Japan. At the time of charging dichlorobenzidine into
the diazotization vessel, atmospheric concentrations of the base were found to be high (2.5
mg/100 cu m). ... The urine of workers involved in charging the diazotization vessel contained 20
mcg/ml (range 10 to 48.5) of aromatic amines measured as dichlorobenzidine, and of those
involved in the drying/cracking process 14.5 mcg/ml (range 9.7 to 23.5). The normal values were
between 12 and 14 mcg/ml ... . [R91]
Workers in the printing and graphic arts professions handling 3,3'-dichlorobenzidine-based azo
dyes. [R15, 229]
SAMP: Collection on a filter, elution with triethylamine in methanol. [R15, 229]
AIR IS DRAWN THROUGH A GLASS-FIBER FILTER FOLLOWED BY A BED OF
SILICA GEL TO COLLECT THESE SUBSTANCES AS EITHER PARTICLES OR VAPORS.
THE COMPOUNDS ARE EXTRACTED FROM THE SAMPLER.
NIOSH Method 5509. Analyte: 3,3'-Dichlorobenzidine. Matrix: Air. Sampler: Filter plus solid
sorbent tube (13 mm glass fiber plus silica gel, 50 mg). Flow Rate: 0.2 l/min. Sample Size: 30
liters. Shipment: Ship 3,3'-dichlorobenzidine samples in dry ice. Sample Stability: 11 days at -15
deg C and 12 days at 23 deg C. [R96]
ALAB: Benzidine and 3,3'-dichlorobenzidine are quantitatively extracted from wastewater and
measured quantitatively through the preparation of their respective pentafluoropropionamides by
using pentafluoropropionamides imidazole. Overall recovery efficiency for benzidines from
wastewater ranges from 91 to 103%. These derivatives are relatively stable cmpd and have
detection limits of 0.2 pg or less, when electron capture detection is used with gas
chromatography. [R97]
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY METHOD IS DESCRIBED FOR
DETERMINATION OF BENZIDINE, 3,3'-DICHLOROBENZIDINE &
1,2-DIPHENYLHYDRAZINE IN AQ MEDIA. THESE CMPD CAN BE ASSAYED EITHER
BY DIRECT INJECTION OR BY SOLVENT EXTRACTION OR RESIN ADSORPTION OF
AQ SAMPLE PRIOR TO ANALYSIS WITH DETECTION LIMITS OF LESS THAN 1 UG/L.
LINEARITY, PRECISION & SPECIFICITY OF METHOD WAS EXCELLENT & NO
INTERFERENCES WERE ENCOUNTERED IN SEVERAL WASTEWATER SAMPLES
ANALYZED. [R98]
NEW AIR-SAMPLING & ANALYTICAL PROCEDURES WERE DEVELOPED FOR
BENZIDINE, 3,3'-DICHLOROBENZIDINE & THEIR SALTS. AIR IS DRAWN THROUGH
A GLASS-FIBER FILTER FOLLOWED BY A BED OF SILICA GEL TO COLLECT THESE
SUBSTANCES AS EITHER PARTICLES OR VAPORS. THE CMPD ARE EXTRACTED
FROM THE SAMPLER & ANALYZED BY HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY WITH SENSITIVITIES IN RANGE OF 3 UG/CU M FOR 48 L AIR
SAMPLES. THE METHOD WAS UNAFFECTED BY TEMP OR HUMIDITY OF THE
SAMPLED ENVIRONMENT. TESTS OF PRECISION, SAMPLE STABILITY &
SEPARATION FROM INTERFERANTS INDICATE THAT THE METHOD SHOULD
PROVIDE RELIABLE RESULTS FOR PERSONAL MONITORING PROCEDURES. [R99]
A CHROMATOGRAPHIC METHOD IS DESCRIBED FOR DETERMINING
3,3'-DICHLOROBENZIDINE IN WASTEWATER. THE LIQUID CHROMATOGRAPHIC
METHOD EMPLOYS A LICHROSORB RP-2 COLUMN, 45% ACETONITRILE (PH 5)
MOBILE PHASE, UV DETECTION AT 280 NM, & ELECTROCHEMICAL DETECTION AT
+0.70 VOLTS APPLIED POTENTIAL. THE THREE-ELECTRODE ELECTROCHEMICAL
CELL DESCRIBED CONTAINS A TUBULAR CARBON-BLACK/POLYETHYLENE
WORKING ELECTRODE THAT PERMITS DETECTION OF SUB-PART-PER-BILLION
LEVELS IN A 50 UL INJECTION WHICH PERMITS DETECTION OF 2 PPB BENZIDINE
& DICHLOROBENZIDINE. RECOVERY FROM WASTEWATER FORTIFIED WITH 3 TO
12 PPB 3,3'-DICHLOROBENZIDINE WAS 87%. [R100]
Gas chromatographic (GC) methods are described for trace analysis of 3,3'-dichlorobenzidine &
its dihydrochloride salt in animal chow & wastewater. /In chow/ extraction of residues as free
amine & cleanup by way of acid-base liq-liq partitioning with benzene followed by silica gel
column. /In/ wastewater, residues are adsorbed by percolating sample through a column of
XAD-2, eluted with acetone, & cleaned up with acid-base partitioning & a silica gel column.
Residues are assayed by gas chromatography either as free amine or after conversion to the
pentafluoropropionyl deriv by using an electron capture or a rubidium-sensitive thermionic-type
detector. Minimum detectable residues in chow & wastewater are about 3 ppb & 18
part-per-trillion, respectively, as determined by electron capture EC-GC of the
pentafluoropropionyl deriv. [R101]
Ground fish tissue is digested with aq sodium hydroxide & extracted with benzene. The extract
is washed with dil acid & cleaned up using gel permeation chromatography. The amines are
separated & quantitated using N-selective gas liquid chromatography. ... [R102]
High performance liquid chromatography with electrochemical detection is used to determine
3,3'-dichlorobenzidine in municipal sludge. The sample is prepared by diluting with phosphate
buffer, extracting with chloroform, extracting again with sulfuric acid, neutralizing with sodium
phosphate, adding methanol & concentrating, followed by diluting with acetate buffer. Limit of
detection is 10 ug/kg. [R103]
Methods for the determination of benzidine, related congeners, and pigments in atmospheric
particulate matter, based on high performance liquid chromatography, were described. The
methods were evaluated using National Bureau of Standards (USA) atmospheric particulate
matter, and freshly collected particulate matter obtained in the vicinity of dyestuff manufacturing
plants. Detection limit was 0.1 ng/cu m for 3,3'-dichlorobenzidine. Recoveries for benzidine and
3,3'-dichlorobenzidine were 30-40% at the 25 ng/cu m (25 ug/g) level and 50-70% at the 10
ng/cu m (100 ug/g) level in atmospheric particulate matter. The method was especially useful as a
qualitative method. [R104]
NIOSH Method 5509. Analyte: 3,3'-Dichlorobenzidine. Matrix: Air. Procedure: High
performance liquid chromatography, ultra violet detection. For 3,3'-dichlorobenzidine this method
has an estimated detection limit of 0.05 ug sample. The overall precision/RSD is less than 0.07.
Applicability: The working range for both analytes is 4 to 200 ug/cu m in for a 50 liter air sample.
Interferences: Aniline; determination of benzidine but may be 4,4'-methylenebis(2-chloroaniline).
[R96]
EPA Method 605. High Performance Liquid and Chromatography with electrochemical
detection for the determination of benzidines including 3,3'-dichlorobenzidine in industrial and
municipal discharges. Under the precribed conditions, for 3,3'-dichlorobenzidine the method has a
detection limit of 0.13 ug/l. Precision and method accuracy were found to be directly related to
the concentration of the parameter and essentially independent of the sample matrix. [R105]
EPA Method 1625. Isotope Dilution Capillary Column Gas Chromatography/Mass
Spectrometry method for the determination of semivolatile organic compounds in municipal and
the industrila discharges. By adding a known amount of a labeled compound to every sample prior
to pruging, a correction of recovery of the pollutant can be made. If labeled compounds are not
available, an internal standard method is used. Under the prescribed conditions, for both labeled,
and unlabeled 3,3'-dichlorobenzidine the method has a minimum detection level of 50 ug/l. The
initial precision is 26 ug/l, the accuracy is 68 to 174 ug/l, and the labled compound recovery is not
specified. [R105]
CLAB: Gas chromatographic (GC) methods are described for trace analysis of
3,3'-dichlorobenzidine & its dihydrochloride salt in human urine. Residues are adsorbed by
percolating sample through a column of XAD-2, eluted with acetone, & cleaned up with
acid-base partitioning & a silica gel column. Residues are assayed by gas chromatography either
as free amine or after conversion to the pentafluoropropionyl deriv by using an electron capture or
a rubidium-sensitive thermionic-type detector. Min detectable residues in human urine are about
60 ppt as determined by electron capture EC-GC of the pentafluoropropionyl deriv. [R101]
The sensitivity of an existing colorimetric method for measurement of 3,3'-dichlorobenzidine in
urine was extended into the low ppb range. The method involves extraction of
3,3'-dichlorobenzidine from urine & then reaction with Chloramine-T. The detection of
3,3'-dichlorobenzidine is linear over range of 1-20 ppb. Linear regression calibration curve for a
group of 44 control urines containing 1-20 ppb of 3,3-'dichlorobenzidine had a relative standard
deviation of 4.6%. Mean extraction recovery was 68%. Spectrophotometric scanning of the soln
containing the color reaction product from urine, concn approx 1-2 ppb, yielded spectra with
distinguishable characteristic max. Such spectra potentially minimize the false-positive results.
[R106]
A method is given for the of detection of trace levels of benzidine, 3,3'-dichlorobenzidine, and
their acetylated and conjugated products in human and hamster urine by liquid or gas
chromatography, after possible exposure to Direct Black 38 and Pigment Yellow 12. Recoveries
from hamster urine were good at greater than or equal to 0.1 mg/l for all cmpds except benzidine
which was approx 54 and 11% at 0.5 and 0.1 mg/l, resp. Recovery for all test compounds in
human urine was 0.1 mg/l. The repeatability of the assay for all levels was approx within 6%.
[R107]
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