Chemical Fact Sheet

Chemical Abstract Number (CAS #) 91941
CASRN 91-94-1
(1,1-Biphenyl)-4,4-Diamine,3,3-dichloro; 3,3-dichloro-4,4-biphenyldiamine
Molecular FormulaC12H10C12N2

Link to the National Library of Medicine's Hazardous Substances
Database for more details on this compound.

UseMFR 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 ColorGray or purple crystalline solid
Boiling Point 402 DEG C
Melting Point 132-133 DEG C
Molecular Weight 253.13
MiscREADILY 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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