OSHA Instruction CPL 2-2.27 FEB 22, 1980 Office of Compliance Programming

Subject: Benzidine-Based Dyes: Direct Black 38, Direct Brown 95 and Direct Blue 6 Dyes

A. Purpose. This instruction provides guidelines to follow when issuing citations under Section 5(a)(1) of the Act, and pertinent standards of a general nature, for employee exposure to Direct Black 38, Direct Brown 95 and Direct Blue 6 benzidine-based dyes.

B. Scope. This instruction applies OSHA-wide.

C. Action. OSHA Regional Administrators/Area Directors shall assure that:

1. The guidelines in paragraphs F., G. and H. of this instruction are followed.

2. The carcinogenic hazard described in Appendix D of this instruction is appropriately considered in issuing citations.

D. Federal Program Change. This instruction describes a Federal program change which affects State programs. Each Regional Administrator shall:

1. Ensure that this change is forwarded to each State designee.

2. Explain the technical content of the change to the State designee as requested.

3. Ensure that State designees are asked to acknowledge receipt of this Federal program change in writing, within 30 days of notification, to the Regional Administrator. This acknowledgment should include a description either of the State's plan to implement the change or of the reasons why the change should not apply to that State.

4. Advise State designees of the following:

a. In order to assure a sound and consistent

OSHA Instruction CPL 2-2.27 FEB 22, 1980 Office of Compliance Programming

national enforcement and litigation strategy in relation to the complex issues addressed by this instruction, it is critical that initial Federal and State implementation of the instruction's purposes be carefully coordinated.

b. State Plan officials are asked to work closely with OSHA Regional staff in carrying out the procedures outlined in this instruction, or comparable State procedures.

c. In addition, it is requested that upon completion of an inspection, each State consult with OSHA on enforcement strategy before proceeding with action on the case. To facilitate such consultation, until otherwise advised:

(1) State designees are asked to send to the OSHA Regional Administrator a copy of each entire case file and any proposed citations and penalties originating from the case, prior to issuance of the citations and penalties.

(2) The Regional Administrator will forward the file and proposed citations and penalties to the Office of Field Coordination and Experimental Programs for review in conjunction with cases originating from other States and from OSHA Area Offices. The Office of Field Coordination and Experimental Programs will consult with the National Office of the Solicitor and the Directorate of Federal Compliance and State Programs in their review.

(3) Upon completion of the review, the Office of Field Coordination and Experimental Programs will arrange, through the Regional Administrator, for consultation with the State designee on the best strategy for handling this case in the light of other related developments.

5. Review policies, instructions and guidelines issued

OSHA Instruction CPL 2-2.27 FEB 22, 1980 Office of Compliance Programming

by the State to determine that this change has been communicated to State program personnel. Routine monitoring activities (accompanied inspections and case file reviews) shall also be used to determine if this change has been implemented in actual performance.

E. Background. Based on available scientific evidence, OSHA has determined that employee exposure to the three benzidine-based dyes covered in this instruction presents a significant risk of cancer. Studies indicate that these dyes are carcinogenic in experimental animals. Additional studies indicate that these dyes are also metabolized in the body into benzidine, a human carcinogen. While OSHA has a specific standard to protect workers exposed to benzidine, these dyes cannot be covered under 29 CFR 1910.1010. Although there are many other benzidine-based dyes, the toxicity data for them are not as complete as for the three dyes covered by this directive. However, the agency recommends that the other benzidine-based dyes also be handled as carcinogens.

1. Health Effects.

a. In 1978 the National Cancer Institute (NCI) completed a 13-week subchronic study of Direct Blue 6, Direct Black 38, and Direct Brown 95 using Fischer 344 rats. The results dem- onstrate that these dyes are both tumorogenic and carcinogenic, inducing both hepatic neoplastic nodules and hepatocellular carcinomas, respectively. (For reference source, see E.7.a. of this instruction.)

b. An epidemiologic study by Yoshida, et al.(1971), demonstrated a probable association between bladder cancer and employee exposure to benzidine-based dyes. (For reference source, see E.7.b. of this instruction.)

2. Biological Response.

a. Metabolism studies in five species of animals and in humans indicate that each of the three

OSHA Instruction CPL 2-2.27 FEB 22, 1980 Office of Compliance Programming

dyes is metabolized to benzidine, a known carcinogen. (See Appendix D of this instruction.)

b. A study of Direct Black 38 demonstrated that this dye is mutagenic in the Salmonella test system. (See Appendix D of this instruction.)

3. Source of the Dyes. In the United States, as of August 1979, Fabricolor Incorporated, Paterson, New Jersey, is the only known manufacturer of Direct Black 38, Direct Brown 95 and Direct Blue 6. However, importation of these three dyes supplements domestic production. The "Health Hazard Alert: Benzidine Derived Dyes" contains a list of importers. The distributors have not been identified by OSHA.

4. Synonyms, Physical Properties, and Structural Formulas. The synonyms, physical properties and structural formulas of these three dyes are detailed in this instruction as follows:

a. Appendix A--Direct Black 38. b. Appendix B--Direct Brown 95.

c. Appendix C--Direct Blue 6.

5. Manufacturing Process.

a. Fabricolor uses hydrazobenzene instead of benzidine as a starting material for manufacturing the dyes. The hydrazobenzene is dumped into a closed tank (reaction vessel) where it undergoes rearrangement to benzidine dihydrochloride by reaction with a strong solution of hydrochloric acid in water.

NOTE: The benzidine standard is applicable to this stage of the manufacturing process and remains applicable up to the stage where the concentration of benzidine dihydrochoride drops below 0.1 percent by weight.

OSHA Instruction CPL 2-2.27 FEB 22, 1980 Office of Compliance Programming

b. The benzidine dihydrochloride solution is then reacted with sodium nitrite solution to form the diazo compound. This reaction could be carried out by adding the sodium nitrite solution to the benzidine hydrochloride solution, or alternatively by pumping the benzidine hydrochloride solution into another closed tank containing the sodium nitrite solution. Synthesis of the desired dye is continued by pumping the diazo compound to other reaction vessels for coupling to other compounds.

c. After the final reaction is completed, the product is isolated from solution and moved to a filter press for filtering and the washing out of further impurities.

d. The dye is taken from the filter press in press cake form and dried. Drying may be performed on a drum dryer after recreating a slurry of the dye; or drying may be done with a tray or a spray dryer.

e. The dried dye may be taken to a hammer mill and ground to a fine powder, or it may be taken directly to a ribbon blender. Color blending, standardization by adding salts and dedusting by adding dedusting oil is performed at the blender. The dye is transferred from the blender into drums for shipping.

6. Uses. Some reported uses for the three dyes (for reference source, see E.7.c. of this instruction) are as follows:

a. Direct Black 38: Dyeing or staining of wool, silk, fibers for rope and matting, hogs hair, cotton and other cellulose, acetate, nylon and biological stains.

b. Direct Brown 95: Dyeing or staining silk, cotton, acetate, cellulose, wool, nylon, leather, paper and certain plastics.

OSHA Instruction CPL 2-2.27 CH-1 JUN 3 1985 Office of Health Compliance Assistance

c. Direct Blue 6: Dyeing or staining silk, wool, cotton, nylon, leather, paper, biological stains and writing inks.

7. References.

a. Thirteen-week Subchronic Toxicity Studies of Direct Blue 6, Direct Black 38 and Direct Brown 95 Dyes. National Cancer Institute. Carcinogenesis Technical Report. DHEW Publication #"NIH" 78-1358, 1978.

b. Yoshida, O., et al.: (Bladder cancer in workers of the dyeing industry ) Igaku No. Ayumi, Vol. 79, No. 7: 421-422, 1971. (Japanese)

c. The Colour Index: 1971, 3rd Ed. Lund, Humphries, Bradford, and London, eds. London, England: The Society of Dyers and Colourists-American Association of Textile Chemists and Colorists.

d. Current Intelligence Bulletin 24: April 17, 1978. Benzidine Derived Dyes. National Institute for Occupational Safety and Health/National Cancer Institute.

PARAGRAPH DELETED

OSHA Instruction CPL 2-2.27 CH-1 JUN 3 1985 Office of Health Compliance Assistance

OSHA Instruction CPL 2-2.27 CH-1 JUN3 1985 Office of Health Compliance Assistance

PARAGRAPH DELETED

9. Methods of Documenting Exposure.

a. The compliance officer must rely heavily on procedures other than sampling and chemical analysis to document exposure to the dyes. For example, compliance officers must take photographs that show poor work practices, lack of or improperly designed engineering controls, improperly maintained engineering controls, improperly maintained systems and equipment for processing or using the dyes, contaminated surfaces and/or soiled employees. Other documentary methods include taking measurements showing inadequate ventilation; interviewing employees, employee representatives, and employers; etc.

b. However, in all cases samples shall be taken. Both bulk and air samples shall be taken when documenting exposure to the dyes by inhalation. When documenting exposure to the dyes by skin contact, bulk and wipe samples shall be taken and evidence demonstrating the contact shall be obtained; e.g., a photograph showing an employee's bare hand in contact with the dyes. When sampling:

OSHA Instruction CPL 2-2.27 FEB 22, 1980 Office of Compliance Programming

(1) The dyes should be sampled on glass fiber filters without organic binder at 2 liters per minute for a sampling time of 4 hours.

(2) Sample handling and shipment to the laboratory shall be that prescribed in the Industrial Hygiene Field Operations Manual. Following receipt of the samples in the laboratory, they will be analyzed by high performance liquid chromatography for the specific dyes requested.

(3) Wipe samples of the dyes on normal working surfaces shall be collected on glass fiber filters without organic binders. For rough surfaces on which a glass fiber filter may tend to fall apart, a fluoropore filter may be used to collect the wipe sample.

(4) Collection, handling and shipping of the wipe samples shall be as prescribed in Chapter VI of the IHFOM except for the type of filter used. The dyes may be sensitive to light; therefore, care should be taken not to expose the samples to high levels of indoor lighting or to direct sunlight.

(5) Regardless of whether air or wipe samples are to be analyzed, be sure to send bulk samples so that SLCAL can make up calibration curves.

c. These dyes have been shown to metabolize to benzidine, which may appear in the urine of exposed employees. Thus, some employers may be monitoring the benzidine in the urine of their employees who work with or around one or more of the dyes. With each employer, determine if this is the case. Where an employer does monitor the benzidine in employees' urine, and at least some results are positive, obtain copies of all results relevant to documenting exposure of employees to one or more of these dyes.

OSHA Instruction CPL 2-2.27 CH-1 JUN 3 1985 Office of Health Compliance Assistance

This page replaces deleted pages 14-16

OSHA Instruction CPL 2-2.27 CH-1 JUN 3 1985 Office of Health Compliance Assistance

PARAGRAPH DELETED

G. National Office Review. Until instructed otherwise by the National Office, Area Offices conducting inspections where possible violations exist shall send copies of the entire case file dealing with violations and proposed citations to their Regional Offices, who will forward this material to the

OSHA Instruction CPL 2-2.27 FEB 22, 1980 Office of Compliance Programming Office of Field Coordination and Experimental Programs prior to the issuance of any citations involving the three dyes covered in this instruction. The Office of Field Coordination and Experimental Programs will then consult with the National Office of the Solicitor in determining whether citations should be issued.

H. Expert Witnesses. The Directorate of Technical Support will assist the Regional Offices in locating expert witnesses. In the event that a 5(a)(1) citation is contested, the case file must contain an expert witness to testify before the review judge as to:

1. The fact that there is sufficient evidence to demonstrate that the dye or dyes involved in the citation present a significant cancer risk to humans.

2. The fact that the hazard is recognized in the industry, by the employer, or by a member of the health community who is associated with the employer or the industry.

3. The fact that methods of abatement presented in the citation are feasible.

Grover C. Wrenn Director, Federal Compliance and State Programs

DISTRIBUTION: National, Regional and Area Offices All Compliance Officers State Designees NIOSH Regional Program Directors

OSHA Instruction CPL 2-2.27 FEB 22, 1980 Office of Compliance Programming

C.I. DIRECT BLACK 38 (a benzidine-based azo dye)

Chemical Abstracts Service Indexing Name:

Disodium 4-amino-3-((4'-((2,4-diaminophenyl)azo)-4- biphenylyl)azo)-5-hydroxy-6-(phenylazo)-2,7-naphthalene disulfonate

Formula: C34H25N907S2Na2

Molecular Weight: 781 g/mole Dry powder at room temperature

OSHA Instruction CPL 2-2.27 FEB 22, 1980 Office of Compliance Programming

IDENTIFIERS AND SYNONYMS FOR DIRECT BLACK 38

Chemical Abstracts Service Registry Number 1937-37-7 NIOSH/RTECS Number JM71700 Chemical Formula C34H25N907S2Na2

Ahco Direct Black GX Chloramine Black EX Airedale Black ED Chloramine Black EXR Aizen Direct Deep Black EH Chloramine Black XO Aizen Direct Deep Black GH Chloramine Carbon Black S Aizen Direct Deep Back RH Chloramine Carbon Black SJ Amanil Black GL Chloramine Carbon Black SN Amanil Black WD Chlorazol Black E Amidine Black GA Chlorazol Black E Apomine Black GX (Biological Stain) Atlantic Black BD Chlorazol Black EA Atlantic Black C Chlorazol Black EN Atlantic Black E Chlorazol Burl Black E Atlantic Black EA Chlorazol Leather Black ENP Atlantic Black GAC Chlorazol Silk Black G Atlantic Black GG Chrome Leather Black E Atlantic Black GXCW Chrome Leather Black EC Atlantic Black GXOO Chrome Leather Black EM Atlantic Black SD Chrome Leather Black G Atul Direct Black E Chrome Leather Azine Deep Black EW Brilliant Black ER Azocard Black EW Coir Deep Black C Azomine Black EWO Columbia Black EP Belamine Black GX Diacotton Deep Black Bencidal Black E Diacotton Deep Black RX Benzanil Black E Diamine Deep Black EC Benzo Deep Black E Diamine Direct Black E Benzo Leather Black E Diaphtamine Black V Benzoform Black BCN-CF Diazine Black E Black 2EMBL Diazine Direct Black E Black 4EMBL Diazine Direct Black G Brasilamina Black GN Diazol Black 2V Brilliant Chrome Leather Diazol Black JXA Double Black H Diphenyl Deep Black G C.I. 30235 Direct Black Methyl C.I. Direct Black 38 Direct Black A Calcomine Black Direct Black BHX Calcomine Black EXL Direct Black BRN Carbide Black E Direct Black CP Chloramine Black C Direct Black CX Chloramine Black EC Direct Black CXR Chloramine Black ERT Direct Black E

OSHA Instruction CPL 2-2.27 FEB 22, 1980 Office of Compliance Programming

INDENTIFIERS AND SYNONYMS FOR DIRECT BLACK 38--Continued

Direct Black EW Formic Black MTG Direct Black EX Formic black TG Direct Black FR Hispamin Black EF Direct Black GAC Interchem Direct Black Z Direct Black GW Kayaku Direct Deep Black EX Direct Black GX Kayaku Direct Deep Black GX Direct Black GXR Kayaku Direct Deep Black S Direct Black JET Kayaku Direct Leather Black EX Direct Black Meta Kayaku Direct Special Black AAX Direct Black Methyl Lurazol Black BA Direct Black N Meta Black Direct Black RX Mitsui Direct Black EX Direct Black SD Mitsui Direct Black GX Direct Black WS NCI-C54557 Direct Black Z Nippon Deep Black Direct Black 3 Nippon Deep Black GX Direct Black 38 Orcomine Direct Black GX Direct Deep Black E Paper Black BA Direct Deep Black E Extra Paper Black T Direct Deep Black EA-CF Paper Deep Black C Elcomine Direct Black CP Peeramine Black GXOO Elcomine Direct Black GXP Phenamine Black BCN-CF Enianil Black CN Phenamine Black CL Erie Black B Phenamine Black E Erie Black BF Phenamine Black E 200 Erie Black GAC Phenamine Black ER-200 Erie Black GXOO Pheno Black EP Erie Black JET Pheno Black SGN Erie Black NUG Pontamine Black E Erie Black RXOO Pontamine Black EBN Erie Brilliant Black S Sandopel Black EX Erie Fibre Black VP Seristan Black B Fenamin Black E Synodirect Black GAC Fibre Black VF Telon Fast Black E Fixanol Black E Tetrazo Deep Black G Formaline Black C Tertrodirect Black E Formic Black C Tetrodirect Black EFD Formic Black CW Union Black EM Formic Black EA Vondacel Black N

OSHA Instruction CPL 2-2.27 FEB 22, 1980 Office of Compliance Programming

C.I. DIRECT BROWN 95 (a benzidine-based azo dye)

IUPAC Name: Disodium (5-((4'-((2,6-dihydroxy-3-((2-

hydroxy-5-sulfophenyl)azo)phenyl)azo)-4-

Chemical Abstracts Service Indexing Name:

Disodium (5-((4'-((2,6-dihydroxy-3-((2-hydroxy -5-sulfophenyl)azo)phenyl)azo)(1,1'-biphenyl) -4-yl)azo)-2-hydroxybenzoato(4-)) cuprate(2-)

Formula: C31H20N609SNa2Cu

Molecular Weight: 761.5 g/mole Dry powder at room temperature

OSHA Instruction CPL 2-2.27 FEB 22, 1980 Office of Compliance Programming

IDENTIFIERS AND SYNONYMS FOR DIRECT BROWN 95

Chemical Abstracts Service Registry Number 16071-86-6 NIOSH/RTECS Number JM 78780

Chemical Formula C31H20N609SNa2Cu

Aizen Primula Brown BRLH Fastolite Brown BRL Aizen Primula Brown PLH Fastusol Brown LBRSA Amanil Fast Brown BRL Fastusol Brown LBRSN Amanil Supra Brown LBL Fenaluz Brown BRL Atlantic Fast Brown BRL Helion Brown BRSL Atlantic Resin Fast Brown BRL Hispaluz Brown BRL Belamine Fast Brown BRLL Intralite Brown BRLL Benzanil Supra Brown BRLL KCA Light Fast Brown BR Benzanil Supra Brown BRLN Kayarus Supra Brown BRS Brown 4EMBL NCI-C54568 C.I. 30145 Paranol Fast Brown BRL C.I. Direct Brown Peeramine Fast Brown BRL Calcodur Brown BRL Potamine Fast Brown BRL Chloramine Fast Brown BRL Pontamine Fast Brown NP Chloramine Fast Cutch Brown PL Pyrazol Fast Brown BRL Chlorantine Fast Brown BRLL Pyrazoline Brown BRL Chrome Leather Brown BRLL Saturn Brown LBR Chrome Leather Brown BRSL Sirius Supra Brown BRL Cuprofix Brown GL Sirius Supra Brown BRS Derma Fast Brown W-GL Solantine Brown BRL Dermafix Brown PL Solar Brown PL Dialuminous Brown BRS Solex Brown R Diaphtamine Light Brown BRLL Solius Light Brown BRLL Diazine Fast Brown RSL Solius Light Brown BRS Diazol Light Brown BRN Sumilight Supra Brown BRS Dicorel Brown LMR Suprazo Brown BRL Diphenyl Fast Brown BRL Suprexcel Brown BRL Direct Brown 95 Synodirect Brown BRL Direct Brown BRL Tertrodirect Fast Brown BR Direct Fast Brown BRL Tetramine Fast Brown Direct Fast Brown LMR BRDN Extra Direct Light Brown BRS Tetramine Fast Brown BRP Direct Supra Light Brown ML Tetramine Fast BRS Durazol Brown BR Triantine Brown BRS Durofast Brown BRL Triantine Fast Brown OG Elcomine Brown BRLL Triantine Fast Brown OR Eliamina Light Brown BRL Triantine Light Brown BRS Enianil Light Brown BRL Triantine Light Brown OG

OSHA Instruction CPL 2-2.27 FEB 22, 1980 Office of Compliance Programming

C.I. DIRECT BLUE 6 (a benzidine-based azo dye)

IUPAC Name: Tetrasodium 3,3'-(4,4'-biphenylylenebis(azo))

bis(5-amino-4-hydroxy-2,7-naphthalene disulfonate)

Chemical Abstracts Service Indexing Name:

Tetrasodium 3,3'-((1,1'-biphenyl)-4,4'-

diylbis(azo))bis(5-amino-4-hydroxy-2,7-

naphthalene disulfonate)

Formula: C32H20014N6S4Na4

Molecular Weight: 932 g/mole Dry powder at room temperature

OSHA Instruction CPL 2-2.27 FEB 22, 1980 Office of Compliance Programming

INDENTIFIERS AND SYNONYMS FOR DIRECT BLUE 6

Chemical Abstracts Service Registry Number 2602-46-2 NIOSH/RTECS Number QJ64000

Chemical Formula C32H20014N6S4N4

Airedale Blue 2BD Diphenyl Blue 2B Aizen Direct Blue 2BH Diphenyl Blue KF Amidine Blue 2B Diphenyl Blue M2B Amanil Blue 2BX Direct Blue A Atlantic Blue 2B Direct Blue 2B Atul Direct Blue 2B Direct Blue 6 Azocard Blue 2B Direct Blue BB Azomine Blue 2B Direct Blue GS Belamine Blue 2B Direct Blue K Bencidal Blue 2B Direct Blue M2B Benzanil Blue 2B Direct Fast Blue 2B Benzo Blue BBA-CF Elcomine Blue 2B Benzo Blue BBN-CF Enianil Blue 2BN Benzo Blue GS Fenamin Blue 2B Blue 2B Fixanol Blue 2B Blue 2B Salt Hispamin Blue 2B Brasilamina Blue 2B Indigo Blue 2B Calcomine Blue 2B Kayaku Direct Chloramine Blue 2B Kayaku Direct Blue BB Chlorazol Blue B Mitsui Direct Blue 2BN Chlorazol Blue BP Naphtamine Blue 2B Chrome Leather Blue 2B Niagara Blue 2B CI 22610 Nippon Blue BB C.I. 22610 Paramine Blue 2B C.I. Direct Blue 6 Phenamine Blue BB C.I. Direct Blue 6, Pheno Blue 2B Tetrasodium Salt Pontamine Blue BB Cresotine Blue 2B Sodium Diphenyl-4,4'-Bis- Diacotton Blue BB Azo-2"-8"-Amino- Diamine Blue 2B 1"-Naphthol-3",6"- Diamine Blue BB Disulphonate Diaphtamine Blue BB Synodirect Blue 2B Diazine Blue 2B Tertrodirect Blue 2B Diazol Blue 2B Vondacel Blue 2B

OSHA Instruction CPL 2-2.27 FEB 22, 1980 Office of Compliance Programming

          Evidence of the Hazard of Direct Black 38,
           Direct Brown 95, and Direct Blue 6 Dyes

Summary

OSHA has reviewed the literature on Direct Black 38, Direct Brown 95 and Direct Blue 6 dyes. The literature indicates that these benzidine-based dyes cause cancer in experimental animals and are converted in animals and humans to benzidine. From the accumulated evidence, OSHA concludes that these dyes are potential human carcinogens.

I. Laboratory Studies: Carcinogenicity

In 1978, the National Cancer Institute published the results of a study demonstrating the carcinogenicity of Direct Black 38, Direct Brown 95, and Direct Blue 6.(1) Analyses to determine contamination of the three dyes indicated that Direct Black 38 was 87.1% pure, Direct Brown 95 was 72.2% pure, and Direct Blue 6 was 59.9% pure. Although the dyes did contain some impurities, neither free benzidine nor its salts were detected as an impurity in any of the dyes.

The bioassay consisted of a 13-week subchronic study during which the three dyes were fed to both sexes of Fischer 344 rats and B6C3F1 mice. (Note: For carcinogenesis bioassay, a 2-year study is considered more sensitive for the identification of carcinogenic response.)

Rats:

Groups of 10 rats of each sex were administered one concentration of one of the three dyes throughout the study. The dyes were mixed in the feed of both male and female rats at concentrations of 190, 375, 750, 1500 and 3000 ppm. All rats receiving 3000 ppm of any dye (or 1500 ppm of Direct Brown 95) died prior to the termination of the study.

OSHA Instruction CPL 2-2.27 FEB 22, 1980 Office of Compliance Programming

Control groups consisted of 10 rats of each sex. Throughout the study period, no deaths were recorded in control animals.

Table D-1 shows the data for the treated and control rats. Eight of 10 male rats fed Direct Blue 6 at a concentration of 1500 ppm and 7 of 9 female rats fed 3000 ppm of the dye developed hepatocellular carcinomas as compared with none in the matched control groups. In the group of rats fed Direct Black 38, 9 of 9 male rats and 5 of 10 female rats developed hepatic neoplastic nodules. Male rats also developed hepatocellular carcinomas.

No hepatocellular carcinomas or neoplastic nodules were found in male rats fed Direct Brown 95. In female rats fed 1500 ppm of this dye, 5 out of 8 developed hepatocellular carcinomas and neoplastic nodules. It is noteworthy that tumors were observed as early as 4-5 weeks after initial exposure.

No tumors were found in any of the matched control groups during the 13-week period or at terminal sacrifice. Historical data indicate that male and female Fischer 344 rats have a spontaneous incidence of liver tumors of 1.8% and 3.1%, respectively.(1) A comparison of the number of tumor-bearing animals in the test groups with the matched control groups demonstrated that the increased incidences of tumors induced by these three dyes were statistically significant by chi-square analysis (Table D-1).

Mice:

Groups of male mice were fed 750,1500, 3000, 6000 and 12,500 ppm of the dyes. The female mice received the same concentra- tions with the exception of those fed Direct Brown 95, which received concentrations of 375, 750, 1500, 3000, and 6000 ppm. The animals were fed for a total period of 91 days before being sacrificed. Untreated controls for mice consisted of groups of 10 animals of each sex. No deaths were recorded in the groups of matched control animals.

OSHA Instruction CPL 2-2.27 FEB 22, 1980 Office of Compliance Programming

       Table D-1. Incidence of tumor-bearing Fischer 344
              rats by bensidine-based dye exposure
                         level and sex.

Exposure

Level Direct Blue 6 Direct Black 38 Direct Brown 95

ppm Males Females Males Females Males Females

1500 8/10 0/10 9/9 5/10 0/9 5/8

p(0.005 p(0.001 p(0.05 p(0.025 3000(2) 1/9 7/9 0/9 0/8 0/9 0/8

p(0.005

Matched 0/10 0/10 0/10 0/10 0/10 0/10 Controls

2/All animals receiving 3000 ppm dye in their diet died

prior to the 13-week sacrifice period.

Source: Adapted from NCI Bioassay Report, 1978.

OSHA Instruction CPL 2-2.27 FEB 22, 1980 Office of Compliance Programming

In B6C3F1 mice fed the three dyes, neoplastic lesions were not observed up to 13 weeks after initial exposure. All of the mice survived for the full 13-week study period.

II. Epidemiology:

One epidemiologic study (Yoshida, 1971) has reported a significant association between exposure to bensidine-based dyes and the development of urinary bladder cancer.(2) In a case-control study of 200 male patients having bladder cancer, 17 (8.5%) worked in the dye industry as contrasted with 1.4% in the matched control group. These workers were exposed to unspecified bensidine-based dyes. The authors did not specify the route of exposure. For 3 of the 17 workers with bladder cancer in the dye industry, the primary route of exposure was probably through oral ingestion, as these individuals were kimono painters who had a habit of licking the tips of their paint brushes. For the remaining 14 cases, routes of exposure may have been through inhalation and/or skin absorption. (Although the three dyes covered in this instruction are not specifically mentioned, the study suggests the potential for benzidine-based dyes to induce bladder cancer in humans.)

III. Metabolism:

Rats:

1. The NCI bioassay (1978) for carcinogenicity included an analysis for the presence of bensidine in the urine of the test animals. Urine was collected from the Fischer 344 rats over 24-hour periods during the fourth and twelveth week of the 13-week subchronic toxicity study of the three benzidine-based dyes. Three male and three female animals were selected from each of the three dosage groups for the three dyes, Direct Black 38, Direct Brown 95, and Direct Blue 6. In addition, urine samples were collected from untreated control rats. All of the urine samples from the exposed rats contained benzidine while the urine samples from the control animals were free of benzidine. Since the original

OSHA Instruction CPL FEB 22, 1980 Office of Compliance Programming

analysis of each of the three dyes did not reveal contamina- tion with either benzidine or benzidine salts, the benzidine detected in the urine of these animals is considered a metabolic product of the direct dyes. Furthermore, a dose response relationship existed between the amount of direct dye fed to the rats and the amount of benzidine detected in the urine.

2. Genin (1977) reported the metabolic conversion of a benzidine-based dye, Direct Black 38, to benzidine in albino mongrel rats.(3) These animals were injected subcutaneously and dosed orally with the dye which contained no benzidine. Benzidine was detected in the urine. The number of albino mongrel rats used in the study was not specified, nor was it stated whether controls were used.

Mice: 3. In the NCI bioassay (1978), benzidine was detected in the urine of mice fed the three benzidine-based dyes.(1) No benzidine was found in the urine of the control group of mice

4. Yoshida (1973) injected four benzidine-based dyes including Direct Black 38 into ligated intestines of rats and mice.(4) All dyes were free of residual benzidine. Direct Black 38, as well as the three additional benzidine-based dyes, metabolized to benzidine.

Monkeys:

5. Rinde and Troll (1975) conducted a study on the metabolism of benzidine-based dyes in rhesus monkeys.(5) The authors reported that the dyes, Direct Black 38, Direct Brown 95, and Direct Blue 6 were free of unreacted amines. Urine samples of the exposed monkeys showed the presence of benzidine and monoacetyl benzidine, unlike the control urine samples. The authors concluded that the dyes were metabolically converted to benzidine and monoacetyl benzidine by the monkeys.

Hamsters:

6. The National Center for Toxicologic Research (1979) reported the metabolic conversion of Direct Black 38 to

OSHA Instruction CPL FEB 22, 1980 Office of Compliance Programming

benzidine, monoacetylbenzidine, diacetylbenzidine, 4-aminobiphenyk, and alkaline hydrolyzable conjugates of benzidine and 4-aminobiphenyl in Syrian golden hamsters.(6) The dye was administered to the animals in a single dose by stomach intubation. The dye was purified extensively prior to use in the animal test. None of the above mentioned metabolite residues were found in control urine samples.

Rabbits:

7. Radioactively labeled Direct Black 38 in an ink mixture and in aqueous solution was applied to the unbroken skin of rabbits.(7) The experiment was conducted as two-step process whereby treatment with a reducing agent followed one hour after dermal application. Extensive amounts of radio-activity were detected in urine and feces collected during the 144 hour study period. The extensive amounts were detected despite the fact that treatment with the reducing agent was reported as effective in removing the dye from the rabbits' skin. These observations led the investigators to conclude that rabbits' skin may be permeable to Direct Black 38 and/or its metabolites. Other researchers are repeating the experiments to confirm these results.

Humans:

8. Genin (1977) reported on urinalyses performed on a group of 22 workers who dried and ground two benzidine-based dyes, one of which was Direct Black 38.(3) Benzidine was detected in the urine of eight of these workers.

9. Boeniger, et al. (1978) conducted a field survey of six facilities involved in the manufacture and use of benzidine- based dyes.(8, 9) The investigators performed urinalyses of employees occupationally exposed to benzidine-based dyes, specifically including Direct Black 38, Direct Brown 95, and Direct Blue 6. Results of the urinalyses indicated that benzidine or its metabolite, monoacetyl benzidine, was identified in the urine of some of the workers.

IV. Mutagenicity:

Due to the correlation between mutagenicity and carcino-

OSHA Instruction CPL 2-2.27 FEB 22, 1980 Office of Compliance Programming

genicity, Direct Black 38 was tested for mutagenicity. The National Center for Toxicologic Research (1979) reported that Direct Black 38 and benzidine were mutagenic with microsomal activation in Salmonella strains TA98 and TA100.(6) The metabolites monoacetylbenzidine and diacetylbenzidine were mutagenic with activation when tested in Salmonella strain TA1538.

V. Discussion and Conclusion:

The results of the NCI 13-week subchronic study demonstrated that all three benzidine-based dyes are carcinogenic in both male and female rats with one exception: Direct Brown 95 failed to produce hepatocellular carcinomas or hepatic neoplastic nodules in male rats. The observation of hepato- cellular carcinomas and/or neoplastic nodules as early as 4-5 weeks after initial exposure demonstrates that these dyes induced tumors in experimental animals in a much shorter time period than documented for benzidine itself. (Studies with benzidine have shown that the earliest appearance of hepatomas in rats injected with solutions of commerical benzidine ranged from 23 to 38 weeks.)

Although tumors were not observed in the mice exposed to these three dyes, the observation period (13 weeks) may not have been long enough to allow the development of cancer in these animals.

Consistent with study results demonstrating cancer in experi- mental animals, the study by Yoshida demonstrated a significant association between bladder cancer and workers exposed to benzidine-based dyes.

In addition, Direct Black 38, benzidine, monoacetylbenzidine and diacetylbenzidine were found to be mutagenic in the Salmonella test system.

Furthermore, each of these three dyes is metabolized to benzidine, a known carcinogen, in five species of animals and in humans. Benzidine and/or its metabolite, monoacetylbenzidine, was recovered in the urine of experimental animals, whether the route of exposure was through oral ingestion or skin absorption. The NIOSH study of workers exposed to benzidine-based dyes also demonstrated benzidine and its metabolite in the urine. Workers exposed to benzidine-based dyes may be subjected to an increased risk of developing cancer due to the fact that the three dyes tested induced cancer in

OSHA Instruction CPL 2-2.27 FEB 22, 1980 Office of Compliance Programming

experimental animals. These dyes also are metabolized to benzidine, a substance known to cause cancer in humans.

On the basis of the NCI bioassay study results, NCI and NIOSH jointly issued a Current Intelligence Bulletin(10) in April of 1978 to inform the industrial health community and workers of the potential carcinogenic risk of individuals exposed to these benzidine-based dyes.

Therefore, OSHA concludes that Direct Black 38, Direct Brown 95, and Direct Blue 6 should be controlled as carcinogens in the workplace.

1. 13-Week Subchronic Toxicity Studies of Direct Blue 6, Direct Black 38 and Direct Brown 95 Dyes, DHEW Publication No. (NIH) 78-1358, U.S. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health, National Cancer Institute, Division of Cancer Cause and Prevention, Carcinogenesis Testing Program, 1978, 127 pp.

2. Yoshida, O., Harada, T., Miyagawa, M., Kato, T.: (Bladder cancer in workers of the dyeing industry.) Igaku No. Ayumi 79:421-22, 1971. (Japanese)

3. Genin, V.A.:(Formation of blastomogenic diphenylamino derivatives as a result of the metabolism of direct azo dyes.) Vopr. Onkol. 23:50-51, 1977. (Russian)

4. Yoshida, O., Miyakawa, M.: Etiology of bladder cancer-- "Metabolic" aspects, in Nakohara, W., Hirayama, T., Nishioka, K., and Sugano, H. (eds.): Proceedings of the 3rd International Symposium of The Princess Takamatsu Cancer Research Fund-- Analytic and Experimental Epidemiology of Cancer. Baltimore, University Park Press, 1973, pp. 31-39.

5. Rinde, E., Troll, W.: Metabolic reduction of benzidine azo dyes to benzidine in the rhesus monkey. J. Natl. Cancer Inst. 55:181-182, 1975.

6. Metabolism of Azo dyes to Potentially Carcinogenic Aromatic Amines, NCTR Technical Report for Experiment Number 196, March, 1979.

OSHA Instruction CPL 2-2.27 FEB 22, 1980 Office of Compliance Programming 7. Absorption and Elimination of 14C-Labeled Direct Black 19 and Direct Black 38 Following Dermal Application to Male New Zealand Rabbits, Midwest Research Institute report to IBM Corporation, MRI Project Number 4612-B(12), October 26, 1979.

8. Special Hazard Review of Benzidine-Based Dyes, Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, November 1979, pp. 77-82.

9. Boeniger, M. : An investigation of the metabolic reduction of benzidine azo dyes to benzidine and its metabolites, and their possible relationship to carcinoma of the bladder in man. Cincinnati, U.S. Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, 1978, 106 pp. (Unpublished)

10. Direct Blue 6, Direct Black 38, and Direct Brown 95-- Benzidine-Derived Dyes , Current Intelligence Bulletin 24, April 17, 1978, DHEW (NIOSH) Publication No. 78-148, Cincinnati, U.S. Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, 1978, 11 pp.

OSHA Instruction CPL 2-2.27 FEB 22, 1980 Office of Compliance Programming

1. Thirteen-week Subchronic Toxicity Studies of Direct Blue 6, Direct Black 38 and Direct Brown 95 Dyes. National Cancer Institute. Carcinogenesis Technical Report. DHEW Publication #"NIH" 78-1358, 1978.

2. Current Intelligence Bulletin 24: April 17, 1978. Benzidine Derived Dyes. National Institute for Occupational Safety and Health/National Cancer Institute.

3. American Industrial Hygiene Association Journal, Vol. 39, No. 8, August 1978, pp. A-18 to A-24. (Reprint of Current Intelligence Bulletin #24).

4. American Industrial Hygiene Association Journal, Vol. 39, No. 12, December 1978, p. A-20. (Reprint of "Industrial Alert" No. 3, issued by the Commonwealth of Virginia.)

5. Jenkins, C.: Textile dyes are potential hazards, J. Environ. Health 40(5), 1978, p. 12.

6. "NCI says three dyes are animal carcinogens, "Chemical & Engineering News, May 15, 1978, p. 12.

7. Yoshida, O., et al.: (Bladder cancer in workers of the dyeing industry.) Igaku No. Ayumi, Vol. 79, No. 7: 421-422, 1971. (Japanese)

8. Comments dated October 6, 1978, by Ecological and Toxicological Association of the Dyestuff Manufacturing Industry (ETAD) on petition by the Amalgamated Clothing and Textile Workers.

9. International Chemical Workers Union Hazard Alert, December 25, 1978.

10. Special Hazard Review of Benzidine-Based Dyes, Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, October, 1979.

OSHA Instruction CPL 2-2.27 FEB 22, 1980 Office of Compliance Programming

11. Appendix I to petition by Amalgamated Clothing and Textile Workers Union, et al. Scientific Basis for the Proposed Regulation of Dyes Derived from the Chemical Substances Benzidine, 3,3'-Dimethylbenzidine, and 3,3'-Dimethoxybenzidine. Catherine L. Jenkins, Ph.D., National Science Foundation Public Service Resident, Center for Occupational Hazards, Inc., New York, New York.

12. Testimony at OSHA Carcinogen Policy Hearing by Harold R. Imbus, M.D., of Burlington Industries, Inc. (OSHA Docket No. H-090).

13. "Benzidine and Its Salts" (Medical Surveillance paragraph). Occupational Diseases: A Guide to Their Recognition (Rev. Ed.), DHEW (NIOSH) Publication No. 77-181, Marcus M. Key, M.D., et al., eds., U.S. Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, June 1977, p. 267.

14. Absorption and Elimination of 14C-Labeled Direct Black 19 and Direct Black 38 Following Dermal Application to Male New Zealand Rabbits, Midwest Research Institute report to IBM Corporation, MRI Project Number 4612-B(12), October 26, 1979.

15. Metabolism of Azo dyes to Potentially Carcinogenic Aromatic Amines, NCTR Technical Report for Experiment Number 196, March, 1979, pp. 27-28, 41-44, 62-63 (mutagenicity); pp 6-7, 26-27 (hamsters).

16. "HEW Institute Implicates Dyes In Cancer In Rats", Textile Chemist and Colorist, June 1978, pp.7-8.

17. "U.S. Links 3 Dyes to Rat Cancer, " Daily News Record, New York, May 10,1978.

E-2