F o r s a le b y th e S u p e rin te n d e n t of D o c u m e n ts , U .S . G o v e rn m e n t P rin tin g O ffic e , W a sh in g to n , D .C . 2 0 402# DISCLAIMER
Mention of company name or product does not constitute endorsement by the National In s titu te for Occupational Safety and Health.

# DHEW (NIOSH) Publication No. 80-109

# PREFACE
The Occupational Safety and Health Act of 1970 emphasizes the need for standards to protect the health and safety of workers exposed to an everincreasing number of p oten tial hazards in th e ir workplace. Consequently, the National In s titu te for Occupational Safety and Health (NIOSH) has implemented a program to evaluate the adverse health effects of widely used chemical and physical agents. This program includes the development of Special Hazard Reviews that serve to support and complement the other major health assessment a c tiv itie s of the In s titu te .
The intent of a Special Hazard Review is to analyze and document, from a health standpoint, the problems associated with a given in d u strial chemical, process, or physical agent. Generally, a Special Hazard Review is concerned with those hazards of a chronic nature such as cancer, mutagenicity, terato gen icity, or effects on reproduction. However, they may also deal with other effects that have been id en tified as being harmful to workers when these effects re su lt from exposure to substances found in the workplace. Special Hazard Reviews also recommend control measures, work p ractices, or other appropriate action to a s s is t employers in protecting the health and w ell-being of workers. This Special Hazard Review evaluates available information concerning the carcinogenicity and metabolism of benzidine-based dyes and concludes that a ll these dyes should be recognized as p o tential human carcinogens. This conclusion is based on evidence that four of the dyes have rapidly induced tumors in animals; that two studies of dye workers demonstrate an association between benzidine-based dye exposure and bladder cancer in workers; that a ll of the benzidine-based dyes thus far tested have been metabolized in animals to the carcinogen, benzidine; and that the enzyme (azoreductase) which breaks down these dyes to benzidine is found in both animals and humans. This enzyme acts upon a m ultitude of azo compounds to chemically reduce and break the azo linkage and, therefore, i t is highly probable that the azo-linkage in the as yet untested benzidine-based dyes is also cleaved forming benzidine. In addition, two carcinogenic im purities, 4-aminobiphenyl (an OSHA regulated carcinogen) and 2,4diaminoazobenzene (an animal carcinogen) have been id en tified in one commercial sampling of a benzidine-based dye and may contribute to the carcinogenic p otential of these dyes.
Anthony
Dyes con stitu te a large and diverse group of chemicals that have many applications for imparting color to diverse products. More than 1,200 chemically unique and stru c tu ra lly d ifferen t dyes are manufactured in the United S tates, and an additional 800 are imported. Dyes are c lassified according to th e ir chemical stru ctu re as well as by the method of application in the dyeing process.
A major class of dyes are those derived from benzidine, an aromatic amine acknowledged by both industry and government as causing bladder cancer in humans.
These recommendations include those dyes containing an unsubstituted benzidine structure in th e ir makeup. Such dyes are largely cla ssifie d as d irect dyes, since they may be applied d irec tly to fabrics or other substrates without pretreatm ent or subsequent processing.
Of some 200 benzidine-based dyes, about 30 are now marketed in the United S tates. The most recent estim ate of employee exposure is about 79,000 workers in 63 occupations. Market trends show a decreased usage from over 7 m illion pounds in 1976 to about 3.3 m illion pounds in 1978. All but one US company has phased out the manufacture of these dyes, but imports have increased to 1.6 m illion pounds in 1978 indicating continued worker exposure. Benzidine-based dyes are used prim arily to color te x tile s , leather, and paper. However, diverse industries consume 20% of the to ta l. Dyes constitute a large and diverse group of chemicals that have diverse applications for imparting color to many types of products. They may be broadly defined as w ater-soluble chemical substances that contain chromophoric structures and thus may be used to color another substance by becoming attached to i t by molecular bonding, adsorption, absorption, or mechanical adherence.
Pigments, in contrast to dyes, are insoluble and impart th eir color to another substance by being physically attached to or incorporated into i t . Detailed information on dyes and pigments may be found in the Colour Index , a compendium of technical information on dyes and th e ir use.
The use of dyes dates as far back as p reh isto ric man, but the synthetic dye industry began in 1856 when William H. Perkin f i r s t synthesized the dye mauve . Since that time, the synthetic dye industry has grown extensively in the United States as well as in other countries.
More than 1,200 chemically unique and stru c tu ra lly d ifferen t dyes are manufactured in the United S tates, and an additional 800 dyes are imported . Dyes are c la ssified according to th e ir chemical stru ctu re as well as by the method of application in the dyeing process. The dyes evaluated in th is review are those containing the benzidine moiety without any su bstitution on the diphenyl portion of the molecule, ie , those having a para diamino diphenyl grouping in th e ir stru c tu ra l configuration. They are hereafter referred to as benzidine-based dyes. Benzidine-based dyes are largely classifie d as d irec t dyes, since they may be applied d irec tly to fabrics or other substrates without pretreatm ent or without subsequent processes that firm ly attach the dye to the substrate (mordanting) . More than 200 benzidine-based dyes are lis te d in the Colour Index or are used commercially (see Appendix I I ) . Over 30 benzidine-based dyes have commercial importance in the United S tates, according to the US International Trade Commission . Seventeen are manufactured in th is country, a ll by a single company; the remaining dyes are a ll imported (see Appendix I I I ) . Benzidine-based dyes are used chiefly in the leather, te x tile , and paper ind u stries , but they are also used by beauticians, cra ft workers, and the general public .
The common sta rtin g m aterial for the manufacture of these dyes, benzidine, is acknowledged by both industry  and government (Federal Register 39 (20):3756-97, January 29, 1974) to cause bladder cancer. This is based on considerable evidence from studies with humans as well as with animals. The carcinogenicity of benzidine was reviewed by Clayson  and by Haley . The evidence presented in those reviews demonstrates that both b rief and prolonged exposures to benzidine have been associated with the development of bladder cancer in workers . Aromatic amines, as a class of chemicals, are generally carcinogenic. Benzidine is an outstanding carcinogen w ithin th is class; i t causes cancer in humans, ra ts , hamsters, and mice . The stru c tu re -a c tiv ity relationship between aromatic amines and carcinogenic potency has been reviewed in d e ta il . In addition, benzidine has also been used as a reference mutagen (a known positive control) for several years by the Huntingdon Research Center . In lig h t of a ll the extensive evidence in both humans and experimental animals, NIOSH recommended in 1973 that benzidine be regulated as a carcinogen; in 1974, the Occupational Safety and Health Administration of the Department of Labor promulgated a regulatory standard for benzidine.
U ntil recently there had been l i t t l e concern regarding the dyes derived from and which contain benzidine as an integ ral portion of th e ir chemical stru ctu re. Benzidine chemically united to other substances was not considered dangerous; in 1959, Billiard-Duchesne  stated there was "no danger in using the finished dyes." Only recently has there been serious consideration given to the likelihood that processes in the body could free the benzidine that was o rig in ally used in the manufacture of the dyes . The process of metabolizing or converting a substance back to a sta rtin g component or to another compound is commonly referred to as biotransform ation.
In A pril 1978, NIOSH  and the National Cancer In s titu te (NCI) jo in tly recommended that three widely used benzidine-based dyes (Direct Black 38, D irect Blue 6, and Direct Brown 95) be handled as if they were carcinogens. The NIOSH/NCI recommendation was based on two major findings. F irs t, NCI had determined in a short-term animal feeding study that rats administered the above dyes developed a high incidence of hepatocarcinomas and neoplastic nodules in 5-13 weeks. No sim ilar lesions developed in control animals . Second, NIOSH field studies demonstrated that some workers who had been exposed to these dyes had benzidine in th e ir urine . The amount found in the urine was greater than could be expected from benzidine contamination in the dyes. This demonstrated that the dye its e lf had broken down, releasing benzidine.
Since that time, NIOSH has examined and evaluated the available lite ra tu re concerning these and related dyes. This Special Hazard Review appraises the carcinogenic p o tential of those dyes known to contain the unsubstituted benzidine moiety.

# II. CHARACTERISTICS OF BENZIDINE-BASED DYES

# Chemical and Physical C haracteristics
At room temperature, benzidine-based dyes are a ll colored solids with negligible vapor pressure. The specific water so lu b ility varies from dye to dye, but a ll are su ffic ien tly w ater-soluble to be used for dyeing in an aqueous solution. They are a ll re lativ e ly stable in a ir or solution at ambient temperatures, and possess considerable fastness to lig h t. Unlike some of the anthraquinone vat dyes, benzidine-based dyes do not accelerate the tendering of cellu lo sic type fabrics under the influence of lig h t . They exhaust onto (combine with) cellu lo sic fib ers from a s a lt bath without other treatm ent. Based on th is property, these dyes are called substantive . They usually contain two or three azo groups. D irect dyes give excellent coverage and generally do not otherwise a lte r the appearance or character of the m aterial to be colored. They cover a wide hue range  . Many d irect dyes now on the market do not contain the benzidine moiety and, therefore, are not considered in th is review. These nonbenzidine-derived d irect dyes provide industry with a wide range of colors and hues which may be considered for use as su b stitu tes for benzidine-based dyes ,
The chemical stru ctu res of benzidine-based dyes currently of commerical importance are shown in Appendix II I; a ll have the ch a ra c teristic diazotized benzidine nucleus as shown in Figure I I -l and d iffe r only in the substituents attached at eith er diazo linkage, ie, -N=N-. This linkage is considered the most la b ile portion of each of these dyes, and, in each case, enzymatic  or thermal breakdown  re su lts in the production of benzidine.

#N = N DIAZOTIZED BENZIDINE MOIETY FIGURE n-1

# Purity and Stability
Benzidine and other carcinogenic substances such as 4-amino biphenyl and 2 ,4-diaminoazobenzene may be present in benzidine-based dyes both as the resu lt of im purities introduced in manufacturing processes or may be present in benzidine-based dyes as products of thermal or enzymatic decomposition. NIOSH has measured the amount of benzidine present in d irect dyes from imported and domestic sources . For 26 samples of 11 types of dyes imported from seven countries, the benzidine concentration ranged from less than 1 ppm to 224 ppm. Only six samples contained greater than 10 ppm of benzidine. Similar re su lts were found for 26 samples from domestic sources. No single type of dye accounted for the high re su lts. Few investigators have attempted to analyze im purities other than benzidine present in d irect dyes. However, in one commercially produced lo t of Direct Black 38 (a benzidine-based dye), 150 ppm of 4-amino biphenyl and 9,200 ppm of 2 ,4-diaminoazobenzene (Basic Orange 2) were found even though only 0.1 ppm of benzidine was detected .
The concentration of im purities such as benzidine can be increased by decomposition of dyes containing benzidine as a portion of the stru ctu re. A measurable increase in the concentration of benzidine and 4-amino biphenyl im purities was found when D irect Black 38 in aqueous solution was stored at 25 or 37.5 C for 48 hours . In hamster urine stored at room temperature, su b stan tial increases in the concentrations of benzidine, 4amino biphenyl, and 2 ,4-diaminoazobenzene were found 48 and 96 hours a fter the addition of D irect Black 38 to the urine; at 5 C the dye in hamster urine was stable at lea st 96 hours .
Although a ll decomposition products have not been id en tified , benzidine-based dyes have been shown to break down in aqueous solution at elevated temperatures . Mel'nikov and K irillova  examined a number of technical grade d irect dyes, including some that were benzidine-based. They found that the rate of decomposition of the dyes was accelerated by increased temperature and greatly accelerated in the presence of iron. For example, at 140 C a fte r 6 hours, 17.3% of Congo Red (Direct Red 28) decomposed, and, under the same conditions in the presence of iron, 83/i decomposed. When a 0.1% aqueous solution of Congo Red (Direct Red 28) was stored at 100 C for 1 hour, no decomposition was observed. The presence of te x tile additives such as urea, triethanolam ine, and cellosolve also increased the rate of decomposition.

# III. CHARACTERISTICS OF EXPOSURE

# Manufacture and Uses
The synthesis of dyes is b rie fly described in the Colour Index . Generally, the diazotized benzidine is reacted (coupled) with suitable secondary components such as hydroxylated aromatics or aromatic amines . Since the manufacture of dyes is directed toward producing a p articu lar color and not to producing a specific chemical e n tity , im purities such as benzidine, 4-amino biphenyl, 2,4-diaminoazobenzene, diphenyline, and semidine find th e ir way into the fin a l dye product. If these substances do not in te rfe re with the dyeing process, o rdinarily they are not removed . Additional substances, including other dyes, may be required to produce the p articu lar shade or hue desired in a product. Thus, dyes in general represent mixtures of substances rather than a p articu lar chemical e n tity . This is further complicated by the fact that manufacturers may use d iffering conditions of synthesis, startin g m aterials of d ifferen t purity, and even d ifferen t methods to manufacture dyes . These facts are important since each dye is depicted as a d efin itiv e chemical stru ctu re in textbooks  and in the Colour Index .
More than 30 manufacturers in the United States now produce various d irect dyes. Only one of the nine US manufacturers that produced benzidine-based dyes in 1974 s t i l l does so in 1979 . Some d istrib u to rs import the benzidine-based dyes for resale in the United States  . The 1979 Buyer's Guide published by the American Association of T extile Chemists and C olorists (AATCC) lis ts 20 unsubstituted benzidinebased dyes for sale in the United States . Not a ll dyes sold in the United States, however, are lis te d in the Buyer's Guide (W M artin, verbal communication, July 1979).
Since 1974, the eight US manufacturers that phased out the synthesis of benzidine-based dyes  have replaced them with phthalocyanine, oto lid in e, o-dianisidine, phenylenediamine, and dioxyazine-type dyes . Nonbenzidine-based su b stitu tes appear to be in demand; five manufacturers now s e ll nonbenzidine-based su b stitu tes for Direct Black 38 . A suitable su b stitu te for D irect Blue 6 in the leather dyeing industry has been d iffic u lt to find . NIOSH considers the need for appropriate toxicologic testin g of a ll dye su b stitu tes to be essen tial before they are used in the workplace.
Benzidine-based dyes are used extensively to color te x tile s , leather, and paper. Ease of application has been a major asset to th e ir general a v a ila b ility and widespread usage. Of the to ta l use, 40% is used to color paper, 25% to color te x tile s , 15% for leather, and 20% for diverse applications in the petroleum, rubber, p la stic s , wood, soap, fu r, and hair dye ind u stries .

# Production Volume
Seventeen benzidine-based dyes are produced in the United States by one manufacturer, Fabricolor, In c ., of New Jersey. Table I I I -l gives the US production figures for benzidine dyestuffs manufactured during 1978. These figures were provided to NIOSH through the courtesy of Fabricolor (E Angstadt, w ritten communication, January 1979).

# IV. BIOLOGIC EFFECTS OF BENZIDINE-BASED DYES

# Human Cancer Studies
Two epidemiological studies have reported that exposure to benzidinebased dyes produces cancer in humans . Yoshida et al  examined the p o ssib ility of a relationship between employment in the dyeing industry and an increased risk of developing bladder cancer. Occupational h isto rie s were available for 200 male p atien ts, a ll of whom had bladder cancer and who resided in Kyoto, Japan. One control group consisted of 148 men, at lea st 45 years old, who had been admitted to hospitals in the area with urinary disorders other than malignant tumors. Of the 200, 17 had worked in the dyeing industry. Bladder cancer p atien ts were 6.8 times more lik ely to have been employed as dyers than p atien ts with other urinary disorders. Ten of the 17 had been Kimono p ain ters. In a subsequent survey of Kimono painters in the Kyoto area, Yoshida and Miyakawa  found that the practice of wetting the brush or spatula on the tongue was common. Of 141 persons interviewed, 47% admitted to th is p ractice. This indicated that ingestion of benzidine-based dyes was lik e ly . No information on specific dyes to which the bladder cancer p atien ts were exposed was available. However, Yoshida and Miyakawa referred to four benzidine-based dyes, D irect Black 38, Direct Green 1, D irect Red 17, and D irect Red 28, that had widespread use in Japan in the early 1970's .
Genin  also studied worker exposure to benzidine-based dyes. A fter prelim inary work in which benzidine or dianisidine had been found in the urine of ra ts given dyes based on these substances, Genin examined the urine of 22 workers engaged in the drying and grinding of d irect azo dyes . Benzidine was present in the urine of eight persons and dianisidine was present in three. The q uantities varied from what were described as trace amounts to 0.3 jig/ml. Although the dyes being worked with at the time of urine sample collection were not described, Genin referred to two benzidine-based dyes, D irect Black 38 and D irect Blue 2, as being of greatest commercial importance in the USSR. Genin then examined the company records and found five cases of bladder tumors. Three had occurred between 1965 and 1968 among workers engaged in drying and grinding of d irect azo dyes. The persons were 68, 70, and 72 years old; had laten t periods of 18, 33, and 43 years; and had been exposed 24, 3, and 18 years, respectively.
Genin concluded that exposure to d irect azo dyes, synthesized from diphenyl amino d erivatives, is p o ten tially a cancer hazard. A dose-response relation sh ip could not be established, since the in ten sity of exposure to the dyes was not measured.

# Animal Cancer Studies
The National Cancer In s titu te (NCI) conducted a 93-day study with mice and ra ts that were fed three commercially available benzidine-based dyesi D irect Black 38, D irect Blue 6, and D irect Brown 95 . The three dyes were technical grade, factory-strength d irect dyes.
The molecular structures are shown in Appendix I I I , and the benzidine moiety can be iden tified in each stru ctu re. D irect Blue 6 was 66% pure, D irect Black 38 was 86% pure, and D irect Brown 95 was 79% pure, according to the manufacturer. Midwest Research In s titu te confirmed sim ilar values upon reanalysis of the m aterials. The balance of the constituents was mostly s a lt and w ater, but analyses by thin-layer chromatography (TLC), which used two d ifferen t solvent systems, demonstrated the presence of 8-15 minor im purities in each dye. No attempt was made to iden tify or quantitate these individual im purities. A specific analysis by high-pressure liquid chromatography was done to determine the presence or absence of benzidine. No benzidine was detected at the lowest detectable lim it of 0.004% (40 ppm) in any of the dyes. Corn o il was added to the dyes at 1.3% to suppress dust formation. While no specific analyses were done for benzidine in the food, spectrophotometric analyses of extracts of the d iet showed that each of the dyes was stable in feed for up to 2 weeks at temperatures up to 45 C.
For the bioassay in Fischer 344 ra ts , each dye was fed to 10 male and 10 female ra ts at 190, 375, 750, 1,500 or 3,000 ppm . Each dye was fed to 10 B6C3F1 mice of each sex at 750, 1,500, 3,000, 6,000, or 12,500 ppm, except that females were given D irect Brown 95 only up to 6,000 ppm. Ten matched mice and ra ts of each sex served as controls at each dose level. All animals except controls received one of the three dyes in th e ir feed for approximately 13 weeks.
The f i r s t observed tumor in the ra ts given dye occurred before 5 weeks in each case, regardless of the type of dye fed . This time-to-tumor interv al is the shortest encountered in the NCI bioassay program thus fa r. Dibromoethane has the next shortest time-to-tumor in te rv a l, which was 10 weeks . The shortest time-to-tumor for benzidine its e lf that has been observed in adult ra ts is 6 months . Tumor incidences in the ra ts given 1,500 ppm of a benzidine-based dye in the food are shown in Table IV
The single most important aspect of th is study  was that sig n ifican t numbers of cancerous and precancerous lesions developed in the ra ts w ithin 93 days; hepatocarcinomas and/or neoplastic nodules developed in a sig n ifican t number of ra ts , except in the males exposed to Direct Brown 95. With th is dye the lesions found in male ra ts were lim ited to those described in the NCI report as precancerous. Neither cancerous nor precancerous lesions occurred in any of the control animals, nor had such lesions ever been observed in any of the controls in previous studies w ithin a 93-day time period.
The investigators  concluded that the cancer observed was caused by the dyes themselves or a m etabolite and was not due to benzidine as an impurity. Since the benzidine concentration in each dye was less than 40 ppm, and since the time-to-tumor interv al for benzidine in ra ts at even higher levels has always been much longer than that observed for the dyes , th is conclusion appears to be ju s tifie d . The possible role of other im purities in the demonstrated carcinogenicity of the dyes cannot be established from the information contained in the re p o rt.  Matched Controls
*At 3,000 ppm in female ra ts, the incidence of hepatocellular carcinoma was 4/10.

# Adapted from reference 29
The Clearinghouse on Environmental Carcinogens, a group representing industry, government, and the public, makes fin a l evaluation of the experimental re su lts on substances tested by the NCI bioassay program. The group concluded that Direct Blue 6 and D irect Black 38 dyes caused cancer in both sexes of Fischer 344 ra ts under the conditions of th is bioassay procedure, and that D irect Brown 95 dye caused cancer only in female ra ts . The Clearinghouse on Environmental Carcinogens concluded that testin g at lower levels for longer durations was unnecessary because of the rapid appearance of the tumors. The premature term ination of the study, originally contracted to be carried out over the normal lifetim es of the ra ts and mice and at nontoxic lev els, precluded demonstration of any carcinogenic effect in the mouse or a dose-response relationship in the ra t.
In contrast to the NCI study, two other investigations of the carcinogenic properties of benzidine-based dyes have given equivocal re su lts. In a 270-day study by F u jita et al , D irect Blue 6 was injected subcutaneously into 20 male and 20 female ra ts of an unspecified stra in . Total doses were 170 and 180 mg, respectively, given at weekly or biweekly in terv als. The daily dose was 1 ml of an aqueous 1% solution of the dye. Total survival at 270 days was 50%. In 2 of the 20 females, injection s ite sarcomas developed at 211 and 216 days, respectively. While atrophy of the parenchymal c e lls of the liv e r and d ila ta tio n of the liv er sinusoids were found during pathological examination of. the ra ts , no pre neoplastic or neoplastic lesions were reported. This study did not support the NCI findings of a rapid neoplastic response. The appearance of local sarcomas at injection site s may or may not indicate carcinogenicity in this study since no tumors were observed at remote site s .
N iitsu  studied the carcinogenicity of two benzidine-based dyes, D irect Blue 6 and D irect Black 38 (sources unspecified). Wistar ra ts were given the dyes via th e ir drinking water (0.04%). Twenty male and twentyfive female ra ts were used to te s t D irect Black 38. Because Direct Blue 6 was found to be p artic u larly toxic to male ra ts , the assay of th is dye was lim ited to 20 female ra ts. The observation period was lim ited to only 14 months, since by that time a large number of animals had developed infections and died. The author  concluded that the immunological competence of the ra ts had been compromised. With D irect Black 38 adm inistration, 14-month survival was 4/20 for males and 2/25 for females. One of the two surviving females had cancer of the breast (pathological designation not specified). With Direct Blue 6 adm inistration, 12/20 female ra ts were aliv e at 12 months; 1 of the 12 surviving Wistar female ra ts had a glandular tumor of the outer ear. No tumors were found in the controls.
N iitsu concluded that the carcinogenicity or lack of carcinogenicity of these two d irect dyestuffs could not be determined from the re su lts of the experiment . D ifferent stra in s of ra ts and d ifferen t sources of dye were used in the N iitsu and the NCI studies. Also, the NCI re su lts were found using concentrations of 1,500 and 3,000 ppm in the food , while the N iitsu study used 400 ppm in the drinking water . The marked su sc e p tib ility of the ra ts to infection noted in the N iitsu 14-month study was not found in the shorter 93-day NCI study, except for one ra t that died of b acterial infection. The 93-day period may have been too short a time to expect the type of infection noted by N iitsu .
Marshall , in 1953, reported that V ital Red (a benzidine-based dye) injected in trap erito n eally (ip) into 27 Wister albino ra ts , aged 3-4 months, at a dosage of 1 ml of a 2% aqueous solution of the dye every 2 weeks for 7 months, produced no pathological changes. A detailed pathology description was not given in the report. Twenty animals maintained on the same d iet were the negative controls for th is study. In contrast, ra ts injected with Trypan Blue or Evans Blue (o-tolidine-based dyes) developed lymphomatous tumors. While th is study cannot be considered a negative lifetim e study, i t is apparent that ip-in jected V ital Red did not, under the experimental conditions stated , rapidly produce liv e r tumors as did oral adim inistration of Direct Black 38, Brown 95, and Blue 6 in the NCI study. Korosteleva et al  reported a study in 1977 designed to te s t the carcinogenicity of D irect Red 10. They u tiliz e d a to ta l of 75 white ra ts of unspecified stra in without statin g the number used as controls. The purity of D irect Red 10 was not described. The dye was administered to the ra ts at 500 mg per ra t daily in the food. Nephrotoxic and hepatotoxic effects were evident by the 100th day of observation. More severe kidney effects were noted at a s t i l l la te r stage. Direct Red 10, a benzidinebased dye, produced tumors in 10 of the 18 male ra ts that had survived 500 days.
The type of neoplasms found in the study were four microcholangiomas (malignancy of mixed masses of liv e r cord c e lls and b ile d u c ts), three leukemias (malignant transform ation at white bood c e lls ) , two plasmacytomas (m ultiple myeloma, a neoplasm of plasma c e lls ) , and one hypernephroma (kidney neoplasm with stru ctu re that resembles c o rtical tissue at the adrenal gland). These types of tumors, which are rare in ra ts , were markedly d ifferen t from the hepatocarcinomas found in the NCI study . Korosteleva et al also designed the study to determine whether D irect Red 10 could be metabolized to benzidine (see section on Animal Metabolic S tu dies).
Another chronic te s t of the carcinogenicity of Deep D irect Black EX (Direct Black 38)  was reported by Okajima et al in 1975 . The dye was administered to male W istar ra ts in the drinking water for 60 weeks. At that time a ll were k illed since a sig n ifican t number had developed neoplasms. Results are given in Table IV-2. This report confirmed that D irect Black 38 is carcinogenic in ra ts when administered at lower doses over a longer period of time than used in the NCI study , Because the 60-week experimental period is much shorter than the r a t 's normal lif e span, a more extended experiment would be expected to re su lt in s t i l l more tumors. As in the NCI bioassay , i t would appear as though i t was not necessary to continue th is experiment  over the lifetim e of the ra ts since a sig n ifican t number of tumors has resulted by 60 weeks.  Okajima et al  and N iitsu  both adm inistered D irect Black 38 to ra ts in the drinking water a t sim ilar concentrations (500 and 400 ppm, resp ectiv ely ). I t is not apparent why carcinogenicity was established only in the study by Okajima et a l . Undoubtedly, the low survival ra te in the N iitsu study was a major facto r, since early deaths may have precluded the development of cancer in th is study. The m ortality ra te in the N iitsu study was 97% for D irect Black 38, while the m ortality ra te in the Okajima study was 13% for the same dye.

# Human Metabolic Studies
Humans as well as other mammals can change (metabolize) the benzidinebased dyes back to benzidine . The major organ in which benzidine-based dyes are metabolized to benzidine is the liv e r, but other organs also can do th is to a greater or lesser degree .
Various bacteria and yeast normally found in the small and large in testin e can also reduce the azo bond in benzidine-based dyes and release benzidine . This occurs both in vivo and in v itro ,
The major enzyme capable of lysing (breaking) the azo linkage of benzidine-based dyes is a cytochrome oxidase. This enzyme, termed azoreductase, is associated with the cytochrome P-450 microsomal fraction of the ce llu la r homogenate. The enzyme was extensively studied with relatio n to the azo dye prontosil , and has been described as an "enzyme par excellance" for lysing the azo linkage of dyes . Azoreductase is an extremely nonspecific enzyme found in a ll mammals tested so far , as well as in various microorganisms . I t is not necessary that the substance be a dye for th is enzyme to exert its action. The only requirement is the diazo linkage, ie , -N=N-. No exceptions have been reported . Knowledge of th is enzymatic capability in humans led to the following study .
In 1977, a NIOSH investigation was undertaken to survey in d u strial hygiene practices in the dye industry and to examine urine samples from workers exposed to benzidine-based dyes  . The study protocol included prior n o tifica tio n of the benzidine-based dye manfacturer and team sampling of environmental levels of to ta l p artic u la te . In most cases, i t also included determ ination of the amount of dye present in the p articu late and analysis of the dye samples for residual benzidine. In addition, determination of the urinary levels of benzidine through, in most cases, controlled dual analytical procedures was also performed. These procedures were developed through the e ffo rts of the C linical and Biomedical Support Section, Division of Biomedical and Behavioral Science, and the Measurement Services Section, Division of Physical Sciences and Engineering, NIOSH. Both procedures are described in the report . One of the procedures is also given in Appendix I of th is Special Hazard Review. The lowest detectable lim it for nonspecific primary aromatic amines by th is method was 1 ppb with the provision that at le a st 100 ml of urine be available for analysis. This method required confirm ation of the nonspecific primary aromatic amines as benzidine or monoacetyl benzidine by TLC. The other procedure used to confirm the s p lit samples was electron-capture gas chromatography as developed by Nony and Bowman . The lowest detectable concentration of benzidine in urine stated by these authors  was 1.4 ppb; for monoacetyl benzidine, i t was 5.8 ppb.
Environmental and urinary samples were collected at six fa c ilitie s where workers were p o ten tially exposed to benzidine-based dyes. These f a c ilitie s were: two benzidine-based dye m anufacturers, two te x tile dyeing p lan ts, a leather tanning plan t, and a specialty paper m ill .
In the f i r s t dye manufacturing fa c ility , two of eight workers p oten tially exposed to benzidine-based dyes had monoacetyl benzidine in th eir urine. The concentration of primary amines was 3 ppb in one case and 7 ppb in the other. These spot samples of urine were positive despite the fact that the workers observed were using cartridge-type face resp irato rs at the time of the sampling and that the environment appeared to be dust free. This fa c ility has since discontinued the manufacture of benzidinebased dyes .
In the other dye manufacturing fa c ility , four workers were monitored for exposure to benzidine-based dyes. The average environmental exposure levels of four of the workers were 4.3, 5.2, 11.7, and 17.4 mg to ta l p articu late/cu m. The corresponding urinary concentrations of benzidine averaged 52, 11, 10, and 112 ppb, respectively. The worker having 112 ppb benzidine in his urine (spray dry operator) also had 590 ppb monoacetyl benzidine in the same sample. This fa c ility has also taken measures since then to control dyestuff exposures and to monitor the urine of workers for benzidine .
Three of the above four workers had benzidine congeners other than monoacetyl and diacetyl benzidine in th e ir urine. Two of these workers had o -tolidin e in th e ir urine at 15 and 50 ppb. The third had o-dianisidine in his urine at 1 ppb. At the time of the inv estig atio n, these three workers were not exposed to o -to lid in e, o-dianisidine, or to dyes derived from these two substances. However, prior exposure to any of these substances could not be ruled out. Therefore, the source of o -tolidin e and odianisidine in the urine of these workers could not be established. Many workers, who were monitored and found to have short-term environmental exposures as high as 92.7 mg to ta l p articu late/cu m, did not provide urine samples to the investigators and thus th e ir urine could not be evaluated for the presence of benzidine .
In one te x tile dye manufacturing fa c ility , 7 p oten tially exposed workers were compared with 23 nonindustrially exposed office workers who were used as controls. D irect Black 38 and Direct Blue 2, both benzidinebased dyes, were being used at th is fa c ility . No benzidine was detected in the urine of any control. Urinary concentrations of benzidine in the seven p oten tially exposed workers ranged from below the lim it of detection to 39 ppb; three had both benzidine and monoacetyl benzidine in the urine (one dye tub operator and two dye-weighers). The four other p oten tially exposed workers had no benzidine or monoacetyl benzidine in the urine. The to ta l airborne p articu late m aterial (measured gravim etrically) ranged from 1 to A mg/cu m. Neither the dye concentration nor the types of dye in the p articu lates were determined.
In the other te x tile plan t, 10 workers were also monitored in the same way. The dyes used were D irect Blue 6, D irect Black 38, D irect Brown 95, and Direct Red 8. All airborne concentrations were less than 2 mg to ta l particu late/cu m. Some exposures were equivalent to those in the f ir s t te x tile dye fa c ility . At th is fa c ility , the amount of benzidine-based dye in the p articu late samples was measured colorm etrically. The amount ranged from 0 to 29% by weight in the d ifferen t samples. Presence of benzidine in the urine was expected but not found .
Dye exposure in the leather finishing fa c ility was lim ited to Direct Black 38 and D irect Brown 95. Time-weighted average (TWA) environmental concentrations were 0.69, 5.79, and 10.65 mg/cu m (three samples each). Each of the three workers p o ten tially exposed wore NIOSH-approved half-face cartridge re sp irato rs. No benzidine was found in the urine of the three workers .
In the sp eciality paper processing fa c ility , 23 environmental samples and 47 urine samples were analyzed. The environmental samples were a ll less than 6 mg/cu m (range, 0.17-5.10 mg to ta l p articu late m atter/cu m). In th is fa c ility , management had recently in itia te d a program in which resp irato r use was s tr ic tly enforced. Approximately 1,667 kg (3,000 lb) of D irect Black 38 were consumed during the 3-day survey. Despite th is heavy consumption, no urine samples contained benzidine . Time lim itatio ns prevented extended and repeated monitoring of the environment and the workers under a v ariety of conditions . The time of day for urine collection , for example, may be c r itic a l, because exposure is most lik e ly during work hours and a major portion of the benzidine m etabolites may have been elim inated in the urine before spot sampling the following day. This p o ssib ility of a cyclic type of excretion pattern is made more apparent by work in experimental animals showing that the benzidine m etabolites of D irect Black 38 were largely excreted w ithin the f i r s t 16 hours a fte r dye intake . Since spot samples were taken during the w orkshift, the peak excretion phase may have been missed.
This study  demonstrated that benzidine can be found in the urine of workers who have contact only with the finished dyes under the present working conditions of the industry. No dye samples contained more than 25 ppm benzidine. C alculations provided in the NIOSH report demonstrated that the amount of benzidine found in urine of the workers was too great to have come only from benzidine impurity in the dye, and thus was a metabolic breakdown product of the dye. Even when i t appeared that standard protective equipment such as cartridge resp irato rs were used, the occurrence of benzidine in the urine was not necessarily prevented. One pulverizer operator who was observed to be using a h alf-face resp irato r had 52 ppb benzidine in his urine. However, th is worker was only observed a short time and i t is not known whether he used the resp irato r throughout the day.
Following id e n tifica tio n of 2,4-diaminoazobenzene (at 9,200 ppm) and 4amino biphenyl as contaminants in D irect Black 38, the National Center for Technical Research (NCTR)  reanalyzed some of the the urine samples from workers studied in the above NIOSH investigation . Although q uantitative data were not given, 2,4-diaminoazobenzene was reported to be present in some urine samples but not 4-amino biphenyl . The International Agency for Research on Cancer (IARC) has reviewed the carcinogenic effect of 2,4-diaminoazobenzene, and designated i t an animal carcinogen . OSHA regulates 4-amino biphenyl as a carcinogen in the same manner as benzidine.
Genin  analyzed the urine of 22 workers who had p o ten tial long-term contact with benzidine-based dyes during the manufacture of the d irect azo dyes D irect Black 38, D irect Blue 2, Direct Blue 15, and Direct Blue 218. He found benzidine in the urine of 8 of the 22 workers p o ten tially exposed and dianisidine in 3. The concentrations of benzidine or dianisidine in the urine ranged from what were described as "trace amounts" to 300 ppb, but the individual levels were not reported. Although th is study demonstrated that workers exposed to benzidine-based or dianisidine-based dyes may have benzidine or dianisidine in th e ir urine, q uantitative exposure could not be measured under the conditions p resen t, and doseresponse data were not reported. Korosteleva et al , in studies from 1966 to 1977, id en tified a benzidine complex in the serum of workers in a te x tile factory. The amount of the benzidine complex in the serum depended on the extent and duration of exposure to d irect dyestuffs in the workplace . In th is study, the author compared the blood serum of female te x tile m ill workers (18-60 years old) with that of nonindustrially-exposed blood donors. He found 22 of 77 workers p o ten tially exposed to any type of dye had benzidine complexed to albumin in the serum, compared with no instances of th is complex in 24 nonindustrially-exposed blood donors. Further, those workers exposed only to d irect dyes showed an incidence of 19 of 40, while 21 workers exposed to dyes that were not d irect dyes, or to other in d u stria l substances, had no benzidine-albumin complex in th e ir serum. Since the major d irect dyes reported were benzidine-based , and the benzidine-albumin complex was only found in the workers exposed to d irect dyes, the only reasonable source of the benzidine in the blood was from the benzidine-based dyes.
Thus, two Russian studies  and one US study  have demonstrated that benzidine or benzidine complexes are present in the body flu id s of humans exposed to benzidine-based dyes.

# Animal Metabolic Studies
As early as 1911, i t was known that azo dyes could be metabolized to simpler components. Sisley and Porcher  reported that when dogs received oral doses of Orange 1, a monoazo dye, the dye was reductively cleaved at the azo linkage, resu ltin g in su lfa n ilic acid production in the urine. Sisley and Porcher further demonstrated that i t was necessary for Orange 1 to pass through the in te stin a l tra c t to be lysed. They suggested at that time that the m icrobial flo ra of the digestive tra c t was essen tial for the reduction of th is dye . In 1970, Walker reviewed the metabolism of azo compounds and concluded that many species of animals, yeast, and bacteria could reduce the azo linkage . The nonspecificity of azoreductase has been repeatedly demonstrated . This enzyme rapidly and e ffic ie n tly breaks the double bond in the N=N linkage regardless of the other e n titie s in the substrate.
The National Cancer In s titu te found that both ra ts and mice can metabolize benzidine-based dyes to benzidine . P rior to feeding Direct Black 38, D irect Brown 95, and D irect Blue 6 in the d ie t, the investigators analyzed each batch of dye, and detected no free benzidine (detection lim it was 0.004%). The amounts of benzidine found in urine of the animals are given in Tables IV-3 and IV-4.
Although there was no d irect correlation between the amount of benzidine excreted in the urine and the incidence of tumors in ra ts , each animal fed dye excreted benzidine, and the amount excreted was dose-related in most cases to the amount of dye administered . Since food consumption was not reported, individual dose-excretion ra tio s could not be calcu lated .
Benzidine was measured in the urine of mice and ra ts 3 and 11 and 4 and 12 weeks, respectively, a fte r the experiment began . The mice excreted approximately the same amount of benzidine at 3 weeks as the ra ts did at 4 weeks, and, in general, the mice excreted considerably more at 11 weeks than ra ts excreted at 12 weeks. There were no tumors found in mice by 93 days, while high incidences were found in ra ts exposed for the same period. Since benzidine alone did not produce tumors in ra ts u n til approximately 6 months of exposure at high doses , the production of tumors in ra ts by 93 days suggested that the parent dye (or a m etabolite other than benzidine) was the active carcinogen and that carcinogenicity did not depend exclusively on the presence of benzidine, per se. The NCI report concluded that the benzidine found in urine was a product of dye biotransform ation and not from a benzidine contaminant in the dye . The role of other known carcinogens in these dyes, such as 4-amino biphenyl or 2,4-diaminoazobenzene , was not examined; since they were not suspected contaminants at that time, no analysis was carried out to estab lish th eir presence or absence. I t should be noted that the analytical techniques used in the NCI study were colorim etric assays sim ilar to the one described in Appendix I. These methods are not specific for benzidine, and i t is possible that m etabolites and/or other aromatic amines were responsible for the colorim etric response .  *Samples from untreated controls taken at weeks 3 and 11 showed no benzidine when spotted on TLC p lates. Numbers in parentheses are standard deviations. If fewer than three samples were averaged, the number of samples is given in parentheses instead.

# Adapted from reference 29
Aromatic amines, such as benzidine, produce tumors only ind irectly . They are f ir s t converted by the body to a more reactive substance, which has been termed the ultim ate (or proximate) carcinogen . The NCI investigators  did not attempt to iden tify specific m etabolites of benzidine, and i t is not known if N-hydroxy diacetyl benzidine (one m etabolite suggested as the ultim ate carcinogen ) was among the m etabolites in eith er the ra t or mouse urine.
Rinde  and Rinde and Troll  reported that when any of four benzidine-based dyes, D irect Blue 6, D irect Black 38, D irect Brown 95, and Direct Red 28 (Congo Red), was administered to rhesus monkeys by gavage, benzidine and its monoacetyl derivative could be detected in the urine on an average of 1.25% of the benzidine moiety in the dyes studied. When benzidine its e lf was fed, free benzidine and its monoacetyl derivative were detected in the urine on the average of 1.45% of the original benzidine fed. The authors  concluded on the basis of the above evidence that nearly to ta l conversion of the dye to benzidine took place. However, th is may not be the case because w ater-soluble m etabolites of benzidine or the dye may con stitu te a d ifferin g proportion of the m etabolites than the sparingly-soluble portion .
Because each dye was administered in dimethyl sulfoxide (DMSO), absorption of the dye from the in te stin e would be expected to be greater than when administered in aqueous solution, since DMSO enhances so lu b ility and absorption. Since no other m etabolites were investigated, NIOSH does not consider the re p o rt's conclusion of complete conversion of benzidinebased dyes to benzidine as appropriate. N evertheless, the fact was established that there is at lea st p a rtia l conversion of each of the four dyes to benzidine under the conditions of the experiment. Black 38 were found to be susceptible to azo reduction in th is study. Thus, b acte rial ly sis of the azo bond in the in te stin e is probably a basic means of producing benzidine from the benzidine-based dyes. This benzidine is then available to be absorbed into the body and excreted by the kidney to produce an effect on the bladder. An increase in the b acterial azoreductase enzyme level in the in te stin e was brought about in Fischer 344 ra ts by feeding a meat-based d iet in place of the normal grain-based diet . I t is not known whether ra ts given benzidine-based dyes together with a meat-based d iet would excrete more benzidine in the urine than when fed the dyes together with a grain-based d iet (such as used in the NCI study), but such an effect would not be unexpected. This is sig n ifican t, since man generally consumes a d iet high in meat protein.
A review a rtic le on benzidine metabolism  called attention to re su lts from various investigations  that showed that a v ariety of azo dyes including benzidine-based dyes were reducible at the azo linkage. In the case of azonaphthols, reduction occurred far more readily in the b acterial system than the liv e r preparation . Direct Blue 6, however, while reducible in vivo , was not reported to be reducible by the in te stin a l bacteria of female mice of a stra in designated as dd .
Yoshida et al reported in 1973 that Direct Black 38 (Direct Deep Black EX) was reducible both by E co li and common so il bacteria .
The I coli used were isolated from humans. The common so il bacteria were those taken from so il as well as from raw riv er w ater. Thin-layer chromatography was used for benzidine detection, and adequate negative and positive controls were used. Benzidine was found to be a reduction product from a ll b acterial samples used. In addition, Yoshida et al demonstrated that 3 g of cotton cloth dyed with D irect Black 38 yielded benzidine when incubated with the b acterial flo ra of raw riv er water for 72 hours. The color of the fabric faded under the action of the bacteria but not when incubated for 2 weeks with d is tille d water. This is the only investigation found in the lite ra tu re that dealt with b ac te rial breakdown of benzidine-based dyes once the dye is attached to a fab ric. The importance of th is investigation can also be appreciated by considering that while in tact benzidine-based dyes may not penetrate the skin  , the benzidine portion of the molecule is readily absorbed through the skin 11,14,16,17,45]. Since E coli is a b acteria commonly found on the skin, i t is lik e ly that the dye attached to a benzidine-based dyed fabric that contacts the skin w ill break down to benzidine. Since benzidine can be absorbed d irec tly through the skin, fabric with benzidine-based dyes can be a source of th is compound. II coli is unusually re sista n t to the b a c te rio sta tic effect of dyes in general and grows at temperatures as low as 20 C .
In 1977, Korosteleva et al  demonstrated that ra ts of an unspecified stra in , given 500 mg of D irect Red 10 orally each day, could metabolize th is benzidine-based dye to benzidine, which then acted as a hapten forming a complex with protein in the liv e r and kidney w ithin 4 days after the in itia l dose. By the 30th day, the benzidine complex was present in the blood. The investigators reported a correlation between the carcinogenicity of D irect Red 10 and its a b ility to form antigens containing the benzidine moiety in vivo.
Metabolism studies on Direct Black 38 were recently completed for NIOSH at the National Center for Toxicological Research, in Jefferson, Arkansas . Sensitive as well as sp ecific analytical chemical methods were developed for assay of the known and the proposed im purities in D irect Black 38 as well as i ts known and proposed m etabolites. Similar studies were carried out for possible m etabolites of the substance Pigment Yellow 12, a pigment containing and deri-ved from 3 ,3 '-dichlorobenzidine. This pigment was not metabolized to benzidine, dichlorobenzidine, 2,4diaminoazobenzene, or 4-amino biphenyl, confirming prior studies in other species .
F ifteen male Syrian golden hamsters weighing approximately 110 g were administered D irect Black 38 at 100 mg/kg by g astric lavage . Urine was collected for analysis at in terv als up to 7 days. Three hamsters were used as controls. The dry, purified dye was analyzed and found to contain 3 ppm benzidine, 6 ppm 4-amino biphenyl, and 670 ppm 2,4-diaminoazobenzene. Further attempts a t p u rificatio n to elim inate the 2,4-diaminoazobenzene were unsuccessful.
The major portion of a ll m etabolites of Direct Black 38 was excreted w ithin 16 hours a fter adm inistration. The average to ta ls of m etabolites excreted by 16 hours are shown in Table IV

# Hydrolyzable benzidine- 257
Hydrolyzable 4-amino biphenyl- 5.1 *Hydrolyzable means that these substances were o rig in ally present as conjugates and were divided by adding sodium hydroxide. Adapted from reference 37 This analysis would account for approximately 10% of the benzidine moiety available in the D irect Black 38 o rigin ally fed to these animals.
The presence of 4-amino biphenyl as a m etabolite in the hamster urine is sig n ifican t in th at 4-amino biphenyl is a substance regulated by OSHA as a carcinogen in the same manner as benzidine . In addition, the presence of re la tiv e ly high concentrations of monoacetyl benzidine and hydrolyzable benzidine in the urine means that at some point the to ta l exposure of each animal to benzidine in th is experiment must have been several orders of magnitude greater than that indicated by the concentration of benzidine its e lf in the urine . In monkeys, Rinde  was able to recover approximately 1.5% of the benzidine contained in each of four benzidine-based dyes as benzidine or monoacetyl benzidine. A major portion of benzidine would be expected to be excreted in a conjugated form and would not be detected unless the urine is f i r s t treated with an a lk a li to hydrolyze the conjugates of benzidine. Methods developed up to th is point have not included a hydrolysis step. Future methods for monitoring human urine should u tiliz e a hydrolysis step to provide the most sensitive indicator of exposure to benzidine-based dyes .
The In ternatio n al Business Machines Corporation recently reported preliminary data to the Environmental Protection Agency on a te st to determine possible skin absorption of Direct Black 38 in rabbits . The diphenyl portion of D irect Black 38 was f i r s t labeled with carbon-14. A proprietary mixture of D irect Black 38 was then applied to the skin of two rab bits. At the end of 144 hours, 91% of the rad io activ ity was recovered in the urine and feces of the rab bits. This indicates that D irect Black 38 or a portion of the molecule had penetrated the skin. Previous work by Rinde in monkeys had not shown skin absorption in that species .

# In another prelim inary study, Matthews  examined the following benzidine-based dyes:
Direct Blue 2, D irect Black 4, D irect Brown 2, D irect Red 28, D irect Orange 8, and D irect Green 1. Each was fed to one of six female mongrel dogs at 100 mg/kg. Benzidine its e lf was fed to a seventh dog as a positive control. The treated dogs were held in individual metabolism cages, where they received food and water ad libitum . The urine was collected daily for 3 days, and analyzed for benzidine. The amount of benzidine measured in each urine varied from 320 to 1,675 /ig, but, in every case, dye adm inistration resulted in the excretion of benzidine. In the case of D irect Brown 2, benzidine excretion after adm inistration of the dye exceeded to ta l urinary benzidine excretion observed in the positive control dog given pure benzidine.
These re su lts increase to 11 the number of dyes that have been demonstrated to be metabolized to benzidine in humans, monkeys, ra ts , hamsters, mice, or dogs .

# V. EVALUATION AND CONCLUSIONS
Benzidine, an interm ediate in the synthesis of most benzidine-based dyes, is controlled as a human carcinogen in the workplace. When a Federal standard for benzidine (29 CFR 1910.1010) was promulgated in 1974, there was l i t t l e evidence to suggest that dyes prepared from benzidine were carcinogenic. Since then, a number of cases of bladder cancer have been reported in two groups of workers with exposure to benzidine-based dyes . These reports are meaningful in that they provide evidence that man is susceptible to the carcinogenic action of these dyes. However, the major evidence for the carcinogenic action of benzidine-based dyes is found in controlled animal studies. Rats fed Direct Blue 6, D irect Black 38, and Direct Brown 95 developed tumors in as l i t t l e time as 5 weeks . By 13 weeks, many exposed ra ts developed hepatocarcinomas or neoplastic nodules. In a separate study in which ra ts were fed D irect Red 10, carcinogenic a c tiv ity was also demonstrated , In yet another study, D irect Black 38 was found to be carcinogenic in ra ts when given at lower doses over a longeri period of time . Since the re su lts in animals support the findings in humans, i t must be concluded that benzidine-based dyes may cause cancer in humans.
Studies on ligated in te stin e  and b acteria commonly present in the in testin e  have shown that the azo linkage can be broken to yield benzidine from Direct Black 38, Direct Red 10, D irect Red 17, D irect Red 28, and D irect Orange 8. While inhalation is a major route of employee exposure to benzidine-based dyes , many of the inhaled dye p articles may be too large to reach and be retained in the lung. They then would be returned to the e p ig lo ttis by the c ilia l action of the bronchial mucosa or trapped by nasal impaction, and then swallowed so that they become available for absorption by the body. In addition, hand to mouth tran sfer, contamination of foods, or poor work p ractices would lead to oral ingestion of the dyes. B acterial reduction in the in te stin e would represent one source of benzidine in such cases.
Other available evidence suggests that cleavage of the azo linkage of the dye also occurs a fte r absorption of the dye, resu ltin g in the release of benzidine in vivo. Benzidine has been found in the urine of workers who handled benzidine-based dyes in the dye manufacturing and te x tile industries . In a Russian study, about h alf of the te x tile m ill workers examined who handled d irect dyes had benzidine-albumin complexes in the blood . In another Russian study, 8  Further research is needed to c la rify the issue of skin absorption of the dyes. However, i t is known that D irect Black 38 can be reduced to benzidine by b acteria commonly found on the skin . This suggests that the dermal route could be a source of employee exposure since benzidine is readily absorbed through the skin . Preliminary work in rabbits supports th is as a possible route of exposure .
The evidence presented above demonstrates that benzidine is a metabolic product of at least .11 benzidine-based dyes. The azoreductase enzyme that breaks down these dyes to benzidine is ubiquitous and generic. I t acts on a m ultitude of azo compounds, containing a large v ariety of individual components and has been observed to cleave the N=N linkage common to these compounds. The a b ility to be metabolized in vivo to a known carcinogen is su fficien t evidence to necessitate regulation of a ll benzidine-based dyes. In addition, animal experiments have suggested that these dyes could have a greater p otential for carcinogenicity than benzidine alone, since the dyes have been reported to form tumors much more quickly than benzidine . Therefore, benzidine-based dyes may be a more robust source of the ultim ate carcinogen. Im purities introduced in the manufacture of the dye may also be a factor. For example, 4-amino biphenyl and 2 ,4-diaminoazobenzene were id en tified in commercially prepared D irect Black 38 .
Both contaminants are important because 2,4-diaminoazobenzene is considered a carcinogen by IARC  and 4-amino biphenyl is regulated as a human carcinogen (29 CFR 1910.1011). Mutagenesis te s ts using the Salmonella typhimurium (TA-98 and TA-100) assay with activation were positive for the following substances: the urine of hamsters fed D irect Black 38, the major m etabolites of th is dye (benzidine, monoacetyl benzidine, diacetyl benzidine, and 4-amino biphenyl), and the dye its e lf .
Studies have reported that four benzidine-based dyes rapidly induce cancer in experimental animals, suggesting that these substances may have a greater carcinogenic p otential than can be attrib u ted to th e ir m etabolite benzidine alone. Other studies have reported a number of cases of bladder cancer in two groups of workers exposed to benzidine-based dyes, but not to benzidine. In addition, a ll of the 11 benzidine-based dyes thus far tested have consistently been metabolized in animals to the carcinogen benzidine. The azoreductase enzyme responsible for formation of benzidine in the body is known to be nonspecific in i ts action and is found in b acteria, animals, and humans. Occupational exposure to benzidine-based dyes has also resulted in benzidine formation in the bodies of workers. This then indicates that there is an extremely high probability that those yet untested benzidine-based dyes can be metabolized to benzidine also. Based on a combination of the above facto rs, NIOSH concludes that a ll benzidinebased dyes, regardless of th e ir physical sta te or proportion in a mixture, should be recognized as p oten tial human carcinogens. In addition, NIOSH recommends that the production, use, storage, packaging, and d istrib u tio n of a ll benzidine-based dyes be discontinued in lig h t of present evidence o p o tential carcinogenic risk s. The replacement of benzidine-based dyes with less toxic su bstitutes should be in itia te d immediately. As an interim measure, stringent controls and work p ractices are recommended to minimize exposure to any of the benzidine-based dyes.
During th is interim period, since the carcinogens benzidine, 4-amino biphenyl, and 2 ,4-diaminoazobenzene have been id en tified as contaminants or breakdown products of a commercially prepared benzidine-based dye , p articu lar attention should be given to the possible increase in concentration of these im purities in the cleanup of sp ills or leaks, waste disposal, and hot dyeing processes.
A number of reports have considered or referred to the use of su b stitu tes to replace benzidine-based dyes . In the absence of additional inform ation, NIOSH recommends that the benzidine congener dyes be handled with extreme care in the workplace and that exposure to these dyes be minimized. Substitution of noncarcinogenic dyes for those that are benzidine-based is e ssen tial. However, i t must be recognized that the stru ctu ral requirements for a compound to impart color leads to the use of dyes containing m oieties that tend to be chemically reactive and toxic. Thus, the p o ssib ility of metabolic conversion to even more toxic compounds, as well as the effects of the dye its e lf , must be considered. In addition, toxic im purities can be introduced in manufacture. Information on the toxic effects of possible su b stitu tes should be taken into account during replacement of benzidine-based dyes. If such information is incomplete or suggests that the dye might also have carcinogenic p o ten tial, other su b stitu tes must be used.

# VI. WORK. PRACTICES AND CONTROL RECOMMENDATIONS
This section evaluates the conditions under which employee exposure to benzidine-based dyes is lik e ly . I t also delineates those operations in which the most intense exposures would be predicted in the absence of controls. Emphasis is placed on work practices and control recommendations to lim it excessive employee exposure to benzidine-based dyes in operations that can be p artic u la rly hazardous. The employer should, in addition, evaluate existing programs for labeling and posting, employee education, cleanup of s p ills , disposal of waste, emergencies, and general plant san itatio n to ensure their adequacy in lig h t of evidence of carcinogenicity of the benzidine-based dyes. If present programs are inadequate, new ones should be implemented to ensure a clean and healthful workplace and to ensure that employees are aware of the hazards involved and of th e ir role in maintaining a safe working environment.
During manufacture, the dyes are generally prepared in a closed system in which benzidine is formed by the reaction of the sta rtin g m aterial, hydrazobenzene, with hydrochloric acid . One dye manufacturer has developed a process th at uses nitrobenzene as the sta rtin g m aterial, thus elim inating the need for employees to handle hydrazobenzene, which forms benzidine in the stomach if ingested . After the dye is p recip itated , however, generally i t is handled in open systems . The dye is filte re d in presses and the press cake is unloaded manually. The press cake is then dried, and the dried dye is ground into a fine powder. This fine powder is then transferred to ribbon blenders where other dyes are often admixed to obtain the desired colors. Sulfonated dedusting o il is usually added at th is point to reduce the tendency for the dye to produce an aerosol when poured or mixed. S alt or sugar is nearly always added to d ilu te the concentrated dye . The fin a l product is then weighed and packaged for marketing.
Processes in which dried dye is handled have the g reatest p otential for employee exposure during the manufacture or repackaging of benzidine-based dyes; such processes should be performed in closed systems. Access to such areas should be re stric te d to authorized employees. When such enclosure is not possible, each operation should be provided with continuous local exhaust v en tilatio n so that a ir movement is always from surrounding work areas to the operation and then through su itab le f ilte r s as described in OSHA safety and health standards (29 CFR 1910), so as to prevent the release of any benzidine-based dye to the work environment.
Handling of moist press cakes and solutions constitutes a lesser source of employee exposure to benzidine-based dyes than handling dry powders, since solutions and moist m aterials are less lik ely to be dispersed into the a ir or d istrib u ted over large areas. N evertheless, closed systems should be used to further lim it employee exposure to benzidine-based dyes. F ilte r presses that can be emptied and decontaminated without opening the f ilte r s have been used for preparation of benzidine su lfate , and the use of th is type of f i l t e r should also be applicable in the manufacture of the dyes.
Various methods of elim inating the dust hazard associated with dyes have been used. In addition to the treatment with sulfonated o ils referred to above, two other methods are presently employed . One method is to form the dye into p ellets so that dusting is minimized. The other procedure is to make up the dyes in unitized double packages. The outer package, which is used for protection during shipment, contains an inner package that dissolves in water. Thus, the worker making up a dye bath would add the appropriate number of units of dye to the bath without opening the inner package. These procedures may be combined for added safety (a dye package of p ellets in double packets).
Pastes rather than dried dyes have been used by the paper industry for some time . In th is case, the paste is added to a large container of an aqueous solution and that solution is metered to each batch of paper as i t is dyed.
Benzidine-based dyes are used in the paper, te x tile , and leather finishing ind u stries. Since the industries are diverse, i t would be expected that the conditions of p o tential exposure to benzidine-based dyes are equally diverse. At le a st 63 occupational categories have been found to be associated with p o tential exposure to benzidine-based dyes . In the f a c ilitie s surveyed by NIOSH , a three-step process for handling the benzidine-based dyes was c h aracteristic of a ll three ind u stries. F irs t, a dye weigher dispensed the m aterial into a vessel. In some cases the weigher also dissolved the dry dyestuffs. Next, the m aterial to be dyed and the dissolved dye were placed in a dyeing v at. F inally, the dyed m aterial was dried and finished. The degree of worker exposure in areas where paper, te x tile s , or leather are being dyed would be expected to vary widely depending on conditions such as the temperature of the dye solution, the amount of manual handling of the dyed m aterial, and the design of the dye v ats. Employees who handle paper, te x tile s , or leather a fte r i t is dyed could also be exposed to benzidine-based dyes since excess dye retained on the finished m aterial would be available for release as dried powder.
During use of benzidine-based dyes, the g reatest p o ten tial for exposure would be expected to be among dye-weighers who handle dry powders . Their operations should be carried out in a hood designed and maintained so as to draw a ir inward at an average linear face velocity of 150 feet per minute (0.76 m/s) with a minimum of 125 feet per minute (0.64 m /s). P articular attention should be paid to the design of such hoods to ensure that the employee can transfer the m aterial from its o rigin al container to the weighing scale without taking any dye outside the enclosure or inserting any part of the body other than hands and arms inside the hood. Containers of benzidine-based dyes should be opened only during the weighing operation. Once opened, they should remain w ithin the hood u n til disposal or u n til a ll the contents have been used. The use of pastes or liquids rather than dried dyes should be considered as a control measure.
The dye weigher should prepare the dye solutions to be placed in the vats. This procedure would elim inate the need to tran sfer the more hazardous dry m aterial from one statio n to another. The dye weighing area should be regulated, and access should be lim ited to authorized employees who are wearing adequate personal protective equipment adequate to prevent skin contact with or inhalation of the dyes. If workers must handle the m aterial in the vats manually, or if adjustments to machinery are necessary while benzidine-based dye is present, the worker must wear impervious clothing and resp irato ry protection to prevent exposure to the dyes.
T extiles are dyed at various stages in th e ir manufacture, including unspun fib ers, unwoven yarn, and finished fab ric. Workers who prepare fabrics from unspun fib ers are of p artic u la r concern, since they could be p oten tially exposed to benzidine-based dyes contained on dusts generated during manufacture. In addition, some benzidine-based dyes possess much poorer fastness to wet treatment than do others; persons who launder such clothing are p o ten tially exposed to the dyes. Employees and employers should be aware that those who launder, weave, or sew fabrics dyed with benzidine-based dyes are p o ten tially exposed to the dyes. If exposure is considered lik e ly , the employer should in s titu te stringent control measures and work p ractices to prevent such exposure.
Several generally acceptable p ractices for the control of hazardous m aterials are recommended wherever there is p o ten tial for exposure . For example, pressure fa ilu re alarms for closed systems and exhaust v en tilatio n can rapidly indicate a system fa ilu re that might re su lt in the release of substan tial q uan tities of benzidine-based dyes. Continuous flow indicators, such as water or o il manometers properly mounted at the juncture of a fume hood and duct throat and marked to indicate acceptable airflow , w ill give a readily observable indication of decreased efficiency in the v en tilatio n system for the hood. Wet methods, vacuum cleaning, or other methods that do not lead to redispersion of se ttle d dust should be used for plant maintenance and san itatio n . Dry sweeping or blowing with compressed a ir should be prohibited. In the cleanup of leaks or s p ills and in maintenance or rep air operations on contaminated systems or equipment, employees should wear clean impervious garments, including at least gloves, boots, and an air-supplied resp irato r with positive pressure in the facepiece.
Benzidine is readily absorbed through the skin, and reuse of protective equipment or work clothing contaminated with benzidine can lead to its dermal absorption . However, evidence for dermal absorption of the benzidine-based dyes is conflicting , As a prudent measure, absorption of benzidine-based dyes through the skin must be considered a real p o ssib ility , Thus, employee exposure through contaminated clothing may be as serious a problem for benzidine-based dyes as i t is for benzidine . P articu late m aterial containing the dyes can be released into the external environment, including the employee's home, unless clothing p oten tially contaminated with the dyes is removed before the employee leaves the exposure area. If an employee's skin is p oten tially contaminated with benzidine-based dyes, the employee should wash or shower as appropriate before leaving the exposure area.

# Workplace Air
At th is time, NIOSH is unaware of a p ra ctic al method for identifying each sp ecific benzidine-based dye workers may be exposed to. I t should be possible to identify classes of azo dyes such as the benzidine-based dyes by the use of high performance liquid chromatography or gas chromatography a fte r reduction of the azo linkage. Additional research is generally needed in th is area.
Although methods to measure the concentration of an individual benzidine-based dye in a ir are not available, a screening method for diazonium sa lts and azo dyes in air has been developed by NIOSH  and is described in Appendix 1(A). The range of the method is lis te d as 0.01-0.4 mg/cu m in a 500-lite r sample of a ir. If only one benzidine-based dye is present and other positive interferences are absent, a q uantitative estim ate of the concentration of the dye present in the a ir can be made. If more than one azo dye of any type is present, the method is not quan titative and the concentration of to ta l azo dye present must be given as a range. However, the method can indicate the adequacy of work practices and engineering controls employed to minimize the concentration of airborne benzidine-based dyes during the period required to phase in su b stitu te dyes.

# Urinary Levels
Aromatic diamines, such as benzidine, are not normally found in the body. However, benzidine, a metabolic product of benzidine-based dyes, has appeared in the urine of employees exposed to the dyes but not to benzidine. This demonstrates systemic absorption of the dye or a portion thereof. Currently, the measurement of urinary benzidine is used more as a diagnostic practice than for use in compliance. The presence of benzidine in the urine of a worker p o ten tially exposed to a benzidine-based dye would v erify that such exposure had, in fa c t, occurred, but its absence cannot be considered v e rific a tio n that no exposure has occurred.
The method recommended in Appendix 1(B) can detect the presence of aromatic amines at 100 ng/100 ml of urine, although recovery efficiency at the lim it of detection is poor. If aromatic amines are found in the urine, thin-layer chromatography (TLC) can be used to confirm (though not rigorously prove) the presence of benzidine. The to ta l volume of the sample must be no less than 100 ml; the minimum detection lim it in such a volume is 300 ng. An Rf value iden tical to th at of benzidine constitutes th is confirm ation. If benzidine is detected by u rin a ly sis, i t would demonstrate that employee exposure to benzidine or benzidine-based dyes has occurred and would suggest inadequacies in eith er engineering controls or work p ractices. Thus, u rinalysis in addition to environmental monitoring is necessary to assure the employer that exposure of employees to benzidine-based dyes has been minimized.
Specific methods for the detection of benzidine in human  and hamster  urine and in in d u stria l effluents  have been developed. Two  are based on electron-capture gas chromatography, one is based on high performance liquid chromatography , and one is a spectrophotofluorim etric technique . Unlike the fluorescamine method  or the method given in Appendix 1(B), they can measure benzidine in the presence of other aromatic amines. While a ll these methods should be readily adaptable to detection of benzidine in an employee's urine, they involve considerably more elaborate instrum entation and analytical techniques than the routine screening method described in Appendix 1(B). Several points need further c la rific a tio n before the amount of benzidine in the urine can be correlated precisely with the concentration of benzidinebased dyes in the a ir. For example, the optimum conditions for sample collection are not known. The NCTR study  recommended alkaline hydrolysis of the urine to measure both free and conjugated benzidine. Since u rin aly sis is presently useful as a q u alitativ e index of exposure only, the less elaborate screening method constitutes the best approach at th is time.   Duplicate samples of airborne p artic u la te m aterial are collected on cellulose acetate membrane f il te r s . One f i l t e r is extracted with d ilu te hydrochloric acid and analyzed for diazonium sa lts spectrophotom etrically at 375 nm. The second f i l t e r is extracted with an appropriate solvent and a spectrophotometric scan of the solution is made in the 400-to 700-nm range. The absorbance maxima are compared with the absorbance maxima of standard solutions prepared from bulk samples of the azo dyes.  c h a r a c t e r i s t i c a b s o r p t i v i t y , the c o n c e n t r a t io n o f t o t a l diazonium s a l t s must be g iv e n as a ra n g e .  (2) Connect the cassette to the vacuum pump. No tubing should be placed in front of the f i l t e r .

# D irect

# The s a l t h avin g the h ig h e s t a b s o r p t i v i t y d eterm in es the upper l i m i t o f the ran ge and th e s a l t h avin g the lo w e st a b s o r p t i v i t y d eterm in es the
(3) Turn on the pump to begin sample collection . Measure the flowrate and time, or volume, as accurately as possible. Sample 500 lite r s or more at 1.5-2 liters/m in .
(4) Take duplicate samples at each sampling s ite . One is for the diazonium s a lt analysis; the other for the azo dye analysis. P rotect the f ilte r s from lig h t to prevent photodecomposition of the diazonium s a lts .
(5) Ship the f ilte r s in a su itab le container designed by NI0SH to minimize contamination and to prevent damage in tra n sit. Include two or more blank f il te r s , which are handled in the same manner as the sample f ilte r s except that no a ir is sampled through them.
(c) Analysis of Samples (1) Diazonium Salts Extract one of the duplicate sample f ilte r s from each s ite with 40 ml of 0.1 N HCl in a 100-ml beaker. A gitate the mixture in an u ltraso nic bath for 5 minutes. Allow the f i l te r and any suspended solids to s e ttle . Extract a blank f i l t e r in the same manner. Perform the extractions with minimum exposure to lig h t. Transfer a portion of each solution to a 1-, 5-, or 10-cm fused s ilic a c e ll, depending on the anticipated diazonium s a lt concentration. Determine the absorbance of the blank and the sample of 375 nm. Use 0.1 N HCl as the reference solution.
(2) Azo Dyes Select an appropriate solvent by experimentation with the bulk samples. (See Advantages and Disadvantages, section (f).) Extract one of the duplicate sample f ilte r s and a blank f i l t e r with the solvent using the extraction procedure outlined in section (1) on Diazonium S alts. Tranfer a portion of each solution to a 1-, 5-, or 10-cm fused s ilic a c e ll, depending on the anticipated concentration. Using the solvent as a reference, scan the 400-to 700-nm range to locate the absorbance maxima. Measure the absorbance at each wavelength where the bulk azo dyes absorb. (2) Transfer a portion of each solution to a 1-, 5-, or 10-cm fused s ilic a c e ll.
(3) Using the 0.1 N HCl solution as the reference solution, determine the absorbance of each standard a t 375 nm.
(4) Construct a standard curve for each diazonium s a lt by plo ttin g the absorbance against the amount (in micrograms) of the individual diazonium s a lt in the corresponding standard.
yielding the largest value. The lower lim it of the range is taken from the curve yielding the lowest value.
(c) Correct the calculated amount or range of amounts of the appropriate diazonium salt(.s) or azo dye(s) for any corresponding value found by the analysis of blank f ilte r s .
(d) The concentration or range of concentrations of diazonium sa lts and azo dyes may be expressed in mg/cu m: mg/cu m = Amount ((ug)g) Vs (in lite rs )
where: Vs = volume (lite rs ) of a ir sampled.

# APPENDIX I -ANALYSIS (CONTINUED) (B) METHOD FOR MONITORING FOR BENZIDINE IN URINE
NIOSH has reviewed the various methods of biological monitoring for benzidine and related chemicals. There are two acceptable methods: the recommended method that follows  and the fluorescamine method given in the o -tolidin e c rite ria document . The recommended method presented here is less expensive and more sen sitive than the fluorescamine method; however, i t requires a longer working time. Neither method is specific for benzidine.
Any other method of at least equal se n sitiv ity and precision may be substituted for the recommended method [37, 59,78 100-20,000 ng/ 100 ml urine Precision:
Not determined C lassificatio n: D (opera tional) Date Issued: 8/3/79 P rinciple of the Method Aromatic amines, including benzidine, are extracted from urine with chloroform a fte r pH adjustment. The chloroform extract is back extracted into 0.1 N HC1 and derivatized with 2 ,4 ,6-trinitrobenzene sulfonic acid (TNBS). The TNBS derivatives are extracted with chloroform to remove in terferin g chromophores and the fin a l chloroform extract quantitated spectrophotom etrically at 400 nm while using benzidine as a standard. The resu ltin g chloroform extract is concentrated to droplet volume for benzidine id e n tific a tio n by thin-layer chromatography (TLC). A ttention is called to the poor s ta b ility of the chloroform solution of the TNBS derivative of benzidine. Q uantitation of th is derivative by colorimetry should be performed w ithin 30-45 minutes a fte r preparation of the fin al chloroform solution.
(a) The detection lim it of th is spectrophotometric procedure is 100 ng of benzidine (aromatic amine) per 100 ml of urine. The detection lim it for benzidine by TLC is 300 ng/100 ml urine.
(b) The range is from the detection lim it up to. 20,000 ng/100 ml of urine.
(c) The UV and/or v isib le detector (maximum absorbance, 400 nm) has a lin ear response up to 0.02 mg of benzidine per 100 ml of urine.

# Range and Sensitivity
Interferences (a) Any other aromatic amine having an absorbance maximum at 400 nm and an Rf value iden tical to that of benzidine when chromatographed by TLC would in te rfe re .
(b) Aromatic amines in substances that are normally present, ingested, or produced by metabolic processes in the worker w ill produce a chromophore (false p o sitiv e ).
(c) Extraction at pH 5 with chloroform and subsequent back extraction into HC1 reduce interference from other compounds.

# Precision and Accuracy
(a) Recovery studies conducted on benzidine indicate 70% at the 500 ng/100 ml level and a marked decrease (about 20%) at the 100 ng/100 ml le v e l.
(b) The precision has not been evaluated at th is time.
Advantages and Disadvantages (a) The principal advantage is that the method is specific for aromatic amines in the nanogram range.
(b) Another advantage is that benzidine at 300 ng/100 ml can be confirmed by TLC in conjunction with the method. I t should be noted that the TLC confirmation data does not prove that the chromophore is benzidine. More rigorous methods must be used for absolute confirm ation.
(c) The disadvantages of the method are the com plexities of the procedure (emulsions and losses in e x tra c tio n s), nonspecificity for benzidine, and the increased time for confirmation by TLC.
(3) The urine is extracted with 10 ml of chloroform for 2 minutes. If an emulsion is formed, then centrifuge to separate the two phases. The chloroform fractio n (organic phase) is collected and saved.
(4) The urine is extracted twice more with chloroform (10 ml), and a ll three of the chloroform fractions are combined.
(5) Reextract the combined chloroform mixture with 2 ml of 0.1 N HC1 for 30 minutes on a ro tato r.
(6) Transfer the aqueous phase (about 2 ml) into a culture tube (16x125 mm) using a Pasteur pipet.
(7) Add 2 ml of pH 5.5 buffer and 0.7 ml of TNBS reagent, mix w ell, and le t stand for 15 minutes at room temperature. A reagent blank is prepared by adding 2 ml of 0.1 N HC1 to 2 ml of pH 5.5 sodium acetate buffer and 0.7 ml TNBS reagent and is treated as a sample.
(8) Add 2 ml of CHC13 and shake for 1 minute.
(9) Measure the absorbance of the organic phase at 400 nm on a spectrophotometer.
(10) Retain the organic phase for the benzidine-TLC confirmation, (c) TLC Confirmation of Benzidine (1) The TNB-derivative (chloroform extract) is concentrated by evaporating with nitrogen to about 0.2-ml aliquot.
(2) Ten m icro liters of the aliquot is spotted on a silic a -g e l TLC plate that was activated at 110 C for 30 minutes.
(3) The p late is then developed in chloroform-formic acid 90:10, volume to volume (prepared d a ily ).
(4) The Rf of the unknown amine derivative is compared with that of a benzidine spiked deriv ativ e, which should always be run as a standard. Benzidine produces a spot on the TLC p late having an Rf = 0.41, visualized by both v isib le and UV lig h t. The spot is yellow in v isib le lig h t and appears as a dark spot under UV lig h t.

# C alibration and Standardization
(a) CAUTION: Benzidine is a known human carcinogen and appropriate precautions should be u tiliz e d to minimize exposure. All wastes including acetone rinsed d irty glassware should be collected and disposed by approved methods.

# (b)
Prepare a working standard solution containing 10 fig of benzidine per ml of methyl alcohol. A series of benzidine spiked urine samples are prepared from a urine pool sample that was previously shown to have less than 100 ng benzidine per 100 ml of urine. The spiked urine samples serve as standards and are analyzed by the colorim etric method. The calibratio n curve is established by plo ttin g benzidine concentration (ng per 100 ml of urine) vs the absorbance at 400 nm.

# Calculations
(a) The concentration of the analyte in the urine sample is compared with a standard curve prepared with benzidine spiked urine samples as described in C alibrations and Standardization.
All samples are read against a reagent blank as described in Procedure. (c) If the calculated concentration exceeds 300 ng/100 ml, the chloroform extract should be analyzed by TLC to ten tativ ely confirm the presence of benzidine.
(d) In th is laboratory, normal rangds of urine specimens from NIOSH employees not exposed to benzidine or aromatic amines are reported below:

# Number of Urine Specimens
Cone, (ng/100 ml) Aromatic Amine

# D ise a se C o n tr o l, N a tio n a l I n s t i t u t e f o r O cc u p a tio n a l S a f e t y and
(4) Determine the absorbance of each standard at the wavelength of its maximum. (5) Construct a standard curve for each azo dye by p lo ttin g the absorbance against the amount (in micrograms) of the individual azo dye in the corresponding standard.

# Calculations
(a) Diazonium Salts (1) If only one diazonium s a lt is present in the sample, read from the appropriate calib ratio n curve the amount (in micrograms) corresponding to the absorbance of the sample.
(2) If more than one diazonium s a lt is present, the re su lts must be given as a range. (See Advantages and Disadvantages (f).) From the calibratio n curve for each diazonium s a lt, read the amount (in micrograms) corresponding to the absorbance of the sample. The upper lim it of the range is taken from the curve yielding the larg est value. The lower lim it of the range is taken from the curve yielding the lowest value. (b) Azo Dyes (1) If only one azo dye is present in the sample, read from the appropriate calib ratio n curve the amount (in micrograms) corresponding to the absorbance of the sample at the wavelength of maximum absorption.
(2) If more than one azo dye is present, the re su lts must be given as a range. (See Advantages and Disadvantages (h).) From the calibratio n curve for each azo dye absorbing at the same wavelength as the sample, read the amount (in micrograms) of azo dye corresponding to the absorbance of the sample. The upper lim it of the range is taken from the curve (d)
A rigorous specific method for benzidine in urine has recently been published . I t uses fluoroanhydride deriv atizatio n and electron capture gas chromatography, but requires considerable more time and equipment to perform.

# Apparatus
(a) Spectrophotometer capable of measuring absorbance at 400 nm and accommodating semimicrocuvettes (1-ml capacity ).
(b) Centrifuge with speed range to 4000 rpm. (c) Rotator for mixing te s t tubes (25x200 mm).
(d) pH meter.
(e) TLC plates precoated with s ilic a gel and without fluorescence indicator (0.5-mm thickness, E. Merck, Darmstadt, Germany).
(f) Chromatographic tank for thin -lay er chromatography.
(g) UV source for reading TLC p lates. (h) Graduated cylinders (100 ml).  (2) Samples of "spot" urine (150 ml) are collected following 6 hours of suspected exposure to benzidine-based azo dyes.
(3) Samples should be collected in polyethylene b o ttle s and, if not analyzed on the same day, should be frozen u n til analysis can be done.

# (b) Analysis of Samples
(1) One hundred m illilite r s of well mixed urine is adjusted to pH 5.0 to 6.0 (1 N HC1 or 1 N NaOH) in a glass b o ttle (180-ml capacity). A control urine sample (100 ml) and a control urine sample (.100 ml) spiked with benzidine (300-1000 ng) should be analyzed concurrently with the unknown samples.
(2) Add 0.2 g NaCl cry stals to the pH adjusted urine.             and reported as produced or imported by the US International Trade Commission (ITC)  or those*$o which potential exposure was found f441.
If less than three manufacturers make a dye, ITC does not publish the production figures. A discussion of limitations of the estimation of worker exposure is contained in reference 44. *This dye may also be synthesized with cresotic acid in place of salicylic acid.
The Colour Index designates both dyes as Direct Orange 1.

# D E P A R T M E N T O F H E A L T H . E D U C A T I O N , A N D W E L F A R E PUBLIC HEALTH SERVICE C E N T E R F O R D I S E A S E C O N T R O L

# T H IR D C L A SS N A T I O N A L I N S T I T U T E F O R O C C U P A T I O N A L S A F E T Y A N D H E A L T H R O B E R T A . T A F T L A B O R