ORNL
Oak Ridge
National
Laboratory
Operated by
Union Carbide Corporation for the
Department of Energy
Oak Ridge, Tennessee 37830
ORNL/EIS-86
EPA
United States
Environmental Protection
Agency
Office of Research and Development
Health Effects Research Laboratory
Cincinnati, Ohio 45268
EPA-600/1-78-024
REVIEWS OF THE ENVIRONMENTAL
EFFECTS OF POLLUTANTS:
II. Benzidine
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
SEARCH series. This series describes projects and studies relating to the toler-
ances of man for unhealthful substances or conditions. This work is generally
assessed from a medical viewpoint, including physiological or psychological
studies. In addition to toxicology and other medical specialities, study areas in-
clude biomedical instrumentation and health research techniques utilizing ani-
mals — but always with intended application to human health measures.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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ORNL/EIS-86
EPA-600/1-78-024
April 1978
REVIEWS OF THE ENVIRONMENTAL EFFECTS OF POLLUTANTS: II. BENZIDINE
by
Carole R. Shriner, John S. Drury, Anna S. Hammons,
Leigh E. Towill, Eric B. Lewis, and Dennis M. Opresko
Information Center Complex, Information Division
Oak Ridge National Laboratory
Oak Ridge, Tennessee 37830
operated by
Union Carbide Corporation
for the
Department of Energy
Technical Reviewer
Howard Klemmer
University of Hawaii
Honolulu, Hawaii 96816
Interagency Agreement No. D5-0403
Project Officer
Jerry F. Stara
Office of Program Operations
Health Effects Research Laboratory
Cincinnati, Ohio 45268
Date Published; May 1978
Prepared for
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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This report was prepared as an account of work sponsored by an agency
of the United States Government. Neither the United States Government nor
any agency thereof, nor any of their employees, contractors, subcontractors,
or their employees, makes any warranty, express or implied, nor assumes any
legal liability or responsibility for any third party's use or the results
of such use of any information, apparatus, product or process disclosed in
this report, nor represents that its use by such third party would not
infringe privately owned rights.
This report has been reviewed by the Health Effects Research Laboratory,
U.S. Environmental Protection Agency, and approved for publication. Approval
does not signify that the contents necessarily reflect the views and policies
of the U.S. Environmental Protection Agency, nor does mention of trade names
or commercial products constitute endorsement or recommendation for use.
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CONTENTS
Figures V
Tables vii
Foreword ix
Acknowledgments xi
Abstract xiii
1. Summary 1
1.1 Introduction 1
1.2 Benzidine Chemistry 1
1.2.1 Physical Properties 1
1.2.2 Chemical Properties 1
1.2.3 Biochemical Reactions 2
1.2.4 Methods of Analysis 2
1.3 Uses and Production 3
1.4 Environmental Distribution and Fate 4
1.4.1 Distribution in Air 4
1.4.2 Distribution in Water 4
1.4.3 Distribution in Soil 4
1.4.4 Mobility and Persistence 5
1.4.5 Food Chain Accumulation 5
1.5 Biological Aspects in Microorganisms, Plants, and Wild
and Domestic Animals 5
1.5.1 Microorganisms 5
1.5.2 Plants 6
1.5.3 Wild and Domestic Animals 6
1.6 Biological Aspects in Humans 6
1.6.1 Metabolism 6
1.6.2 Effects 8
1.7 The Problem 11
1.8 Conclusions 11
2. Physical and Chemical Properties and Analysis 14
2.1 Summary 14
2.2 Physical and Chemical Properties 15
2.2.1 Benzidine and Salts 15
2.2.2 3,3'-Dichlorobenzidine and Salts 29
2.2.3 3,3'-Dimethylbenzidine and Salts 33
2.2.4 3,3'-Dimethoxyben7.idine and Salts 35
2.2.5 4-Aminobiphenyl 37
2.2.6 4-Nitrobiphenyl 39
2.2.7 Other Benzidine Congeners 41
2.3 Analysis for Benzidine 41
2.3.1 Sampling and Sample Preparation 46
2.3.2 Methods of Analysis 50
2.3.3 Comparison of Analytical Procedures 64
3. Biological Aspects in Microorganisms 74
4. Biological Aspects in Plants 77
5. Biological Aspects in Wild and Domestic Animals 79
6. Biological Aspects in Humans 81
6.1 Summary 81
iii
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iv
6.2 Metabolism 82
6.2.1 Uptake 82
6.2.2 Distribution and Accumulation 83
6.2.3 Biotransformation and Elimination 89
6.3 Effects 97
6.3.1 Physiological Effects 100
6.3.2 Toxicity 100
7. Environmental Distribution and Transformation 123
7.1 Summary 123
7.2 Production and Use 123
7.2.1 Benzidine 123
7.2.2 3,3'-Dichlorobenzidine 123
7.2.3 3,3'-Dimethylbenzidine 124
7.2.4 3,3'-Dimethoxybenzidine 124
7.3 Distribution in the Environment 124
7.3.1 Distribution in Soils 124
7.3.2 Distribution in Water 124
7.3.3 Distribution in Air 125
7.4 Environmental Fate 125
7.4.1 Mobility and Persistence 125
7.4.2 Accumulation in Food Chains 125
8. Environmental Assessment 128
8.1 Production, Uses, and Potential Environmental
Contamination 128
8.1.1 Production 128
8.1.2 Uses 128
8.1.3 Losses to the Environment. 128
8.2 Environmental Persistence 129
8.2.1 Physical and Chemical Degradation 129
8.2.2 Biodegradation 130
8.3 Effects on Aquatic and Terrestrial Organisms 131
8.3.1 Nonlaboratory Organisms 131
8.3.2 Laboratory Animals 131
8.4 Effects on Human Health 132
8.4.1 Toxic Effects 132
8.4.2 Teratogenic Effects 132
8.4.3 Mutagenic Effects 132
8.4.4 Carcinogenic Effects 132
8.5 Potential Health Hazards 133
8.5.1 Industrial Workers 133
8.5.2 Laboratory Workers 134
8.5.3 General Population 134
8.6 Potential Environmental Hazards 134
8.7 Regulations and Standards 135
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FIGURES
2.1 Formation of reactive esters and aminonium cations from
2-acetylaminofluorene (AAF) and N-methyl-4-aminoazobenzene
(MAB) 27
2.2 The in vitro reaction of N-acetoxy-AAF and methionylglycine. . . 28
2.3 The in vitro reaction of N-acetoxy-AAF with guanosine 29
2.4 Early version of all glass Greenburg-Smith and midget impingers. 47
2.5 Types of dry, cascade impingers . 48
2.6 Procedure for pretreatment of sample prior to analysis by gas-
liquid chromatography (GLC), thin-layer chromatography (TLC),
or colorimetry 49
2.7 Schematic diagram of a gas chromatograph 53
2.8 Resolution of four aromatic amines by the column mixture OV-17
(4.7%), QF-1 (5.0%), and DC-200 (0.5%) on 80- to 100-mesh
Gas-chrom Q, using PTGC (150-250°C at 3°/min, holding 12 min
at 150°C) 54
2.9 Spectrum of benzidine oxidation product 60
6.1 Concentration in blood after intravenous administration of
0.2 mg of 1'*C-labeled 3,3'-dichlorobenzidine or benzidine per
kilogram body weight to rats 84
6.2 Concentration in blood after intravenous administration of
0.2 mg of lilC-labeled 3,3 '-dichlorobenzidine or benzidine per
kilogram body weight to dogs 85
6.3 Trends of mean urinary concentration of quinonizable substance
(expressed as benzidine) at different times of day over
two-week periods in June and December 1950 92
6.4 Comparative excretion of N-oxidation products of 4-amino-
biphenyl by monkeys and dogs 98
6.5 Graph of time from initial exposure to benzidine to diagnosis
of tumors 106
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TABLES
2.1 Commercial dyes derived from benzidine 20
2.2 Cotton dyes derived from benzidine 20
2.3 Possible mechanisms of chemical carcinogenesis 30
2.4 Physical properties of 3,3'-dichlorobenzidine dihydrochloride. . 31
2.5 Some dyes or pigments manufactured from 3^'-dichloro-
benzidine in 1971 32
2.6 Commercial dyes derived from 3,3'-dime thy Ibenzidine 34
2.7 Some dyes manufactured from 3,3'-dimethoxybenzidine in 1971. . . 36
2.8 Other benzidine congeners 42
2.9 Methods for determining benzidine 51
2.10 Solvents used for chromatographic separation of
4,4'-dinitrobiphenyl, benzidine, and their respective
metabolic products . 56
2.11 Composition of spraying reagents used for chromatographic
separation of 4,4'-dinitrobiphenyl, benzidine, and
their respective metabolic products 56
2.12 Chromatographic behavior of 4,4'-dinitrobiphenyl, benzidine,
and their metabolites 57
2.13 Retardation factor values of 4,4'-dinitrobiphenyl, benzidine,
and their most important metabolites 58
2.14 Wavelengths used and relative intensities observed during
analysis of benzidine and its congeners 61
2.15 Analysis of two biological growth media spiked with benzidine,
two congeners, and their salts at 0, 0.10, and 1.0 ppm .... 62
2.16 Analysis of wastewater spiked with benzidine, two congeners,
and their salts at 0 and 20 ppb. 63
3.1 Mutagenic activity of benzidine analogues to Salmonella
typhimuviwri in the presence or absence of liver mixed
function oxidase 75
6.1 Relationship between environmental conditions and benzidine
concentrations in urine of six pressroom workers 83
vii
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viii
6.2 Distribution pattern 1 and 4 hr after intravenous administration
of 0.2 mg of 14C-labeled 3,3'-dichlorobenzidine or benzidine
per kilogram body weight to rats and dogs 86
6.3 Distribution pattern 7 and 14 days after intravenous administra-
tion of 0.2 mg of li'C-labeled 3,3'-dichlorobenzidine or
benzidine per kilogram body weight to rats, dogs, and
monkeys 87
6.4 The concentration and distribution of free and total diazo-
tizable material in rat tissues following a single intra-
peritoneal injection of 100 mg benzidine per kilogram of
body weight 88
6.5 Metabolites formed by biotransformation of benzidine and
benzidine derivatives in animals 90
6.6 The urinary and biliary metabolites of benzidine produced by
various experimental animals 94
6.7 Excretion in the first seven days after intravenous
administration of 0.2 mg of lilC-labeled 3,3'-dichloro-
benzidine or benzidine per kilogram body weight to rats,
dogs, and monkeys 96
6.8 Effects of benzidine, its congeners, and metabolites on
various animal species 99
6.9 Development of proteinuria and anemia in four female rats
fed a diet containing N,N'-diacetylbenzidine 101
6.10 Survival and hyperplastic changes of epithelium in embryonic
kidney tissue cultures treated transplacentally with
3 ,3'-dichlorobenzidine and 3,3'-dimethylbenzidine 103
6.11 Bladder and liver changes produced in Albino Delph mice
administered benzidine or dihydroxybenzidine by subcutaneous
injection 108
6.12 Tumors induced in rats receiving 20 mg of diorthotolidine
either by subcutaneous injection or subcutaneous implantation
of pellets Ill
6.13 Comparison of tumor incidence between workers exposed to
dichlorobenzidine and those exposed to dichlorobenzidine
plus benzidine 113
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FOREWORD
A vast amount of published material is accumulating as numerous
research investigations are conducted to develop a data base on the
adverse effects of environmental pollution. As this information is
amassed, it becomes continually more critical to focus on pertinent,
well-designed studies. Research data must be summarized and interpreted
in order to adequately evaluate the potential hazards of these substances
to ecosystems and ultimately to public health. The Reviews of the Environ-
mental Effects of Pollutants (REEPs) series represents an extensive com-
pilation of relevant research and forms an up-to-date compendium of the
environmental effect data on selected pollutants.
Reviews of the Environmental Effects of Pollutants: II. Bensidine
includes information on chemical and physical properties; pertinent
analytical techniques; transport processes to the environment and sub-
sequent distribution and deposition; impact on microorganisms, plants,
and wildlife; toxicologic data in experimental animals including metabo-
lism, toxicity, mutagenicity, teratogenicity, and carcinogenicity; and an
assessment of its health effects in man. The large volume of factual
information presented in this document is summarized and interpreted in
the final chapter, "Environmental Assessment," which presents an overall
evaluation of the potential hazard resulting from present concentrations
of benzidine in the environment.
The REEPs are intended to serve various technical and administrative
personnel within the Agency in the decision-making processes, i.e., in
the development of criteria documents and environmental standards, and
for other regulatory actions. The breadth of these documents makes them
a useful resource for public health personnel, environmental specialists,
and control officers. Upon request these documents will be made available
to any interested individuals or firms, both in and out of the government.
Depending on the supply, the document can be obtained directly by writing
to:
Dr. Jerry F. Stara
U.S. Environmental Protection Agency
Health Effects Research Laboratory
26 W. St. Glair Street
Cincinnati, Ohio 45268
R. J. Garner
Director
Health Effects Research Laboratory
ix
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ACKNOWLEDGMENTS
The authors are particularly grateful to R. A. Griesmer, National
Cancer Institute, J. K. Selkirk and C. H. Ho, Oak Ridge National Laboratory
(ORNL), and Thomas J. Haley, National Center for Toxicological Research,
for reviewing preliminary drafts of this report and for offering helpful
comments and suggestions. The advice and support of Gerald U. Ulrikson,
Manager, Information Center Complex, and Jerry F. Stara, EPA Project Officer,
and the cooperation of the Toxicology Information Response Center, the
Environmental Mutagen Information Center, and the Environmental Resource
Center of the Information Center Complex, Information Division, ORNL, are
gratefully acknowledged. The authors also thank Carol Brumley, editor,
and Donna Stokes and Patricia Hartman, typists, for preparing the manu-
script for publication.
Appreciation is also expressed to Bonita M. Smith, Karen L. Blackburn,
and Donna J. Sivulka for EPA in-house reviews and editing and for coordinat-
ing contractual arrangements. The efforts of Allan Susten and Rosa Raskin
in coordinating early processing of the reviews were important in laying
the groundwork for document preparation. The advice of Walter E. Grube
was valuable in preparation of manuscript drafts. The support of R. John
Garner, Director of Health Effects Research Laboratory, is much appreciated.
Thanks are also expressed to Carol A. Haynes and Peggy J. Bowman for typing
correspondence and corrected reviews.
XI
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ABSTRACT
This study is a comprehensive, multidisciplinary review of the health
and environmental effects of benzidine and specific benzidine derivatives.
More than 200 references are cited.
Benzidine and its congeners are used in industry primarily for the
synthesis of azo dyes. Available data indicate that U.S. production and
importation of benzidine and benzidine compounds may exceed 3.5 million
kilograms per year. Benzidine and its derivatives are not naturally occur-
ring compounds and are not thought to be generally dispersed in the environ-
ment; however, little information is available for documentation.
In most animal species tested, the greatest damage from benzidine expo-
sure occurred to the liver, kidney, and urinary bladder. Following adminis-
tration of sublethal doses, tumors have developed in almost all species
tested. Little information is available on the effects of benzidine on
plants or on domestic and wild animals.
Epidemiological evidence leaves no doubt that exposure to benzidine can
lead to bladder cancer in humans. An exact quantitative relationship between
the degree of exposure to benzidine, the length of the exposure period, and
the incidence of bladder cancer has not been established. Benzidine com-
pounds are believed to become active carcinogens only after metabolic altera-
tions. Since 1974, when the Occupational Safety and Health Administration
of the U.S. Department of Labor instituted more stringent industrial stand-
ards, the risk of cancer from industrial exposure to benzidine has been
substantially reduced. There is no evidence that benzidine or its congeners
act as human teratogens; however, some tests suggest that these compounds
may be mutagenic. Benzidine compounds are also known to cause dermatitis,
cystitis, and hematuria.
This report was submitted in partial fulfillment of Interagency
Agreement No. D5-0403 between the Department of Energy and the U.S. Environ-
mental Protection Agency. The draft report was submitted for review
June 1976. The final report was completed in October 1977.
xiii
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SECTION 1
SUMMARY
1.1 INTRODUCTION
Benzidine is an aromatic amine that does not occur naturally; it
was first prepared by Zinin in 1845 and is now used in the synthesis of
commercial dyes. 3,3'-Dichlorobenzidine, 3,3'-dimethylbenzidine, and
3,3'-dimethoxybenzidine are benzidine derivatives widely used in the
United States and therefore are included in this discussion. Two ben-
zidine congeners, 4-aminobiphenyl and 4-nitrobiphenyl, are discussed
because of structural and chemical similarities to benzidine.
1.2 BENZIDINE CHEMISTRY
1.2.1 Physical Properties
Benzidine is a colorless, crystalline compound with a molecular
weight of 184.23, a density of 1.250 (20/4°C), a melting point of 115°C
to 120°C (slow heating) or 128°C (fast heating), and a boiling point of
400°C. It is soluble in alcohol and ether and is slightly soluble in
water. 3,3'-Dichlorobenzidine, a colorless, crystalline compound, has
a molecular weight of 253.1 and a melting point of 132°C to 133°C. It
is soluble in ethanol, benzene, and glacial acetic acid, is slightly
soluble in dilute hydrochloric acid, and is almost insoluble in cold
water. 3,3'-Dimethylbenzidine is a white to reddish crystalline com-
pound which has a molecular weight of 212.28 and a melting point of 131°C
to 132°C; it is highly soluble in ethanol, ethyl ether, and acetone and
is slightly soluble in water. 3,3'-Dimethoxybenzidine, a colorless,
crystalline compound that turns violet on standing, has a molecular
weight of 244.3 and a melting point of 137°C to 138°C. It is soluble in
ethanol, ethyl ether, acetone, benzene, and chloroform and is poorly
soluble in water. 4-Aminobiphenyl is also a colorless, crystalline
compound that darkens on oxidation. The molecular weight is 169.2, the
melting point is 53°C to 54°C, and the boiling point is 191°C. It is
soluble in lipids, hot water, and nonpolar solvents and is slightly
soluble in cold water. The compound 4-nitrobiphenyl occurs as white to
yellow needles. It has a molecular weight of 199.2, a boiling point of
340°C, and a melting point of 114°C. 4-Nitrobiphenyl is soluble in
benzene, chloroform, and ether, is slightly soluble in cold alcohol, and
is almost insoluble in water.
1.2.2 Chemical Properties
Benzidine is both a primary aromatic amine and a diphenyl derivative;
it undergoes chemical reactions which are characteristic for each class
of compounds as well as some which are peculiar to it alone (Section
2.2.1.2). Benzidine forms stable salts with mineral acids and complexes
with a large number of metallic salts. It is sensitive to oxidation and
can be readily acetylated, alkylated, and diazotized. The acetylated
-------
product is readily nitrated and halogenated. The chemical reaction lead-
ing to the diazotized product has commercial value since this product
couples with many different aromatic amines and phenols to yield a wide
variety of azo dyes.
The benzidine derivatives 3,3'-dichlorobenzidine, 3,3'-dimethylben-
zidine, 3,3'-dimethoxybenzidine, and 4-aminobiphenyl (Sections 2.2.2.1,
2.2.3.1, 2.2.4.1, and 2.2.5.1) undergo chemical reactions similar to
those displayed by benzidine. They can be acetylated, alkylated, and
diazotized. The latter reaction is commercially important as the first
step in their use in the synthesis of azo dyes. Because 4-nitrobiphenyl
(Section 2.2.6.1) contains no reactive amino group, its chemical reactions
differ. However, it is readily reduced to 4-aminobiphenyl.
1.2.3 Biochemical Reactions
In a manner similar to the biochemical reactions of aromatic amines
in general, benzidine is conjugated through the functional amino groups
(usually only one group is conjugated) and hydroxylated in the ortho
position with respect to the amine. Hydroxylation of benzidine requires
mediation by an enzyme system consisting of a hepatic microsomal enzyme,
oxygen, and reduced nicotinamide adenine dinucleotide phosphate. The
hydroxy groups of the hydroxybenzidine thus formed may be conjugated
with sulfate and with glucuronic acid through mediation of other required
enzyme systems.
Less attention has been given to the biochemical reactions that occur
with benzidine derivatives, but, with the exception of 3,3'-dichloroben-
zidine, the reactions thought to occur are similar to those for benzidine -
namely, acetylation, sulfation, and orthohydroxylation. Because of the
in vivo stability of the chlorine atom linkage to the benzidine molecule,
3,3'-dichlorobenzidine apparently does not form 3-hydroxy products. This
compound appears to be relatively stable in vivo.
Although the process is still somewhat speculative, it appears that
the azo dyes prepared from benzidine and its derivatives may be reduced
in vivo to release the parent materials. A recent report on this possible
reduction is discussed in Section 2.2.1.4.
1.2.4 Methods of Analysis
Several methods of analysis of benzidine, its derivatives, and their
metabolites are used for qualitative and quantitative determinations in
environmental and biological media. These methods are gas-liquid chroma-
tography, thin-layer chromatography, spectrophotometry (chloramine-T),
spectrophotometry (diazo dye), and spectrophotofluorometry (Section 2.3.2).
None of these is officially sanctioned for determining low levels of
benzidine in environmental or biological samples, but several appear ade-
quate for assessment of gross amounts of aromatic amines. Gas-liquid
chromatography seems to be the most promising method of benzidine analysis
because of its high degree of sensitivity, selectivity, versatility, and
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speed. This is the only available method which easily determines ben-
zidine in the presence of its derivatives.
The oldest and best developed analytical methods are the diazo and
chloramine-T spectrophotometric methods. Recent refinements allow sensi-
tivity in the parts-per-billion range by instrumentation available in
most laboratories. However, neither method can be used to distinguish
between benzidine and benzidine derivatives in mixed samples. Furthermore,
the diazo method is responsive to aromatic amines in general, while the
chloramine-T method is photosensitive. Nevertheless, the U.S. Environ-
mental Protection Agency has designated the chloramine-T method as the
interim method for determining benzidine.
The thin-layer chromatographic method is simple to perform and fre-
quently is used qualitatively for separating and identifying benzidine,
benzidine derivatives, and metabolites. Although it can be used quantita-
tively, it is relatively insensitive.
Spectrophotofluorometry has had only limited use with benzidine
compounds. The method combines inherent sensitivity with simplicity and
rapidity. However, the fluorescent spectra produced by benzidine and its
derivatives tend to overlap and vary greatly in their intensity, thus
preventing the method from being used to quantitatively distinguish such
compounds in mixed samples.
1.3 USES AND PRODUCTION
Benzidine and its salts are used principally in the manufacturing
of dyestuffs based on the coupling of tetrazotized benzidine with phenols
and aromatic amines. At least 250 commercial dyes are based on benzidine.
Many of these dyes are economically important because of their ability to
dye cotton without the use of mordants. In analytical chemistry, benzi-
dine is used to detect inorganic ions (Section 2.2.1.3), as a chromogenic
spray reagent in chlorinated organic pesticide thin-layer chromatography,
and as a reagent for determining hydrogen peroxide, nicotine, sugars,
occult blood, and bacterial cytochromes. Benzidine is also used as a
hardener for polyurethane and as an agent to reveal bank check altera-
tion. Production in the United States was 680.9 metric tons in 1972.
The benzidine derivatives 3,3'-dimethylbenzidine, 3,3'-dimethoxy-
benzidine, and 3,3'-dichlorobenzidine are used in the manufacture of
dyestuffs. These dyestuffs are used to color plastics, lacquer, paint,
rubber, leather, paper, and textiles. They are used in the detection
and determination of various inorganic ions. 3,3'-Dichlorobenzidine
and 3,3'-dimethoxybenzidine are used as curing agents for polyurethane
elastomers; the latter of these two compounds is also used in the manu-
facturing of the diisocyanate compound for isocyanate-based adhesive
systems. 4-Aminobiphenyl has been used as an antioxidant in rubber.
4-Nitrobiphenyl is an intermediate in the manufacture of 4-aminobiphenyl.
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Production in the United States of benzidine, 3,3'-dichloro.benzidine,
3,3'-dimethylbenzidine, and 3,3'-dimethoxybenzidine is believed to amount
to many millions of kilograms annually. Benzidine and 3,3'-dichlorobenzi-
dine are produced in greatest quantities. Large-scale production of
4-aminobiphenyl and of 4-nitrobiphenyl in the United States is believed
to have ceased following the general recognition in 1955 of the carcino-
genicity of the former compound; however, small amounts are still synthe-
sized for research and pilot plant use.
1.4 ENVIRONMENTAL DISTRIBUTION AND FATE
Since the melting points of benzidine and its derivatives are above
ambient environmental temperatures, these compounds exist in the undis-
solved state as aerosols, dusts, or crystals and in the dissolved state
in aqueous or organic solutions.
1.4.1 Distribution in Air
At one time, benzidine and its derivatives were manufactured and
used in open systems which permitted loss of the compounds to the atmo-
sphere, the worker, and the work site. In some operations, average con-
centrations of 2.5 to 17.6 mg/m3 of air were observed. Large-scale
manufacturers are now believed to utilize closed systems which prevent
this hazard, except possibly during cleaning of the closed-system equip-
ment. At present, small-scale manufacturing may be proceeding without
the use of closed systems.
The use by the general public of benzidine yellow pigments in pres-
surized containers of lacquers and paints presents an exposure of cur-
rently unknown potential and hazard. This pigment is derived from
3,3'-dichlorobenzidine.
1.4.2 Distribution in Water
In the Sumida River of Japan, benzidine has been measured downstream
from dye factories. In the United States, benzidine and its derivatives
have been detected in manufacturing and dye factory wastes but not in the
drainage areas into which these wastes feed; however, few attempts at
actual measurements seem to have been made. Lack of detection of these
compounds is thought to result from degradation, mostly before the release
of the wastes, and from dilution of residual concentrations to below the
detection limit. Unfortunately, the chloramine-T method of detection
used evidently will measure only the parent benzidine compounds and may
therefore have failed to measure the possible presence of slightly de-
graded products, including those of carcinogenic potential. Newer analyt-
ical methods now under study should be more versatile (Section 1.2.4).
1.4.3 Distribution in Soil
No data were found concerning the distribution of benzidine compounds
in soil. Benzidine has been shown to be adsorbed to some clays and,
because of its physical properties, is probably immobilized rapidly in
soils and sediments.
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1.4.4 Mobility and Persistence
Benzidine is thought to be volatile and soluble enough to have the
potential for wide dispersion. According to several investigators, it
probably persists for a time in the environment. The principal reaction
of benzidine in air and water is believed to be oxidative degradation by
free radical, enzymatic, or photochemical processes, but actual data on
reaction rates are not available. Half-life reaction rates have been
estimated to be 1 to 60 days in air and 100 days in water for benzidine
and 3,3'-dichlorobenzidine. Under experimental water waste treatment
conditions, air oxidation of benzidine proceeded readily, and there was
also some evidence of biological oxidation.
1.4.5 Food Chain Accumulation
Since benzidine and its derivatives are soluble in many organics,
movement of these compounds may take place within the food chain. Accu-
mulation is expected to occur, and recent laboratory findings indicated
that bluegill fish concentrated li'C-benzidine to a level 44 times that
present in water.
1.5 BIOLOGICAL ASPECTS IN MICROORGANISMS, PLANTS, AND WILD AND DOMESTIC
ANIMALS
Very little information is available concerning the metabolism and
effects of benzidine and its derivatives in these classes of organisms.
1.5.1 Microorganisms
The metabolism of benzidine by microorganisms has received little
attention. It is known, however, that benzidine-based azo dyes are
reduced to benzidine by certain bacteria.
Little is known about the toxicity of benzidine and its derivatives
to microorganisms. At low concentrations, benzidine appears to be oxi-
dized by microorganisms present in the activated sludge of waste treatment
plants, but at higher concentrations it may inhibit these microorganisms
and prevent general biooxidation of the organic material present. Benzi-
dine and some of its derivatives have been shown to be mutagenic to strains
of Salmonella typh-imuY"Liffn during testing by an assay method developed for
measuring the mutagenic and carcinogenic potential of a wide variety of
chemicals.
The benzidine-based azo dye Congo Red has been used as a bacterio-
static agent in the selective culture of the tubercle bacillus and as an
indicator dye in the culture of other species of bacteria. Congo Red and
other azo dyes have been shown to have antiviral activity and to inhibit
phage production by one species of bacteria.
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1.5.2 Plants
No data are available on benzidine metabolism or on effects from
benzidine exposure in higher plants. Benzidine has not received much
attention in this area since it is not a natural environmental constit-
uent and is not believed to be widely dispersed from industrial sources.
Benzidine (0.3 mg/liter) stimulates growth of Anti£hamni,on plumula (red
algae). It is oxidized by peroxidases from several plant species, in-
cluding horseradish and green algae.
1.5.3 Wild and Domestic Animals
Information pertaining to wild and domestic animals and benzidine
compounds does not exist except for laboratory research on a few species
under experimental conditions. Benzidine has been shown to be toxic to
the red shiner and fathead minnow at TLSO values of 2.5 mg/liter and
20 mg/liter, respectively, and to cause liver damage, including hyper-
plasia, in guppies fed a diet containing 30 mg benzidine per 100 g of
dry feed. When injected into eggs, benzidine caused morphological abnor-
malities in immature chick embryos. Other research findings with the
more usual laboratory animals (e.g., rat, mouse, and dog) are summarized
in Section 1.6 since such data provide useful comparative information for
studying the metabolism and toxicity of benzidine compounds in humans.
Because benzidine and its derivatives may possibly find their way
into the open environment, much more research is needed to determine
species differences in the uptake, accumulation, metabolism, and excre-
tion of these compounds and in the toxic effects that may occur through
exposure.
1.6 BIOLOGICAL ASPECTS IN HUMANS
Benzidine exposure in humans is primarily an industrial problem.
Because benzidine and its derivatives are not natural constituents of
the environment and the general population is therefore not exposed to
these chemicals, information concerning human exposure is restricted to
industrial exposure.
1.6.1 Metabolism
1.6.1.1 Uptake, Distribution, and Accumulation — Benzidine compounds
enter the body through the respiratory tract, by penetration of the skin,
or by ingestion. The inhalation of these compounds is believed to be
minimized by the use of closed systems during manufacture and use
(Section 1.4.1); a concentration of 0.02 mg benzidine per cubic meter of
air has been suggested as a safe level of exposure. Penetration through
the skin is believed to be the most important avenue of uptake. Penetra-
tion is increased at elevated air temperatures and humidities which cause
sweaty skin. Some compounds penetrate more readily than others (benzidine
> 3,3'-dimethoxybenzidine > 3,3'-dichlorobenzidine). Ingestion via
contaminated food is not generally thought to be an important route of
uptake, but ingestion is believed to be responsible for some cancer
-------
induction among Japanese kimono painters because of their habit of
moistening brushes that have been dipped in benzidine-based azo dyes
with their lips.
Information is lacking on the distribution or accumulation of benzi-
dine and its derivatives in human tissues. Indirect evidence afforded
by repeated measurements of benzidine in urine samples from exposed
workers indicated a lack of any appreciable accumulation.
Data collected from experimental animals is more complete. In the
rat, dog, and monkey, administered concentrations of benzidine and
3,3'-dichlorobenzidine were distributed within hours to various tissues.
Prior to the elimination of these compounds, which was virtually complete
in one week, concentrations were noticeably higher in liver and lung
tissues than in other tissues. In similar testing, 3,3'-dimethylbenzidine
administered to the rat was found in highest concentration in the Zymbal's
gland. The liver and Zymbal's gland tissues of the rat are among those
tissues most prone to tumor induction by benzidine and its derivatives,
perhaps because of the tendency of these compounds to concentrate within
such tissues (Section 6.3.2.2).
There is indirect evidence for possible placental transfer of benzi-
dine or its derivatives since some of these compounds have been found to
affect embryonic kidney tissue following subcutaneous injection of preg-
nant mice and, later, explantation and culture of the embryonic tissue
(Section 6.3.2.1.4).
Although there is no evidence of any significant bioaccumulation of
benzidine or its derivatives in humans or in the usual experimental
animals, it has been reported recently that edible portions of bluegill
fish contained a 44-fold increase in residues of 14C-benzidine over those
present in water during a radiometric study.
1.6.1.2 Eiotransformation and Elimination — Benzidine and its derivatives
are biotransformed in humans and in experimental animals. The metabolites
as well as the parent materials are eliminated via the urine, bile, and
feces. Some of the metabolites produced are thought to be the actual
carcinogenic forms of these compounds (Sections 2.2 and 6.2.3).
Humans metabolize benzidine primarily to 3-hydroxybenzidine and
secondarily to monoacetylbenzidine, diacetylbenzidine, and N-hydroxy
acetyl amino benzidine. These compounds, as well as benzidine, are
eliminated largely through urinary excretion. Urinary excretion in ex-
posed workers appears to vary directly and in proportion to the amount
of benzidine absorbed. Thus, a safe air concentration of benzidine has
been suggested as 0.2 mg or less of benzidine per cubic meter of air
since this amount, inhaled over an 8-hr period, is calculated to lead to
a urinary output not exceeding 0.26 mg benzidine per liter of urine.
3,3'-Dimethylbenzidine appears to be biotransformed and eliminated in a
manner similar to that of benzidine. Workers exposed to benzidine yellow
have reportedly excreted 3,3'-dichlorobenzidine in their urine, probably
because of the metabolic reduction of the dye and release of this benzi-
dine derivative.
-------
The metabolites of benzidine found in humans are also found in some
experimental animals, along with additional products resulting from sul-
fate and glucuronide conjugations. Species differences occur among the
test animals in the type of metabolites produced and in the main routes
of elimination. In comparative tests, the dog was unable to acetylate
benzidine, although this metabolic reaction occurred in the mouse, rat,
rabbit, and guinea pig. Metabolites of benzidine with available amino
groups were produced in larger amounts by the dog than by the rabbit.
Benzidine was found to be eliminated largely through the urine of the
dog, through the feces of the rat, and in about equal amounts through
the urine and feces of the monkey. Benzidine metabolites found in bile
appear to be similar to those found in urine but are present in only
about one-third the amount found in urine.
Monkeys are capable of metabolically reducing a number of benzidine-
based azo dyes and thereby freeing the benzidine molecule. Substantial
amounts of benzidine and of monoacetylbenzidine were found in the urine
of the animals following single oral doses of the dyes.
The metabolism and excretion of benzidine derivatives have been less
extensively studied in experimental animals. In one experiment, admin-
istered doses of 3,3'-dichlorobenzidine in dogs were only partially
accounted for by measured urinary products, some of which were the non-
metabolized parent materials. No metabolites of 3,3'-dichlorobenzidine
were found, suggesting that this compound, by virtue of the chlorine
substitutions at the 3,3' positions, is more inert to biotransformation
than are the methyl or methoxy substitutions. A separate study showed
that 3,3'-dichlorobenzidine in dogs is excreted in greater quantity by
the feces than by the urine, in direct contrast to the excretion pattern
of benzidine. This finding indicated that the route of excretion was
altered by substitution of the benzidine molecule in the 3,3' positions.
Dogs and monkeys fed 4-aminobiphenyl excreted N-hydroxy products of
this compound more rapidly in their urine than they excreted the parent
compound. The urinary yield of a 3-sulfuric acid conjugate of 4-amino-
biphenyl varied widely among five species of animals, ranging from 0% in
the guinea pig to 25% to 40% in the dog.
1.6.2 Effects
1.6.2.1 Physiological Effects — A possible relationship was found during
in vitro studies between potential carcinogenicity of benzidine and its
derivatives and their ability to reduce cytochrome o. 4-Aminobiphenyl,
considered to be strongly carcinogenic, did not reduce cytochrome o,
whereas 3,3'-diaminobenzidine, considered to be only weakly carcinogenic,
did reduce this enzyme. Aminobiphenyls, which are easily oxidized by
mitochondrial systems, may be detoxified in this manner and excreted in
combination with sulfuric or amino acids.
Exposure to benzidine has been found to alter enzyme activity in
vivo. The activity of 3-glucuronidase was higher in the urine of exposed
workers than in nonexposed workers. Cessation of exposure caused a
-------
decrease in activity to a level more nearly normal. Blood phenolase
activity in rabbits was decreased by benzidine but not by 3,3'-dimethyl-
benzidine. Catalase and peroxidase activity was decreased in rats by
benzidine.
1.6.2.2 Toxic Effects — In addition to their carcinogenic potential,
benzidine compounds are known to cause dermatitis, cystitis, and hema-
turia in humans and a number of poisoning symptoms, including glomerulo-
nephritis, in laboratory animals (Section 6.3.2). Dermatitis has been
reported among workers in dye manufacturing plants. Benzidine and ben-
zidine-based dyes have been implicated. Benzidine and 3,3'-dimethylben-
zidine have caused dermatitis among laboratory-exposed chemists.
Individuals appear to vary in sensitivity to these compounds.
Toxic effects to experimental animals are varied. For example,
mice fed a daily diet of benzidine lost weight in proportion to the
amount of compound ingested, but the weight of the spleen and thymus
increased. Morphological changes included a cloudy swelling of the
liver, vascuolar degeneration of renal tubules, and hyperplasia of the
myeloid elements in bone marrow and of the lymphoid cells in the spleen
and thymic cortex. Several investigators have reported glomerulonephri-
tis in rats exposed to N,N'-diacetylbenzidine. The nephrotic syndrome
induced resembles glomerulonephritis in humans. At the high dosage rates
applied to test animals in carcinogenicity studies, liver pathology is
severe and may lead to death prior to any tumor development.
There is convincing evidence that benzidine and certain other aromatic
amines are responsible for the high rate of occurrence of bladder cancer
among occupationally exposed persons. It is generally accepted that metab-
olites of these aromatic amines are the actual carcinogens. Evidence sug-
gests that N-hydroxylation of the parent amine produces the precarcinogen
and that the sulfate or glucuronide conjugates are the carcinogenically
active forms in vivo. The latter metabolites appear to have an affinity
to and exert a carcinogenic action on certain target organs. Cancer induc-
tion in humans by benzidine appears to be limited to the bladder.
Since benzidine and its derivatives are not naturally occuring
chemicals and are not thought to be dispersed in the environment, the
thrust of studies on their carcinogenicity has been directed at groups
of occupationally exposed workers in manufacturing and user industries
of various countries. Tumor development in these workers has been reported
following exposures to benzidine ranging from 2 to 16 years and averaging
8 years. The latent period between first exposure and tumor induction
ranges from 8 to 32 years and averages 18 years. Thus, workers who have
been exposed to benzidine for as little as two years have developed bladder
cancer several years later. The degree of exposure and the number of years
of survival beyond the exposure period are thought to influence the rate of
occurrence of bladder tumors. High rates of occurrence averaging 11% to
50% of the exposed workers have been reported from benzidine manufacturing
plants, dyestuff factories, and coal tar dye plants. Awareness of this
hazard has prompted improved work conditions and work habits, which have
resulted in reduced exposure. The introduction of closed systems in the
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10
synthesis of benzidine has greatly reduced atmospheric contamination.
Unfortunately, there is a general lack of information on actual exposures
encountered in various plants. More data should be collected and made
available on air concentrations, on respiratory and dermal absorption, and
on the physical conditions and health of the workers.
In some earlier studies on the rate of occurrence of bladder cancer
in manufacturing and dyestuff plants, the role of benzidine in the induc-
tion of the cancer was confused by simultaneous exposures to other chem-
icals, particularly to B-naphthylamine, which is considered to be a
potent carcinogen. Later comparative studies indicated that such dual
exposures may significantly increase the risk of bladder cancer induc-
tion. One study, for example, showed an increase from 21.3% among
workers exposed only to benzidine to 45.5% among workers exposed addi-
tionally to B-naphthylamine.
In humans, benzidine has been implicated only in the induction of
bladder cancer. In contrast, it induces several different types of
cancer in experimental animals. The evidence of cancer induction by
benzidine and its derivatives suggests that induction by these compounds
depends on both species and compound.
3,3'-Dichlorobenzidine, unlike benzidine, has not been reported to
have produced bladder tumors in humans, but it has been implicated
recently in the occurrence among exposed workers of tumors of the lung,
bone marrow, rectum, sigmoid colon, prostate, and breast muscle. These
workers were exposed for 35 years. Since dichlorobenzidine is nonvola-
tile and poorly absorbed by the skin, respiratory and dermal intake can
be expected to be less than that which would occur with benzidine for an
equivalent period. Experimentally, a variety of tumors have been induced
by dichlorobenzidine in rats and mice, including bladder tumors in rats.
It is possible that this compound can induce bladder cancer in humans,
but only after a latent period longer than that for benzidine.
There is no evidence that 3,3'-dimethylbenzidine or 3,3'-dimethoxy-
benzidine induce any type of cancer in humans. However, both compounds
have been found to be carcinogenic in rats and have induced a variety
of tumors. They must therefore be considered as potentially carcinogenic
in humans.
4-Aminobiphenyl is considered to be a potent carcinogen in humans.
Bladder tumors have developed in workers following as little as 133 days
of exposure as well as after more extended exposure periods ranging up
to 19 years. The latent period appears to range from 5 to 19 years.
This compound causes a variety of tumors in experimental animals, includ-
ing bladder cancer. However, its production and use in the United States
are believed to have been greatly reduced by the early recognition of
its carcinogenicity.
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11
1.7 THE PROBLEM
Benzldine presents a serious carcinogenic hazard to exposed indus-
trial workers because of its association with the high rate of occurrence
of bladder cancer among these workers. It is a potent carcinogen in
experimental animals. Derivatives of benzidine also cause cancer in
experimental animals, but only 4-aminobiphenyl has been associated with
bladder cancer in humans; usage of this compound is thought to have de-
creased markedly in recent years. 3,3'-Dichlorobenzidine has recently
been implicated in causing cancer other than bladder cancer in humans,
but no reports have implicated either 3,3'-dimethylbenzidine (diortho-
tolidine) or 3,3'-dimethoxybenzidine (dianisidine). Nevertheless, these
derivatives must be viewed as potentially hazardous to humans. The use
of closed systems and better hygiene by workers have resulted in a
decrease in the level of exposure to these compounds and thereby have
reduced the hazard involved.
Benzidine and its derivatives are not naturally occurring compounds
and are not thought to be dispersed in the environment, although docu-
mentation on this is lacking. Judging from their toxic effects to
research animals, these compounds, if present in the environment, can be
expected to be hazardous to wildlife. Recent laboratory information
indicating appreciable bioaccumulation in one species of fish increases
the hazard potential.
1.8 CONCLUSIONS
1. Benzidine and its derivatives are not naturally occurring compounds
and are probably not found in the general environment, although
adequate documentation is lacking.
2. Analysis of benzidine and its derivatives can be performed best by
use of gas-liquid chromatography or spectrofluorometry; these
methods provide needed sensitivity, selectivity, and versatility.
3. The chloramine-T method of analysis is designated as the interim
method of benzidine analysis by the U.S. Environmental Protection
Agency, but, unfortunately, it does not adequately identify or
distinguish between benzidine and its derivatives and the important
metabolites.
4. Benzidine and the derivatives 3,3'-dichlorobenzidine, 3,3'-dimethyl-
benzidine, and 3,3'-dimethoxybenzidine are manufactured in the
United States by three major firms which employ closed systems to
reduce air contamination and exposure by the workers. There is
reason to believe that some manufacturing, including limited
production of 4-aminobiphenyl, is being undertaken by smaller firms
without the use of the closed-system approach.
5. Benzidine, 3,3'-dichlorobenzidine, 3,3'-dimethyIbenzidine, and 3,3'-
dimethoxybenzidine are used primarily in the production of dyes and
pigments for the coloring of textiles, paper, leather, rubber,
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12
plastics, inks, paints, lacquers, and enamels. Other less extensive
uses of these compounds are as reagents in the detection of inorganic
ions, organic molecules, and occult blood, as stains in microscopy,
and as a curing agent for polyurethane elastomers. 4-Aminobiphenyl
has been used as a rubber antioxidant.
6. Data are not available concerning the distribution of benzidine
compounds in the atmosphere or soil.
7. Benzidine and its derivatives are thought to present a hazard in
water only near factories which use or manufacture these compounds.
No data exist on benzidine in drinking water.
8. No data are available on accumulation in and movement through food
chains, except for a recent radiometric study indicating that blue-
gill fish can bioconcentrate benzidine to a 44-fold increase over
concentrations in water.
9. Toxic effects to plants and to wild and domestic animals are not
known except for recent laboratory studies on a very limited number
of species.
10. The principal avenues of benzidine absorption by humans are dermal
and pulmonary.
11. Benzidine does not appear to accumulate in human body tissues, but
the evidence is indirect and is based on measurements of urinary
concentrations in exposed workers.
12. Benzidine compounds do not accumulate in tissues of the usual
laboratory animals, although unequal concentrations appear in
different organs following administration. Concentrations tend
to be high in those organs prone to later tumor development.
13. Although there is no direct evidence of the placental transfer of
these compounds, studies in mice and rats point to this possibility.
14. Benzidine compounds are believed to become active carcinogens only
after metabolic transformations that involve N-hydroxylation to
produce the proximate carcinogens, followed by esterification and
possibly conjugation with sulfate or glucuronate to produce the
ultimate carcinogens.
15. Benzidine compounds and metabolites are eliminated from the animal
body via the urine, bile, and feces.
16. Benzidine compounds cause dermatitis, cystitis, and hematuria in
humans.
17. Benzidine is believed to be carcinogenic in humans and a major
cause of bladder cancer among exposed industrial workers.
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13
18. A recent report also Implicated 3,3'-dlchlorobenzldine as a cause
of certain types of cancer among exposed industrial workers, but
there are no reports of its association with bladder cancer.
19. Although there are no reports of any associations between cancer
in humans and exposure to 3,3'-dimethylbenzidine or 3,3'-dimethoxy-
benzidine, these compounds induce a variety of cancers in experi-
mental animals and must therefore be considered as potentially
hazardous to humans.
20. Although 4-aminobiphenyl is believed to cause bladder cancer in
humans, its manufacture and use in the United States today are
considered to be very low.
21. Areas of research needed include: (]) epidemiological studies
designed to determine relative risks and incidence rates of bladder
cancer and other forms of cancer among adequate numbers of exposed
and nonexposed workers, with careful consideration of confounding
factors such as age, smoking history, and exposure to other potenti-
ally hazardous chemicals; (2) plant monitoring studies to better
establish the degree of skin and pulmonary involvement in absorp-
tion; (3) pharmacokinetic studies to better substantiate mechanisms
and extent of body transport and storage; (4) more extensive moni-
toring and surveillance of users of these compounds outside of
large industrial plants, including those who use aerosol formula-
tions of paints, lacquers, or enamels containing benzidine yellow;
(5) environmental monitoring studies involving attempts to measure
these compounds in air, water, sediment, soil, and selected species
of aquatic and terrestrial life; and (6) laboratory studies aimed
at determining the extent of toxic effects and possible bioaccumula-
tion in a variety of aquatic and terrestrial species.
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SECTION 2
PHYSICAL AND CHEMICAL PROPERTIES AND ANALYSIS
2.1 SUMMARY
Benzidine is a synthetic aromatic diamine not known to occur naturally.
It was first synthesized in 1845 and has since become a versatile and impor-
tant chemical coupling agent in the manufacture of commercial azo dyes.
These dyes form the largest and most useful class of all commercial dyes;
many of them are of great economic importance in the textile industry
because of their ability to dye cotton directly without the use of mordants.
Benzidine is also widely used in analytical chemistry and biological lab-
oratories where its versatile chemistry makes it a useful reagent in the
detection and determination of many inorganic ions as well as hydrogen
peroxide, nicotine, sugars, occult blood, and bacterial cytochromes. Benzi-
dine is sometimes used in security printing because it reacts with ink
erasers to give colored products which reveal attempted alterations. About
680,400 kg of benzidine was produced in the United States in 1972.
Despite its versatile chemistry and widespread use, benzidine is a
hazardous substance which is believed to be a potent bladder carcinogen.
Apparently, a metabolite, rather than the aromatic amine per se, is the
ultimate carcinogen. Most investigators have therefore been concerned with
identifying and testing the carcinogenicity of the metabolic products of
aromatic amines in various species. Results from early studies gave rise
in 1952 to the hypothesis that carcinogenicity is due to the formation of
ort/zo-hydroxy metabolites of aromatic amines. Later results suggested the
generally held current theory that formation of an N-hydroxy metabolite is
the first step in the activation of aromatic amines and amides for carcino-
genesis. According to this view, subsequent esterification of the N-hydrox-
ylated metabolite produces a highly reactive electrophile, the ultimate
carcinogen, that attacks nucleophilic tissue components such as DNA and RNA.
Experimental work verifying this chemical sequence in the metabolism of
benzidine has not been performed, but firm evidence exists for the presence
of an acetyl-N-hydroxy metabolite in the urine of persons exposed to
benzidine.
Several benzidine derivatives are also used extensively in the United
States. 3,3'-Dichlorobenzidine is employed as a coupling agent in the
manufacture of dyes and pigments which color plastic resins, rubber, print-
ing inks, metal finishes, textiles, and wallpaper prints. During 1971,
about 1.6 million kg of this compound was manufactured in the United States
and an additional 635,000 kg was imported. Other widely used derivatives of
benzidine include 3,3'-dimethylbenzidine and 3,3'-dimethoxybenzidine.
These compounds are components of nearly 100 different dyes listed in the
Colour Index. More than 44,000 kg of 3,3'-dimethylbenzidine and 122,000 kg
of 3,3'-dimethoxybenzidine were imported into the United States during
1970 and 1971 respectively. Only limited information about the metabolic
chemistry of these compounds is available. Other pertinent congeners of
benzidine include the nitro, alkoxy, sulfonic acid, and carboxylic acid
14
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15
derivatives as well as 4-aminobiphenyl and 4-nitrobiphenyl. Few metabolic
or carcinogenic studies have been performed with these benzidine deriva-
tives. 4-Aminobiphenyl is an established carcinogen whose large-scale
commercial production in the United States apparently ceased in 1955 when
its carcinogenic character was generally recognized. 4-Nitrobiphenyl is
readily reduced in vitro and in vivo to 4-aminobiphenyl; therefore, it is
as hazardous as 4-aminobiphenyl. 4-Nitrobiphenyl was used chiefly as an
intermediate in the synthesis of 4-aminobiphenyl. It apparently has not
been produced in large quantities in the United States since the use of
4-aminobiphenyl was discontinued; however, small amounts of both 4-amino-
biphenyl and 4-nitrobiphenyl are still manufactured for research and pilot
plant operations.
Approved analytical methods for determining benzidine were first estab-
lished by the Association of British Chemical Manufacturers in 1954. These
colorimetric methods were used with occasional innovations for almost all
of the quantitative analyses of benzidine which were subsequently reported.
Recently, federal legislation in this country provided an impetus for the
development of more sensitive and precise methods for quantifying benzidine
and its congeners; the end results of this stimulation are not yet apparent.
Gas chromatographic and spectrophotofluorometric methods appear to be
attractive alternatives to the older colorimetric procedures, but further
experience will be needed to fully evaluate the new methods. Meanwhile,
until an adequately tested method is officially sanctioned, the U.S. Environ-
mental Protection Agency has designated a recently revised version of the
older chloramine-T colorimetric technique as the interim method of determin-
ing benzidine.
2.2 PHYSICAL AND CHEMICAL PROPERTIES
In January 1974, the U.S. Department of Labor issued occupational
safety and health standards for 14 chemical compounds known to cause, or
suspected of causing, human cancer from occupational exposure (federal
Register, 1974). Benzidine and 3,3'-dichlorobenzidine were included among
these compounds. Pertinent physical and chemical properties of benzidine
and relevant congeners are discussed in the following sections; established
biotransformation products and urinary metabolites are also characterized.
2.2.1 Benzidine and Salts
First prepared by Zinin in 1845 (Stecher, 1968), benzidine is now a
versatile and important intermediate in the synthesis of commercial dyes
(International Agency for Research on Cancer, 1972, p. 80). Its composi-
tion and structure are indicated by the formulas Cl2Hi2N2 and
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16
the Chemical Abstracts identification number of benzidine is 92875; it is
also known as biphenyl, 4,4'-diamino-, 4,4'-biphenyldiamine, 4,4'-diamino-
biphenyl, 4,4'-diphenylenediamine, 4,4'-diaminodiphenyl, p-diaminodiphenyl,
Colour Index (CI) azoic diazo component 112, benzidine base, and Fast
Corinth Base B (Christensen and Luginbyhl, 1974). The numbering system
commonly used in describing benzidine and its derivatives is indicated in
the following diagram:
2.2.1.1 Physical Properties — Benzidine is a colorless, crystalline com-
pound which has a molecular weight of 184.23, a density of 1.250 (20/4°C),
a melting point of 115°C to 120°C (slow heating) or 128°C (rapid heating),
and a boiling point of about 400°C at 740 torr (Stecher, 1968). It is
soluble in both alcohol and ether but only slightly soluble in water (0.52
mg/ml at 25°C) (Bowman, King, and Holder, 1976). Benzidine forms color-
less, crystalline salts with hydrochloric acid (Ci2Hi2N2»2HC1) and sulfu-
ric acid (Ci2Hi2N2»H2SOi.); the former is soluble in water (61.7 mg/ml at
25°C) (Bowman, King, and Holder, 1976) and alcohol, but the latter is only
slightly soluble in those solvents.
2.2.1.2 Chemical Properties — Benzidine is both a primary aromatic amine
and a diphenyl derivative; it undergoes chemical reactions characteristic
of each of these classes of compounds as well as some which are peculiar
to it alone. Although weakly basic (pK-^ = 12.13), benzidine forms stable
salts with mineral acids (Karrer, 1950, p. 453); it also forms complexes
with a large number of metallic salts (Welcher, 1947, p. 277). Like other
primary and secondary amines, benzidine is sensitive to oxidation, and
pure colorless crystals darken with decomposition products during storage
unless protected from atmospheric oxygen (Fieser and Fieser, 1956, p. 604).
Benzidine is easily acetylated,
NHCOCHj
NHCOCH,
(CH3CO)20
(CH3CO)20
NH2
BENZIDINE
4-ACETAMIDO-
4'-AMINOBIPHENYL
NHCOCHj
4,4'-DIACETAMIDOBIPHENYL
-------
alkylated,
17
NHCH,
CH3I
NaOH
CH3I
NaOH
NH2 NHCH3 N(CH3)2
BENZIDINE N,N'-OIMETHYLBENZIDINE N,N,N',N'-
TETRAMETHYLBENZIDI NE
and diazotized (International Agency for Research on Cancer, 1972, p. 80),
NH,
BENZIDINE
MONO
HCI
2CI
BENZIDINE DIAZONIUM CHLORIDE
The latter compound, tetrazotized benzidine, couples with many different
aromatic amines and phenols to yield a wide variety of commercial dyes
which can be used to color cotton fabrics without the use of mordants.
When the amino groups of benzidine are protected by acetylation,
benzidine is also easily nitrated,
NHCOCH,
HN03
NHCOCHj
4, 4'-DIACETAMIDOBIPHENYL
NHCOCH,
NO,
NHCOCHj
3 ,3'-DINITRO-
4,4'-DIACETAMIDOBIPHENYL
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18
and halogenated,
NHCOCH,
CI2
NHCOCHj
4,4'- DIACETAMIOOBIPHENYL
NHCOCH,
NHCOCH,
3,3 -DICHLORO-
4, 4'-DIACETAMIDOBIPHENYL
On treatment with sulfuric acid, benzidine forms a variety of sulfonic
acids, benzidine sulfone, and sulfonic acids of the sulfone, depending on
the concentration and amount of acid and the reaction temperature. Other
benzidine reactions and reaction products have been reported in the litera-
ture (Lurie, 1964, p. 408); however, they are mainly of academic interest.
2.2.1.3 Occurrence, Synthesis, and Use — Benzidine is a synthetic sub-
stance not known to occur in nature, although it can occur in waste streams
of facilities where it is produced or used. Benzidine can also be formed
in situ by the introduction of benzidine-based azo dyes into waste streams
containing hydrogen sulfide or sulfur dioxide (Takemura, Akiyama, and
Nakajima, 1965); the latter compounds reduce the azo linkage of the dye,
liberating the aromatic amine.
Benzidine is manufactured on a commercial scale by treating an alkaline
solution of nitrobenzene with a mild reducing agent — usually zinc dust.
The resulting solution contains a mixture of stepwise reduction products,
azoxybenzene and azobenzene, but it consists primarily of the intermediate
product, hydrazobenzene:
NITROBENZENE AZOXYBENZENE
01 fO
\/
AZOBENZENE HYDRAZOBENZENE
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19
The addition of mineral acids to the hydrazobenzene solution causes
spontaneous rearrangement of this compound into the desired product,
benzidine:
ACID
HYDRAZOBENZENE BENZIDINE
DIPHENYLINE
This reaction is the well-known benzidine rearrangement (Karrer, 1950, p.
498). A small amount, usually 3% to 15%, of the o,p'-diaminobiphenyl
(diphenyline) isomer is usually formed. Nitrobenzene also can be reduced
with zinc amalgam, iron powder, noble metal or nickel catalysts, or electro-
lytically (Lurie, 1964, p. 411).
The principal use of benzidine and its salts is in the manufacture of
dyestuffs based on the coupling of tetrazotized benzidine (Section 2.2.1.2)
with phenols and aromatic amines. More than 250 commercial dyes based on
benzidine have been reported (ColouT Index, 1956); the most important of
these are listed in Table 2.1. The composition and color of several impor-
tant benzidine-based cotton dyes are listed in Table 2.2. The symbol -> in
Table 2.2 stands for "diazotized and coupled with" and is a shorthand des-
ignation commonly used by dye chemists. The chemical structure of Congo
Red, one of the more important dyes in Table 2.2, is shown below (Johnson,
Zenhauser, and Zollinger, 1963):
H—NH
-------
20
TABLE 2.1. COMMERCIAL DYES DERIVED FROM BENZIDINE
Colour Index name
Mordant Yellow 36
Pigment Red 39
Direct Red 28
Direct Orange 8
Direct Red 10
Direct Red 13
Direct Red 37
Direct Red 1
Direct Brown 2
Direct Orange 1
Direct Violet 1
Direct Blue 2
Co}°ur Colour Index name
Index No .
14135
21080
22120
22130
22145
22155
22240
22310
22311
22370
22375
22430
22570
22590
Direct Blue 6
Direct Brown 1
Direct Brown 1A
Direct Brown 154
Direct Brown 6
Direct Brown 95
Direct Black 38
Direct Black 4
Direct Green 1
Direct Green 6
Direct Green 8
Direct Brown 31
Direct Brown 74
Colour
Index No .
22610
30045
30110
30120
30140
30145
30235
30245
30280
30295
30315
35660
36300
Source: Adapted from U.S. Tariff Commission, 1963, cited in
Lurie, 1964, Table 1, p. 413. Reprinted by permission of the publisher.
TABLE 2.2. COTTON DYES DERIVED FROM BENZIDINE
Composition
Color
Salicylic acid -«- benzidine -> naphthionic acid Orange
Naphthionic acid •«- benzidine -> naphthionic acid Congo Red
H acid •«- benzidine -> H acid Blue
Salicylic acid «- benzidine -> gamma acid Brown
Salicylic acid •*- benzidine ->• H acid -*- p-nitroaniline Green
m-Phenylenediamine •«-> benzidine -»• H acid -*- aniline Black
Source: Adapted from Lurie, 1964, Table 2, p. 414. Reprinted
by permission of the publisher.
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21
Although the manufacture of dyestuffs accounts for the major portion
of the benzidine consumed in the United States, there are many other uses
for this compound. Benzidine is employed in analytical chemistry to detect
and determine inorganic ions such as cadmium, copper, manganese, chloride,
fluoride, cyanide, ferricyanide, ferrocyanide, phosphate, silica, sulfate,
tungstate, hypohalites, permanganate, nitrate, nitrite, and phosphomolybdate
(Welcher, 1947). Benzidine is used as a chromogenic spray reagent in the
thin-layer chromatography of chlorinated organic pesticides (Adamovic, 1966).
It is used as a reagent for determining hydrogen peroxide, nicotine, sugars,
occult blood, and bacterial cytochromes. Benzidine is sometimes used in
security printing because it reacts with ink erasers to give colored prod-
ucts (International Agency for Research on Cancer, 1972, p. 81).
2.2.1.4 Biochemical Reactions and Metabolites — The long-held suspicion
that benzidine is a bladder carcinogen (Hueper and Conway, 1964, p. 268)
was established statistically by Case et al. (1954) and was subsequently
verified by experiments with rats, dogs, hamsters, and mice (Federal Reg-
-ister, 1974). Benzidine is also regarded as mutagenic (Ames et al., 1973).
The specific biochemical reactions responsible for these physiological
effects are not yet known (Miller and Miller, 1971a, p. 84); some doubt
even remains about the identity of certain metabolites (Haley, 1975).
Nevertheless, an appreciable store of metabolic chemistry exists, and the
patterns of biotransformations for aromatic amines pointed out by Williams
(1959, p. 428) probably are generally applicable to benzidine. These char-
acteristic reactions will be used as the basis of the biochemistry discussed
below. However, the reader is cautioned that most reactions described in
this section remain largely unsubstantiated in the literature since very
little work has been done to characterize biochemical reactions of benzidine.
Unlike their aliphatic analogues, aromatic amines are not deaminized
in vivo; instead, the amino group is conjugated and the aromatic ring is
hydroxylated. Conjugates of aromatic amines are more polar and less lipid
soluble than their precursors and consequently are more readily excreted in
the urine (Parke, 1968, p. 4). When a molecule, such as benzidine, has more
than one functional group, only one group is usually conjugated. Double
conjugates are formed in multifunctional substrates only if conjugation of
a single functional group does not increase the polarity of the molecule
sufficiently to promote rapid excretion. The most common conjugation of
the amino group is acetylation (Section 2.2.1.2), a reaction in which the
amino group is converted to an acetamido group:
NH2 NHCOCH3 NHCOCH,
ACETYL COENZYME A
ARYL AMINE ACETYL
TRANSFERASE
ACETYL COENZYME A
ARYL AMINE ACETYL
TRANSFERASE
BENZIDINE 4-ACETAMIOO- 4. 4'-DIACETAMIDOBIPHENYL
4'-AMINOBIPHENYL
-------
22
Acetylation of benzidine is thought to require the cofactor acetyl coenzyme
A as well as an enzyme, aryl amine acetyl transferase.
The aromatic amino group also conjugates with glucuronic and sulfuric
acids to form N-glucuronides,
GLUCURONIC ACID
BENZIDINE
NH2
BENZIDINE N-GLUCURONIDE
NHC6H906
BENZIDINE
N,N'-DIGLUCURONIDE
and N-hydrogen sulfates,
NH-0-S03H
NH-0-S03H
ARYL AMINE
SULFOKINASE
PAPS
ARYL AMINE
SULFOKINASE
»-
PAPS
NH2 NH2 NH-0-S03H
BENZIDINE BENZIDINE BENZIDINE
N-HYDROGEN SULFATE N, N-DI (HYDROGEN SULFATE)
Conjugation with glucuronic acid, the most important conjugation mechanism,
occurs in all mammals and most vertebrates except fish (Parke, 1968, p. 77).
Aryl amine glucuronides form spontaneously from glucuronic acid and aryl
amine, but glucuronides of phenols, alcohols, and carboxylic acids (0-
glucuronides) are formed only with the aid of appropriate enzymes. Simi-
larly, formation of the benzidine hydrogen sulfates requires the adenosine
coenzyme 3'-phosphoadenosine-5'-phosphosulfate (PAPS) and the appropriate
aryl amine sulfokinase.
The second type of biochemical behavior of aromatic amines, hydroxyla-
tion of the ring, occurs extensively unless there is an alternative meta-
bolic reaction involving another substituent in the ring. Usually, the
-------
23
hydroxyl group enters OTtho or para to the amino groups, depending on the
presence of other substituents. Benzidine hydroxylation occurs ovtho to
the amino group since the para position is blocked in the parent compound
(Sciarini and Meigs, 1961a):
MICROSOMAL ARYL
HYDROXYLASE
^.
NADPH2
°2
MICROSOMAL ARYL
HYDROXYLASE
NADPH2
°2
BENZIDINE 3-HYDROXYBENZIDINE 3,3'-DIHYDROXYBENZIDINE
A hepatic microsomal enzyme, oxygen, and reduced nicotinamide adenine
dinucleotide phosphate (NADPH2) are required for the hydroxylation reaction.
At least two distinct mechanisms can operate; the above reaction appears to
account for the reported metabolites of benzidine (Haley, 1975; Parke, 1968,
p. 40).
The hydroxybenzidines shown in the previous reactions may be excreted
.changed; however, they are normally conjugated with glucuronic acid,
un
URIDINE DIPHOSPHATE COENZYME
URIOINE DIPHOSPHATE
TRANSGLUCURONYLASE
NH2
3-HYDROXYBENZIDINE
OCgHgOg
NH2
3-GLUCURONYLBENZIDINE
-------
24
NH2
NH2
OC6H906
OC6H906
NH2
NH2
3,3'-DIHYDROXYBENZIDINE 3,3'-DIGLUCURONYLBENZIDINE
3-HYDROXY-3'-GLUCURONYLBENZIDINE
or sulfuric acid,
NH2
OH
SULFOTRANSFERASE
^-
PAPS
NH2
0-S03H
NH2 NH2
3-HYDROXYBENZIDINE BENZIDINE 3-HYDROGEN SULFATE
NH2
NH2
0-SOjH
0-S03H
I I
NH2
3,3-DIHYDROXYBENZIDINE BENZIDINE 3,3'-DI (HYDROGEN SULFATE)
BENZIDINE 3-HYDROXY-3'-HYDROGEN SULFATE
-------
25
Conjugation of the hydroxyl groups of hydroxybenzidine with glucuronic
acid is not spontaneous as was the previously described reaction of the
amino groups in benzidine. Uridine diphosphate coenzyme and uridine diphos-
phate transglucuronylase are required. An enzyme is also required for forma-
tion of the sulfate esters. These compounds and the N-hydrogen sulfates
mentioned previously are often identified in the literature as etheral sul-
fates because of their solubility in, and easy extraction into, ether
solvents.
In 1960, an in vivo biochemical reaction was discovered in which aro-
matic amines are oxidized to hydroxylamines (Cramer, Miller, and Miller,
1960). This process, called N-hydroxylation, occurs widely (Hathway, 1970,
p. 271) and is now considered by some investigators to be the first step in
the activation of aromatic amines and amides for carcinogenesis (Miller and
Miller, 1971a):
NHOH
OJ 10
MICROSOMAL ENZYME
NADPH2
02
BENZIDINE
NH2
N-HYDROXYBENZIDINE
The N-hydroxylating system requires a microsomal enzyme, NADPH2, and
oxygen; the necessary enzyme occurs in the liver, lungs, and bladder (Parke,
1968, p. 47).
Another type of metabolic process, different from those discussed
above, is the in vivo chemical reaction by which benzidine is produced from
a previously ingested source material such as an azo dye. It has long been
known that humans and other mammals can metabolically reduce certain dyes
to free carcinogenic aromatic amines (Fuller, 1937; Williams, 1949, pp.
150-154). Rinde and Troll (1975) established that metabolic reduction of
benzidine-based azo dyes can occur in the monkey. They found substantial
amounts of benzidine and monoacetylbenzidine in the urine of animals which
were fed single doses of benzidine-based dyes (Direct Red 28, Direct Blue 6,
Direct Black 38, and Direct Brown 95) dissolved in dimethyl sulfoxide. In
view of their findings, these investigators recommended restricting the pro-
duction of azo dyes to those which could be manufactured from noncarcino-
genic aromatic amines, such as aniline. Discussions of metabolic transfor-
mations in general and of aromatic amines in particular may be found in
Arrhenius (1974), Hathway (1970), Parke (1968), and Walker (1970).
-------
26
The simple metabolic products indicated in the various biochemical
reactions described above are not necessarily the compounds excreted when
benzidine is ingested by the host; successive operations on a metabolite by
different biochemical processes may result in a complex end product.
Furthermore, use of a particular biotransformation reaction varies among
animal species and with experimental conditions. It is necessary, therefore,
to identify the metabolites of each animal species experimentally.
2.2.1.5 Chemical Structure Associated with Carcinogenic Activity of
Aromatic Amines — Chemicals were first used to produce experimental tumors
more than 50 years ago (Yamagiwa and Ichikawa, 1918). Since then, carcino-
genic activity has been demonstrated for a wide variety of chemical com-
pounds (Clayson, 1962; Hueper and Conway, 1964; International Agency for
Research on Cancer, 1972, 1973as 1973&, 1974a, 19743). These compounds
are structurally diverse and have no common metabolic patterns (Miller and
Miller, 1966); no general explanation for their carcinogenic activity is
available. Some advances have been made, however, in understanding the
carcinogenic activity of certain classes of compounds. The following dis-
cussion deals with aromatic amines — the class to which benzidine belongs.
Not all aromatic amines are potent carcinogens; to qualify, the amino
group apparently must be attached to an aromatic system containing more
than one ring. Furthermore, the point of attachment of the amino group to
the ring must be such that the position para to the amino group is occupied
by another substituent or by part of the conjugated system (Clayson, 1962,
p. 211). Unlike carcinogenic hydrocarbons, carcinogenic aromatic amines
generally induce tumors at tissue sites distant from the point of administra-
tion. This behavior suggests that aromatic amines are not carcinogenic per
se but acquire this characteristic only after metabolic activation.
Since hydroxylation of the ring occurs extensively in these compounds
(Section 2.2.1.4), hydroxylated derivatives were among the first metabolites
suspected as carcinogens (Hueper, 1938). In addition, since the amino group
is an ortho-para director and since the para position in carcinogenic
aromatic amines is usually blocked, hydroxylation of these compounds char-
acteristically occurs in the ortho position. Early workers thus developed
the ort/zo-hydroxylation hypothesis (Clayson, 1962, p. 219) as a guide to
identifying potentially carcinogenic aromatic amines. Acceptance of the
theory diminished, however, when some o-hydroxy amines failed to produce
tumors and others failed to produce neoplasms when administered by a
different route (Miller and Miller, 1966).
Although strong evidence exists for the carcinogenicity in mice of
o-hydroxyanthranilic acid (Ehrhart, Georgii, and Stanislawski, 1959) and
some o-hydroxyamine metabolites of tryptophan (Bryan, Brown, and Price,
1964), the ort/zo-hydroxylation hypothesis was largely displaced in 1960 by
the discovery of the in vivo reaction of N-hydroxylation (Section 2.2.1.4)
in which the nitrogen of aromatic amines is oxidized enzymatically to
hydroxylamine (Cramer, Miller, and Miller, 1960). Troll, Belman, and Rinde
(1963) established that this metabolite of benzidine occurs in humans.
N-Hydroxylation of aromatic amines and amides occurs widely and N-hydroxy
metabolites of carcinogenic aromatic amines are at least as active as the
-------
27
parent carcinogen (Boyland, Dukes, and Grover, 1963; Miller, Enomoto, and
Miller, 1962; Miller, Miller, and Enomoto, 1964; Poirier et al., 1965).
Also, unlike the o-hydroxy compounds, the N-hydroxy metabolites can induce
tumors at sites where the parent compounds are inactive (Miller and Miller,
1966). As a result of these studies, N-hydroxylation is now widely recog-
nized as the first step in the activation of aromatic amines and amides for
carcinogenesis (Miller and Miller, 1971a).
Although carcinogenic, N-hydroxy aromatic amines lack the electrophilic
properties of ultimate carcinogens which enable the latter to bind to
nucleophilic sites of proteins and nucleic acids at physiological pH (Miller,
1970). This deficiency indicates that N-hydroxy aromatic amines are prox-
imate rather than ultimate carcinogens. The chemical reactivity of esters
of aromatic hydroxylamines and hydroxamic acids suggests that esterification
(Section 2.2.1.4) of N-hydroxylated metabolites is the next step in the
metabolic sequence leading to the ultimate carcinogen (Miller and Miller,
1972). No experimental work establishing this chemical sequence for the
metabolism of benzidine has been performed; however, such experimental
evidence has been obtained for other aromatic amines, namely, 2-acetylamino-
fluorene (AAF) and N-methyl-4-aminoazobenzene (MAB). Miller and Miller
(1972) reported that after treating male rats with AAF, the major reactive
metabolites in the liver were sulfuric acid esters which, when desulfatized,
yielded an aminonium cation that reacted readily with nucleophilic tissue
components such as proteins, RNA, and DNA (Miller and Miller, 1971a). The
principal steps leading to the formation of the reactive sulfates and the
aminonium cations of AAF and MAB are shown in Figure 2.1.
ORNL-DWG 77-13499
MAB
=\../CH3
I
\ VM \
lO-Cp-R, 0-£-»0, 0-S-*0, xl-p-D-GLUCURONYL
L 0 OH J
T=S /COCHjICHjl
Figure 2.1. Formation of reactive esters and aminonium cations from
2-acetylaminof luorene (AAF) and N-methyl-4-aminoazobenzene (MAB). Source:
Miller and Miller, 1969a, Figure 5, p. 297. Reprinted by permission of
S. Karger AG, Basel.
A variety of amino acid residues in proteins and bases in nucleic acids
are attacked by electrophilic reactants derived from aromatic amine carcino-
gens. Among nucleic acids and proteins, the principal targets appear to be
-------
28
guanine, phosphate, methionine, cysteine, tyrosine, and histidine (Miller
and Miller, 1969Z?). The manner in which two of these, methionine and
(deoxy)guanosine, react in vitro with N-acetoxy-AAF is shown in Figures 2.2
and 2.3. In each instance, identical metabolites are also obtained from
the liver proteins or hepatic UNA of rats fed AAF. As shown in Figure 2.2,
the methionine adducts of N-acetoxy-AAF decompose readily to form 1- and 3-
methylmercapto-AAF, a process which can result in cleavage of methionyl
peptide bonds and in modification of the reacted protein (Miller and Miller,
1969Z?). Such reactions could have significant consequences in terms of
enzymatic activity of alterations in substrate specificity. The reaction
of N-esters of AAF with guanine residues, the major site of attack in
nucleic acids (Miller and Miller, 197l£>), has obvious implications relative
to both genetic and epigenetic mechanisms of chemical carcinogenesis. The
alteration of guanine bases in DNA by insertion of bulky AAF groups in the
8-position could impede the progress of this enzyme along the double helix
and lead to deletion mutants. Indeed, deletion mutants are the principal
type observed when Drosophila is treated with N-acetoxy-AAF (Fahmy and
Fahmy, 1967). However, single base pair changes occur predominantly when
Bacillus subt-il-is is treated with the same mutagen (Miller and Miller, 1969&)
From the foregoing discussion it is apparent that considerable prog-
ress has been made in recent years in identifying proximate and ultimate
aromatic amine carcinogens and the mechanisms by which they are formed.
ORNL-DWG 77-12898
CH>:
,COO\ =\ ,COCH3
>J^>
=\ ,COCH, ^.-^ ,COCH,
^N
N-ACETOXY-AAF
—'NH-CH2COOH
NH2
N-HYDROXY-AAF
+ METHIONINE- S
--1C—> HEPATIC PROTEIN-BOUND 'AAF'
L- OJC
Figure 2.2. The in vitro reaction of N-acetoxy-AAF and methionylglycine.
Source: Miller and Miller, 1969&, Figure 3, p. 244. Reprinted by permission
of the publisher.
-------
29
ORNL-DWG 77-12900
=\ /COCH3-citjCOO"
* " /X0 COCHIN—
N-ACETOXY-AAF
N-(GUANIN-8-YL)-AF
GUANOSINE
N-lGUANOSIN-e-YD-AF
C-DERIVATIVE B
-CHjCOO
OH"
\IDFNTir.AI '
coCHj
HO OH
N-(GUANOSIN-8-YL)-AAF
14
C-DERIVATIVE A
PHOSPHOMONO- AND Dl-
ESTERASES
14 R AT
N-HYDROXY-AAF-9- C > HEPATIC RNA-BOUND
Figure 2.3. The in vivo reaction of N-acetoxy-AAF with guanosine.
Source: Miller and Miller, 19692?, Figure 4, p. 244. Reprinted by permission
of the publisher.
Evidence is also accumulating relative to the nucleophilic targets of
these compounds; however, there is no definitive information concerning
the cellular chemistry of the carcinogenic process itself — no understand-
ing of the carcinogenic mechanism in molecular terms. Future work must
address this difficult aspect of the problem. Commonly postulated mecha-
nisms of chemical carcinogenesis are outlined in Table 2.3. Chemical
structures associated with the carcinogenic activity of aromatic amines
are discussed further by Clayson (1975), Conney and Levin (1974), and
Heidelberger (1970).
2.2.2 3,3'-Dichlorobenzidine and Salts
3,3"-Dichlorobenzidine is a widely used chemical intermediate which
has been produced commercially for more than 35 years (U.S. Tariff Com-
mission, 1938, cited in International Agency for Research on Cancer,
1974cz). Its composition and structure are indicated by the formulas
C12H10C12N2 and
ci
-------
30
TABLE 2.3. POSSIBLE MECHANISMS OF CHEMICAL CARCINOGENESIS
I. Genetic mechanisms — heritable changes in DNA genome
via:
1. Direct modification of existing DNA
2. Modification of RNA which is subsequently tran-
scribed into DNA that becomes integrated into host
DNA
3. Alterations which decrease, at least temporarily,
the fidelity of copying of DNA
II. Epigenetic mechanisms — nongenomic changes leading to:
1. Quasi-permanent changes in the transcription of DNA
(including integrated virus genomes and oncogenes)
2. The preferential proliferation of previously
existing preneoplastic or neoplastic cells
Source: Miller and Miller, 197L&, Table 1, p. x.
The Chem-Loal Abstracts identification number of 3,3'-dichlorobenzidine is
91-94-1. It is also known as CI 23060, DCB, 4,4'-diamino-3,3'-dichloro-
biphenyl, o,o'-dichlorobenzidine, dichlorobenzidine, dichlorobenzidine base,
3,3'-dichloro-4,4'-biphenyldiamine, and 3,3'-dichlorobiphenyl-4,4'-diamine.
2.2.2.1 Physical and Chemical Properties — 3,3'-Dichlorobenzidine is a
colorless, crystalline compound which has a molecular weight of 253.1 and
a melting point of 132°C to 133°C, It is nearly insoluble in cold water,
slightly soluble in dilute hydrochloric acid, and readily soluble in etha-
nol, benzene, and glacial acetic acid (International Agency for Research
on Cancer, 1974a). 3,3'-Dichlorobenzidine forms salts with hydrochloric
acid. The principal physical properties of the dihydrochloride salt are
shown in Table 2.4.
The chemistry of 3,3'-dichlorobenzidine is similar to that of aromatic
primary amines in general and benzidine in particular (Section 2.2.1.2).
Its most important chemical reaction is the formation of the tetrazonium
compound which couples with aromatic amines and phenols to form a variety
of dyes and pigments. In general, the chlorine atoms of 3,3'-dichloro-
benzidine are passive and do not participate in these reactions (Gerarde
and Gerarde, 1974; Karrer, 1950, p. 414).
2-2.2.2 Occurrence, Synthesis, and Use - 3,3'Dichlorobenzidine is not
known to occur naturally, although it may be present in the waste streams
from production facilities where it is manufactured or used (International
Agency for Research on Cancer, 1974a). There is also evidence that 3,3'-
dichlorobenzidine is regenerated metabolically in rabbits and humans from
an organic pigment (benzidine yellow) in which it is used as a coupling
agent (Akiyama, 1970).
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31
TABLE 2.4. PHYSICAL PROPERTIES OF
3,3'-DICHLOROBENZIDINE DIHYDROCHLORIDE
Formula
Molecular weight
Flash point (Cleveland open cup)
Auto and ignition temperature
Corrosiveness
Physical state
Solubility
Odor
Vapor pressure
Particle size
C12H10C12N2-2HC1
326.1
No flammable concentration of
vapors evolved at 200°C
No exothermic reaction recorded
up to 300°C
Contains free mineral acidity
or hydrochloric acid
Light grayish semidry powder
(approx 5% water); white to
light gray semidry paste
Slightly soluble in water and
organic solvents; soluble in
acids
Slightly pungent
Zero at room temperature
Salt crystals; similar to table
salt
Source: Adapted from Gerarde and Gerarde, 1974, Table 2,
p. 324. Reprinted by permission of the publisher.
3,3'-Dichlorobenzidine is synthesized on a commercial scale by chem-
ical reactions similar to those used for benzidine, except that the start-
ing material is o-nitrochlorobenzene rather than nitrobenzene:
NaOH
Zn DUST
Cl
c-NITROCHLOROBENZENE 2,2'-DICHLOROHYDRAZOBENZENE
-------
32
On treatment with sulfuric or hydrochloric acid, the intermediate 2,2'-
dichlorohydrazobenzene undergoes the benzidine rearrangement (Karrer, 1950,
p. 409) to form the 3,3'-dichlorobenzidine salt:
2, 2'-DICHLOROHYDRAZOBENZENE 3 ,3'-DICHLOROBENZI DINE
DIHYDROCHLORIDE
As with benzidine, the principal use of 3,3'-dichlorobenzidine is in
the manufacture of dyestuffs based on the coupling of the tetrazotized com-
pound with phenols and aromatic amines; 13 such dyes are described in the
third edition of the Colour Index (1971). The 1971 production rates for
six of these dyes are listed in Table 2.5. These dichlorobenzidine com-
pounds are used chiefly as pigments in plastic resins, lacquer, rubber,
printing inks, metal finishes, and textile and wallpaper prints (Colour
Index, 1971) and in interior grade, lead-free finishes (paints), toy
enamels, and floor coverings (Martens, 1968, p. 367).
Dichlorobenzidine is also used as a curing agent for liquid-castable
polyurethane elastomers (Woolrich and Rye, 1969). However, compared to
dye production, this use of dichlorobenzidine is relatively minor; it
currently amounts to only a few hundred thousand kilograms per year in the
United States (International Agency for Research on Cancer, 1974a).
TABLE 2.5. SOME DYES OR PIGMENTS MANUFACTURED
FROM 3,3'-DICHLOROBENZIDINE IN 1971
Colour
Index No .
21090
21095
21105
21110
21115
21120
Dye or pigment
Pigment Yellow 12
Pigment Yellow 14
Pigment Yellow 17
Pigment Orange 13
Pigment Orange 34
Pigment Red 38
Production
(103 kg)
2547
1034
260
65
55
60
Source: Adapted from U.S. Tariff Commission,
1972, cited in International Agency for Research
on Cancer, 1974a, p. 51.
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33
2.2.2.3 Biochemical Reactions and Metabolites — On a purely chemical
basis, the biochemistry of 3 ,3'-dichlorobenzidine could be expected to
differ appreciably from that of its parent compound, benzidine. First,
loss of halogens from the benzene ring apparently does not occur in vivo
(Gerarde and Gerarde, 1974); thus, there is little probability that the
chlorinated compound could form the 3-hydroxy derivatives characteristic of
benzidine metabolites. Secondly, with the 1, 1', 3, and 3' positions in
the chlorinated compound already blocked, the ortho-para directing influ-
ence of the amino groups should cause any additional substitutions to occur
at the 5 or 5' positions. Such substitutions would result in metabolites
appreciably different from those of benzidine.
The metabolism of 3,3'-dichlorobenzidine differs among species (Sciarini
and Meigs , 1961£>). In most investigations the excreted chlorinated compound
appeared unchanged from its administered form. This apparent inertness of
the chloro derivative may reflect the deactivating influence of halogen sub-
stituents on electrophilic aromatic substitution (Morrison and Boyd, 1974,
p. 342).
2.2.3 3,3'-Dimethylbenzidine and Salts
3, 3'-Dimethylbenzidine is a versatile, widely used dye intermediate;
its elemental and structural composition is indicated by the formulas
Cii.HisNz and
NH
NH,
The Chemical Abstracts identification number of 3,3'-dimethylbenzidine is
119937- It is also known as diorthotolidine, 4,4'-diamino-3,3'-dimethyl-
biphenyl, 4,4'-diamino-3,3'-dimethyldiphenyl,3,3'-dimethy1-4,4'-biphenyl-
diamine, 3,3'-dimethyl-4,4'-diphenyldiamine, 3,3'-dimethylbiphenyl-4,4'-
diamine, 3,3'-dimethyldiphenyl-4,4'-diamine, 3 ,3'-tolidine, 4,4'-bi-o-
toluidine, 4,4'-di-<9-toluidine, diaminoditolyl, Fast Dark Blue Base R, and
CI Azoic Diazo Component 113.
2.2.3.1 Physical and Chemical Properties — 3,3'Dimethylbenzidine is a
white to reddish crystalline compound which has a molecular weight of
212.28 and a melting point of 131°C to 132°C. It is slightly soluble in
water (1.3 mg/ml at 25°C) (Bowman, King, and Holder, 1976) and very soluble
in ethanol, ethyl ether, and acetone (International Agency for Research on
Cancer, 1972). 3,3'-DimethyIbenzidine is a weak base which readily forms
-------
34
salts with strong mineral acids such as hydrochloric and sulfuric. The
monohydrochloride salt is soluble to the extent of 1 part in 112.4 parts
of water at 12°C; the dihydrochloride, 1 part in 17.3 parts. The monosul-
fate salt is moderately soluble; the disulfate is difficultly soluble in
water. The monohydrated, monacetyl derivative of 3,3'-dimethylbenzidine
melts at 103°C; the diacetyl and tetraacetyl compounds at 315°C and 211 C
respectively. 3,3'-Dimethylbenzidine forms an intense blue color with chlo-
rine or bromine vapor (Lurie, 1964, p. 414). Like benzidine, its parent
compound, it can also be hydroxylated, tetrazotized, and acetylated
(Section 2.2.1.2).
2.2.3.2 Occurrence, Synthesis, and Use - 3,3'-Dimethylbenzidine is a chem-
ical intermediate used in the preparation of more than 95 dyes listed in
the Colour Index (International Agency for Research on Cancer, 1972, p. 88).
Those manufactured in the United States during 1962 are listed in Table 2.6.
3,3'-Dimethylbenzidine is prepared in the manner described for benzi-
dine (Section 2.2.1.3) except that o-nitrotoluene, rather than nitrobenzene,
is the starting material (Lurie, 1964, p. 414). In addition to its use in
the dye industry, 3,3'-dimethylbenzidine is also used extensively in quali-
tative and quantitative analyses for the detection and determination of
cobalt, copper, gold, lead, manganese, mercury, silver, tungsten, iodide,
nitrate, nitrite, and sulfate (Welcher, 1947, p. 462).
2.2.3.3 Biochemical Reactions and Metabolites — Little attention has been
given thus far to the metabolic chemistry of 3,3'-dimethylbenzidine. In
limited studies of dogs and humans, only the expected metabolic processes
such as ortho hydroxylation, sulfation, and acetylation have been observed.
TABLE 2.6. COMMERCIAL DYES
DERIVED FROM
3,3'-DIMETHYLBENZIDINE
Colour Index Colour
name Index No.
Direct Red 2 28500
Direct Red 39 23630
Acid Red 114 23635
Direct Blue 25 23790
Direct-Blue 14 23850
Direct Blue 26 31930
Azoic Coupling
Component 5 37610
Source: Adapted from
U.S. Tariff Commission, 1963,
cited in Lurie, 1964, Table 3,
p. 415. Reprinted by permis-
sion of the publisher.
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35
Since aliphatic side chains attached to a benzene ring are quite sus-
ceptible to oxidation in vitro (Morrison and Boyd, 1974, p. 384), Dieteren
(1966) suggested that 3,3'-benzidinedicarboxylic acid may be a metabolic
product of 3,3'-dimethylbenzidine:
NH2
NH2
NH2
COOH
NH2
3, 3'-BENZIDINEDICARBOXYLIC ACID
3-METHYL-3-BENZIDINECARBOXYLIC ACID
However, no experimental evidence exists to support this suggestion.
2.2.4 3, 3'-Dimethoxybenzidine and Salts
3,3'-Dimethoxybenzidine is a well-established commercial dye inter-
mediate whose composition and structure are represented by the formulas
Cii.H16N202 and
NH2
OCH,
OCH,
NH2
The Chemical Abstracts number of 3,3'-dimethoxybenzidine is 119-90-4. The
compound is also known as bianisidine, 4,4'-diamino-3,3'-dimethoxybenzidine,
di-p-amino-di-m-methoxydiphenyl, dianisidine, and 3,3'-dimethoxy-4,4'-
diaminobiphenyl.
2.2.4.1 Physical and Chemical Properties — 3,3'-Dimethoxybenzidine is a
colorless, crystalline compound which turns violet on standing and has a
molecular weight of 244.3 and a melting point of 137°C to 138°C. The com-
pound is poorly soluble in water (0.06 mg/ml at 25°C) (Bowman, King, and
-------
36
Holder 1976), but it is soluble in ethanol, ethyl ether, acetone, benzene,
chloroform, and probably most organic solvents and lipids (International
Agency for Research on Cancer, 1974a, p. 41). 3,3'-Dimethoxybenzidine is
a weak base and forms salts with mineral acids; it has the chemical char-
acteristics of primary aromatic amines in general and of benzidme in
particular (Section 2.2.1.2).
2.2.4.2 Occurrence, Synthesis, and Use - 3,3'-Dimethoxybenzidine is an
important dye intermediate which has been produced commercially for at
least 50 years; it is not known to occur naturally, although it may some-
times be present in waste streams from facilities where it is manufactured
or used (International Agency for Research on Cancer, 1974a, p. 43).
3,3'-Dimethoxybenzidine is prepared in the manner described for benzi-
dine (Section 2.2.1.3) except that the methyl ether of o-nitrophenol is
used as the starting material instead of nitrobenzene. Current U.S.^pro-
duction data on 3,3'-dimethoxybenzidine are unavailable. In 1967, five
domestic producers manufactured 167,000 kg (U.S. Tariff Commission, 1968,
cited in International Agency for Research on Cancer, 1974a). By 1971,
only two domestic producers were reported; imports in that year totaled
124,000 kg (U.S. Tariff Commission, 1972, cited in International Agency
for Research on Cancer, 1974a).
3,3'-Dimethoxybenzidine is the coupling agent in 89 dyes listed in
the Colour Index (1971). Seven of these, for which 1971 U.S. production
data are available, are listed in Table 2.7. In addition, about 87,000 kg
of a 3,3'-dimethoxybenzidine-based pigment, Pigment Blue 25 (CI 21180),
was also manufactured in the United States in 1971 (U.S. Tariff Commission,
1972, cited in International Agency for Research on Cancer, 1974a). 3,3'-
Dimethoxybenzidine-based dyes and pigments are used primarily to color
leather, paper, plastics, rubber, and textiles.
TABLE 2.7. SOME DYES MANUFACTURED FROM
3,3'-DIMETHOXYBENZIDINE IN 1971
Colour
Index No .
24401
21160
24410
24400
24140
24411
23155
Dye
Direct Blue 218
Pigment Orange 16
Direct Blue 1
Direct Blue 15
Direct Blue 8
Direct Blue 76
Direct Blue 98
Production
(103 kg)
479
153
136
94
64
53
39
Source: Adapted from U.S. Tariff Commission,
1972, cited in International Agency for Research
on Cancer, 1974a, p. 43.
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37
Smaller quantities of 3,3'-dimethoxybenzidine are used in applications
unrelated to dyes or pigments. The most important of these is probably the
manufacture of 3,3'-dimethoxy-4,4'-diphenylene diisocyanate, an ingredient
in isocyanate-based adhesive systems and a component of polyurethane elas-
tomers. 3,3'-Dimethoxy-4,4'-diphenylene diisocyanate is prepared by dis-
solving the hydrochloride salt of 3,3'-dimethoxybenzidine in an aromatic
solvent and heating the resulting solution with phosgene (Woolrich and Rye,
1969) . The annual rate of production for this compound is not known but is
estimated to be less than 500,000 kg (International Agency for Research on
Cancer, 1974a, p. 43). 3,3'-Dimethoxybenzidine is also used in analytical
chemistry to detect and determine cobalt, copper, gold, vanadium, thio-
cyanate, and nitrite (Welcher, 1947, p. 342).
2.2.4.3 Biochemical Reactions and Metabolites — Laham (1971) fed the mono-
substituted compound 3-methoxybenzidine to rats and detected 3-methoxy-4-
amino-4'-acetamidobenzidine in freshly collected urine. In vivo acetylation
of this amine in the 4' rather than the 4 position indicates steric hin-
drance from the methoxy group in the 3 position.
2.2.5 4-Aminobiphenyl
4-Aminobiphenyl is a derivative of biphenyl rather than benzidine.
It is discussed here because of its structural and chemical similarities
to benzidine and its established carcinogenicity in animals and humans
(Federal Register, 1974). The elemental and structural composition of
4-aminobiphenyl is indicated by the formulas C12HnN and
The Chemical Abstracts identification number of 4-aminobiphenyl is 92671.
It is also known as 4-biphenylamine, p-aminobiphenyl, p-aminodiphenyl,
4-aminodiphenyl, p-biphenylamine, p-phenylaniline, and xenylamine.
2.2.5.1 Physical and Chemical Properties — 4-Aminobiphenyl is a colorless,
crystalline compound that darkens on oxidation. It has a molecular weight
of 169.2, a boiling point of 191°C (15 torr), and a melting point of 53°C
to 54°C (Weast, 1970). 4-Aminobiphenyl is very slightly soluble in cold
water and is soluble in hot water and nonpolar solvents and in lipids.
Like benzidine (Section 2.2.1.2), it forms salts with mineral acids, is
oxidized by air, and its amino group is diazotized, acetylated, and alkyl-
ated (International Agency for Research on Cancer, 1972).
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38
2.2.5.2 Occurrence, Synthesis, and Use — 4-Aminobiphenyl does not occur
naturally; however, it can form inadvertently as an impurity during the
synthesis of certain aromatic amines. In fact, 4-aminobiphenyl was first
isolated in 1862 from residues remaining after the distillation of crude
aniline (Hueper and Conway, 1964, p. 265). The formation of 4-amino-
biphenyl in aniline is probably due to the reduction of 4-nitrobiphenyl
produced by the nitration of diphenyl contained in impure benzene. Occur-
rence of 4-aminobiphenyl in aniline synthesized in modern plants has been
greatly minimized by improved distillation techniques (International Agency
for Research on Cancer, 1974a, p. 114).
4-Aminobiphenyl was produced on a large scale in the United States
from 1935 to 1955 for use as a rubber antioxidant. It was prepared by
nitrating diphenyl and treating the resulting intermediate with a mild
reducing agent:
HN03
H2S04
o
\/
DIPHENYL
REDUCING
AGENT
4-NITROBIPHENYL
4-AMINOBIPHENYL
Production of the compound in the United States is thought to have ceased
following the general recognition in 1955 of its carcinogenicity (Inter-
national Agency for Research on Cancer, 1972, p. 74). However, it still
is being manufactured, but on a smaller scale than formerly (Haley, 1977).
2.2.5.3 Biochemical Reactions and Metabolites — Studies seeking 4-amino-
biphenyl metabolites have been performed by several investigators. Miller
et al. (1961) observed that rats convert 4-diphenylacetamide to the
N-hydroxy derivative:
NHCOCH?
01 10
^^
-DIPHENYLYLACETAMIDE N-HYDROXY-4-DIPHENYLYLACETAMIDE
COCH3
-------
39
Rabbit liver microsomes also metabolize 4-acetamidobiphenyl in this manner
(Booth and Boyland, 1964). In dogs, 4-aminobiphenyl is metabolized to
4 amino-3-biphenylyl hydrogen sulfate (Bradshaw and Clayson, 1955),
4-AMINOBIPHENYL 4-AMINO-3-BIPHENYLYL HYDROGEN SULFATE
and 4-amino-3-biphenylyl glucuronic acid (Gorrod, 1971),
4-AMINOBIPHENYL 4-AMINO-3-BIPHENYLYL GLUCURONIC ACID
2.2.6 4-Nitrobiphenyl
4-Nitrobiphenyl is also a derivative of biphenyl rather than benzidine.
Like 4-aminobiphenyl, it has structural and chemical similarities to benzi-
dine and is an established carcinogen in dogs (Federal Register, 1974).
The composition and structure of 4-nitrobiphenyl are indicated by the for-
mulas Ci2H9N02 and
-------
40
The Chemical Abstracts identification number of 4-nitrobiphenyl is 92-93-3.
Other names for the compound are p-nitrobiphenyl, p-nitrodiphenyl, 4-nitro-
diphenyl, PNB, 4-phenyl-nitrobenzene, and p-phenyl-nitrobenzene.
2.2.6.1 Physical and Chemical Properties — 4-Nitrobiphenyl occurs as
white to yellow needles which have a molecular weight of 199.2, a boiling
point of 340°C, and a melting point of 114°C. It is almost insoluble in
water, slightly soluble in cold alcohol, and readily soluble in benzene,
chloroform, and ether (Weast, 1970). 4-Nitrobiphenyl is easily reduced
both in vitro and in vivo to 4-aminobiphenyl. Oyt/zo-hydroxylation occurs
on prolonged warming of 4-nitrobiphenyl with powdered potassium hydroxide
in benzene (International Agency for Research on Cancer, 1974cr).
2.2.6.2 Occurrence, Synthesis, and Use — 4-Nitrobiphenyl does not occur
naturally. Prior to 1900, it formed inadvertently during the commercial
preparation of nitrobenzene because of the presence of biphenyl in impure
benzene. This source of 4-nitrobiphenyl has apparently been eliminated by
improved distillation techniques which completely remove biphenyl from the
benzene feedstock (International Agency for Research on Cancer, 1974a).
Presently, there appears to be no large-scale industrial production of
4-nitrobiphenyl in the United States. The only known commercial application
of the compound is as a chemical intermediate in the preparation of 4-amino-
biphenyl (Section 2.2.5). When use of the latter material as a rubber anti-
oxidant ceased in 1955, the commercial production of 4-nitrobiphenyl also
stopped (Deichmann et al., 1958); however, small amounts are still manufac-
tured for research and pilot plant use. The method used to prepare the
4-nitrobiphenyl consumed between 1934 and 1955 has not been reported (Inter-
national Agency for Research on Cancer, 1974a). An obvious technique
involves the nitration of biphenyl:
o,
HN03
CH3OOH
o
\^-
BIPHENYL 4-NITROBIPHENYL
Synthesis and use of 4-nitrobiphenyl is prohibited in the United
Kingdom under the Carcinogenic Substances Regulations, 1967, Statutory
Instrument No. 487. The preparation and use of 4-nitrobiphenyl in the
United States is regulated by standards set by the Occupational Safety and
Health Administration (Federal Register, 1974).
-------
41
fate
Biochemical Reactions and Metabolites - Rats metabolically reduce
611!1 to.4~aminobiPheny1 and ^-aminobiphenyl-3-yl hydrogen sul-
o
^/
4-NITROBIPHENYL
OSO»H
4-AMINOBIPHENYL 4-AMINOBIPHENYL-3-YL
HYDROGEN SULFATE
A similar reduction of 4-nitrobiphenyl to 4-aminobiphenyl occurs in vitro
if rat liver enzymes, the necessary cofactors, and anaerobic reaction
conditions are present (Uehleke and Nestel, 1967).
2.2.7 Other Benzidine Congeners
In addition to the benzidine compounds discussed in the preceding
sections, many other substituted products are possible. Nitro, alkoxy,
sulfonic acid, and carboxylic acid derivatives are the most important.
Few, if any, carcinogenic or metabolic studies have been performed with
these materials. Their general characteristics are summarized in Table 2.8.
2.3 ANALYSIS FOR BENZIDINE
Although alarm over "aniline tumors" was expressed by Rehn as early as
1895, general concern over the carcinogenic role of benzidine and its con-
geners did not develop for nearly half a century (Spitz, Maguigan, and
Dobriner, 1950). Approved analytical methods for determining benzidine
were first established by the Association of British Chemical Manufacturers
in 1954 (Butt and Strafford, 1956). These colorimetric methods were used,
with innovations, for almost all of the quantitative analyses of benzidine
which were subsequently reported. Recently, the manufacture and use of
benzidine and 3,3'-dichlorobenzidine in this country became subject to the
regulations of the Williams-Steiger Occupational Safety and Health Act of
1970 as amended in the Federal Register, January 29, 1974. Analytical
techniques or instruments for environmental monitoring are not specified in
the adopted standards, but the administration requested the National
Institute for Occupational Safety and Health to develop qualitative and
quantitative methods for determining the listed carcinogens (Federal Reg-
ister, 1974). Approved methods for determining benzidine have not yet
been published, but an interim procedure (Section 2.3.2.3) was suggested
(Federal Register, 1975). In the absence of formally sanctioned analytical
procedures, the discussions which follow reflect the existing state of the
art as described in the current literature.
-------
TABLE 2.8. OTHER BENZIDINE CONGENERS
_ , Empirical Structural
Compound , . ,.
formula formula
2-Nitrobenzidine C12HuN302 NH,
N02
NH2
3-Nitrobenzidine C12H11N302 NH2
NH2
2,2'-Dinitrobenzidine C12H10N<,0,, NH2
(o^
^S/N02
NH,
Molecular Form and Melting Method of
weight color point (°C) Se preparation
229.2 Red needles 143 Dye intermediate Nitration of ben-
zidine sulfate
in sulfuric
acid
229.2 Red crystals 208-210 Careful reduction
of 4-amino-3,4 '-
dinitrobiphenyl
274.2 Yellow 214 Dye intermediate Nitration of ben-
crystals zidine in exces
sulfuric acid
3,3'-Dinitrobenzidine
274.2
Red needles
281-282
Dye intermediate
Hydrolysis of its
N,N'-diacetyl
or N,N'-di-p-
toluene sulfonyl
derivative
(continued)
-------
TABLE 2.8 (continued)
„ j
Compound
Empirical
-
formula
Structural
formula
Molecular
weight
Form and
color
Melting
point (°C)
Method of
preparation
3-Ethoxybenzidine
Ci<.H16N20
4,4'-Diamlno-3-
biphenylsulfonic
acid
Benzidine-3,3'-
disulfonic acid
C12H12N203S
C12H12N206S2
228.3
oc2H5
Shiny, flat
needles
134-135
264.3
Dye intermediate
344.4
Small, white
plates
Dye intermediate
Coupling benzene-
diazonium chlo-
ride with phenol-
4-sulfonic acid,
followed by
ethylation of
the hydroxyl
group, reduction
to the hydrazo
compound, rear-
rangement with
mineral acid,
and desulfona-
tion with dilute
acid at 170°C
Heating benzidine
sulfate to
170°C for 24 hr,
extracting the
product in al-
kali, and preci-
pitating the
monosulfonic
acid with acetic
acid
Baking benzidine
sulfate with
excess sulfuric
acid at 210°C
for 36 to 48 hr,
extracting the
product in alka-
li, and precip-
itating the
product with
mineral acid
-O
OJ
(continued)
-------
TABLE 2.8 (continued)
Compound
Benzidine-2,2'-
disulfonic acid
Benzidine sulfone
o-Tolidine-6,6'-
disulfonic acid
Empirical Structural
formula formula
C,2H12N206S2 NH,
S03H
S03h
NH2
Ci2H10N202S NH2
Col
y^^soj
X^^S°2
C$J
NH2
Cn1H16N206S2 NH2
H3C^^\
TO!
\x^S03H
/•k^SOjH
foT
ri3c^\|xj
NH2
Molecular Form and Melting Method of
weight color point (°C) preparation
344.4 Prisms Dye intermediate Reducing m-nitro-
benzenesulfonic
acid with zinc
dust and caustic
soda at 70°C,
followed by
neutralization ,
further reduc-
tion with zinc
at 80°C, filtra-
tion, and rear-
rangement in
mineral acid at
20°C
246.3 Light yellow >350 Dye intermediate Treating benzidine
plates sulfate with
excess 20% oleum
100°C
372.4 Needles Dye intermediate Reducing 2-nitro-
toluene-4-
sulfonic acid
with zinc and
caustic soda,
followed by
rearrangement
with mineral
acid
(continued)
-------
TABLE 2.8 (continued)
Compound
Empirical
formula
Structural
formula
Molecular
weight
Form and
color
Melting
point (°C)
Use
Method of
preparation
Benzidine-3,3'-
dicarboxylic acid
272.3
Needles
275
Dye intermediate
Reducing o-nitro-
benzoic acid
with zinc dust
and alkali at
100°C to 105°C,
precipitating
the hydrazo
compound with
acetic and hydro-
chloric acids,
and rearranging
the product in
concentrated
hydrochloric
acid at 95°C to
100°C
Source: Compiled from Lurie, 1964, pp. 416-419.
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46
2.3.1 Sampling and Sample Preparation
The melting points of benzidine and its common derivatives are well
above ambient environmental temperatures; consequently, these compounds
occur in the undissolved state as aerosols, dusts, or crystals and in dis-
solved form as aerosols, aqueous solutions, or organic solutions. The
principal considerations involved in handling the various sample types are
discussed in the following sections.
2.3.1.1 Benzidine in Air — Formerly, benzidine and its derivatives were
manufactured and used in open systems which permitted loss of the compound
to the atmosphere, the worker, and the work site. In some operations,
average concentrations in the air of 2.5 to 17.6 mg/m3 were observed
(Akiyama, 1970; Zavon, Hoegg, and Bingham, 1973). Under regulations
adopted in the United States in 1974, only closed .systems are permitted.
Loss of benzidine or other regulated carcinogens to the atmosphere is
expected to be reduced to such an extent that analytical techniques having
a sensitivity at the parts-per-billion level will be needed for monitoring
(Anonymous, 1974). However, some exposure by inhalation can occur when
equipment is being cleaned (Haley, 1975).
Another source of exposure can occur in the use by the general public
of pressurized spray containers of paints, lacquers, and enamels containing
benzidine yellow, an azo dye derived from 3,3'-dichlorobenzidine. The
various uses of this derivative of benzidine are noted in Section 2.2.2.2.
The amount of air contamination and dispersion of benzidine yellow afforded
by the use of aerosol products is not known.
Benzidine in the air has previously been collected with standard
Greenburg-Smith impingers (Figure 2.4) containing hydrochloric acid (Inter-
national Union of Pure and Applied Chemistry, 1962; Zavon, Hoegg, and Bing-
ham, 1973) or water (Classman and Meigs, 1951) and with high-volume vacuum
samplers equipped with fiberglass filters (Akiyama, 1970; Zavon, Hoegg, and
Bingham, 1973). When necessary, the collected sample was dissolved in
hydrochloric acid or extracted with diethyl ether in preparation for analy-
sis by spectrophotometry. Under the new federal regulations, highly effi-
cient samplers will be required in many instances since the level of
contamination will be greatly reduced relative to previously existing con-
ditions. In this case, countercurrent scrubbers, spray columns, and multi-
stage impingers can be used. Some examples of dry, cascade impingers are
shown in Figure 2.5. Sampling devices and procedures for collecting dusts
from the air are further discussed in the Air Pollution Manual (American
Industrial Hygiene Association, 1972) and by Hendrickson (1967).
2.3.1.2 Benzidine in Wastewaters — Wastewaters can be contaminated with
benzidine or its congeners not only by direct addition of these substances
through careless or accidental operating procedures during manufacture, but
also by the discharge of benzidine-coupled dyes. These dyes are subsequently
converted to the parent amine by reductants such as sulfur dioxide or hydro-
gen sulfide which are present in, or are later added to, the wastewater
(Takemura, Akiyama, and Nakajima, 1965). Samples are normally taken in
glass or polyethylene bottles. No data are available concerning storage
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47
ORNL-DWG 77-9689
-mm HOLE
WATER OR HYDROCHLORIC ACID
Figure 2.4. Early version of all glass Greenburg-Smith and midget
impingers. Source: Adapted from American Industrial Hygiene Association,
1972, Figure 9-22, p. 133. Reprinted by permission of the publisher.
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48
ORNL-DWG 77-(2899
SMALL
'PARTICLE
IM FACTION
SLIDE
Figure 2.5. Types of dry, cascade impingers. Source: American Indus-
trial Hygiene Association, 1972, Figure 9-23, p. 134. Reprinted by per-
mission of the publisher.
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49
losses. Samples of raw sewage, secondary effluents, rivers, and industrial
waste streams usually require pretreatment prior to analysis for benzidine
figure 2.6). Typically, the sample is adjusted to pH 11 by addition of
sodium hydroxide, filtered to remove suspended solids, and extracted with
diethyl ether. For colorimetric analyses, the resulting solution is extrac-
ted with hydrochloric acid, followed by neutralization with sodium hydroxide.
it the sample is to be analyzed chromatographically, a second ether extrac-
tion and a concentration step are performed. Jenkins and Baird (1975)
reported photodecomposition of benzidine during concentration of the final
ether phase but observed that this can be prevented by the addition of
methanol.
ORNL-OWG 77-H647
SAMPLE PRETREATMENT
RAW SEWAGES, SECONDARY
EFFLUENTS. RIVERS
ADJUSTMENT OF pH?ll
WITH 10/y NoOH/FILTRATlON
EXTRACTION OF FILTRATE
WITH DIETHYL ETHER
DISCARD AQUEOUS PHASE
EXTRACTION OF ETHER
PHASE WITH 2 /VHCI
NEUTRALIZATION OF ACID
PHASE WITH 10/VNaOH
DISCARD ETHER PHASE
COLORIMETRIC
ANALYSIS
EXTRACTION OF AQUEOUS
PHASE WITH DIETHYL ETHER
CONCENTRATION OF ETHER
PHASE
DISCARD AQUEOUS PHASE
GLC AND/OR TLC
ANALYSIS
Figure 2.6. Procedure for pretreatment of sample prior to analysis by
gas-liquid chromatography (GLC), thin-layer chromatography (TLC), or colorim-
etry. Source: Jenkins and Baird, 1975, Figure 2, p. 441. Reprinted by
permission of the publisher.
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50
2.3.1.3 Benzidine in Biological Media — Since benzidine is primarily a
bladder carcinogen, urine is by far the most common biological medium in
which benzidine is sought; however, tissue and fecal samples are also occa-
sionally analyzed. Urine samples should be freshly voided. If immediate
analysis is not feasible, the samples should be refrigerated or frozen to
avoid loss of benzidine, which occurs after only a few hours at room tem-
perature (Sciarini and Mahew, 1955). Typically, urine samples are alka-
lized with sodium hydroxide to pH 8.5 to 11, filtered or centrifuged, and
extracted one or more times with diethyl ether or an ethyl acetate-acetone
mixture. When concentration of the sample is necessary, the organic phase
is evaporated. The resulting organic extract can be used directly in deter-
mining benzidine by chromatographic techniques or it can be treated with an
aqueous mineral acid solution for colorimetric analysis (Classman and Meigs,
1951; Sciarini and Mahew, 1955). Tissues are usually homogenized and ex-
tracted with acetone. After evaporation of the solvent, the residues are
washed with hot glacial acetic acid and the resulting solution is cooled
and filtered. This liquid is then analyzed for benzidine by the preferred
colorimetric or chromatographic technique. Typically, fecal, samples are
homogenized and extracted with water. After centrifugation, the aqueous
phase is extracted with ether; the latter is washed with aqueous sodium
hydroxide, concentrated, and back-extracted with hydrochloric acid. The
resulting aqueous solution is then analyzed for benzidine by the preferred
method (Sciarini and Meigs, 1961a).
2.3.2 Methods of Analysis
Benzidine, its congeners, and their metabolites can be determined in
environmental and biological media by a variety of procedures. Those which
are currently important or which show promise of future usefulness are
described in this section. The performance and limitations of each method
are emphasized rather than minute operational details. The methods are
summarized in Table 2.9. Sensitivity, precision, and accuracy vary not
only among different methods, but also among various models of analytical
instruments and among different operators (Karasek, 1975); the tabulated
data should therefore be considered representative rather than definitive.
Performance data obtained from laboratories concerned with developing a new
method usually reflect optimized conditions. Interlaboratory comparisons,
when they exist, offer more realistic evaluations of the various methods.
2.3.2.1 Gas-Liquid Chromatography — Gas-liquid chromatography is an analyt-
ical process in which components of the sample are physically partitioned
between two phases — a stationary bed of large surface area and a gas which
percolates through and along the stationary bed. Typically, the stationary
bed is a finely divided column packing which is covered with a suitable
liquid sorbent. An inert gas, such as helium, argon, or nitrogen, is
usually used as the carrier for the volatile phase. When the sample is
introduced into the chromatographic column, the unadsorbed carrier gas moves
the various constituents of the sample through the column at a rate deter-
mined by the interaction of each constituent with the liquid sorbent.
Since each constituent of the sample has a slightly different affinity for
the sorbent, each fraction of the sample usually emerges from a well-
designed column completely resolved from other components after the passage
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TABLE 2.9. METHODS FOR DETERMINING BENZIDINE
Analytical
method
Gas-liquid
chromatography
Thin-layer
chromatography
Important
applications
Air, water, urine,
blood
Simple, inexpen-
sive method of
Precision
T . • ^ n / -i • Accuracy
Limit of (relative , .
detection standard
j . . s error)
deviation)
2-3 yg/liter
(waste-
water)12
0.4-0.5 yg
(5-yl spot)a
Interfering .
. Selectivity
substances
Substituted benzidine
compounds which in-
terfere in other
methods can usually
be separated and
quantized if pro-
grammed- temperature
operation is used.
Selective for benzi-
dine
Comments
Method of choice
for determining
benzidine and
its congeners
Often used to
identify the
Spectrophotometry
(chloramine-T
method)
Spectrophotometry
(diazo dye
method)
separating com-
plex mixtures
of benzidine,
its metabolites,
and congeners;
basically a
qualitative
procedure
Air, water, urine,
blood
Air, water, bio-
logical samples
0.2 yg/liter
(1-liter
sample of
wastewater)
5 ppb (waste-
water^
100 ppb
(natural
waters)0
5.1% (1.8
yg/liter of
river water)'
31% (1.8
yg/liter
of river
water)b
Organic bases
0.5%-6%
(20-100 yg
amine)
5%-30%
(unchlorinated
wastewater)
Hypochlorous
acid (from
chlorinated
wastewaters)
Compounds similar to
benzidine (e.g.,
dichlorobenzidine,
o—tolidine, and
dianisidine) inter-
fere with quantifi-
cation.
Selective for aromatic
amines in general
but does not dis-
tinguish benzidine
from its derivatives
or many other aro-
matic amines
metabolites of
benzidine and
its congeners
but only infre-
quently to
quantify such
results
Sample color fades;
must be measured
rapidly and repro-
ducibly for con-
sistent results;
nevertheless,
popular procedure
Frequently used
(continued)
-------
TABLE 2.9 (continued)
Analytical Important Limit of
method applications detection
Spectrophoto- Trace analysis of 2 ng/ml (as
fluorometry benzidine, 3,3'- amine)<^
dimethoxybenzi- 10 ng/ml (as
dine and their salt)^
dihydrochloride
salts in micro-
biological
growth media,
potable water
and waste-
waters , human
urine, and rat
blood
^Jenkins and Baird, 1975.
Longbottom, 1974.
°El-Dib, 1971.
"T> IT- -: — „ ,-. ~ J u ^ "1 ^ ^ -^ 1 Q "7 f.
Precision
(relative
standard
deviation)
0.0%-5.0%
(0.10 ppm) ,
biomedia"
0.0%-1.2%
(1.0 ppm),
biomedia"
0.0%-5.. 0%
(20 ppb),
wastewater"
l%-25%
(10 ppm),
rat blood"
5%-18% (20
ppb ) human
urine"
Accuracy interfering
(relative substances
error)
32% (0.10 ppm), None reported
biomedia"
20% (1.0 ppm),
I 1
biomedia"
25% (20 ppb),
(wastewater)"
86% (10 ppm) ,
rat blood
without
hydrolysis"
81% (10 ppm) ,
rat blood with
alkaline
hydrolysis"
37% (10 ppm),
rat blood
with acid
hydrolysis"
10% (20 ppb),
human urine
without
hydrolysis"
32% (20 ppb),
human urine
with alkaline
hydrolysis"
Selectivity Comments
Good for benzidine, Although not ade-
3, 3 '-dimethyl- quately tested
benzidine, 3,3'- under field con-
dimethoxybenzidine, ditions, appears^
and their salts when attractive for
present singly analysis of trace
levels of these
compounds in
wastewater, pota-
ble waters,
urine, blood,
and selected bio-
logical media
Ul
to
-------
53
of a characteristic volume of carrier gas. Under standardized operating
conditions, each component can be identified by its characteristic elution
time. The composition of the original sample is determined by identifying
and measuring each component. Various kinds of detectors are available for
quantifying the fractions; electron capture, flame ionization, and thermal
conductivity types are commonly used. A schematic diagram of a typical sys-
tem is shown in Figure 2.7.
Unlike colorimetric methods, gas-liquid chromatography readily distin-
guishes individual components of aromatic amine mixtures, including closely
related derivatives. For example, Jenkins and Baird (1975) used gas-liquid
chromatography to analyze samples of wastewater containing benzidine, 3,3'-
dimethoxybenzidine, diphenylamine, and 1-napthylamine in the parts-per-
billion concentration range. Their analytical system consisted of a Perkin-
Elmer Model 900 gas chromatograph equipped with dual 6 ft x 1/8 in. inside
diameter glass columns packed with a mixture of OV-17 (4.7%), QF-1 (5%),
and DC-200(0.5%) on 80/100 mesh Gas-chrom Q. Dual flame ionization detec-
tors were operated in the reference/sample mode and nitrogen was used as
the carrier gas. Ten microliters of prepared sample (Section 2.3.1.2) were
analyzed either isothermally at 200°C or programmed from 150°C to 250°C.
Detection limits for benzidine were 0.1 mg/liter with the former method and
0.5 mg/liter with the latter mode of operation. Although programmed ther-
mal operation gave decreased sensitivity for benzidine when compared with
isothermal analysis, resolution was improved and complete separation of
benzidine, 3,3'-dimethoxybenzidine, diphenylamine, and 1-napthylamine was
obtained (Figure 2.8).
ORNL-DWG 77-9690
VENT
INJECTION
PORT
GAS
FLOW
' 11 ITU ITT
HEATER
COLUMN
OVEN
/
Pi
<
\
^
1
^-
MANI-
FOLD
DETECTOR
0
o
o
o
0
0
AMPLIFIER
^
0
o
o
o
0
0
COLUMN RECORDER
Figure 2.7. Schematic diagram of a gas chromatograph.
from Environmental Instrumentation Group, 1973.
Source: Adapted
-------
54
ORNL-DWG 77-9691
Q
M
UJ
03
X
o
I
LJ
55
50
45
40
10
0
TIME (min)
Figure 2.8. Resolution of four aromatic amines by the column mixture
OV-17 (4.7%), QF-1 (5.0%), and DC-200 (0.5%) on 80- to 100-mesh Gas-chrom Q,
using PTGC (150-250°C at 3°/min, holding 12 min at 150°C) . Source: Adapted
from Jenkins and Baird, 1975, Figure 1, p. 440. Reprinted by permission of
the publisher.
Published data concerning the chromatographic determination of benzi-
dine and its congeners are thus far very limited. Standardized procedures
have not been established and statistics by which the precision and accu-
racy of the method can be evaluated are unavailable. Nevertheless, the
superior sensitivity and selectivity of the gas chromatographic method make
it very attractive; it seems likely to become the method of choice for
determining mixtures of benzidine and its congeners in the parts-per-billion
concentration range. The method is suitable for all types of samples if
prescribed preliminary preparations are performed. The principles and ana-
lytical applications of gas chromatography are discussed extensively by
Pauschmann and Bayer (1974, pp. 143-165) and by Krugers (1968).
-------
55
2.3.2.2 Thin-Layer Chromatography — Thin-layer chromatography (TLC) is a
separation technique in which the solution to be analyzed is deposited on
a sorbent surface along which it is caused to migrate by a mobile liquid
phase. As the sample moves, the various components of the sample interact
with the sorbent surface in a characteristic manner and usually fractionate
into distinct spots or zones which can be identified by comparison with
standard chromatograms. For analysis of benzidine, a typical sorbent sur-
face is Silica Gel G (0.25-mm thickness) or a sheet of prepared paper such
as Whatman No. 1. After the sample is deposited, the chromatogram is devel-
oped by enclosing the sorbent layer in a cylindrical or sandwich-type chro-
matographic tank and allowing a prepared solvent to diffuse through the
sorbent surface by capillary action. Usually, the direction of diffusion
is upward. The solvent is a liquid or, more frequently, a mixture of
liquids chosen on the basis of a trial performance. For a given sample,
the more polar the solvent, the greater will be the distance of migration.
After components of the sample have migrated sufficiently, usually about
10 cm above the spot origin, development is stopped and the spots are made
visible by irridation with ultraviolet light or by spraying with a suitable
reagent. For a given system, identification of particular compounds is
given by characteristic retardation factor (R-p) values, the ratio of the
distances traveled by the component and the solvent. Frequently, more
positive identification is obtained by spraying the separated components
with reagents which give characteristic identifying colors.
In a recent application of TLC to the determination of benzidine in
wastewaters, Jenkins and Baird (1975) used 0.25-mm-thick Silica Gel G for
the adsorbent, benzene-methanol (95/5) for the solvent, and ultraviolet
light or 0.01% ethanolic fluorescein dye for visualization. They observed
the following Rf values: diphenylamine, 85; 1-napthylamine, 70; 3,3'-dimeth-
ylbenzidine, 51; and benzidine, 37. Detection limits were 0.4 to 0.5 yg
when an initial 5-yl spot was used. A similar TLC system also was described
by Jakovljevic, Zynger, and Bishara (1975) for the determination of trace
amounts of 4-aminodiphenyl present in 2-aminodiphenyl. The free amines
were isolated by TLC, extracted from the plates, and determined fluorometri-
cally. More recently, Holder, King, and Bowman (1976) utilized Silica Gel
GF (0.25-mm-thick) to obtain TLC Rf values in ten different solvent systems.
Diphenyl was used as the reference compound. The TLC system used by these
authors is thus sufficiently selective to isolate benzidine from the other
components of the sample, but the overall sensitivity of the method is poor.
Laham, Farant, and Potvin (1970) studied a variety of TLC systems to
isolate benzidine, 4,4'-dinitrobiphenyl, and the most important biotrans-
formation products of these compounds from human and animal urine. They
used Whatman No. 1 paper sheets, ten different solvents (Table 2.10), and
five different detection reagents (Table 2.11) in their work. The char-
acteristic identifying colors of benzidine, 4,4'-dinitrobiphenyl, and their
metabolites are shown in Table 2.12; the Rf values for each compound and
each solvent are given in Table 2.13. The results show that the chromato-
graphic behavior of the various compounds is related to their structure and
that the migration trends of the various compounds in the selected solvents
can be used to identify the biotransformation products of benzidine and
4,4'-dinitrobiphenyl.
-------
56
TABLE 2.10. SOLVENTS USED FOR CHROMATOGRAPHIC SEPARATION OF
4,4'-DINITROBIPHENYL, BENZIDINE, AND THEIR
RESPECTIVE METABOLIC PRODUCTS
Solvent system
Volume to
volume ratio
n-Butanol, water
n-Butanol, acetic acid, water
n-Butanol, ethanol, N NHAOH
see-Butanol, 3% NHAOH (in water)
Toluene, methanol, water
Cyclohexane, tert-butanol, pyridine, water
Ligroine, toluene, methanol, N NH<,OH
Ligroine, benzene, methanol, N NH<,OH
Ligroine, methanol, water
Ligroine, methanol, water
1:1
4:1:5
2:1:1
3:1
4:3:1
16:2:2:1
33:17:30:20
33:17:40:20
9:2:1
20:17:3
Source: Adapted from Laham, Farant, and Potvin, 1970,
Table 1, p. 17. Reprinted by permission of the publisher.
TABLE 2.11. COMPOSITION OF SPRAYING REAGENTS USED FOR CHROMATOGRAPHIC
SEPARATION OF 4,4'-DINITROBIPHENYL, BENZIDINE, AND
THEIR RESPECTIVE METABOLIC PRODUCTS
No.
Name
Reagent composition
Group(s)
detected
Ehrlich
Gibbs
3-Resorcylaldehyde
Folin ciocalteu
FeCl3
p-Dimethylaminobenzaldehyde, 2 w/v %
in 95% EtOH - 8 vol
HC1, 6 N - 2 vol
First spray: sodium carbonate, 0.125 M
Second spray: N-2,6-trichloro-p-quinone
imine in 100 w/v % EtOH — 0.4%
6-Resorcylaldehyde — 1 g
Glacial acetic acid — 1 ml
Ethanol (100%) - 100 ml
First spray: Folin ciocalteu
Second spray: sodium carbonate, 20 w/v %
Potassium ferricyanide, w/v %
Ferric chloride, 1 w/v %
NH2, NHCOCHa
OH
NHS03H, OS03H
OH
OH
Source: Adapted from Laham, Farant, and Potvin, 1970, Table 2, p. 18.
Reprinted by permission of the publisher.
-------
TABLE 2.12. CHROMATOGRAPHIC BEHAVIOR OF 4,4'-DINITROBIPHENYL, BENZIDINE, AND THEIR METABOLITES
Identifying
Chemical
4,4' -Dinitrobiphenyl
4-Amino-4 '-nitrobiphenyl
4-Acetamido-4 '-nitrobiphenyl
4-Amino-4 ' -nitro-3-hydroxybiphenyl
4-Amino-4 '-nitro-3-biphenylyl potassium sulfate
4-Acetamido-4 '-nitro-3-biphenylyl potassium sulfate
4 ,4 '-Diaminobiphenyl
4-Acetamido-4 •' -aminobiphenyl
4,4 '-Diacetamidobiphenyl
4,4'-Diamino-3-biphenylyl sodium sulfate
Untreated
paper
DLfc
NC
Y
FtY
Y
Y
FtY
NC
NC
NC
NC
UV°
DB1
FY
FtPi
Br
Br
Pi
FBI
NC
NC
FBI
colors
With spray reagents
Ehrlich
DL
NC
Or
FtOr
Or
Or
FtOr
R-Or
Or
NC
Pi
UV
NC
G-Br
Br-Pi
Br
FtBr
Br
FOr
FY
NC
FtBr
Gibbs
DL
NC
Y
NC
G
NC
NC
NC
NC
NC
NC
UV
DB1
Br-Pi
NC
DB1
Br
Br
DB1
FBI
NC
DB1
g-Resorcylal-
dehyde
DL
NC
Y
NC
Y
FtY
NC
Y
FtY
NC
Y
UV
NC
DG
Pi
Br
G
Pi
FGr
FGr
NC
FGr
Folin
ciocalteu
DL
NC
Y
NC
01
Y
NC
Gr
Gr
NC
Gr
UV
NC
Br
FY
FGr
FtBr
FY
DB1
FGr
NC
DB1
FeCl3
DL
NC
G
NC
Gr
G
NC
G
Bl
NC
Bl
UV
DB1
Br
FY
Br
Br
Br
FBI
FBI
NC
FBI
Bl — blue; Br — brown; G — green; Gr — grey; Or — orange; Pi — pink; R — red; Y — yellow; 01 — olive; Tu — turquoise; F — fluorescent;
NC — no colour; D — dark; Ft — faint.
^Examined in daylight.
^Examined under ultraviolet light (2540 nm).
Llarkened after exposure to ultraviolet light.
Source: Adapted from Laham, Farant, and Potvin, 1970, Table 4, p. 20. Reprinted by permission of the publisher.
-------
TABLE 2.13. RETARDATION FACTOR VALUES OF 4,4'-DINITROBIPHENYL, BENZIDINE,
AND THEIR MOST IMPORTANT METABOLITES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Rf x 100 in solvent
123456789 10
4,4 '-Dinitroblphenyl o2N-/Q\ — /O/-N°2 91 90 8S 85
4-Amino-4 '-nitrobiphenyl 02N— /Q/ (O/~NH2 91 S9 88 86
4-Acetamido-4' -nitrobiphenyl o2N— (O/ — /O/-NHCOCH3 90 90 86 86
/OH
4-Amino-4'-nitro-3-hydroxybiphenyl 02N-(O/ (O)-NH2 S6 86 78 79
\ / \ /
,OS03K
/ \ / \
4-Amino-4 '-nitro-3-biphenylyl °ZN— \O ) — ( O)~NH2
potassium sulfate — — 60 62 77 70
4-Acetamido-4 '-nitro-3-biphenylyl o2N— \Q} — \O/ NHC°CH3
potassium sulfate \ — ' ^ — ' 72 77 77 80
4,4'-Diaminobiphenyl HjN-/O/ — (O/-NH2 84 70 79 78
4-Acetamido-4 '-aminobiphenyl H2N— ^O/ — \ O/~NHCOCH3 89 83 83 82
4-4'-Diacetamidobiphenyl HJCOCHN— (O^> — ^OVNHCOCH3 83 85 83 84
88 80 88 89 57 48
88 58 59 57 9 9
63 39 24 15 3 0
37 34 7 4 0 0
000000
000000
43 10 2 35 4 0
16 6 2 2 1 0
22 5 0 4 0 0
4,4 '-Diamino-3-biphenylyl ri2™— \O/ — \O/~NH2
sodium sulfate N — ' N — ' 38 40 59 40 0 0 0 0 0 0
Ln
00
Source: Adapted from Laham, Farant, and Potvin, 1970, Table 3, p. 19. Reprinted by permission of the publisher.
-------
59
From these examples, it is apparent that TLC is a convenient separa-
tion procedure which is readily adapted to the qualitative analysis of
benzidine and its metabolites in environmental and biological samples.
Semiquantitative estimates of sample components can also be made from thin-
layer chromatograms by comparing sample spots with those on previously pre-
pared standard chromatograms. Although convenient, this technique is not
as sensitive or dependable as other methods. Better data can usually be
obtained by removing identified spots from thin-layer chromatograms for
quantitative determination by an appropriate conventional analytical tech-
nique. An extensive discussion of recent chromatographic developments is
available in Ckpomatography (1974).
2.3.2.3 Spectrophotometry: Chloramine-T Method — The chloramine-T variant
of the spectrophotometric method for determining benzidine depends on the
formation of a yellow oxidation product when an acid solution of benzidine
is treated with toluene-p-sulphonchloroamide (chloramine-T). The quantity
of benzidine in the sample is determined by extracting the yellow oxidation
product with an organic solvent and measuring the amount of light it absorbs
under standardized conditions (Butt and Strafford, 1956; Classman and Meigs,
1951). The color reaction is not specific for benzidine; all p,p'-diamino-
diphenyl derivatives form the yellow oxidation product to some degree.
Furthermore, the yellow oxidation product is not permanent; it is photo-
sensitive and gradually fades to a colorless state, possibly a quinone
complex, over a 2-hr interval. The light absorption of the sample must
therefore be measured immediately after the color is developed.
In spite of these inadequacies, the chloramine-T method is convenient,
and it is frequently used to determine benzidine and its derivatives. In
the most recent and sensitive version of the method (Longbottom, 1974),
aqueous samples are adjusted to pH 8.5 to 9.0 with sodium hydroxide or
hydrochloric acid and extracted with ethyl acetate. The latter is back-
extracted with hydrochloric acid and the resulting aqueous solution is
refrigerated in the dark until the remaining steps of the procedure can be
performed rapidly on one sample at a time. Chloramine-T is then added to
the aqueous solution, with shaking, to develop the yellow oxidation product.
The product is extracted with ethyl acetate, filtered, and scanned with a
double-beam spectrophotometer over the spectral range of 510 to 370 nm.
Benzidine is identified by its absorbance maximum at 436 nm (Figure 2.9).
The spectrum of dichlorobenzidine is similar, but it has an absorbance
maximum at 445 nm. The detection limit of the method is 0.2 yg/liter if
1-liter samples are used. The average relative standard deviation (n = 8)
is 5.1% for analyses of 1-liter samples of river water containing 1.8 yg
benzidine. The relative error under these conditions is large — 31.1%.
Organic bases interfere with the analysis, as do compounds having a struc-
ture very similar to benzidine, such as dichlorobenzidine, 3,3'-dimethyl-
benzidine, and 3,3'-dimethoxybenzidine. This method can also be applied to
benzidine or its derivatives in samples of air, urine, feces, and blood if
suitable sample preparation steps are taken.
2.3.2.4 Spectrophotometry: Diazo Dye Method — This analytical method is
based on the conversion of benzidine to its diazo salt and subsequent
coupling to an aromatic phenol to produce a diazo dye which absorbs light
-------
60
ORNL-DWG 77-9692
—I 50
100
500 480 460 440 420
WAVELENGTH (nm)
400
380
Figure 2.9. Spectrum of benzidine oxidation product.
from Longbottom, 1974, Figure 1.
Source: Adapted
in the visible portion of the electromagnetic spectrum. The amount of ab-
sorbed light is related to the benzidine concentration in the sample by
comparison with a previously determined calibration plot.
In a typical spectrophotometric analysis of benzidine in wastewater
(Jenkins and Baird, 1975), the sample is adjusted to pH 11 by addition of
10 M sodium hydroxide solution, filtered to remove suspended solids,
diazotized with sodium nitrite and hydrochloric acid, treated with sulfamic
acid to destroy excess nitrite, and coupled with resorcinol. Absorption
of the resulting diazo dye is determined at 550 nm with a double-beam
spectrophotometer. When 10-cm absorption cells are used, the detection
limit for benzidine is about 5 ppb. For unchlorinated wastewaters, the
relative error of spiked samples varies from 5% to 30%. Recovery of benzi-
dine from chlorinated wastewater is poor, however, apparently because hypo-
chlorous acid reacts with benzidine to yield a chloramine-type compound
which does not give a positive benzidine test. Although this procedure
readily distinguishes between aromatic and alkyl amines, it is not selective
for benzidine in the presence of other aromatic amines; accordingly, the
method is unsuitable for samples containing mixed aromatic amines. Despite
these shortcomings, the diazo dye technique is a useful analytical method
for determining benzidine in the absence of other aromatic amines and is
frequently used for all types of samples when this condition is satisfied.
2.3.2.5 Spectrophotofluorometry — Spectrophotofluorometry is based on the
measurement of fluorescence radiation emitted by a sample previously ex-
cited by ultraviolet or visible light. The emitted radiation results from
-------
61
the transition of the excited molecule from the first excited singlet state
to the ground state; the frequency of the emitted light is therefore char-
acteristic of the analyte. The intensity of the emission is proportional
to the concentration of the analyte as well as the intensity of the excit-
ing radiation. Because of the latter relationship, spectrophotofluorometry
is inherently very sensitive; under favorable conditions, it can be four
orders of magnitude more sensitive than molecular absorption spectrophotom-
etry (Mancy, 1971, p. 70). In a typical application of spectrophotoflu-
orometry to the determination of benzidine in environmental or biological
media, samples are prepared as described in Section 2.3.1.2 or 2.3.1.3
except that methanol, rather than ether, is used as the final solvent. The
prepared sample is then irradiated with monochromatic light from mercury or
xenon lamps and a selected wavelength of the resulting fluoresence is meas-
ured with a photodetector tube. The intensity of the measured fluoresence
is then related to the amount of benzidine in the original sample by means
of a predetermined calibration curve. Bowman, King, and Holder (1976)
determined benzidine, 3,3'-dimethylbenzidine, 3,3'-dimethoxybenzidine, and
their dihydrochloride salts in a variety of matrices with a spectrophoto-
fluorometer equipped with a xenon lamp, a 1P28 photodetector, and 1-cm
square cells. The frequencies of the incident (XEx) and emitted (XEm)
radiations varied for each compound and are shown in Table 2.14. The
relative intensities of the fluorescence of each compound are also listed.
The detection limits of the amines and their salts were 2 ng/ml and 10 ng/ml
respectively. Good precision was obtained in a variety of sample types;
relative standard deviations of 0.0% to 5.0% were reported for trypticase
soy dextrose and brain heart infusion medium containing 0.1 ppm and 1.0 ppm
benzidine (Table 2.15) and wastewater containing 20 ppb benzidine (Table
2.16). Somewhat poorer results (1% to 25%) were obtained with human urine
(20 ppb) and rat blood (10 ppm). The relative errors in the above analyses
varied from 10% to 35%, except for the samples of rat blood, which averaged
about 85%. This level of accuracy is generally adequate for all samples
mentioned other than rat blood.
TABLE 2.14. WAVELENGTHS USED AND RELATIVE INTENSITIES OBSERVED
DURING ANALYSIS OF BENZIDINE AND ITS CONGENERS
Compound
Benzidine
Benzidine- 2HC1
3,3' -Dimethylbenzidine
3,3' -Dimethylbenzidine • 2HC1
3,3' -Dimethoxybenzidine
3,3' -Dimethoxybenzidine* 2HC1
XEx
(nm)
295
302
300
310
312
318
X_ Relative intensity
Em , J
(nm) Per yg Per ml
396
410
384
410
380
422
33.5
7.45
51.8
12.2
64.3
8.25
Source: Adapted from Bowman, King, and Holder, 1976, p. 211.
Reprinted by permission of the publisher.
-------
62
TABLE 2.15. ANALYSIS OF TWO BIOLOGICAL GROWTH MEDIA SPIKED WITH BENZIDINE,
TWO CONGENERS, AND THEIR SALTS AT 0, 0.10, and 1.0 ppm
Compound
Benzidine
Benzidine-2HCl
3
3
, 3 ' -Dime thy Ibenzidine
, 3'-Dimethylbenzidine«2HCl
3, 3 '-Dimethoxybenzidine
3,
3 ' -Dimethoxybenzidine • 2HC1
„ , . a
Medium
BHI
TSD
BHI
TSD
BHI
TSD
BHI
TSD
BHI
TSD
BHI
TSD
Amount
added*
(ppm)
0
0.10
1.00
0.00
0.10
1.00
0.00
0.10
1.00
0.00
0.10
1.00
0.00
0.10
1.00
0.00
0.10
1.00
0.00
0.10
1.00
0.00
0.10
1.00
0.00
0.10
1.00
0.00
0.10
1.00
0.00
0.10
1.00
0.00
0.10
1.00
(yg)
0
1.0
10.0
0.0
1.0
10.0
0.0
1.0
10.0
0.0
1.0
10.0
0.0
1.0
10.0
0.0
1.0
10.0
0.0
1.0
10.0
0.0
1.0
10.0
0.0
1.0
10.0
0.0
1.0
10.0
0.0
1.0
10.0
0.0
1.0
10.0
Amount
(x
recovered
± SE)e
(ppm) (%)
0.040
0.070
0.780
0.019
0.075
0.787
0.040
0.073
0.795
0.019
0.073
0.778
0.037
0.074
0.839
0.029
0.073
0.902
0.045
0.070
0.777
0.036
0.064
0.710
0.031
0.078
0.923
0.019
0.055
0.829
0.030
0.057
0.715
0.027
0.052
0.770
± 0.000
± 0.000
± 0.004
± 0.000
± 0.000
± 0.012
± 0.000
± 0.002
± 0.000
± 0.002
± 0.001
± 0.004
+ 0.001
+ 0.005
± 0.010
+ 0.004
± 0.002
+ 0.009
+ 0.004
± 0.002
+ 0.006
± 0.003
± 0.001
± 0.003
+ 0.003
± 0.001
+ 0.007
± 0.001
± 0.000
± 0.008
+ 0.003
± 0.005
± 0.002
± 0.003
± 0.001
± 0.006
70.0
78.0
75.0
78.7
73.0
79.5
73.0
77.8
74.0
83.9
73.0
90.2
70.0
77.7
64.0
71.0
78.0
92.3
55.0
82.9
57.0
71.5
52.0
77.0
± 0.0
± 0.4
± 0.0
± 1.2
± 2.0
± 0.0
± 1.0
± 0.4
± 5.0
± 1.0
± 2.0
±0.9
± 2.0
± 0.6
± 1.0
± 0.3
± 1.0
± 0.7
± 0.0
± 0.8
± 5.0
± 0.2
± 1.0
± 0.6
,BHI — brain heart infusion; TSD — trypticase soy dextrose.
Per 10 ml of sample.
Mean and standard error from triplicate assays; spiked samples are corrected for
controls. Controls and 0.10-ppm samples contained 1-g equivalent of medium per milli-
liter for spectrophotofluorometric reading; the 1.0-ppm samples contained 0.2-g
equivalents per milliliter.
Source: Adapted from Bowman, King, and Holder, 1976, Table I,
by permission of the publisher.
216. Reprinted
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63
TABLE 2.16. ANALYSIS OF WASTEWATER SPIKED WITH BENZIDINE,
TWO CONGENERS, AND THEIR SALTS AT 0 AND 20 ppb
Amount
Amount ,
a recovered
Compound added (Zc ±
(ppb) (yg) (ppb)
Benzidine
Benzidine* 2HC1
3,3' -Dimethylbenzidine
3,3' -Dimethylbenzidine»2HCl
3,3' -Dimethoxybenzidine
3,3' -Dimethoxybenzidine* 2HC1
0
20
0
20
0
20
0
20
0
20
0
20
0.0
2.0
0.0
2.0
0.0
2.0
0.0
2.0
0.0
2.0
0.0
2.0
4 + 1
17 ± 0
4 ± 1
15 ± 1
4 ± 1
15 ± 0
3 ± 1
13 ± 1
3 ± 0
16 ± 0
3 + 0
14 + 1
85
75
75
65
80
70
,Per 100 ml of sample.
Mean and standard error from triplicate assays; spiked
samples are corrected for controls. Samples contained 10-g
equivalents of water per milliliter for spectrophotofluoro-
metric reading.
Source: Bowman, King, and Holder, 1976, Table II, p. 217,
Reprinted by permission of the publisher.
In continued research in the same laboratory (Holder, King, and Bowman,
1976), 4-aminobiphenyl was determined utilizing the same substrates and
techniques. Recovery rates ranged from 81% to 95% in biological growth
media and wastewater, from 45% to 95% in human urine, and from 6% to 76% in
mouse whole blood. Best recovery from urine occurred without prior hydroly-
sis, and acid hydrolysis led to an unsuitable purple discoloration. However,
best recovery from blood occurred after acid hydrolysis (67% to 72%).
Although the spectrophotofluorometric method is very specific for indi-
vidual benzidine compounds because of the particular wavelengths involved,
an effort by Bowman, King, and Holder (1976) to determine three amines or
their three salts in admixture by measuring the relative intensities at
each excitation and emission maxima was unsuccessful because of large dif-
ferences in the specific responses and extensive overlap of the spectra.
Further experience is needed to define the useful limits of this application,
but the method appears attractive for the determination of trace levels of
benzidine and its congeners in a variety of environmental samples.
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64
2.3.3 Comparison of Analytical Procedures
Although benzidine has been known since 1845 and has been used exten-
sively in analytical chemistry to determine other substances, relatively
little attention has been given to analytical procedures by which the rea-
gent itself can be determined, particularly at the low concentrations
which are of current interest. Indeed, the International Union Against
Cancer concluded that "present analytical methods are satisfactory for the
assessment of gross aromatic amine contamination of the human environment"
but that "new techniques will have to be designed for the measurement of
lower degrees of contamination" (Shubik, Clayson, and Terracini, 1970,
p. 16). More recently, the U.S. Environmental Protection Agency stated,
"Adequately tested methods for benzidine are not available," and recommended
the chloramine-T method described in Section 2.3.2.3 as an interim method
(Federal Eegi-ster, 1975). Thus, there are no officially sanctioned analyt-
ical procedures for determining low levels of benzidine in environmental
and biological samples.
The most likely candidate for official sanction appears to be gas
(or gas-liquid) chromatography. Although only a limited number of gas
chromatographic analyses of benzidine have been performed, the technique
has already demonstrated a high degree of sensitivity, selectivity, versatil-
ity, and speed, and better performance can be expected with further use.
Gas chromatography is the only analytical technique available which readily
determines benzidine in the presence of its congeners. The chief dis-
advantages of the gas chromatographic method are its relatively high capital
investment and maintenance costs and the somewhat higher level of sophis-
tication required in the operating and maintenance personnel compared with
other methods of analysis.
The determination of benzidine by spectrophotofluorometry has been even
more limited than the use of gas chromatography. Nevertheless, spectro-
photofluorometry is potentially important as an analytical method for benzi-
dine and its congeners. These compounds emit distinctive fluorescent
spectra by which each can be identified and quantified. The method is char-
acterized by inherent sensitivity, simplicity, and speed. However, quanti-
tative determinations of mixed benzidine congeners are not yet feasible
because of spectral overlap and large differences in relative intensities
(Bowman, King, and Holder, 1976). The importance of this deficiency depends
on the composition and type of sample to be analyzed — in many instances it
is not significant. In any event, marked improvement in the sensitivity and
selectivity of this technique may be expected with continued use.
The diazo and chloramine-T spectrophotometric methods of analyzing
benzidine and its derivatives are the oldest, most widely used methods
described. Each method has benefited from a series of improvements over
the years; recent versions of each method have sensitivities in the parts-
per-billion range, good precision, and moderate accuracy. However, neither
method distinguishes between benzidine and its congeners; the diazo tech-
nique is responsive to aromatic amines in general and the chloramine-T
method responds generally to p,p'-diaminodiphenyl derivatives. In addition,
the chloramine-T method is photosensitive, requiring rapid reading of the
samples to prevent inaccurate results (Butt and Strafford, 1956). Despite
-------
65
these shortcomings, both spectrophotometric procedures are useful. For
example, a modern variation of the chloramine-T method was designated by
the U.S. Environmental Protection Agency as the interim method of determin-
ing benzidine (Federal Register, 1975).
The thin-layer chromatographic technique is a simple, frequently used
qualitative procedure for separating and identifying benzidine, its congeners.
and their metabolites. Although the procedure can be applied to the quanti-
tative determination of these compounds by comparing the area or fluorescence
of separated spots with that of standard chromatograms, it is relatively in-
sensitive and is not the method of choice.
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66
SECTION 2
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SECTION 3
BIOLOGICAL ASPECTS IN MICROORGANISMS
No data were found on the metabolism of benzidine in microorganisms.
However, some duodenal bacteria, soil bacteria, and Esoheviohia aoli can
reduce the azo linkage of benzidine-based azo dyes to liberate benzidine
(Yoshida and Miyakawa, 1973).
Antiviral activity has been demonstrated among benzidine-based dyes.
Of 36 such dyes tested, 5 showed antiviral activity and 26 showed hemag-
glutinating activity (Dettori, 1964). No relationship occurred between
the intensities of the hemagglutinating and antiviral activities. Congo
Red (derived from benzidine) and benzopurpurin (derived from 3,3'-dimeth-
ylbenzidine) inhibited TI and T7 phage production in a strain of Escheriehia
aoli (Dettori and Neri, 1964). In this study, the dyes showed no bacteri-
cidal activity and did not inhibit respiration or anaerobic glycolysis.
Congo Red has been used as a bacteriostatic agent in the isolation of
the tubercle bacillus from sputum (Frobisher, 1946) and as an indicator dye
in the mixed culture growth of Rhizobiwn and related species of bacteria
(Vincent, 1970).
The Federal Register (1976) noted that benzidine inhibited biooxida-
tion in activated sludges at 500 mg/liter, caused some inhibition at 1 to
5 mg/liter, and was thought to be oxidized at 0.1 mg/liter or less. Howard
and Saxena (1976), in reporting on work done by the Synthetic Organic Chem-
ical Manufacturers Association in 1975, noted that Warburg tests showed
benzidine inhibition of oxygen uptake at concentrations of 40 to 80 mg/liter
in unacclimated sludge and at concentrations of 60 to 120 mg/liter in accli-
mated sludge.
Benzidine and some of its congeners have been found to be mutagenic
to strains of Salmonella typhimuriym through the use of an assay method
recently developed for the testing of the mutagenic and carcinogenic
potential of a wide variety of chemicals (McCann and Ames, 1976). Ames
et al. (1973) first showed that benzidine was mutagenic to this bacte-
rium, although only after it had been activated by a rat liver homogenate.
Since the assay involved the detection of reversals of frameshift muta-
tions, it was suggested that benzidine could cause frameshift mutations.
More recently, Garner, Walpole, and Rose (1975) have used tester strain
TA 1538 of Salmonella typhimuriwn in determining the mutagenic capability
of benzidine and a number of congeners in the presence and absence of a
preparation of liver mixed function oxidase. The assay measured the
production of histidine prototrophs (revertants). As noted in Table 3.1,
3,3'-dichlorobenzidine and 3,3'-dichlorobenzidine sulfate (technical
grade) were the only compounds which exhibited weak mutagenic activity
in the absence of the liver preparation. 3,3',5,5'-Tetramethylbenzidine
produced no mutagenic activity even in the presence of the liver prepara-
tion. The presence of the liver preparation caused weak mutagenic activity
by 3,3'-dianisidine and strong mutagenic activity by 3,3',5,5'-tetrafluoro-
benzidine, 3,3'-dichlorobenzidine, and 3,3'-dichlorobenzidine sulfate.
74
-------
75
TABLE 3.1. MUTAGENIC ACTIVITY OF BENZIDINE ANALOGUES TO SALMONELLA TYPHIMURIUM
IN THE PRESENCE OR ABSENCE OF LIVER MIXED FUNCTION OXIDASE
_ , Amount added Liver
Compound / / -, \ .a
(yg/plate) preparation
Benzidine
3,3' ,5,5' -Tetraf luorobenzidine
3,3' ,5,5'-Tetramethylbenzidine
3,3' -Dichlorobenzidine
3,3 '-Dichlorobenzidine sulfate
(technical grade)
3,3' -Dianisidine
(3, 3'-dimethoxybenzidine)
Dimethylsulfoxide (control)
50 +
100 +
50
100 -
50 +
100 +
50
100 -
50 +
100 +
50 -
100 -
50 +
100 +
50 -
100 -
50 +
100 +
50 -
100 -
50 +
100 +
50 -
100 -
+
Mutagenic
activity
(His+ revertants
per plate)
430
640
5
15
560
1040
20
29
15
15
5
9
3360
7520
114
131
5490
8350
127
129
63
82
6
11
16
8
a
'+ indicates liver homogenate present; — indicates liver homogenate absent.
Source: Adapted from Garner, Walpole, and Rose, 1975, Table 1, p. 41.
Reprinted by permission of the publisher.
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76
SECTION 3
REFERENCES
1. Ames, B. N., W. E. Durston, E. Yamasaki, and F. D. Lee. 1973.
Carcinogens Are Mutagens: A Simple Test System Combining Liver
Homogenates for Activation and Bacteria for Detection. Proc. Natl.
Acad. Sci. U.S.A. 70:2281-2285.
2. Dettori, R. 1964. Attivita Antivirale di Coloranti Azoici: I.
Correlazione Tra Attivita Emoagglutinate e Attivita Antivirale
(Antiviral Activity of Dyes: I. Correlation between Haemoaggluti-
nating and Antiviral Activities). G. Microbiol. (Italy) 12:135-144.
3. Dettori, R., and M. G. Neri. 1964. Attivita Antivirale di Coloranti
Azioci: II. Azione di Alcuni Derivati Benzidinici sul Sistema
Batteriofago-Cellula Batterica (Antiviral Activity of Dyes: II.
Action of a Group of Benzidine Derivatives on a Series of Phage-
Bacterium Systems). G. Microbiol. (Italy) 12:145-152.
4. Federal Register. 1976. 41:27012-27017.
5. Frobisher, M. 1946. Fundamentals of Bacteriology, 3rd ed.
W. B. Saunders Company, Philadelphia, pp. 126-127.
6. Garner, R. C., A. L. Walpole, and F. L. Rose. 1975. Testing of
Some Benzidine Analogues for Microsomal Activation to Bacterial
Mutagens. Cancer Lett. (Netherlands) 1:39-42.
7. Howard, P- H., and J. Saxena. 1976. Persistence and Degradability
Testing of Benzidine and Other Carcinogenic Compounds. EPA
560/5-76-005, U.S. Environmental Protection Agency, Washington, D.C.
p. 14.
8. McCann, J., and B. N. Ames. 1976. A Simple Method for Detecting
Environmental Carcinogens as Mutagens. Ann. N.Y. Acad. Sci.
271:5-13.
9. Vincent, J. M. 1970. A Manual for the Practical Study of Root
Nodule Bacteria. Blackwell Scientific Publishers, Oxford, England.
pp. 4-5.
10. Yoshida, 0., and M. Miyakawa. 1973. Etiology of Bladder Cancer:
"Metabolic" Aspects. In: Analytic and Experimental Epidemiology
of Cancer, W. Nakahara, T. Hirayama, K. Nishioka, and H. Sugano,
eds. University Park Press, Baltimore, pp. 31-39.
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SECTION 4
BIOLOGICAL ASPECTS IN PLANTS
The only reference found in the literature concerning benzidine metab-
olism in higher plants involved the in vitro oxidation of 3,3'-diamino-
benzidine by a commercial preparation of horseradish peroxidase (Barbotin
and Thomas, 1974). The oxidative activity occurred when the peroxidase
was in solution or immobilized in an artificial membrane. Oxidation has
also been shown to occur with peroxidase from a variety of green algae,
but not with peroxidases from varieties of red and brown algae (Siegel
and Siegel, 1970). Diaminobenzidine oxidation in blue-green algae occurs
via the respiratory and photosynthetic electron transport systems
(Lauritis et al., 1975).
Diaminobenzidine is used in electron microscopy as a histochemical
stain. It apparently is oxidized by the cytochromes of the mitochondrial
electron transport chain, polymerizes and remains tightly bound to the
oxidizing heme protein, and reacts with osmium tetroxide to give an
electron-dense deposit (Opik, 1975). Diaminobenzidine can also be used
to localize sites of peroxidase and catalase activity (Nougarede, 1971;
Silverberg, 1975).
No information was found in the literature on any effects to higher
plants produced by benzidine exposure. However, Boney (1974) has noted
that benzidine (0.3 rag/liter) provided a 12% stimulation in growth over
control culture conditions in the growth of the red alga Antithamnion
plwrrula.
11
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78
SECTION 4
REFERENCES
1. Barbotin, J. N., and D. Thomas. 1974. Electron Microscopic and
Kinetic Studies Dealing with an Artifical Enzyme Membrane; Applica-
tion to a Cytochemical Model with the Horseradish Peroxidase-3,3'-
Diaminobenzidine System. J. Histochem. Cytochem. 22(11):1048-1059.
2. Boney, A. D. 1974. Aromatic Hydrocarbons and the Growth of Marine
Algae. Mar. Pollut. Bull. (Great Britain) 5:185-186.
3. Lauritis, J. A., E. L. Vigil, L. Sherman, and H. Swift. 1975.
Photosynthetically-Linked Oxidation of Diaminobenzidine in Blue-
Green Algae. J. Ultrastruct. Res. 53:331-344.
4. Nougarede, A. 1971. Observations Nouvelles sur les Lieux de
Peroxydation de la 3,3'-Diaminobenzidine (DAB) dans les Gals et
les Suspensions Cellulaires d'un Mutant Chlorophyllien Obtenu a
partir de la Souche Sauvage de iMcer pseudoplatanus L. C. R.
Seances Acad. Sci. (France) 273:864-867.
5. Opik, H. 1975. The Reaction of Mitochondria in the Coleoptiles of
Rice (Ovyza sativa L.) with Diaminobenzidine. J. Cell Sci. (Great
Britain) 17:43-55.
6. Siegel, B. Z., and S. M. Siegel. 1970. Anomalous Substrate Spe-
cificities among the Algal Peroxidases. Am. J. Bot. 57(3):285-287.
7. Silverberg, B. A. 1975. 3,3'-Diaminobenzidine (DAB) Ultra-
structural Cytochemistry of Microbodies in Chlorogonium elongatum.
Protoplasma (Austria) 85:373-376.
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SECTION 5
BIOLOGICAL ASPECTS IN WILD AND DOMESTIC ANIMALS
No data were found concerning the metabolism of benzidine or its
derivatives by wild or domestic animals. Metabolism by research animals
is discussed in Section 6; there are differences among species of animals.
Therefore, it is reasonable to expect some variation among diverse aquatic
and terrestrial fauna.
Apparently, the only information available on the toxicity of benzi-
dine or its derivatives to wild or domestic animals is that which has been
obtained through laboratory research on a limited number of species,
including the commonly used research animals. Most of this information
is covered in Section 6 since it provides comparative data useful in deter-
mining the toxicity potential of these compounds to humans, including their
carcinogenic potential. Some of the data are also useful in projecting the
toxicity potential among wild and domestic animals, should they be exposed
in their natural environment. Thus, recent bioassays of benzidine in fish
have shown TL50 values ranging from 2.5 mg/liter for red shiner to 20
mg/liter for the fathead minnow (Federal Register, 1976). Pliss and
Khudoley (1975) found liver damage in guppies fed 30 mg benzidine per 100
g of dry diet material. At two to four weeks of exposure, liver tissue
developed foci of necrosis and fatty dystrophy. Hyperplasia of the hepa-
tocytes occured by four to eight weeks. None of the fish had developed
tumors by the 56-week termination of the experiment.
Benzidine has not been found to occur in the aquatic environment of
the United States (Federal Register, 1976), although it has been detected
in the Sumida River of Japan (Takemura, Akiyama, and Nakajima, 1965).
Should contamination occur, the possibility exists for heightened toxicity
among some species because of the potential of bioaccumulation within food
chains. The Federal Register (1976) reported that bioaccumulation occurred
in bluegill fish in a radiometric study using 14C-benzidine. Edible
tissues contained a 44-fold increase in concentration over that in water.
Benzidine injected into the blastoderm of early chick embryos at 18
to 24 hr of incubation was found by Noto (1967) to cause morphological
abnormalities. The closure of the cephalic neural tube was inhibited.
79
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80
SECTION 5
REFERENCES
1. Federal Register. 1976. 41:27012-27017-
2. Noto, T. 1967. The Effects of Some Carcinogens on the Morphogenesis
and Differentiation in the Early Chick Embryo. Sci. Rep. Tohoku
Univ. Ser. 4 (Japan) 33:65-69.
3. Pliss, G. B., and V. V. Khudoley. 1975. Tumor Induction by Carcino-
genic Agents in Aquarium Fish. J. Natl. Cancer Inst. 55(1):129-136.
4. Takemura, N., T. Akiyama, and C. Nakajima. 1976. A Survey of the
Pollution of the Sumida River, Especially on the Aromatic Amines
in the Water. Int. J. Air Water Pollut. (Great Britain) 9:665-670.
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SECTION 6
BIOLOGICAL ASPECTS IN HUMANS
6.1 SUMMARY
Benzidine exposure is primarily an industrial problem among workers
in plants which manufacture or use benzidine and its derivatives. Uptake
of these compounds is by skin absorption, inhalation, and ingestion. Skin
absorption and inhalation are the major uptake routes; ingestion adds an
insignificant portion, except for Japanese kimono painters who use their
lips to moisten brushes dipped in benzidine dyes. Uptake is positively
correlated with industrial hygiene and the particular benzidine compound
to which the worker has been exposed. Volatile compounds, compounds of
small particle size, and bases easily penetrate the skin. High air tem-
perature and relative humidity also aid skin penetration.
Benzidine does not appear to accumulate in human blood or other tis-
sues for any appreciable length of time. Studies made with warm-blooded
experimental animals also showed no significant accumulation by benzidine
or its derivatives. However, there appears to be rapid distribution by
the blood to other tissues and temporarily increased concentrations in
some organs, notably the liver, lung, and kidney, although this is not a
consistent finding; findings vary with the compound tested and the test
animal used. Although the usual experimental animal does not appear to
accumulate benzidine in its tissues, a recent study has demonstrated a
44-fold accumulation of this compound in bluegill fish.
Benzidine and its derivatives are biotransformed into several metab-
olites in the animal body; the metabolites produced are species dependent.
The dog differs from other animal species in that it cannot acetylate ben-
zidine. The major benzidine metabolite in both humans and dogs appears
to be 3-hydroxybenzidine. In humans, benzidine and 3,3'-dimethylbenzidine
(diorthotolidine) seem to be metabolized similarly since hydroxylation of
diorthotolidine occurs in the ortho position with respect to the amino
group.
Benzidine, its derivatives, and metabolites are eliminated in the
urine and feces. Biliary secretion contributes to fecal excretion. Uri-
nary excretion in humans increases with increased exposure. There is
evidence that urinary excretion in exposed workers is higher in warmer
periods of the year because of increased uptake. Excretion in experi-
mental animals varies with the compound administered and the species used.
The rat, for example, excretes benzidine primarily in the feces, whereas
benzidine is excreted primarily in the urine by the dog and monkey. In
contrast, 3,3'-dichlorobenzidine is excreted primarily in the feces by
all three animals. Bile appears to contain the same metabolites as urine,
but in only one-third the quantity.
Benzidine and its derivatives produce toxic effects in humans and
test animals, including the induction of cancer. In humans, strong
81
-------
82
evidence implicates benzidine and the related compound 4-aminobiphenyl as
a cause of bladder cancer among workers occupationally exposed to these
compounds. Although exposure to 3,3'-dimethylbenzidine, 3,S'-dimethoxy-
benzidine, or 3,3'-dichlorobenzidine remain suspect, there is no present
evidence that bladder cancer is induced in humans. However, a recent
report has suggested that 3,3'-dichlorobenzidine may be responsible for
other types of tumor production in humans other than bladder cancer, and
all three compounds, as well as benzidine and 4-aminobiphenyl, have pro-
duced various tumors in experimental animals. Interestingly, evidence for
the induction of bladder cancer in test animals is very meager for all of
these compounds except 4-aminobiphenyl. It is generally accepted that the
actual carcinogenic compounds are metabolites of the parent aromatic amines.
There is mounting evidence that the precarcinogen may be produced through
N-hydroxylation of the parent compound, but esterification may be required
as a further activation step. The sulfate and glucuronidate conjugation
products of the aromatic amines may also be carcinogenic. Benzidine and
its derivatives cause dermatitis, cystitis, nephritis, and hematuria in
humans and various effects in test animals.
Benzidine and its derivatives also influence the activity of some
enzymes. Workers exposed to benzidine had higher levels of 3-glucuronidase
in their urine than unexposed workers. Cessation of exposure led to more
nearly normal urinary levels. Benzidine also has been shown to decrease
blood phenolase activity in rabbits and catalase and peroxidase activity
in rats. In vitro studies have shown that benzidine, its derivatives, and
related compounds exert differing degrees of ability to reduce cytochrome
o. This reducing power appears to relate inversely with the strength of
the compounds as carcinogens. Thus, a compound such as diaminobenzidine,
believed to be a weak carcinogen, displays strong reducing power, whereas
a potent carcinogen such as p-aminobiphenyl displays weak reducing power.
6.2 METABOLISM
6.2.1 Uptake
Exposure to benzidine and benzidine derivatives is of concern mainly
to employees in factories which manufacture and use these compounds. Ben-
zidine uptake by exposed persons may be through the respiratory tract, from
the air, by contaminated food into the gastrointestinal tract, and by pene-
tration of the skin through direct contact or from contaminated clothing
(Meigs, Brown, and Sciarini, 1951). All steps in benzidine synthesis as
carried out by large manufacturers in the United States are now conducted
in closed systems. Even under such conditions, some exposure can occur
when equipment is being cleaned (Haley, 1975). There is also a question
as to whether closed systems are being used by firms within the dye industry
who may be manufacturing their own benzidine and derivatives (Haley, 1977).
Ingestion of benzidine or its derivatives has not been reported as a
significant source of exposure with the exception of painters of silk
kimonos in Japan who "point" or moisten their brushes with their lips and
thereby ingest some of the benzidine dyes on the brushes (Yoshida and
Miyakawa, 1973).
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83
Skin penetration is the most important avenue of entry into the body.
Both benzidine and 3,3'-dimethylbenzidine readily penetrate intact skin
(Meigs, Brown, and Sciarini, 1951). 3,3'-Dichlorobenzidine, which is non-
volatile and has a large particle size, presents little inhalation hazard
and less skin absorption hazard than benzidine (Gerarde and Gerarde, 1974;
Rye, Woolrich, and Zanes, 1970). Barsotti and Vigliani (1952) pointed out
that workers occupationally exposed in the manufacture of benzidine risk
development of tumor lesions because the light, fluffy, powdery composition
of the benzidine base provides for ready skin contact and absorption. The
order of acute toxicity of benzidine and some of its derivatives appears
to follow the order of ease of skin penetration: benzidine, 3,3'-dimethoxy-
benzidine, and 3,3'-dichlorobenzidine (Rye, Woolrich, and Zanes, 1970).
Meigs, Sciarini, and Van Sandt (1954) suggested that environmental
conditions of high air temperature and high relative humidity influence
the quantity of benzidine, 3,3'-dimethoxybenzidine, 3,3'-dichlorobenzidine,
and 3,3'-dimethylbenzidine which penetrates the skin. Men who perspired
freely, thus having wet skin, had higher amounts of benzidine in their urine,
indicating a more rapid skin penetration, than men whose skin was less wet.
Benzidine skin penetration was higher on hot days than on cool days, as
indicated by increased benzidine urine concentrations during high temper-
atures (Table 6.1).
6.2.2 Distribution and Accumulation
No information was found on direct measurements made to determine the
distribution and accumulation in human tissue of benzidine or its deriva-
tives. However, indirect evidence afforded by the measurements of benzi-
dine in urine samples from occupationally exposed people (Meigs, Brown,
and Sciarini, 1951) indicates that no appreciable accumulation occurs with
this compound.
TABLE 6.1. RELATIONSHIP BETWEEN ENVIRONMENTAL CONDITIONS AND BENZIDINE
CONCENTRATIONS IN URINE OF SIX PRESSROOM WORKERS
Environmental
conditions
Cool, dry days
Hot, humid days
Mean
temperature
23
21
34
25
Mean
relative
humidity
63
58
85
78
Mean urinary
specific gravity
1.026
1.024
1.027
1.025
Mean
quinonizable
substance
expressed as
benzidine
(mg/liter)
0.313
0.362
0.668
0.895
Source: Adapted from Meigs, Sciarini, and Van Sandt, Arch. Ind. Hyg.
Occup. Med., Vol. 9, Table 6, p. 129. Copyright 1954, American Medical
Association.
-------
84
Studies made with experimental animals, discussed below, suggest that
benzidine and its derivatives are distributed fairly rapidly into various
tissues following uptake but do not remain for any great length of time
before elimination. Prior to elimination, these compounds or their metab-
olites appear to concentrate in some tissues more than others.
According to Solimskaya (1968), benzidine was rapidly absorbed within
1 hr of injection into rats. Maximum concentrations of free and bound ben-
zidine were detected at 2 and 3 hr, respectively, after injection. In
tissues other than blood, concentrations were higher in liver and kidney
than in spleen, heart, and lung.
Kellner, Christ, and Lotzsch (1973) followed the disappearance of
^C-labeled benzidine and 3,3'-dichlorobenzidine in the blood of rats and
dogs after single, intravenous injections of each compound at the rate of
0.2 mg/kg body weight. Half-lives of the labeled compounds in blood were:
benzidine, 65 hr in the rat, 88 hr in the dog; dichlorobenzidine, 68 hr in
the rat, 86 hr in the dog. As shown in Figures 6.1 and 6.2, blood concen-
trations of both compounds decreased relatively steadily, but the most
rapid decreases occurred during the first 24 hr after injection.
In the same study, several measurements were made of the concentra-
tions of the two compounds in various tissues of the dog, rat, and monkey.
ORNL-DWG 77-9684
i BENZIDINE (n = 8)
$ 3,3'-DICHLOROBENZIDINE (n = 8)
1234567
TIME (days)
Figure 6.1. Concentration in blood after intravenous administration of
0.2 mg of 1AC-labeled 3,3'-dichlorobenzidine or benzidine per kilogram body
weight to rats. Mean values and 95% confidence intervals. Source: Adapted
from Kellner, Christ, and Lotzsch, 1973, Figure 1, p. 65. Reprinted by
permission of the publisher.
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85
ORNL-DWG 77-9685
J BENZIDINE (n = 3)
5 3,3'-DICHLOROBENZIDINE (n = 3)
23456
TIME (doys)
Figure 6.2. Concentration in blood after intravenous administration of
0.2 mg of li'C-labeled 3,3'-dichlorobenzidine or benzidine per kilogram body
weight to dogs. Mean values and 95% confidence intervals. Source: Adapted
from Kellner, Christ, and Lotzsch, 1973, Figure 2, p. 65. Reprinted by
permission of the publisher.
The results obtained are summarized in Tables 6.2 and 6.3. The only sig-
nificant difference between the tissue concentrations of benzidine and
dichlorobenzidine was in the urinary bladder wall of dogs at 4 hr after
administration; this difference was thought to be caused by contamination
of the wall with urine. At 4 hr after administration, concentrations of
both compounds tended to be elevated in lung tissue of rats and in liver
tissue of dogs. At 7 to 14 days after administration, elevated concentra-
tions of benzidine were found in liver tissue of the rat, dog, and monkey
and in lung tissue of the monkey. A somewhat similar pattern of distri-
bution was found for dichlorobenzidine; elevated concentrations occurred
in liver tissue of the rat, dog, and monkey and in lung tissue of the dog
and monkey.
Baker and Deighton (1953) gave rats intraperitoneal injections of
benzidine, 100 mg/kg body weight, and measured the concentrations found
in various tissues and urine at 4 and 12 hr after injection. Concentra-
tions, expressed as diazotizable amino groups, are given in Table 6.4.
Highest concentrations of total diazotizable material were found in the
stomach, stomach contents, and small intestine at 4 hr and in the small
intestine and small intestine contents at 12 hr. Lowest concentrations
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86
TABLE 6.2. DISTRIBUTION PATTERN 1 AND 4 hr AFTER INTRAVENOUS ADMINISTRATION OF 0.2 mg OF
"C-LABELED 3,3'-DICHLOROBENZIDINE OR BENZIDINE PER KILOGRAM BODY WEIGHT TO RATS AND DOGS
Concentration in rat tissues
Tissue
Stomach
Small intestine
Large intestine
Kidney
Ureter
Urinary bladder
Liver
Bile
Pancreas
Spleen
Adrenals
Gonads , male
Lung
Heart
Skeletal muscle
Retroperitoneal fat
Subcutaneous fat
Brain
Mesentery lymph nodes
Blood
Plasma
Mean
value
0.13
0.26
0.13
0.27
0.005
0.22
0.81
0.13
0.14
0.24
0.088
0.91
0.14
0.093
0.061
0.070
0.033
0.10
0.16
0.22
1 hr
95%
confidence
interval
0.02
0.11
0.02
0.03
to 0.088
0.11
0.22
0.02
0.02
0.05
0.012
0.18
0.03
0.019
0.010
0.015
0.005
0.03
0.06
0.07
Mean
value
Benzidine
0.050
0.26
0.26
0.13
0.001
0.18
0.53
0.044
0.067
0.086
0.030
0.90
0.053
0.030
0.021
0.024
0.011
0.036
0.066
0.092
4 hr
95%
confidence
interval
0.010
0.05
0.12
0.03
to 0.035
0.08
0.11
0.011
0.017
0.024
0.011
0.29
0.017
0.006
0.008
0.006
0.004
0.013
0.027
0.031
Concen
tissue
No. 4
0.094
0.20
0.46
0.24
0.32
2.2
0.79
30
0.036
0.12
0.081
0.031
0.11
0.044
0.030
0.044
0.060
0.013
0.061
0.076
0.12
tration
s, 4 hr
No. 5
0.15
0.23
0.24
0.32
0.28
2.3
0.78
19
0.053
0.16
0.096
0.041
0.12
0.046
0.026
0.088
0.076
0.015
0.068
0.10
0.16
in dog
(yg/g)
No. 6
0.057
0.080
0.22
0.21
0.24
3.3
0.56
7.9
0.043
0.11
0.080
0.063
0.068
0.043
0.054
0.085
0.043
0.013
0.053
0.058
0.094
3,3'-Dichlorobenzidine
Stomach
Small intestine
Large intestine
Kidney
Ureter
Urinary bladder
Liver
Bile
Pancreas
Spleen
Adrenals
Gonads , male
Lung
Heart
Skeletal muscle
Retroperitoneal fat
Subcutaneous fat
Brain
Mesentery lymph nodes
Blood
Plasma
0.020
0.55
0.032
0.096
0.11
0.030
0.20
0.045
0.024
0.24
0.037
0.41
0.032
0.016
0.12
0.12
0.014
0.057
0.025
0.041
0.006
0.18
0.008
0.024
0.02
0.007
0.05
0.018
0.005
0.13
0.010
0.16
0.010
0.002
0.05
0.04
0.005
0.012
0.004
0.007
0.015
1.1
0.33
0.078
0.04
0.049
0.22
0.013
0.012
0.086
0.018
0.28
0.014
0.007
0.018
0.017
0.005
0.014
0.016
0.028
0.006
0.8
0.29
0.011
0.02
0.043
0.09
0.004
0.002
0.037
0.003
0.10
0.004
0.003
0.012
0.008
0.003
0.003
0.005
0.008
0.025
0.087
1.0
0.078
0.072
0.068
0.35
38
0.018
0.013
0.050
0.018
0.073
0.017
0.017
0.093
0.092
0.035
0.013
0.026
0.096
0.56
0.80
0.046
0.074
0.081
0.28
36
0.032
0.022
0.032
0.020
0.044
0.015
0.016
0.056
0.044
0.013
0.049
0.016
0.031
0.024
0.23
0.49
0.041
0.089
0.032
0.23
68
0.070
0.014
0.045
0.017
0.057
0.021
0.013
0.18
0.089
0.012
0.12
0.010
0.019
Source: Adapted from Kellner, Christ, and Lotzsch, 1973, Table 4, pp. 75-76. Reprinted by
permission of the publisher.
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TABLE 6.3. DISTRIBUTION PATTERN 7 AND 14 DAYS AFTER INTRAVENOUS ADMINISTRATION OF 0.2 mg OF ^C-LABELED
3,3'-DICHLOROBENZIDINE OR BENZIDINE PER KILOGRAM BODY WEIGHT TO RATS, DOGS, AND MONKEYS
Concentration in wet tissues (pg/g)
Rat
Tissue
7
Mean
value
days
95%
confidence
interval
Mean
value
14 days
Dog, 7 days
95%
confidence
interval
No. 1
No. 2
No. 3
Monkey
7 days
No. 1
No. 2
14 days,
No. 3
Benzidine
Kidney
Ureter
Urinary bladder
Liver
Bile
Spleen
Adrenals
Gonads , male
Lung
Kidney
Ureter
Urinary bladder
Liver
Bile
Spleen
Adrenals
Gonads , male
Lung
0.009
0.001
0.005
0.042
0.005
0.004
<0.001
0.005
0.006
0.004
0.013
0.004
0.015
0.002
0.004
<0.001
<0.001
<0.001
0.002
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
0.003
<0.001
<0.001
0.006
0.002
0.006
0.022
0.010
0.003
0.001
0.005
3
0.002
0.002
0.004
0.003
0.006
<0.001
0.002
<0
<0
<0
0
0
<0
<0
.001
.001
.001
.004
.001
.001
.001
0.012
0.001
0.007
0.12
0.086
0.029
0.016
0.002
0.007
0.013
0.002
0.007
0.19
0.13
0.029
0.010
0.002
0.010
0.020
0.003
0.007
0.087
0.13
0.030
0.016
0.008
0.011
0.007
0.027
0.003
0.012
0.008
0.001
0.003
0.010
0.007
0.002
0.002
0.005a
0.010
0.004
0.002
0.002
0.011
0.004
0.003
0.003
0.001
0.011
, 3 ' -Dichlorobenzidine
0
<0
<0
<0
<0
<0
.001
.001
.001
.001
.001
.001
0.014
0.004
0.005
0.062
0.60
0.004
0.004
0.002
0.036
0.010
0.003
0.003
0.040
0.18
0.005
0.005
0.002
0.023
0.007
0.003
0.003
0.044
0.13
0.004
0.005
0.002
0.031
0.018
0.011
0.015
0.042
0.055
0.012
0.016
0.006
0.012
0.009
0.003
0.018
0.032
0.019
0.006
0.004
0.001
0.013
a.
Female.
Source: Adapted from Kellner, Christ, and LStzsch, 1973, Table 2, pp. 72-73. Reprinted by permission of the
publisher.
oo
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TABLE 6.4. THE CONCENTRATION AND DISTRIBUTION OF FREE AND TOTAL DIAZOTIZABLE MATERIAL IN RAT TISSUES FOLLOWING
A SINGLE INTRAPERITONEAL INJECTION OF 100 mg BENZIDINE PER KILOGRAM OF BODY WEIGHT
Concentration after 4
Tissue
Whole blood
Plasma
Blood cells
Liver
Spleen
Kidney
Stomach
Stomach contents
Small intestine
Small intestine contents
Caecum
Caecum contents
Colon
Urine
Miscellaneous
Carcass
Total
(ug/g)
16.2
32.5
8.7
16.2
21.2
21.2
275.8
310.0
81.2
2.5
2.0
3.7
5.4
1.7
81.2
Free
(% of total)
0.3
1.1
1.7
0.2
0.2
8.3
15.6
13.0
0.1
0.1
0.2
0.3
0.3
28.8
69.2
(ug/g)
35.0
70.0
11.2
45.0
46.2
46.2
310.0
315.0
157.5
21.2
11.4
21.2
10.2
21.2
82.5
hr
Total
(% of total)
0.7
0.1
4.8
0.5
0.5
9.3
15.8
25.2
0.6
0.6
1.6
0.5
3.8
29.3
93.3
Concentration after 12
(Pg/g)
<1.0
<1.0
3.8
11.3
6.3
2.5
20.0
54.0
26.3
134.0
13.8
0.1
1.0
28.0
1.0
8.8
Free
(% of total)
<0.1
<0.1
1.6
0.1
0.1
0.6
2.7
2.2
6.7
0.3
<0.1
0.1
1.4
0.2
3.2
19.20
(yg/g)
17.5
15.0
5.0
25.0
17.5
11.2
80.0
85.0
135.0
560.0
75.0
15.0
25.0
50.0
11.2
36.2
hr
Total
(% of total)
0.2
0.1
3.5
0.2
0.2
2.4
4.3
11.5
28.0
1.9
0.8
1.3
2.5
2.0
13.5
72.4
The miscellaneous sample consisted of brain, diaphragm, bladder, gonads, esophagus, heart, thymus, lungs, and adrenals.
Source: Adapted from Baker and Deighton, 1953, Table 1, p. 531. Reprinted by permission of the publisher.
oo
oo
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89
were found in the red blood cells at both times. The measured, total diaz-
otizable material accounted for 93% of the administered dose at 4 hr and
72% at 12 hr. Conjugated forms of the diazotizable material rose from 24%
at 4 hr to 53% at 12 hr, indicating the formation of appreciable levels of
metabolites. The content of total diazotizable material in urine was con-
siderably higher at 12 hr than at 4 hr, with proportionately higher amounts
of conjugated amines present. It was suggested that the difference between
the amount of material injected and that measured during the 12-hr period
was caused by in vivo oxidation to products not detectable by the method of
measurement used. Since there is evidence that benzidine causes tumors in
the liver of some animals, it may be of importance that measured concentra-
tions in the liver were relatively high and remained fairly constant over
the 12-hr period.
Pliss and Zabezhinsky (1970) reported on the distribution of free and
bound amines in the tissues of rats given 20 mg of 3,3'-dimethylbenzidine
subcutaneously once per week for eight weeks. Three days after the final
injection the highest amine content was found in the Zymbal's gland with
lesser amounts found in the kidney, omentum, spleen, and liver. A higher
incidence of tumor development in the Zymbal's gland was observed in rats
administered 3,3'-dimethyIbenzidine.
Although no data that directly concern placental transfer of benzi-
dine or its derivatives were found, studies made by Golub (1969) and Shabad
et al. (1972) indicated transplacental effects of 3,3'-dichlorobenzidine
and 3,3'-dimethyIbenzidine on cultured embryonic kidney tissue. The tis-
sue was obtained from pregnant mice treated with the benzidine derivatives
at concentrations of 8 to 10 mg. Both derivatives promoted a longer sur-
vival rate of tissue and induced epithelial cell hyperplasia.
Although there is no evidence in the literature of any significant
bioaccumulation of benzidine or its derivatives in human tissue or in
tissues of the usual experimental animals, the Federal Register1 (1976)
recently reported that edible portions of bluegill fish contained a 44-
fold increase in residues of 14C-benzidine over those present in water
during a radiometric study.
6.2.3 Biotransformation and Elimination
Benzidine and its derivatives are wholly or partly transformed in
humans and experimental animals to a number of metabolites, some of which
are thought to act as proximate carcinogens in the induction of tumor
formation. The metabolites, as well as the nontransformed parent
compounds, are eliminated from the animal body via the urine and feces.
A summary of the biotransformation products of benzidine and its
derivatives is provided in Table 6.5. The closely related compounds 4-
aminobiphenyl and 4-nitrobiphenyl are included because of their structural
and chemical similarities to benzidine and similar carcinogenic behavior
in humans and animals. The specific chemistries of benzidine, its deriva-
tives, and related compounds are covered in Section 2.2.
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90
TABLE 6.5. METABOLITES FORMED BY BIOTRANSFOKMATION OF BENZIDINE AND BENZIDINE DERIVATIVES IN ANIMALS
Compound
Species
Metabolite
Reference
Benzidine
Human Acetyl N-hydroxy compound
Human N-Hydroxy acetylaminobenzidine
Human Monoacetylbenzidine and
diacetylbenzidine
Human 3-Hydroxybenzidine
Human 3,3'-Dihydroxybenzidine
Monkey Monoacetylbenzidine
Dog 3-Hydroxybenzidine sulfate
and glucuronide
Dog 3-Hydroxybenzidine hydrogen sulfate
Dog 3-Hydroxybenzidine
Dog 4,4'-Diamino-3-diphenylyl hydrogen
sulfate
Dog 4-Amino-4-hydroxybiphenyl
Dog Monoacetylbenzidine and
diacetylbenzidine
Guinea pig 4'-Acetamido-4-aminodiphenyl
Guinea pig 4'-Acetamido-4-amino-3-diphenylyl
hydrogen sulfate
Guinea pig 4'-Amino-4-diphenylyl sulfamic acid
Guinea pig N-Glucuronides
Guinea pig 4'-Acetamldo-4-diphenylyl sulfamic
acid
Rabbit 3-Hydroxybenzidine sulfate and
glucuronide
Rabbit 4'-Acetamido-4-aminodiphenyl
Rabbit 3-Hydroxybenzidine
Rabbit 4'-Acetamido-4-amino-3-diphenylyl
hydrogen sulfate
Rabbit 4'-Amino-4-diphenylyl sulfamic acid
Rabbit 4'-Acetamido-4-diphenylyl sulfamic
acid
Rabbit N-Glucuronides
Rat 3,3'-Dihydroxybenzidine
Rat N-Glucuronides
Rat 4'-Acetamido-4-aminodiphenyl
Rat 3-Hydroxybenzidine
Rat 4,4'-Diamino-3-diphenylyl hydrogen
sulfate
Rat 4'-Acetamido-4-amino-3-diphenylyl
hydrogen sulfate
Rat 4'-Amino-4-diphenylyl sulfamic acid
Rat 4'-Acetamido-4-diphenylyl sulfamic
acid
Mouse Monoacetylbenzidine and
diacetylbenzidine
Mouse Monoacetylated 3-hydroxybenzidine
glucuronide and/or ethereal sulfate
Mouse N-Hydrogen sulfate and/or glucuronide
Mouse 3-Hydroxybenzidine glucuronide
Mouse 4'-Acetamido-4-aminodiphenyl
Mouse 4,4'-Diamino-3-diphenylyl hydrogen
sulfate
Mouse 4'-Acetamido-4-amino-3-diphenylyl
hydrogen sulfate
Mouse N-Glucuronides
Troll, Belman, and Rinde, 1963
Haley, 1975
Haley, 1975
Haley, 1975
Haley, 1975
Rinde and Troll, 1975
Troll and Nelson, 1958
Sciarini and Meigs, 1958
Bradshaw and Clayson, 1955
Sciarini, 1957
Clayson, Ward, and Ward, 1959
Haley, 1975
Haley, 1975
Clayson, Ward, and Ward, 1959
Clayson, Ward, and Ward, 1959
Clayson, Ward, and Ward, 1959
Clayson, Ward, and Ward, 1959
Clayson, Ward, and Ward, 1959
Troll and Nelson, 1958
Clayson, Ward, and Ward, 1959
Clayson, Ward, and Ward, 1959
Clayson, Ward, and Ward, 1959
Clayson, Ward, and Ward, 1959
Clayson, Ward, and Ward, 1959
Clayson, Ward, and Ward, 1959
Haley, 1975
Elson, Goulden, and Warren,
1958
Clayson, Ward, and Ward, 1959
Clayson, Ward, and Ward, 1959
Clayson, Ward, and Ward, 1959
Clayson, Ward, and Ward, 1959
Clayson, Ward, and Ward, 1959
Clayson, Ward, and Ward, 1959
Clayson, Ward, and Ward, 1959
Sciarini and Meigs, 1961<2
Sciarini and Meigs, 1961a
Sciarini and Meigs, 1961a
Sciarini and Meigs, 1961a
Clayson, Ward, and Ward, 1959
Clayson, Ward, and Ward, 1959
Clayson, Ward, and Ward, 1959
Clayson, Ward, and Ward, 1959
(continued)
-------
91
TABLE 6.5 (continued)
Compound
3,3'-dimethyl-
benzidine
(orthotolldine)
3,3'-Dimethoxy-
benzidine
(dianisidlne)
3-Methoxybenzidine
(mono-substituted
dianisidine)
4-Aminobiphenyl
4-Diphenylacet amide
4-Nitrobiphenyl
Species
Human
Human
Human
Dog
Dog
Rat
Monkey
Monkey
Monkey
Dog
Dog
Dog
Dog
Rat
Monkey
Monkey
Dog
Dog
Metabolite
Diacetyl-o-tolidine
5-Hydroxy-o-tolidine
Monoacetyl-o-tolidine
5-Ethereal sulfate of o-tolidine
Unidentified diamine metabolite
4-Amino-4 '~acetamido-3-methoxybiphenyl
N-Hydroxy-4-aminobiphenyl
4-Nitrosobiphenyl
N-Hydroxy-4-acetylaminobiphenyl
glucuronide
4-Amino-3-diphenylyl hydrogen sulfate
N-Hydroxy-4-aminobiphenyl
4-Nitrosobiphenyl
4-Amino-3-biphenylyl glucuronic acid
N-Hydroxy-4-diphenylacetamide
N-Hydroxy-4-aminobiphenyl
4-Nitrosobiphenyl
N-Hydroxy-4-aminobiphenyl
4-Nitrosobiphenyl
Reference
Dieteren, 1966
Dieteren, 1966
Dieteren, 1966
Sciarini and Meigs , 196L&
Sciarini and Meigs, 1961&
Laham, 1971
Radomski et al. , 1973
Radomski et al., 1973
Radomski et al. , 1973
Bradshaw and Clayson, 1955
Radomski et al . , 1973
Radomski et al., 1973
Gorrod, 1971, as cited in
International Agency for
Research on Cancer, 1972
Miller et al., 1961, as cited
in International Agency for
Research on Cancer, 1972
Radomski et al . , 1973
Radomski et al., 1973
Radomski et al. , 1973
Radomski et al. , 1973
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92
6.2.3.1 Human Studies — Englebertz and Babel (1953, as cited by Haley,
1975) identified free benzidine and its monoacetylated and diacetylated
metabolites in the urine of a person receiving a single oral dose of
100 mg of benzidine. Not all of the dose received was recovered in the
urine, indicating some additional fecal excretion or body accumulation.
Troll, Belman, and Rinde (1963) identified free benzidine and N-hydroxy
acetyl amino benzidine in the urine of persons receiving 200 mg of benzi-
dine orally. In the analysis of urine from plant workers exposed to
benzidine, Sciarini and Meigs (1961a) also found free benzidine and the
monoacetylated and diacetylated derivatives and, in addition, found 3-
hydroxybenzidine. The latter metabolite was the major excretion product,
amounting to 78.5% to 89.7% of the total.
Total urinary excretion in the study by Sciarini and Meigs (1961a)
was greater in August than in March, indicating greater uptake of benzi-
dine by the workers during the warmer month of August. These results con-
firmed an earlier study made by Meigs, Brown, and Sciarini (1951) of plant
workers exposed to benzidine. As shown in Figure 6.3, urinary excretion in
this study was higher in June (mean for 12 men was 1.48 mg/liter) than in
December (mean for 12 men was 0.433 mg/liter). Control urine from univer-
sity or plant personnel entirely away from benzidine operations contained
LJ
z
Q
N
Z
iLJ
03
co
co
co
UJ
cr
Q.
X
ORNL-OWG 77-9686
•s -
CO
CD
CO
Id
CD
z
o
z
o
1.5
~ 1.0
0.5
JUNE 1950
MEAN
/ \
DECEMBER 1950 / \
6:30 AM
NOON 4 PM
TIME OF DAY
PM
Figure 6.3. Trends of mean urinary concentration of quinonizable
substance (expressed as benzidine) at different times of day over two-
week periods in June and December 1950. Source: Adapted from Meigs,
Brown, and Sciarini, Arch. Ind. Hyg. Occup. Med. , Vol. 4, Figure 4,
p. 539. Copyright 1951, American Medical Association.
-------
93
no detectable benzidine. The higher urinary concentrations detected in
June possibly were caused by greater concentrations of benzidine in the
work area air. In a later study, Meigs, Sciarini, and Van Sandt (1954)
suggested that air contaminated with 0.018 mg of benzidine compounds per
cubic meter would lead, if inhaled for 8 hr, to a urinary concentration of
not more than 0.026 mg/liter of diamines in urine voided at the end of a
work shift. Hence, the authors concluded that a safe level of air expo-
sure was probably 0.02 mg or less of benzidine per cubic meter of air.
Workers exposed to benzidine in a dyestuff factory were found by
Vigliani and Barsotti (1962) to excrete, via the urine, benzidine and
smaller amounts of 4-amino-4-oxybiphenyl and monoacetylbenzidine. No
significant differences in urinary excretion were found between summer
and winter periods of exposure.
Dieteren (1966) analyzed urine from chemical plant workers exposed
to 3,3'-dimethylbenzidine (diorthotolidine) indicated the presence of free
diorthotolidine, diacetyl-o-tolidine, and a hydroxyamino compound thought
to be 5-hydroxy-o-tolidine. Although monoacetyl-o-tolidine could not be
identified, there remained the possibility of its presence. These results
indicate that in humans hydroxylation of diorthotolidine occurs in the
ortho position with respect to the amino group and that, in this respect,
the metabolisms of diorthotolidine and benzidine are similar.
Akiyama (1970) reported that workers handling benzidine yellow excreted
3,3'-dichlorobenzidine in their urine. This derivative of benzidine is part
of the benzidine yellow molecule and presumably is liberated upon reductive
metabolism of the dye.
6.2.3.2 Comparative Experimental Animal Studies — The biotransformation
of benzidine to 3-hydroxybenzidine and to monoacetylated and diacetylated
derivatives, as found in humans, also occurs in some experimental animals.
Sciarini and Meigs (1961a) found that mice given benzidine intraperitone-
ally (100 mg/kg body weight) excreted in their urine the free benzidine,
the monoacetylated and diacetylated metabolites, and, at somewhat higher
concentrations, the ethereal sulfates and glucuronates of 3-hydroxybenzi-
dine. Earlier, Sciarini (1957) found that dogs given benzidine intraperi-
toneally (100 mg/kg body weight) excreted free benzidine and conjugated
3-hydroxybenzidine. Sciarini and Meigs (1958) identified the conjugated
metabolite as the ethereal sulfate of 3-hydroxybenzidine, which was recov-
ered from the urine of dogs at rates of 25% to 50% of the intraperitoneal
dose. Troll and Nelson (1958) reported that the major metabolites of ben-
zidine in the dog and rabbit were the sulfate and glucuronide of 3-hydroxy-
benzidine. No unesterified hydroxy derivatives were found in dogs injected
intraperitoneally with 1 g benzidine dihydrochloride or in rabbits given
100 to 300 mg of this compound. The metabolites were thought to result
largely from ortho oxidation.
Differences appear among species of experimental animals in the metab-
olism and excretion of benzidine and its derivatives. Clayson, Ward, and
Ward (1959) gave benzidine and 4'-acetamido-4-aminodiphenyl orally to dogs
and rabbits and intraperitoneally to rats, mice, and guinea pigs. A sum-
mary of the metabolites produced by these species is given in Table 6.6.
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94
TABLE 6.6. THE URINARY AND BILIARY METABOLITES OF BENZIDINE PRODUCED
BY VARIOUS EXPERIMENTAL ANIMALS
Compound
Dog
Dog
Rat
Mouse
Rabbit
Cavy
Urinary
Benzidine
4'-Acetamido-4-aminodiphenyl
3-Hydroxybenzidine (ether extracts)
4,4'-Diamino-3-diphenylyl hydrog-en sulfate
4'-Acetamido-4-amino-3-diphenylyl hydrogen
sulfate
4'-Amino-4-diphenylyl sulfamic acid
4'-Acetamido-4-diphenylyl sulfamic acid-
N-Glucuronides
Acid stable unknowns
Benzidine
4'-Acetamido-4-aminodiphenyl
4,4'-Diamino-3-diphenylyl hydrogen sulfate
4'-Acetamido-4-amino-3-diphenylyl hydrogen
sulfate
4'-Amino-4-diphenylyl sulfamic acid
4'-Acetamido-4-diphenylyl sulfamic acid
N-Glucuronides
Acid stable unknowns
Biliary
+
+
+
+
4'-Acetamido-4-aminodiphenyl administered.
Source: Adapted from Clayson, Ward, and Ward, 1959, Tables I and II, pp. 582-583.
Reprinted by permission of the publisher.
Dog urine contained free benzidine, 3-hydroxybenzidine, 4,4'-diamino-3-
diphenylyl hydrogen sulfate, and N-glucuronides. Rat urine contained the
same metabolites as well as 4?-acetamido-4-aminodiphenyl, 4'-acetamido-4-
amino-3-diphenylyl hydrogen sulfate, 4'-amino-4-diphenylyl sulfamic acid,
and three acid stable unknowns. Mouse urine contained only free benzidine
and 4'-acetamido-4-amino-3-diphenylyl hydrogen sulfate. Rabbit urine con-
tained the latter compound as well as 4'-amino-4-diphenylyl sulfamic acid,
4'-acetamido-4-diphenylyl sulfamic acid, and N-glucuronides. Guinea pig
urine contained only 4'-acetamido-4-amino-3-diphenylyl hydrogen sulfate
and N-glucuronides. These results suggest two major differences in benzi-
dine metabolism among the species tested: (1) the dog was the only species
tested unable to acetylate benzidine, and (2) the rabbit, rat, and guinea
pig could convert benzidine to 4'-amino- and 4'-acetamido-4-diphenylyl
sulfamic acid, whereas the dog and mouse could not.
-------
95
Troll and Nelson (1958) found differences between dogs and rabbits
in the excretion of amine metabolites of benzidine following intraperi-
toneal injection of benzidine. Dogs excreted 80% or more of the dose as
compounds with an available aromatic amino group, whereas rabbits excreted
only 50% of the dose as the same compounds. Also, dogs excreted relatively
more of the sulfate conjugate and an unidentified amine fraction that was
ether extractable.
Some differences in the amounts of excretion products found may result
from varying periods of in vivo oxidation following administration. Thus,
Baker and Deighton (1953) gave rats 100 mg benzidine per kilogram of body
weight by intraperitoneal injection and, by analysis of the diazotizable
material in tissues and urine 4 and 12 hr later, found that recovered amino
groups rose from 24% to 49% in tissues over the 8-hr period and from 0.5%
to 2.5% in urine over the same period. Sciarini and Meigs (1958) found
that with decreasing dosage levels of benzidine given intraperitoneally
to dogs the detection time in urine was comparatively decreased. A dose
of 100 mg/kg body weight was detected in the urine for as long as seven
days, whereas a dose of 20 yg/kg body weight was detected only in the
first morning void.
Differences appear in the type and quantity of metabolites eliminated
by different routes (i.e., urine, feces, and bile). In the work discussed
above by Clayson, Ward, and Ward (1959), dog bile contained qualitatively
the same metabolites as urine but in one-third the amount. Dog feces did
not contain 3-hydroxybenzidine or the N-glucuronides present in the urine
and bile.
Kellner, Christ, and Lb'tzsch (1973) determined that substitution of
benzidine in the 3,3' positions affects the route of excretion in several
animal species. Rats, dogs, and monkeys were injected intravenously with
^C-labeled benzidine and 3,3'-dichlorobenzidine (2 mg/kg body weight). As
shown in Table 6.7, differences in the main route of excretion were more
apparent for benzidine than for dichlorobenzidine. In the rat, both benzi-
dine and dichlorobenzidine were excreted in greater quantities in the feces
than in the urine. In the dog, fecal excretion of dichlorobenzidine was
again greater than urinary excretion, but fecal excretion of benzidine was
much less than urinary excretion. In the monkey, neither route decisively
excreted more of either compound, although there was a trend toward greater
excretion of both compounds via the urine. Sciarini and Meigs (1961&) also
found that dichlorobenzidine excretion by dogs was greater (by some ten
times) in feces than in urine, and, in an earlier study (Sciarini and Meigs,
1958), they found just the opposite pattern for benzidine (i.e., some ten-
fold greater amounts excreted via the urine).
Biliary secretion is an important route of elimination. A portion of
the products of benzidine metabolism in rats given intraperitoneal injec-
tions, 100 mg/kg body weight, was excreted by the bile to the stomach and
intestinal tract (Baker and Deighton, 1953). As shown in Table 6.6, Clayson,
Ward, and Ward (1959) detected some six metabolites of benzidine in bile of
several animal species, but in only one-third the quantity found in urine.
Kellner, Christ, and Lb'tzsch (1973) found benzidine and dichlorobenzidine
in the bile of dogs and monkeys seven days after intravenous injection,
-------
TABLE 6.7. EXCRETION IN THE FIRST SEVEN DAYS AFTER INTRAVENOUS ADMINISTRATION OF 0.2 mg OF 1;
-------
97
but not in the bile of rats after an identical administration. However,
the biliary concentrations found were low as compared with those found in
urine or feces.
Rinde and Troll (1975) reported that the rhesus monkey can metabol-
ically reduce a number of benzidine-based azo dyes to free the amine. They
found substantial amounts of benzidine and monoacetylbenzidine in the urine
of animals fed single doses of these dyes. This finding is in agreement
with the finding by Akiyama (1970) (Section 6.2.3.1) that workers occupa-
tionally exposed to benzidine yellow (derived from 3,3'-dichlorobenzidine)
excreted dichlorobenzidine in their urine, presumably through metabolic
reduction of this dye.
The metabolism and excretion of benzidine derivatives and related
compounds have been less extensively studied than that of benzidine.
Sciarini and Meigs (19612?) injected dogs intraperitoneally with 3,3'-
dimethylbenzidine (diorthotolidine), 3,3'-dimethoxybenzidine (dianisidine),
and 3,3'-dichlorobenzidine. Administered levels of these compounds were
only partly accounted for by measured, urinary excretory products. All
three diamines were recovered partly in their nonmetabolized form. A
metabolite of diorthotolidine, thought to be the 5-ethereal sulfate, was
recovered at concentrations ranging from 15% to 40% of the administered
dose. A metabolite of dianisidine was also recovered, but at a relatively
lower percentage yield. However, no metabolite of dichlorobenzidine was
found. Laham (1971) detected an acetyl derivative, 4-amino-4'-acetamido-
3-methoxybiphenyl, in the urine of rats following injection of 3-methoxy-
benzidine, the mono-substitute of dianisidine. Radomski et al. (1973)
found 4-nitrosobiphenyl and N-hydroxy-4-aminobiphenyl in the urine of dogs
and rhesus monkeys fed 5 mg of 4-aminobiphenyl per kilogram body weight.
Monkey urine contained a third metabolite, N-hydroxy-4-acetylaminobiphenyl
glucuronide. Urinary excretion of the N-hydroxy metabolites was more rapid
in both animals than that of the administered 4-aminobiphenyl, with the max-
imum excretion occurring at 4 to 6 hr (Figure 6.4). Bradshaw and Clayson
(1955) fed dogs 200 mg/day of 4-aminobiphenyl and recovered the parent com-
pound and a metabolite, 3-hydroxy-4-aminobiphenyl from the urine. Bradshaw
(1959) calculated recoveries in the urine of several species of animals fed
4-aminobiphenyl. The yield of the 3-sulfuric acid conjugate measured in
the urine was 25% to 40% in dogs, 3% to 7% in rats, 1% to 2% in hamsters,
1% in mice, and 0% in guinea pigs.
6.3 EFFECTS
As noted by Clayson (1976) , there is convincing evidence that benzi-
dine is a bladder carcinogen in humans and that benzidine and several deriv-
atives are carcinogenic in experimental animals. In addition to their
carcinogenic potential, there is also evidence that these compounds cause
dermatitis, cystitis, and hematuria in humans and a number of additional
effects in experimental animals. A summary of these effects is provided
in Table 6.8.
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98
ORNL-DWG 77-9687
2 -
en
E
Q
LU
H
iLl
o:
o
x
o
s
<
1 -
0
N-HYDROXY-4-
AMINOBIPHENYL
N-HYDROXY-4-
ACETYLAMINOBIPHENYL
0
10
12
TIME ( hr!
Figure 6.4. Comparative excretion of N-oxidation products of 4-amino-
biphenyl by monkeys and dogs. Source: Adapted from Radomski et al., 1973,
Figure 2, p. 992.
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99
TABLE 6.8. EFFECTS OF BENZIDINE, ITS CONGENERS,
AND METABOLITES ON VARIOUS ANIMAL SPECIES
Species
Carcinogen
Effect
Mouse Benzidine
3,3'-Dihydroxybenzidine
Rat
Hamster
Rabbit
Dog
Benzidine and its sulfate
3,3'-Dihydroxybenzidine
Dianisidine
o-Ditoluidine
3,3'-Benzininedioxyacetic
3,3'-Dichlorobenzidine
N,N'-Diacetylbenzidine
Benzidine
O-Ditoluidine
Benzidine
Benzidine
Monkey Benzidine
Human Benzidine
Hepatoma, lymphoma, bile duct
proliferation
Hepatoma, lymphoma, bile duct
proliferation, benign blad-
der papilloma
Cirrhosis of liver, hepatomas,
carcinoma of Zymbal's gland,
adenocarcinoma, degeneration
of bile ducts, sarcoma, mam-
mary gland carcinoma
Hepatoma, adenocarcinoma of
colon, carcinoma of fore-
stomach, Zymbal's gland
carcinoma, bladder carcinoma
Zymbal's gland carcinoma,
ovarian tumor
Papilloma of stomach, Zymbal's
gland carcinoma, mammary
tumor, leukemia
Papilloma of bladder, hepatic
sarcoma
Extensive cancer
Chronic glomerulonephritis
Hepatoma, liver carcinoma,
cholangiomas
Bladder cancer
Proteinuria, hematuria, liver
cirrhosis, myocardial atro-
phy, bladder tumor, gall
bladder tumor
Recurrent cystitis, bladder
tumor, convulsions, liver
cirrhosis, hematuria
No pathological changes
Bladder tumor, papilloma,
chronic cystitis, hematuria
a
,3,3'-Dimethoxybenzidine.
3,3'-Dimethylbenzidine.
Source: Adapted from Haley, 1975, Table 5, p. 24.
permission of the publisher.
Reprinted by
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100
6.3.1 Physiological Effects
During in vitro studies on benzidine, its derivatives, and related
compounds, Hirai and Yasuhira (1972) found a possible relationship between
the potential carcinogenicity of these compounds and their ability to reduce
cytochrome o. 3,3'-Dichlorobenzidine and p-aminobiphenyl failed to reduce
cytochrome o; benzidine, 3,3'-dimethylbenzidine, and 3,3'-dimethoxybenzidine
were moderate reductants; and 3,3'-diaminobenzidine was a strong reductant.
Gammer and Moore (1973) also reported that 3,3'-diaminobenzidine markedly
reduced cytochrome o. Thus, a compound such as p-aminobiphenyl, considered
to be strongly carcinogenic, displayed weak reducing power, whereas 3,3'-
diaminobenzidine, considered to be weakly carcinogenic, displayed strong
reducing power. The authors suggested that aminobiphenyls which are easily
oxidizable by mitochondrial systems may be detoxified in this manner and
excreted in combination with sulfuric and amino acids.
Exposure to benzidine alters the activity of several enzymes. The
activity of 3-glucuronidase in urine of exposed workers is significantly
increased when compared with activity levels in healthy unexposed workers
(Kleinbauer et al. , 1969; Popler, Selucky, and Vlasak, 1964). Removal of
workers from benzidine exposure decreased the enzyme activity; however,
the decrease did not reach the level in unexposed workers.
Exposure to benzidine also decreases enzyme activity. Subcutaneous
administration of benzidine to rabbits caused a decrease in blood pheno-
lase activity (Nakajima, 1955), whereas diorthotolidine did not cause this
decrease. Since blood phenolase activity decreases prior to the appear-
ance of anemia, phenolase activity may be a means of early diagnosis of
poisoning by aromatic derivatives. Catalase and peroxidase activity was
decreased in rats injected with benzidine (Soloimskaya, 1968). Along with
the decreased enzyme activity, there was a decrease in erythrocytes and
thrombocytes and an increase in leucocytes.
Along with decreasing enzyme activity in rats, an increase in male
rat liver glutathione occurred with exposure to benzidine (Neish, 1967).
The compound was injected intraperitoneally at a dose of 12.7 mg/100 g
body weight. The control glutathione value was 182 mg %, whereas the
value 24 hr following benzidine injection was 272 mg %.
6.3.2 Toxicity
6.3.2.1 General Toxicity — Benzidine and benzidine derivatives cause poi-
soning in laboratory animals resulting in glomerulonephritis and nephrotic
syndrome. Indirect evidence of transplacental effects on embryonic tissues
also has been observed. In humans, benzidine compounds have been associ-
ated with dermatitis, cystitis, and hematuria.
6.3.2.1.1 Dermatitis — Schwartz, Tulipan, and Peck (1947) reported cases of
dermatitis among workers in several dye manufacturing plants and attributed
a portion of these cases to benzidine. Both benzidine and 3,3'-dimethylben-
zidine have caused dermatitis in chemists who work in laboratories. Various
benzidine color dyes have been implicated as causing dermatitis; however,
not all exposed workers are irritated by these compounds. Individual sensi-
tivity plays a role in determining dermatitis development.
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101
6.3.2.1.2 Glomerulonephritis and nephrotic syndrome — N,N'-Diacetylbenzi-
dine has been used to produce a nephrotic syndrome in rats which morpholog-
ically resembles a rapidly progressive human glomerulonephritis (Harman et
al., 1952; Harman, 1971). Both sexes of Sprague-Dawley rats were fed a
grain diet containing 0.043% N,N'-diacetylbenzidine. Proteinuria developed
in both sexes within three to four weeks. In females, proteinuria rapidly
became severe until as much as 0.1 g of protein was excreted within a 24-hr
period following two months on the diet. Severe anemia in females appeared
following proteinuria (Table 6.9), whereas in the male rats anemia was rare
and never severe. Females, after developing proteinuria, displayed hypo-
proteinemia, hyperlipemia, generalized edema, and, as previously mentioned,
anemia. Glomerular lesions which consisted of florid epithelial crescents,
progressive sclerosis, and obliteration of many glomeruli appeared in
females. Similar renal lesions developed in males; however, their appear-
ance was slower and less extensive. Only a mild nephrotic syndrome was
found in male rats. Atrophy of the testes developed in all males which
showed the syndrome. Morphological similarities between the nephrotic
syndrome produced in rats by N,N'-diacetylbenzidine and the human syndrome
included extracapillary cell proliferation, formation of luxuriant cres-
cents in 80% of the glomeruli, intact tufts in almost all glomeruli, and
the prevalence of many normal glomeruli in advanced grades of the syndrome.
Rats fed N,N'-diacetylbenzidine and 4,4,4',4'-tetramethylbenzidine
at equal molar levels of 0.25% and 0.27%, respectively, developed lipemia
and glomerular lesions consisting of fat-filled spaces in the glomerular
tuft from 2 to 4.5 months after beginning of treatment (Dunn, Morris, and
Wagner, 1956). No difference in the effects of the two compounds was
observed. Two groups of inbred mice fed these compounds at the same con-
centration showed no effects. N,N'-Diacetylbenzidine introduced by sub-
cutaneous (100 mg) or intraperitoneal (100 or 200 mg) injection produced
severe glomerulonephritis in 5 rats and mild lesions in 12 rats (Bremner
and Tange, 1966). Renal lesion severity may have varied with the dose of
TABLE 6.9. DEVELOPMENT OF PROTEINURIA AND ANEMIA IN FOUR
FEMALE RATS FED A DIET CONTAINING
N,N'-DIACETYLBENZIDINE
Mean value after feeding
Observation
0-3 4-8 9-10 11
weeks weeks weeks weeks
Red cell count, 106/yl 8.23 7.69 6.81 3.51
Daily protein loss in
urine, mg 16.54 378.67 314.38 250.63
Source: Adapted from Harman, 1971, Table III, p. 123.
Reprinted by permission of the publisher.
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102
the compound. In another study, Christopher and Jairam (1970) reported a
low-grade type of glomerulonephritis present in white rats fed 4,4'-
diaminobiphenyl.
6.3.2.1.3 Subacute toxicity — Mice daily fed 0.01% to 0.08% benzidine
dihydrochloride exhibited various toxic symptoms (Rao et al., 1971). The
mice lost weight in proportion to the dose of benzidine received, with
males losing up to 20% and females up to 7% of their body weight. The
weights of the carcass, liver, and kidney decreased with an increase in
dose, whereas the spleen and thymus weights increased with dose increases.
Among morphological effects were cloudy swelling of the liver, vacuolar
degeneration of renal tubules, and hyperplasia of the myeloid elements in
bone marrow and of lymphoid cells in the spleen and thymic cortex. Male
mice appeared more sensitive to benzidine than did female mice. This find-
ing seems to be in disagreement with that of Harman (1971) , who reported
female rats more sensitive than male rats. However, this difference may
be a species difference, a difference in benzidine compounds, or a combi-
nation of these two possibilities.
6.3.2.1.4 Indirect evidence of transplacental effects — Golub (1969) and
Shabad et al. (1972) found that 3,3'-dichlorobenzidine and 3,3'-dimethyl-
benzidine increased survival time of embryonic mouse kidney tissue cultures
and produced hyperplastic changes in the epithelium. Eight to 10 mg of
either compound injected subcutaneously into pregnant mice produced hyper-
plastic growth that was nodular or diffuse or that formed solid, compact
areas in explanted embryonic kidney tissue five to six days after adminis-
tration (Table 6.10). Longer survival of the explanted kidneys was pro-
moted by treatment with these two compounds during pregnancy. Administration
of a total dose of 8 to 10 mg per mouse of 3,3'-dimethylbenzidine by subcu-
taneous injection to BALB/c mice during the last week of pregnancy produced
an increased incidence of tumors in the progeny (Golub, Kolesnichenko, and
Shabad, 1974). Eight of 16 mice had tumors; 6 with adenomas of the lungs
and 5 with tumors of the mammary gland. The tumors found in the progeny
could have been induced by transplacental action or by transmission of
3,3'-dimethylbenzidine through milk of the lactating mothers.
6.3.2.2 Carcinogenicity — Benzidine and several other aromatic amines
have been implicated as carcinogens of the urogenous organs, particularly
the bladder (Hueper, 1954). However, it is generally accepted that metab-
olites of these aromatic amines, referred to as proximate carcinogens, are
the active compounds (Clayson, 1969). The metabolites are formed by con-
version of the aromatic amines to ring hydroxylated compounds which then
are N-hydroxylated, acylated and deacylated, and conjugated with sulfate
and glucuronic acid (Haley, 1975). Evidence suggests that the N-hydroxyl-
ation produces the precarcinogen and that the sulfate or glucuronidate
conjugates are the carcinogenically active forms in vivo. Metabolites
produced by experimental animals apparently have an affinity to and exert
a carcinogenic action on organs other than the bladder (Hueper, 1961).
The carcinogen apparently must be administered to the animal a consider-
able time before tumors are produced.
There is strong experimental evidence that benzidine and related com-
pounds induce various tumors in laboratory animals. As noted in Table 6.8,
-------
TABLE 6.10. SURVIVAL AND HYPERPLASTIC CHANGES OF EPITHELIUM IN EMBRYONIC KIDNEY TISSUE CULTURES TREATED
TRANSPLACENTALLY WITH 3,3'-DICHLOROBENZIDINE AND 3,3'-DIMETHYLBENZIDINE
3,3' -Dichlorobenzidine-treated
Duration of
experiment
(days)
3-4
6-7
14-15
18-20
Total
Total survival, %
Total percentage
of hyperplastic
growth
Number of
surviving explants
in control
cultures /total
13/13
12/12
0/23
0/7
25/55
45.5
Number of
surviving
explants /total
3/3
6/6
48/56
9/13
68/78
84.9
culture
Hyperplastic
changes
(outgrowths)
2
15
3
20
30.4
3,3 '-Dimethylbenzidine-treated
Number of
surviving
explants /total
6/6
8/8
12/16
26/30
86.9
Hyperplastic
Hyperplasia
of tubules
2
1
3
11.5
culture
changes
Outgrowths
1
1
4
6
23.1
Control cultures did not reveal any hyperplastic changes.
Source: Adapted from Shabad et al., 1972, Table 6, p. 621. Reprinted by permission of the publisher.
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104
many of these are located in the liver. In single experiments without con-
firmation, bladder cancer induction in experimental animals was limited to
the dog and rabbit.
As pointed out by Haley (1975), a serious problem exists with the
animal experimentation so far accomplished in that experiments have always
been carried out at toxic doses that resulted in liver failure and death of
the majority of the animals prior to tumor development. He suggested that
one means of correcting this problem would be the performance of a 90-day
subacute study in several species at dosage levels below those known to
produce liver pathology.
Despite the substantial evidence accumulated indicating a strong
association between occupational exposure to benzidine and the develop-
ment of bladder cancer in humans, a cause-effect relationship has not
been established in the epidemiological sense. (See MacMahon and Pugh,
1970, for epidemiological procedure and meaning.) A note of caution,
therefore, seems warranted. In the following discussion of a number of
studies made on occupationally exposed workers, the proportions of per-
sons with cancer are noted for specific periods. Such proportional rates
do not express the real magnitude of the risk associated with the expo-
sures encountered. However, this magnitude can be determined by compar-
ing the rate of bladder cancer among the exposed workers with the rate of
bladder cancer among the unexposed population at large. This concept of
risk is useful in identifying high-risk groups. For example, Case et al.
(1954) reported that the risk of developing bladder cancer was 14 times
greater among workers exposed to benzidine than in persons without such
exposure.
6.3.2.2.1 Benzidine — Benzidine has been associated with a high occur-
rence of bladder cancer in exposed industrial workers (International Agency
for Research on Cancer, 1972; Riches, 1972; Sax, 1975). Malignancies have
been reported following only a few weeks of exposure and a latent period
of several years (Deichmann and Gerarde, 1969). Hamblin (1963) reported
an average latent period of 18.6 years for bladder tumors. The develop-
ment of tumors from benzidine exposure seems to be related to the degree
of control of exposure afforded by work habit and personal hygiene pro-
grams (Rye et al., 1970). The occurrence of tumor formation is affected
by initial exposure concentration, exposure duration, and years of sur-
vival following exposure. However, there is a lack of data on the benzi-
dine concentrations to which workers have been exposed.
Zavon, Hoegg, and Bingham (1973) reported 13 of 25 exposed men devel-
oped tumors of the bladder. This unusually high occurrence rate may have
been due to the long exposure period. The workers were exposed to other
chemicals; however, benzidine was the chemical common to all and was indi-
cated as the carcinogen. Those who developed tumors were exposed to ben-
zidine more than 13 years, whereas the nontumor group was exposed less than
9 years. The tumor recurrence rate was 62% (Wendel, Hoegg, and Zavon,
1974). Samples of benzidine atmospheric concentrations taken from vari-
ous locations in the benzidine manufacturing plant ranged from less than
0.005 to 0.415 mg/m , except in one location where the concentration was
17.6 mg/m3.
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105
Bladder tumors in benzldine-exposed workers are found in all coun-
tries in which this chemical is manufactured and used. In France, 85
cases of bladder tumors in men working in aromatic amine manufacturing
plants were found before 1960 (Billiard-Duchesne, 1960). Fifty-one cases
originated in a factory in Normandy; 17 cases from this factory occurred
prior to 1947 and 34 subsequent to 1947- Eighteen of those 34 had symp-
toms of hematuria and stranguria. Seven of the 18 were not directly
exposed to the amines, but they had worked in the factory an average of
25 years. Kuzelova, Kunor, and Hurt (1969) reported positive findings
in 17% of those examined for urinary system tumors in workers employed
in benzidine production. The average length of exposure was eight years;
the highest rate of positive findings was among those with six to ten
years of exposure.
Gehrman (1936) reported occurrence of bladder tumors among workers
in the American dye industry. A total of 24 cases of carcinoma appeared
in workers exposed to aromatic amines, including benzidine. The average
exposure time necessary to produce tumors was 12 years. Case et al. (1954)
found that workmen exposed to benzidine in the dyestuff industry in England
had more bladder tumors than unexposed workers. The induction period aver-
aged 16 years; however, some tumors appeared in less than 2 years. Report-
ing on the occurrence of bladder tumors in a British dyestuff factory, Scott
(1952) found 66 cases of bladder tumors in which 30 were of workers exposed
to benzidine. The latent period averaged 15.9 years and ranged from 8 to
32 years (Figure 6.5). The intensity of exposure in the benzidine manufac-
turing was thought to be a factor in producing earlier tumors among these
workers than among workers engaged in benzidine handling. Of 23 cases of
bladder tumors in benzidine-exposed workers in manufacturing, 14 had papil-
loma, 7 had carcinoma, and 2 had papilloma which recurred as carcinoma.
Studies on the occurrence of bladder tumors among workers in Italian
dyestuff factories indicated a higher proportion among benzidine-exposed
workers than among unexposed workers. Vigliani and Barsotti (1962) found
24 cases of bladder tumors in a total of 109 workers exposed to benzidine
or benzidine and g-naphthylamine. From 1931 to 1960, 47 bladder tumors
were found in workers exposed to benzidine. Of these, 21 were carcinomas
and 16 were papillomas. From 1931 to 1948, 13 cases of carcinoma of the
bladder were detected in 83 workers exposed to benzidine in a dyestuff
plant (Barsotti and Vigliani, 1952). Men working in filtration, pressing,
drying, and milling in the benzidine processing had the greatest exposure.
Workers with tumors had been exposed to benzidine from 5 to 16 years with
an average exposure of 8 years. The maximum time between cessation of
benzidine contact and tumor development was 16 years. Over a period of
six years (1964 to 1970), Forni, Ghetti, and Armeli (1972) examined 858
workers in five dyestuff factories and found 17 cases of bladder tumor.
Of these, ten were papillomas and seven were carcinomas.
Tsuchiya, Okubo, and Ishizu (1975) studied 100 cases of occupational
bladder cancer reported from 1949 to 1970 by dye-producing companies in
Japan. Only a few of these cases were reported prior to 1955 because of
the long latent period involved. The proportion of bladder tumors was
11.25% among workers involved in benzidine production and 1.45% among
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106
ORNL-DWG 77-9688
10 15 20 25
LATENT PERIOD (years)
30
35
Figure 6.5. Graph of time from initial exposure to benzidine to
diagnosis of tumors. Source: Adapted from Scott, 1952, Figure 3, p. 129,
Reprinted by permission of the publisher.
those concerned with benzidine use. Eight of the 100 cases developed
tumors in the upper urinary tract that did not involve the bladder. The
average latent period for those exposed to benzidine was 16.25 years with
a standard deviation of 7.38 years. A high risk of bladder tumors among
silk kimono painters in Kyoto was reported by Yoshida and Miyakawa (1973).
These painters moistened brushes with the tongue and accidentally ingested
benzidine dyes, probably causing the bladder tumors.
Workers in the coal tar dye industry also have a high occurrence of
bladder tumors. Goldwater, Rosso, and Kleinfeld (1965) reported 21.3% of
employees exposed to benzidine contracted malignant bladder tumors in a
coal tar dye plant. Those exposed to benzidine plus 3-naphthylamine had a
higher rate of 45.5%. The average latent period until tumor development
was 18.4 years for papillomas and 18.7 years for malignancies. In a later
report, Kleinfeld, Rosso, and Goldwater (1966) also reported a higher pro-
portion of bladder tumors in coal tar dye plant workers exposed to benzi-
dine plus 3-naphthylamine than among workers exposed to only benzidine.
Mancuso and El-Attar (1966, 1967) found a higher proportion of blad-
der and kidney cancer in benzidine-exposed workers in a. plant manufactur-
ing benzidine and g-naphthylamine than that found among unexposed workers.
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107
Of 171 deaths among the exposed workers, 18 were reportedly due to bladder
and kidney cancers. There was also a higher rate of malignant neoplasms
of the digestive system. Exposure to benzidine plus 3-naphthylamine was
apparently more potent than exposure to benzidine alone.
In a report of carcinogenic hazards in microbiology laboratories,
Wood and Spencer (1972) suggested that benzidine exposure in the labora-
tory could cause bladder cancer. Benzidine is used in clinical biochemi-
cal laboratories and presents a carcinogenic hazard to technicians (Collier,
1974; Veys, 1972). Congo Red, a benzidine-based azo dye, has been used as
a bacteriostatic agent in the isolation of tubercle bacilli from sputum
(Frobisher, 1946) and is still employed as an indicator dye in the mixed
culture of Rhizobi-ivn species and related species of bacteria (Vincent,
1970, pp. 4-5).
Occupational tumors of the bladder from benzidine exposure have a
long latent period averaging 18 years. Age is not considered a factor in
disease development (Hamilton and Hardy, 1974). These occupational blad-
der tumors appear to be similar to bladder tumors found in the general
public and have a tendency for high recurrence.
Benzidine is carcinogenic in several animal species under varying
conditions (Casarett and Doull, 1975). There appears to be, however, some
controversy about the induction of bladder tumors by benzidine in dogs.
Haley (1975) stated that benzidine does in fact produce bladder tumors in
dogs. Spitz, Maguigan, and Dobriner (1950) found a papillary carcinoma
of the urinary bladder in one of seven dogs fed benzidine for five years.
The tumor was found seven and one-half years after the start of the exper-
iment. Benzidine administered orally to dogs has not produced bladder
cancer (Marhold et al., 1968). Deichman et al. (1965) fed female beagle
dogs 1 mg/kg of benzidine per dog five times a week for three years; no
tumors were found. The carcinogenic actions of 2-naphthylamine, benzi-
dine, 4-aminobiphenyl, and 4-nitrobiphenyl were not additive when the
compounds were fed together.
Saffiotti et al. (1967) failed to induce bladder tumors in 30 male
and female Syrian golden hamsters fed benzidine at 0.1% of the diet through-
out their life spans. Carcinogenic effects were produced in other organs;
extensive bile duct proliferations and cysts appeared, with cholangiofi-
brosis and liver cell carcinoma and hepatomas.
Benzidine injected subcutaneously into rats at 15 mg/week produced
hepatic injury followed by cirrhosis (Spitz, Maguigan, and Dobriner, 1950).
Sebaceous gland carcinomas were found as well as hepatomas and adenocar-
cinomas of the rectum. No bladder tumors were produced. Baker (1950)
subcutaneously injected Albino Delph mice with 300 mg benzidine or 300 mg
dihydroxybenzidine, a suspected benzidine metabolite produced in humans.
Both bladder and liver changes were found, including benign papillomas of
the bladder. Table 6.11 summarizes these effects. Dihydroxybenzidine
produced benign papillomas in the bladders of the mice, whereas mice re-
ceiving benzidine did not show these effects. Later, Baker (1953) found
changes in the liver, including cirrhosis and hepatomas, adenocarcinoma
-------
TABLE 6.11.
BLADDER AND LIVER CHANGES PRODUCED IN ALBINO DELPH MICE ADMINISTERED BENZIDINE
OR DIHYDROXYBENZIDINE BY SUBCUTANEOUS INJECTION
Group
Al
A2
Bl, B2
Cl, C2
Chemical
administered
300 mg benzidine base
300 mg 3,3'-dihydroxy-
4,4 '-diaminodiphenyl
Olive oil
Untreated
surviving
45 weeks
9
9
19
17
.. . Papillomas
Normal TT .,
bladder . ,. n .
Benign Malignant
9
215
17 2
17
„ -, T, • n j Hepatomas
Normal Bile duct
liver
1
5
13
12
proliferation
Benign
3 4
1 3
3 3
3
Malignant
I
2 ?
1— '
o
00
Source: Adapted from Baker, 1950, Tables II and III, p. 49. Reprinted by permission of the publisher.
-------
109
of the colon, carcinoma of the sebaceous gland next to the external audi-
tory canal (Zymbal's gland), and squamous-cell carcinoma of the stomach
in rats fed 5 to 8 g/day of dihydroxybenzidine (0.125% of a normal basic
diet). In three of the seven surviving animals, one sessile papilloma
and two keratinized squamous-cell carcinomas of the bladder wall appeared.
Wistar rats injected intraperitoneally or subcutaneously with 100 or 200
mg of N,N'-diacetylbenzidine developed tumors primarily in the external
auditory canals and mammary glands 6 to 15 months following injection.
Twenty-four tumors were formed in 14 of 30 rats. Severe glomerulonephri-
tis was found in 5, and mild lesions were found in 12 animals. Renal
lesion severity probably varied in relation to the size of the dose.
Griswold et al. (1968) also reported carcinomas of the mammary gland in
female Sprague-Dawley rats given 12 to 50 mg of benzidine per rat orally.
One fibroadenoma was also found in the mammary gland. However, Marhold
et al. (1967, 1968) found no tumors in Wistar rats fed 2 mg of benzidine
per rat per day for their life spans.
Rats given benzidine (the amount of dose was not specified) by sub-
cutaneous injection for six months developed cirrhosis early in the study
(Pliss, 1963). Hepatomas, tumors of the Zymbal's gland, and sarcomas at
the injection site were found. These tumors made up 70% of those found
in the rats (Pliss, 1964). Benzidine was more toxic to female rats than
to male rats. Injection of 3,3'-benzidine dicarboxylic acid in mice and
rats over a period of a year produced tumors of the Zymbal's gland and
the liver (Pliss, 1969). Four of 28 rats injected weekly with 5 mg ben-
zidine developed intestinal tumors, and 4 of 37 rats injected weekly with
5 mg benzidine plus 10 mg orthotolidine had intestinal tumors (Pliss,
Volfson, and Jogannsen, 1973). The activity of benzidine plus orthotol-
idine did not appear to be additive.
Of 22 rats given an accumulative dose of 0.75 g/kg body weight of
benzidine for 15 days, 20 rats developed tumors, including 19 hepatomas,
18 cholangiomas, 7 intestinal tumors, and 4 carcinomas of the sebaceous
gland (Holland et al., 1974). Tetramethylbenzidine (4.15 to 8.3 g/kg
body weight) given subcutaneously produced only benign tumors at the injec-
tion site and, hence, was not considered carcinogenic.
Bonser, Clayson, and Jull (1956) subcutaneously administered benzi-
dine or 3,3'-dihydroxybenzidine in weekly doses of 6 mg for 52 weeks to
mice until exitus. Benzidine induced hepatomas and lymphomas, whereas
3,3'-dihydroxybenzidine induced lymphomas and benign intestinal polyps.
Induction periods were 70 weeks. One-third of the control animals devel-
oped spontaneous lymphomas, which made significant interpretation diffi-
cult. 3,3'-Dihydroxybenzidine was a weak carcinogen in mice, causing
tumors of the liver and mammary gland and also causing leucosis when it
was given subcutaneously (Pliss, 1961). When 3,3'-dihydroxybenzidine was
applied to the skin, the mice developed tumors of inner organs. Benzi-
dine administered subcutaneously in weekly doses of 6 mg to mice of line
C3HA produced hepatomas in 31 of 46 mice (Prokofjeva, 1971). One mouse
had a pulmonary adenocarcinoma. The first tumors appeared 15 to 16 months
following the beginning of benzidine administration.
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110
N,N'-Diacetylbenzidine produced tumors in female Wistar rats given
a single intraperitoneal (100 or 200 ing) or subcutaneous (100 mg) injec-
tion (Bremner and Tange, 1966). In 14 of 30 rats, 24 tumors developed
principally in the external auditory canals and mammary glands 6 to 15
months after injection. Interestingly, tumors did not develop in the 6
animals given 200 mg intraperitoneally, whereas they did develop in 11
out of 18 animals given 100 mg by the same route.
6.3.2.2.2 3,3'-Dimethylbenzidine (Diorthotolidine) — There is no evi-
dence that diorthotolidine is carcinogenic in humans (Rye, Woolrich, and
Zanes, 1970). It is a weaker carcinogen than benzidine in animals (Sax,
1975), and this difference may carry over to humans. Transient hematuria
is a common symptom in workers exposed to diorthotolidine (Hamblin, as
cited in Rye, Woolrich, and Zanes, 1970). Since diorthotolidine follows
a metabolic pathway similar to benzidine (Rye, Woolrich, and Zanes, 1970),
it should be treated as a potential industrial carcinogen (Sax, 1975).
The only data concerning diorthotolidine carcinogenicity are obtained
from studies dealing with rats and mice. Rats treated with an accumula-
tive dose of 5.4 g/kg body weight of diorthotolidine for 241 days devel-
oped 11 gastrointestinal tract tumors, 7 hepatomas, 4 tumors of bone and
associated tissues, and 4 carcinomas of the Zymbal's gland. Twenty female
Sprague-Dawley rats were given diorthotolidine orally up to a total dose
of 500 mg per rat. At the end of nine months, 3 of 16 surviving rats had
a total of four mammary carcinomas (Griswold et al., 1968). The method
of administration seems to influence where tumors develop. Pliss and
Zabezhinsky (1970) administered diorthotolidine to rats either by subcu-
taneous injection (20 mg per rat) or by subcutaneous implantation of pel-
lets (20 mg per rat) for 13 to 14 months. Tumors, mainly of the skin,
large sebaceous glands, and mammary glands, developed in 60% to 70% of the
rats (Table 6.12). Implantation of pellets of diorthotolidine induced
subcutaneous sarcomas in 2 of 68 rats and hepatocellular carcinomas in 4;
this effect was not found when diorthotolidine was injected.
6.3.2.2.3 3,3'-Dimethoxybenzidine (Dianisidine) — Dianisidine is another
benzidine derivative in which there is no evidence of carcinogenicity in
humans (Rye, Woolrich, and Zanes, 1970). However, several studies have
provided evidence that it is carcinogenic to rats. Dianisidine adminis-
tered by subcutaneous injection induced tumors of the Zymbal's gland in two
rats and an ovarian tumor and fibroadenoma of the mammary glands in another
rat (Pliss, 1963). The incidence rate was low since only 3 of 18 rats
developed tumors. After 260 doses of dianisidine (10 mg per dose) given
orally, both male and female Fischer rats developed tumors in the gastro-
intestinal tract, skin, breast, and ear duct (Weisburger et al. , 1967).
Tumors developed as early as 293 days after the first administration.
6.3.2.2.4 3,3'-Dichlorobenzidine — No cases of bladder cancer in humans
attributable to dichlorobenzidine have been reported (Rye, Woolrich, and
Zanes, 1970). However, tumor induction by this compound has been demon-
strated in rats and mice, and there is some evidence for its complicity
in the occurrence of nonbladder tumors in humans.
-------
TABLE 6.12. TUMORS INDUCED IN RATS RECEIVING 20 mg OF DIORTHOTOLIDINE EITHER BY SUBCUTANEOUS INJECTION
OR SUBCUTANEOUS IMPLANTATION OF PELLETS
, r- m. r Number of rats , , . . . , . . a
Number of Time of Number of rats with tumors at specific sites
IXLetnoa ot rats at iirst tumor . ..
, . . ,_ . ... . Survived , , . ,
administration beginning of appearance . ,. With Zymbal s
/ , , to time or , ,
experiment (months) tumors gland
v first tumor &
Injection 27 males 8 25 17 14
(8-22)
26 females 8 25 13 6
(8-20)
Total 53 8 50 30 20
Implantation 24 males 12 16 11 6
(12-18)
24 females 12 20 12 5
(12-18)
Total 48 12 36 23 11
Mammary
gland
5
(13-22)
5
7
(12-23)
7
Skin
2
(18-23)
1
(16)
3
1
(17)
1
(18)
2
Preputial T . Fore—
, , Liver , i
gland stomach
213
(20-22) (23) (21-25)
1
(13)
313
3
(15-20)
3
at site of Other
mplantation
S6
1°
6
3d
(20)
I8
(20)
1 4
^Numbers in parentheses indicate time of tumor appearance in months.
In one rat, tumor of small intestine; in two rats, tumors of hematopoietic system (in 12 and 23 months respectively); in one rat,
thyroid tumor (in 25 months); in one rat, lung tumor (in 22 months).
JJterine tumor.
In one rat, angiosarcoma in area of anterior extremity (in 14 months); in one rat, neurosarcoma in area of the ear (in 15 months); in
one rat, tumor of hematopoietic system (20 months).
Lymphangioma in neck region.
Source: Adapted from Pliss and Zabezhinsky, 1970, Tables 1 and 2, p. 285.
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112
Gerarde and Gerarde (1974) found no bladder tumors in employees at
a New York plant of Hercules, Inc. The workers were exposed to dichloro-
benzidine for 35 years. However, 17 workers developed neoplasms in other
locations: 2 in the lung, 1 in bone marrow, 6 lipomas, 3 papillomas of
the rectum, 2 carcinomas of the sigmoid colon, 1 prostate carcinoma, 1
myoblastoma of the breast muscle, and 1 basal cell epithelioma. At a
British plant using dichlorobenzidine for 30 years, no cases of bladder
cancer were found among more than 200 exposed employees (Maclntyre, 1975).
Most of the exposed workers had been exposed for less than 16 years, which
indicates that the latent period may not have been reached.
A comparison of tumor incidence was made between workers exposed to
dichlorobenzidine and those exposed to both benzidine and dichlorobenzidine
at a Clayton Aniline plant (Gadian, 1975). Workers exposed to benzidine
plus dichlorobenzidine developed three bladder tumors, whereas those exposed
to dichlorobenzidine alone did not develop any bladder tumors (Table 6.13).
The employees had been exposed to dichlorobenzidine for 10 to 15 years,
during which time occasional bladder tumor cases should have appeared if
the latent period were similar to benzidine. It is possible, however, that
the latent period is longer. If dichlorobenzidine is a human carcinogen,
it would appear to be weaker than benzidine.
Dichlorobenzidine is carcinogenic to several animal species. Pliss
(1963) injected 3,3'-dichlorobenzidine subcutaneously into rats and found
tumors in 74% of the animals. Tumors developed in the skin, sebaceous and
mammary glands, intestines, bones, and urinary bladder. Tumors developed
in 48 of 64 D-line mice that survived to the appearance of the first tumor
after receiving dichlorobenzidine by ingestion or injection into the under-
lying fat (Pliss, 1959). Sarcomas were found at the injection site in
seven rats. Two rats ingesting the compound had adenocarcinomas of the
small intestine. Tumors were found also in the mammary and sebaceous
glands, and three rats had papillomas of the bladder.
Griswold et al. (1968) did not find any tumors in rats given dichlo-
robenzidine dihydrochloride (300 mg per rat total dose) by oral feeding.
There was a significant increase in tumor number in progeny of BALB/c mice
given dichlorobenzidine (8 to 10 mg per mouse total dose) by subcutaneous
injection (Golub, Kolesnichenka, and Shabad, 1974). Tumors developed in
13 of 24 mice: 4 mice had adenocarcinomas of the mammary gland, 5 had
adenomas of the lung, and 7 had lymphatic leukemia. Rats receiving 1000
ppm dichlorobenzidine in the diet had tumors of the mammary gland in both
males and females and tumors in the Zymbal's gland and hematopoietic tumors
in males (Stula, Sherman, and Zapp, 1971; Stula et al., 1975).
6-3.2.2.5 4-Aminobiphenyl — Melick et al. (1955) studied the workers in a
plant manufacturing 4-aminobiphenyl from 1935 to 1955. Of the 171 exposed
employees, 11.1% had developed bladder tumors from 5 to 19 years after ini-
tial exposure. The duration of exposure was 1.25 to 19 years. A metabo-
lite of 4-aminobiphenyl was suspected as being the carcinogen. In a follow-
up study to the Melick et al. (1955) study, bladder tumors were found in
workers exposed to 4-aminobiphenyl in two plants manufacturing this com-
pound (Melick, Naryka, and Kelly, 1971). The minimum exposure time to
-------
TABLE 6.13.
COMPARISON OF TUMOR INCIDENCE BETWEEN WORKERS EXPOSED TO DICHLOROBENZIDINE
AND THOSE EXPOSED TO DICHLOROBENZIDINE PLUS BENZIDINE
Number
Group of
men
(hr)
Tumor incidence
Urinary Other
tract
Deaths from
other causes
Segregated dichloro-
benzidine workers
Mixed benzidine and
dichlorobenzidine
workers
35
14
68,505
16,200'
a
None
One papilloma
of bladder, two
carcinomas of
bladder
None
One carcinoma
of bronchus
One coronary thrombosis,
one cerebral hemorrhage
One coronary thrombosis
a.
Exposure to approximately 60% benzidine and 40% dichlorobenzidine.
Source: Adapted from Gadian, 1975, Table 1, p. 828. Reprinted by permission of the publisher.
-------
114
tumor induction was 133 days; the longest time interval for tumor develop-
ment to date was 35 years. The incidence rate of bladder tumors between
1957 and 1970 increased from 2.3% to 18.5% in one plant, probably because
of workers reaching the latent period required for tumor development. A
strong association may exist between bladder cancer and occupational expo-
sure to 4-aminobiphenyl (International Agency for Research on Cancer, 1972).
Bladder tumors have been induced in dogs given 4-aminobiphenyl. Two
beagle dogs administered 4-aminobiphenyl (30 to 34 g total) by mouth devel-
oped advanced bladder tumors and were killed 33 months after first dosing
(Walpole, Williams, and Roberts, 1954). Since the tumors appeared early
in the treatment relative to tumor induction time of benzidine, 4-amino-
biphenyl was more effective than benzidine as a bladder carcinogen in dogs.
All female beagle dogs fed 4-aminobiphenyl (0.66 to 0.74 g/kg body weight)
for three years developed from one to several papillomas or carcinomas of
the bladder (Deichmann et al., 1965). 4-Nitrobiphenyl was fed to beagle
dogs at the same dose level, and at the end of the three-year period the
bladders of all treated dogs were normal. 4-Aminobiphenyl was more car-
cinogenic than 4-nitrobiphenyl in the urinary bladder of dogs. Deichmann
and MacDonald (1968) found that a single dose of 50 mg 4-aminobiphenyl per
kilogram body weight administered by mouth to beagle dogs did not induce
precancerous lesions of the bladder mucosa or tumors in the urinary blad-
der. Since the dose size was much smaller than that used by others in
producing tumors, tumor development would not necessarily be expected.
4-Aminobiphenyl is carcinogenic in rats and mice, although the effec-
tive carcinogen is probably an o-hydroxyamine formed in the body's metabolic
hydroxylation (Walpole, Williams, and Roberts, 1952). Rats subcutaneously
injected with 4-aminobiphenyl at total dose levels of 5.8, 4.4, 4.2, and
3.6 g/kg body weight developed tumors of the liver, intestines, mammary
glands, kidney, and uterus. Newborn male and female Swiss mice were given
200 yg of 4-aminobiphenyl by subcutaneous injection during the first three
days of life (Gorrod, Carter, and Roe, 1968). At the termination of the
study, 48 to 52 weeks after injection, greater than 90% of the male mice
and about 18% of the females developed hepatomas. One pulmonary adenoma,
one thymic lymphoma, and one lymphosarcoma of the spleen also were found.
Testing three hydroxylated derivatives of 4-aminobiphenyl (4-amino-3-
hydroxybiphenyl, 4-hydroxylaminobiphenyl, and 4-amino-4'-hydroxybiphenyl)
in the same manner produced a significant increase of hepatomas in males
above the controls and a probable significant increase of hepatomas in
females in response to three of the compounds, but not to 4-amino-3-
hydroxybiphenyl.
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115
SECTION 6
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75. Riches, E. 1972. Industrial Cancers. Nurs. Mirror (Great Britain)
134(16):21-25.
76. Rinde, E., and W. Troll. 1975. Metabolic Reduction of Benzidine
Azo Dyes to Benzidine in the Rhesus Monkey- J. Natl. Cancer Inst.
55(1)=181-182.
77. Rye, W. A., P. F. Woolrich, and R. P. Zanes. 1970. Facts and Myths
concerning Aromatic Diamine Curing Agents. J. Occup. Med. 12(6):
211-215.
78. Saffiotti, U., F. Cefis, R. Montesano, and A. R. Sellakumar. 1967.
Induction of Bladder Cancer in Hamsters Fed Aromatic Amines. In:
Bladder Cancer; a Symposium, W. Deichmann and K. F. Lampe, eds.
Aesculapis, Birmingham, Ala. pp. 129-135.
79. Sax, N. I. 1975. Dangerous Properties of Industrial Materials,
4th ed. Van Nostrand Reinhold Company, New York. pp. 281, 442.
80. Schwartz, L., L. Tulipan, and S. M. Peck. 1947. Dermatitis in
Synthetic Dye Manufacture. In: Occupational Diseases of the Skin.
Lea and Febiger, Philadelphia, pp. 268-280.
81. Sciarini, L. J. 1957- 3-Hydroxybenzidine, a Metabolite of Benzi-
dine. Arch. Biochem. Biophys. 71:437-441.
82. Sciarini, L. J., and J. W. Meigs. 1958. The Biotransformation of
Benzidine (4,4'-Diaminobiphenyl), an Industrial Carcinogen, in the
Dog: I. AMA Arch. Ind. Health 18:521-530.
83. Sciarini, L. J., and J. W. Meigs. 1961a. The Biotransformation
of Benzidine: II. Studies in Mouse and Man. Arch. Environ.
Health 2:423-428.
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84. Sciarini, L. J., and J. W. Meigs. 1961&. Biotransformation of the
Benzidines: III. Studies on Diorthotolidine, Dianisidine, and
Dichlorobenzidine: 3,3' Disubstituted Congeners of Benzidine (4,4'-
Diaminobiphenyl). Arch. Environ. Health 2:584-588.
85. Scott, T. S. 1952. The Incidence of Bladder Tumours in a Dyestuffs
Factory. Br. J. Ind. Med. (Great Britain) 9:127-132.
86. Shabad, L. M., J. D. Sorokina, N. I. Golub, and S. P- Bogovski.
1972. Transplacental Effect of Some Chemical Compounds on Organ
Cultures of Embryonic Kidney Tissue. Cancer Res. 32:617-627.
87. Soloimskaya, E. A. 1968. The Distribution of Benzidine in Rat
Organs and Its Effect on the Peripheral Blood. Vopr. Onkol. (U.S.S.R.)
14(7):51-53.
88. Spitz, S., W. H. Maguigan, and K. Dobriner. 1950. The Carcinogenic
Action of Benzidine. Cancer 3:789-804.
89. Stula, E. F., H. Sherman, and J. A. Zapp, Jr. 1971. Experimental
Neoplasia in ChR-CD Rats with the Oral Administration of 3,3'-
Dichlorobenzidine, 4,4'-Methylenebis(2-chloroaniline), and 4,4'-
Methylenebis(2-methylaniline) (abstract). Toxicol. Appl. Pharmacol.
19:380-381.
90. Stula, E. F., H. Sherman, J. A. Zapp, Jr., and J. W. Clayton, Jr.
1975. Experimental Neoplasia in Rats from Oral Administration of
3,3'-Dichlorobenzidine, 4,4'-Methylene-bis(2-chloroaniline), and
4,4'-Methylene-bis(2-methylaniline). Toxicol. Appl. Pharmacol.
31:159-176.
91. Troll, W., S. Belman, and F. Mukai. 1969. Studies on the Nature
of the Proximal Bladder Carcinogens. J. Natl. Cancer Inst. 43(1):
283-286.
92. Troll, W., S. Belman, and E. Rinde. 1963. N-Hydroxy Acetyl Amino
Compounds, Urinary Metabolites of Aromatic Amines in Man (abstract).
Proc. Am. Assoc. Cancer Res. 4(1):68.
93. Troll, W., and N. Nelson. 1958. Studies on Aromatic Amines: I.
Preliminary Observations on Benzidine Metabolism. Am. Ind. Hyg.
Assoc. J. 19:499-503.
94. Tsuchiya, K., T. Okubo, and S. Ishizu. 1975. An Epidemiological
Study of Occupational Bladder Tumours in the Dye Industry of Japan.
Br. J. Ind. Med. (Great Britain) 32:203-209.
95. Veys, C. A. 1972. Aromatic Amines: The Present Status of the
Problem. Ann. Occup. Hyg. (Great Britain) 15(1):11-15.
96. Vigliani, E. C., and M. Barsotti. 1962. Environmental Tumors of
the Bladder in Some Italian Dye-stuff Factories. Acta Unio Int.
Contra Cancrum (Belgium) 18:669-675.
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97. Vincent, J. M. 1970. A Manual for the Practical Study of Root
Nodule Bacteria. Blackwell Scientific Publishers, Oxford, England.
pp. 4-5.
98. Walpole, A. L., M.H.C. Williams, and D. C. Roberts. 1952. The
Carcinogenic Action of 4-Aminodiphenyl and 3:2'-Dimethyl-4-amino-
diphenyl. Br. J. Ind. Med. (Great Britain) 9:255-263.
99. Walpole, A. L., M.H.C. Williams, and D. C. Roberts. 1954. Tumours
of the Urinary Bladder in Dogs after Ingestion of 4-Aminodiphenyl.
Br. J. Ind. Med. (Great Britain) 11:105-109.
100. Weisburger, J. H., N. Mantel, E. K. Weisburger, Z. Hadidian, and
T. Fredrickson. 1967. New Carcinogenic Naphthalene and Biphenyl
Derivatives. Nature (Great Britain) 213:930-931.
101. Wendel, R. G., U. R. Hoegg, and M. R. Zavon. 1974. Benzidine: A
Bladder Carcinogen. J. Urol. 111:607-610.
102. Wood, J. M., and R. Spencer. 1972. Carcinogenic Hazards in the
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103. Yoshida, 0., and M. Miyakawa. 1973. Etiology of Bladder Cancer:
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eds. University Park Press, Baltimore, pp. 31-39.
104. Zavon, M. R., U. Hoegg, and E. Bingham. 1973. Benzidine Exposure
As a Cause of Bladder Tumors. Arch. Environ. Health 27:1-7.
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SECTION 7
ENVIRONMENTAL DISTRIBUTION AND TRANSFORMATION
7.1 SUMMARY
Benzidine and its related compounds (3,3'-dichlorobenzidine, 3,3'-
dimethylbenzidine, and 3,3'-dimethoxybenzidine) are used primarily as
intermediates in the production of azo dyes. Additionally, they are used
in polyurethane production and in chemical analyses.
Benzidine is not thought to be commonly distributed in the environ-
ment. In water, it is a probable hazard only near dye factories. Water
samples collected below Japanese dye factories have contained up to 0.233
ppm benzidine. No data concerning benzidine in air and soil were found.
Information regarding degradation of benzidine under normal environ-
mental conditions is not available, except for one survey of the Buffalo
and Niagara river areas. Laboratory studies indicated that benzidine
tends to resist biological and physical degradation. Estimated half-lives
for benzidine are 1 day for reaction with OH or 03 in air and 100 days for
reaction with R02 in water.
Based on its solubility in water and various organics, benzidine is
suspected to accumulate within food chains. However, no specific data
are available other than a laboratory study indicating bioaccumulation in
bluegill fish to a factor of 44 times the level in water.
7.2 PRODUCTION AND USE
7.2.1 Benzidine
Benzidine is used primarily as an intermediate in the production of
azo dyes. It is also used as a hardener for polyurethane, in qualitative
and quantitative chemical analyses, in the detection of occult blood, as
a microscopy stain, and as an agent to reveal bank check alteration
(International Agency for Research on Cancer, 1972; Lurie, 1963; Sciarini,
1969; Welcher, 1947). Benzidine production in the United States was 680.9
metric tons (750 tons) in 1972 (Anonymous, 1974).
7.2.2 3,3'-Dichlorobenzidine
3,3'-Dichlorobenzidine, a dye intermediate and curing agent for poly-
urethane, gives azo dyes which are bluer and more stable to acids than
those from benzidine (Radding et al., 1975). More than 95 dyes are derived
from 3,3'-dichlorobenzidine (Colour- Index, 1957); the primary one is benzi-
dine yellow (Hancock, 1975). United States production of 3,3'-dichloro-
benzidine and its salts was 1,600,000 kg (3,500,000 Ib) in 1971 (U.S.
Tariff Commission, 1973, cited in International Agency for Research on
Cancer, 1974). Imports in 1971 were 658,000 kg (1,450,890 Ib) (Interna-
tional Agency for Research on Cancer, 1974).
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7.2.3 3,3'-Dimethylbenzidine
More than 90 dyes are derived from 3,3'-dimethylbenzidine (diortho-
tolidine) (Colour Index, 1957). Additionally, diorthotolidine is used
in detection of various ions (Welcher, 1947). United States production
was approximately 110,200 kg (243,000 Ib) in 1962 (Lurie, 1964). In 1969
and 1970, U.S. imports amounted to 36,600 kg (80,600 Ib) and 44,400 kg
(97,800 Ib) respectively (International Agency for Research on Cancer,
1972).
7.2.4 3,3'-Dimethoxybenzidine
3,S'-Dimethoxybenzidine (dianisidine) is used primarily in dye pro-
duction, with 88 dyes derived from this chemical (Colour Index, 1957).
It is also used to detect copper, cobalt, gold, thiocyanate, and vanadium
(Welcher, 1947). The United States produced 163,000 kg (360,000 Ib) of
dianisidine in 1960 (U.S. Tariff Commission, cited in Lurie, 1964).
7.3 DISTRIBUTION IN THE ENVIRONMENT
7.3.1 Distribution in Soils
No data concerning benzidine measurements in soils were located in
the literature. Due to its physical properties, benzidine probably is
immobilized rapidly in soils and sediments (Radding et al. , 1975).
Furukawa and Brindley (1973) found that benzidine was adsorbed to some
clays.
7.3.2 Distribution in Water
Benzidine in water is probably a hazard only in the vicinity of dye
and pigment factories from which wastes escape or are discharged. Gener-
ally, processes in azo dye production are conducted within a closed
system. Contamination can occur, however, during cleanup (Haley, 1975).
Howard and Saxena (1976, pp. 16-17) reported on a field survey made
by the Synthetic Organic Chemical Manufacturers Association Task Force on
Benzidine. The survey was made to determine whether benzidine could be
detected in the Buffalo and Niagara river areas, where some benzidine
release by upriver plants had been thought to occur. No benzidine was
detected in either water or sediment samples when they were analyzed by
the chloramine-T method, which had a limit of detection of 0.2 yg/ml
(for 1-liter water samples). As pointed out by the authors, lack of
detection of benzidine in these samples may have resulted either from
dilution to below the limit of detection or from oxidative degradation
into compounds not detectable by this method of analysis.
Benzidine was detected (samples containing 0.082, 0.140, and 0.233
ppm) in the Sumida River (Japan) below dye factories (Takemura, Akiyama,
and Nakajima, 1965). These authors speculated, however, that the benzi-
dine may not have been discharged as such but was produced by reduction
of azo dyes in the wastes by H2S or S02 present in the river.
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7.3.3 Distribution in Air
Although benzidlne in closed air systems is potentially hazardous
(Zavon, Hoegg, and Bingham, 1973), no data were located concerning its
distribution in the atmosphere. The azo dye benzidine yellow, derived
from 3,3'-dichlorobenzidine, is used in various paints, enamels, and
lacquers and has been found in pressurized containers designed for the
hobbyist and householder market. Such usage in the home and work environ-
ment may pose a hazard to the user as well as provide an avenue of air
contamination and dispersion.
7.4 ENVIRONMENTAL FATE
7.4.1 Mobility and Persistence
Benzidine is volatile and soluble enough to have a potential for
wide dispersion. According to several authors, benzidine resists physical
and biological decomposition (Lutin et al., 1965; Malaney et al., 1967;
Radding et al. , 1975) and probably persists for a time in the environ-
ment. The principal chemical reaction of benzidine in air and water is
oxidative degradation by free radical, enzymatic, or photochemical pro-
cesses (Radding et al. , 1975). Data concerning the reaction rate of
benzidine with radicals and ozone and on photochemical reactions under
normal environmental conditions are not available. However, estimated
half-lives are 1 day for reaction with OH or 03 in air and 100 days for
reaction with R02 in water (Radding et al., 1975).
In a study of benzidine degradation under experimental water treat-
ment conditions, Howard and Saxena (1976, pp. 16-17) noted that air
oxidation of benzidine seemed to have occurred readily and that some
biological oxidation may also have occurred.
3,3'-Dichlorobenzidine is thought to be bound tightly to humic mate-
rials (Radding et al. , 1975). Due to the halogen substitution, 3,3'-
dichlorobenzidine probably degrades more slowly than benzidine. Its
estimated half-lives are 1 day with OH radicals, 1 to 10 days with 03,
and 100 days with R02 radicals.
7.4.2 Accumulation in Food Chains
Benzidine and 3,3'-dichlorobenzidine are more soluble than DDT in
water and are quite soluble in many organics (Sections 2.2.1.1 and
2.2.2.1). Therefore, it is suspected that these chemicals can move with-
in the food chain. Accumulation is expected to occur, and recent labora-
tory findings indicated that bluegill fish concentrate residues of
14C-benzidine to a level 44 times that present in water (Federal Register,
1976).
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SECTION 7
REFERENCES
1. Anonymous. 1974. Final Rules Set for Exposure to Carcinogens.
Chem. Eng. News 52:12-13.
2. Colour Index. 1957. 2nd ed. , Vol. 3. The Society of Dyers and
Colourists, Bradford, England, and The American Association of
Textile Chemists and Colorists, Lowell, Mass. 3824 pp.
3. Federal Register. 1976. 41:27012-27017.
4. Furukawa, T., and G. M. Brindley. 1973. Adsorption and Oxida-
tion of Benzidine and Analine by Montmorillonite and Hectrite.
Clay Miner. 21:279-288.
5. Haley, T. J. 1975. Benzidine Revisited: A Review of the Litera-
ture and Problems Associated with the Use of Benzidine and Its
Congeners. Clin. Toxicol. 8(1):13-42.
6. Hancock, E. G. 1975. Benzidine and Its Industrial Derivatives.
John Wiley and Sons, New York. p. 516.
7- Howard, P. H., and J. Saxena. 1976. Persistence and Degradability
Testing of Benzidine and Other Carcinogenic Compounds. EPA 560/5-
76-005, U.S. Environmental Protection Agency, Washington, D.C.
pp. 16-17.
8. International Agency for Research on Cancer. 1972. Benzidine.
In: IARC Monographs on the Evaluation of Carcinogenic Risk of
Chemicals to Man, Vol. 1. Lyon, France. pp. 80-91.
9. International Agency for Research on Cancer. 1974. 3,3'-Dichloro-
benzidine. In: IARC Monographs on the Evaluation of Carcinogenic
Risk of Chemicals to Man, Vol. 4. Lyon, France, pp. 49-55.
10. Lurie, A. P. 1964. Benzidine and Related Diaminobiphenyls. In:
Encyclopedia of Chemical Technology, R. E. Kirk and D. T. Othmer,
eds. John Wiley and Sons, Inc., New York. pp. 408-420.
11. Lutin, P. A., J. J. Cibulka, and G. W. Malaney. 1965. Oxidation
of Selected Carcinogenic Compounds by Activated Sludge. Proc. Ind.
Waste Conf. 20:131-145.
12. Malaney, G. W., P. A. Lutin, J. J. Cibulka, and L. H. Hickerson.
1967. Resistance of Carcinogenic Organic Compounds to Oxidation
by Activated Sludge. J. Water Pollut. Control Fed. 39(12):2020-2029.
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13. Radding, S. B., B. R. Holt, J. L. Jones, D. H. Liu, T. Mill, and
D. G. Hendry. 1975. Review of the Environmental Fate of Selected
Chemicals. EPA 560/5-75-001, U.S. Environmental Protection Agency,
Washington, B.C. 37 pp.
14. Sciarini, L. J. 1969. Benzidine. In: Industrial Toxicology,
L. T. Fairhall, ed. Hafner Publishing Company, New York.
pp. 165-166.
15. Takemura, N., T. Akiyama, and C. Nakajima. 1965. A Survey of the
Pollution of the Sumida River, Especially on the Aromatic Amines
in the Water. Int. J. Air Water Pollut. (Great Britain) 9:665-670.
16. Welcher, F. J. 1947. Organic Analytical Reagents, Vol. 2. D. Van
Nostrand Company, Inc., New York. p. 275.
17. Zavon, M. R., U. Hoegg, and E. Bingham. 1973. Benzidine Exposure
as a Cause of Bladder Tumors. Arch. Environ. Health 27:1-7-
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SECTION 8
ENVIRONMENTAL ASSESSMENT
8.1 PRODUCTION, USES, AND POTENTIAL ENVIRONMENTAL CONTAMINATION
8.1.1 Production
Although exact figures for current U.S. production and importation of
benzidine and benzidine compounds are unavailable, an examination of records
of the U.S. Tariff Commission for past years indicates that U.S. production
and importation of these substances may exceed 3.5 million kilograms per
year. In 1972, benzidine production was 680,000 kg, while that of dichloro-
benzidine was 2,300,000 kg in 1971. Records for the 1960s indicate that
total production and importation of 3,3'-dimethylbenzidine and 3,3'-dimethoxy-
benzidine ranged from 200,000 to 500,000 kg per year.
8.1.2 Uses
Benzidine and its congeners are used in industry primarily for the
synthesis of azo dyes. More than 250 dyes are derived from benzidine, 95
from dichlorobenzidine, 90 from 3,3'-dimethylbenzidine, and 88 from 3,3'-
dimethoxybenzidine. The leather, paper, and textile industries utilize more
than 10 million kilograms of such dyes every year. In addition, benzidine,
3,3'-dimethylbenzidine, and 3,3'-dimethoxybenzidine are widely used in chem-
ical and biochemical laboratories as analytical reagents. Dichlorobenzidine
and 3,3'-dimethoxybenzidine are also important in the polyurethane industry—
the latter compound for the production of 3,3'-dimethoxy-diisocyanates and
the former as a curing agent.
8.1.3 Losses to the Environment
There is little published information available to indicate the extent
to which benzidine, benzidine congeners, and benzidine-based dyes are inad-
vertently released into the environment. The manufacturing and processing
operations for benzidine salts and azo dyes are now contained within closed
systems; however, some losses to the environment undoubtedly still occur,
especially during cleaning operations.
In the past, in the absence of evidence that any of these compounds
might be health hazards, routine sampling for air or water contamination
was not practiced. As epidemiological data began to suggest that benzidine
might be a human carcinogen, a greater effort was made to determine levels
of atmospheric contamination at benzidine manufacturing plants. Several
studies revealed that atmospheric concentrations of benzidine in such
plants normally ranged from 0.002 to 0.152 mg/m3 (Meigs, Sciarini, and Van
Sandt, 1954; Zavon, Hoegg, and Bingham, 1973) and could reach as high as
17 mg/m3 (Zavon, Hoegg, and Bingham, 1973).
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Since 1974, when more stringent industrial standards were instituted
by the Occupational Safety and Health Administration (OSHA) of the U.S.
Department of Labor (Federal Register, 1974), the risk of cancer from indus-
trial exposure to benzidine has been substantially reduced. Although the
OSHA standards did not recognize a maximum safe level of exposure, they did
attempt to reduce exposure "to the maximum extent possible consistent with
continued use." Mixtures of compounds containing less than 0.1% of benzidine
or its salts were excluded from the regulation in an attempt to avoid hinder-
ing scientific research on these substances. However, this exclusion clause
in effect permitted a 0.1% level of contamination in compounds, such as vari-
ous azo dyes, which are produced from benzidine. Currently, dye manufacturers
warrant their products to contain not more than a certain level of free benzi-
dine. This level (in one case 20 ppm) is generally much lower than the OSHA
standard; however, it still represents a potential source of environmental
contamination. Consequently, traces of benzidine salts probably occur in the
wastewater effluents of the textile, paper, and leather manufacturing plants
using azo dyes. Such manufacturing plants generally use several gallons of
dye per pound of fabric processed, and the total amount of wastewater gener-
ated is often more than one million gallons per day.
There is increasing evidence that azo dyes, some of which may be toxic
themselves, are capable of being reconverted by chemical or biological mech-
anisms into the toxic parent compounds (Rinde and Troll, 1975; Takemura,
Akiyama, and Nakajima, 1965; Yoshida and Miyakawa, 1973). It may therefore
be very important to determine the extent of environmental contamination by
azo dyes. Published information on this subject is, however, quite limited.
In the azo dye plants themselves, removal of benzidine contaminants
from wastewater effluents is sometimes accomplished by tetrazotization.
Whether this process leads to a safe degradable end product or whether benzi-
dine or some other toxic substance might be resynthesized by later chemical
or microbial processes has not been fully examined. However, the question
is important in light of the fact that benzidine wastes are often piped
directly into municipal sewage systems.
8.2 ENVIRONMENTAL PERSISTENCE
8.2.1 Physical and Chemical Degradation
Very little information is currently available on the environmental
fate of benzidine, its congeners, or benzidine-based dyes. Although benzi-
dine is a moderately volatile substance, naturally occurring photochemical
and chemical processes would probably reduce atmospheric concentrations to
insignificant levels; however, the rates of such reactions are not well
known. Benzidine is sensitive to various oxidative processes. With 03 or
HO radicals the half-life of benzidine in a gaseous state has been calculated
to be about one day (Radding et al., 1975), and that of dichlorobenzidine is
about ten days.
If there should prove to be any environmental accumulation of benzidine
or its congeners, it might be expected that this would occur in natural
bodies of water receiving effluents from manufacturing and processing plants.
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According to Radding et al. (1975), the half-lives of benzidine and dichloro-
benzidine in an aqueous state would be about 100 days if the compounds reacted
primarily with R02 radicals. However, because the R02 rate constants used for
these calculations were only estimates, the actual half-lives may be as little
as 10 days or as much as 1000 days. With a relatively long half-life, the
processes of biological degradation may be more important in reducing benzi-
dine levels in aquatic environments. Because of its halogen substitution,
dichlorobenzidine may be more resistant to biodegradation (Radding et al.,
1975).
The solubility of the benzidine compounds would also have a significant
effect on their environmental fate. Although benzidine is only slightly
soluble in water (0.52 mg/ml at 25 ± 2°C) , its chloride salts, which are used
quite extensively in the azo dye industry, are very soluble in water (61.7
mg/ml) (Bowman, King, and Holder, 1976). In a similar manner, the benzidine
congeners 3,3'-dimethylbenzidine and 3,3'-dimethoxybenzidine are much less
soluble (1.3 and 0.06 mg/ml respectively) than their corresponding chloride
salts (76.7 mg/ml and 41.4 mg/ml).
The chloride salt of benzidine has also been reported to be more resist-
ant to degradation than the free amine. Bowman, King, and Holder (1976)
found that over a 16-day period, the concentration of an aqueous solution of
benzidine declined 11% while that of its salt declined only 2%. The con-
centration of both 3,3'-dimethylbenzidine and its salt declined 9% during
the test period, and that of 3,3'-dimethoxybenzidine and its salt declined
64% and 9% respectively.
8.2.2 Biodegradation
It is known that benzidine and its congeners undergo in vivo oxidation
in higher organisms; however, there is also evidence that benzidine is
resistant to decomposition by microorganisms. Lutin, Cibulka, and Malaney
(1965) and Malaney et al. (1967) found that benzidine at concentrations of
500 mg/liter was usually inhibitory or even toxic to sludge microorganisms,
thus preventing normal biodegradative processes. The studies of Jenkins
and Baird (1975) suggest that the toxic substance may not have been benzi-
dine itself, but a reaction product resulting from chlorination. These
investigators found that in unchlorinated wastewater samples spiked with
benzidine 70% to 95% of the chemical could be removed analytically, but in
chlorinated samples only a very small amount could be recovered. This sug-
gested that benzidine reacted with hypochlorous acid to produce a chloramine-
type compound. Chloramines are known to be toxic to a great number of
aquatic organisms.
It has been reported by several investigators that some azo dyes can
be reduced to free benzidine by soil and intestinal bacteria (Yoshida and
Miyakawa, 1973). Presumably, similar biochemical transformations occur in
bacteria in aquatic systems.
Numerous nonbenzidine azo food dyes are known to undergo reduction to
various metabolites by way of the hepato-azo reductase system of higher
organisms (see Walker, 1970, for a recent review). Metabolic reduction of
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benzidine dyes to benzidine has recently been demonstrated In the rhesus
monkey (Rinde and Troll, 1975), and benzidine dyes have been implicated as
carcinogenic agents in painters accidently ingesting these dyes (Yoshida
and Miyakawa, 1973).
8.3 EFFECTS ON AQUATIC AND TERRESTRIAL ORGANISMS
8.3.1 Nonlaboratory Organisms
Although there is a substantial amount of information on the effects
of benzidine on standard laboratory research animals such as rats, mice,
and dogs, very little is known about the toxicity of benzidine to other
kinds of organisms. That benzidine can be toxic to some plants and wild
animals is shown by the studies of Fitzgerald, Gerloff, and Skoog (1952)
and Pliss and Khudoley (1975). In analyzing the toxicity of several organic
compounds to the blue-green alga MicTOoystis aerug-inosa, Fitzgerald,
Gerloff, and Skoog (1952) found that benzidine, at concentrations of 20
mg/liter, was lethal to 50% of five-day algal cultures. Pliss and Khudoley
(1975) examined the effects of benzidine on the freshwater guppy. They
found that although the chemical did not produce tumors in the fish, its
toxic effects included extensive liver damage. Since the benzidine dosage
used by these investigators was calculated in terms of food intake (30 mg of
benzidine per 100 g of dry food), there is no way to correlate these results
with potential benzidine concentrations in natural bodies of water.
8.3.2 Laboratory Animals
The toxic effects of benzidine and its congeners have been found to
depend on several factors: (1) physiochemical form of the compound admin-
istered, (2) size of the dose, (3) mode of administration, (4) length of
exposure, and (5) species of animal undergoing the tests. In most species
tested these substances tended to cause the greatest damage to the liver,
kidney, and urinary bladder. The gall bladder, intestines, and lymph glands
can also be affected to a lesser extent. Toxic levels of benzidine often
lead to extensive liver damage. In many laboratory studies, excessive deaths
due to the poisoning have obscured the carcinogenic potential of these com-
pounds. However, following administration of sublethal doses tumors have
formed in almost all species studied. In various species of rodents, hepato-
mas, lymphomas, and adenocarcinomas have resulted from exposure to benzidine
and 3,3'-dihydroxybenzidine. In contrast, in hamsters, rabbits, and dogs the
carcinogenic activity of benzidine is centered almost solely in the bladder.
The benzidine congeners are also known to be carcinogenic in rats and mice;
the target organs depend considerably on the mode of administration and con-
centration of the compound. Tumors have been produced in the rat and mouse
in the skin, gastrointestinal tract, liver, bone, ovaries, Zymbal's gland,
sebaceous glands, mammary glands, and urinary bladder.
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8.4 EFFECTS ON HUMAN HEALTH
8.4.1 Toxic Effects
Schwartz, Tulipan, and Peck (1947) have reported on the occurrence of
dermatitis among workers in azo dye manufacturing plants. In several
instances it was found that the dermatitis resulted from exposure to benzi-
dine or benzidine-based dyes. Laboratory exposure to 3,3'-dimethylbenzidine,
as well as to benzidine, has also been known to cause dermatitis among chem-
ists. Hematuria occasionally occurs in workers exposed to 3,3'-dimethyl-
benzidine or benzidine, and the latter compound is also known to cause
chronic cystitis.
8.4.2 Teratogenic Effects
There is no evidence that benzidine or its congeners act as human
teratogens. In one of the few laboratory studies having any possible rele-
vance to this subject, Noto (1967) found that albumen suspensions of benzi-
dine (3 mg) injected into a chicken egg resulted in morphological abnormalities
in the developing embryo. The chemical was thought to inhibit morphogenetic
movements, thus causing the unclosure of the neural tube. Other known carcino-
gens such as 4-nitroquinoline-N-oxide and o-aminoazotoluene caused similar
abnormalities.
8.4.3 Mutagenic Effects
Ames et al. (1973) proposed that many known carcinogens, including
benzidine, owe their carcinogenic activity to the fact that they cause
somatic mutations. In tests using mutant strains of the bacteria Salmonella
typh-i.mur"ium and a rat or human liver homogenate to provide the necessary
enzyme systems responsible for activating the carcinogen, these workers
found that benzidine caused a significant increase in the number of revertant
bacteria colonies. The carcinogen was thought to have caused frameshift
mutations involving addition or depletion of DNA base pairs. Such a change
in the structure of DNA might produce an inheritable change in cell regula-
tion which could thereby lead to the formation of a tumor.
8.4.4 Carcinogenic Effects
Epidemiological evidence leaves no doubt that exposure to benzidine can
lead to bladder cancer in humans. Although there is no evidence to indicate
that the benzidine congeners 3,3'-dimethylbenzidine, 3,3'-dimethoxybenzidine,
°r 3,3'-dichlorobenzidine can cause bladder cancer in humans, all three com-
pounds have been implicated as carcinogens in animal studies. Tumors of other
human organ systems (intestinal tract, lungs, bone, and muscles) have been
found in some dichlorobenzidine workers (Gerarde and Gerarde, 1974), but the
significance of this finding has not yet been established.
Various studies have shown that the actual chemical substance causing
bladder cancer following benzidine exposure is not benzidine itself but a
metabolic by-product of the compound. Enzyme systems that are found pri-
marily in the liver presumably convert benzidine into N-hydroxylated
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133
derivatives which may then require esterfication to become further activated.
The ultimate carcinogens may be sulfate or glucuronidate conjugates of the
metabolically altered parent compound (see Section 2.2.1.5).
An exact quantitative relationship between the degree of exposure to
benzidine, the length of the exposure period, and the incidence of bladder
cancer has not been established. Studies with laboratory animals are not
very indicative in this regard; there appears to be species-specific differ-
ences in the relative toxicity and carcinogenicity of the compounds, rate
and route of uptake and excretion, and metabolic pathways. Studies at benzi-
dine manufacturing plants indicated that workers had been exposed to atmos-
pheric concentrations of benzidine from 0.002 to 17.3 mg/m3 (Meigs, Sciarini,
and Van Sandt, 1954; Zavon, Hoegg, and Bingham, 1973), yet there is no way
of accurately estimating the level of exposure and the amount taken up by
individual workers. Laboratory studies on animals led Meigs, Sciarini, and
Van Sandt (1954) to speculate that approximately 5% of the amount of benzi-
dine taken up is excreted as the free amine. Presumably the remaining 95%
undergoes metabolic transformation. Whether all the by-products are excreted
or whether some might be stored in tissues has not been shown. The degree to
which in vivo oxidation renders the compounds harmless is also unknown.
According to Zavon, Hoegg, and Bingham (1973) there is a nonlinear
relationship among exposure levels, exposure periods, and incidence of
bladder tumors. Supposedly, a minimum exposure will be followed by a pro-
longed latent period, but increased exposure will not necessarily result in
decreased latency or increased incidence of the disease (Scott, cited in
Zavon, Hoegg, and Bingham, 1973). This hypothesis would seem to be con-
firmed by the fact that the latent period is quite prolonged (average time
16 to 20 years) in benzidine workers developing bladder tumors, regardless
of exposure histories.
Meigs, Sciarini, and Van Sandt (1954) calculated that an atmospheric
concentration of 0.02 mg/m3 might be safe for industrial workers. They based
this assumption on the fact that workers exposed to this level of benzidine
(estimated from the concentration of benzidine in their urine, which was
thought to be 5% of the amount taken up) were reportedly unlikely to develop
bladder tumors, whereas workers with higher exposures were more susceptible.
The validity of these calculations has not been verified by other investigators.
8.5 POTENTIAL HEALTH HAZARDS
8.5.1 Industrial Workers
Since the late 1950s when it was definitely established that benzidine
was carcinogenic, there has been a general effort to reduce the levels of
exposure to which benzidine workers are subjected. In 1974 the U.S. Depart-
ment of Labor announced specific regulations governing the conditions under
which benzidine and the benzidine congeners could be manufactured and pro-
cessed (Federal Register, 1974). Presumably, these regulations have reduced
benzidine concentrations in industrial environments to safe levels; however,
these regulations do not recognize maximum permissible levels of contamination.
In the absence of published information on the current monitoring practices
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134
in the benzidine industry, of data regarding current levels of exposure,
and of a proven minimum exposure threshold, it is difficult to evaluate
current health hazards in industry; however, the reduction in tumor inci-
dence would suggest that the risks for benzidine workers have been sub-
stantially reduced in recent years.
8.5.2 Laboratory Workers
A number of reports have emphasized the potential dangers of benzidine
and the benzidine congeners in chemical, biochemical, and microbiological
laboratories where these compounds are often used as analytical reagents
(Collier, 1974; Veys, 1972; Wood and Spencer, 1972). Levels of contamination
would undoubtedly vary from laboratory to laboratory, and it is very likely
that a broad epidemiological study of laboratory workers would be necessary
to determine if laboratory exposure to these compounds constitutes a signif-
icant hazard. In view of the potential danger, suitable alternative analyti-
cal reagents should be recommended, and if no such alternatives exist, users
of these compounds should be adequately warned of their potential
carcinogenicity.
8.5.3 General Population
There is no evidence that benzidine or any of its congeners represent
a health hazard to the general public. The little information that is
available suggests that any benzidine released into the atmosphere would
undergo rapid physiochemical degradation. It is more likely that if danger-
ous environmental levels of benzidine are found, these would be restricted
to water supplies near manufacturing and processing plants. However, these
compounds have not been reported in any natural bodies of water in the
United States. Whether this negative evidence is due to a lack of adequate
monitoring data or to a real absence of these substances is unknown. It is
quite possible that biological decomposition in water may proceed at a rapid
rate; however, it has also been shown that benzidine is resistant to bio-
degradation in water treatment plants — possibly due to the formation of
toxic chloramines. The contamination of potential water supplies is a
distinct possibility as is indicated by a Japanese study showing that benzi-
dine levels were quite high in a river downstream from a series of benzidine
dye plants (Takemura, Akiyama, and Nakajima, 1965). This also raises the
question as to whether environmental contamination by benzidine-based dyes
is a greater potential hazard than that associated with benzidine alone.
It has been reported that such dyes can be broken down into benzidine by soil
and intestinal bacteria (Yoshida and Miyakawa, 1973); it is very likely that
bacteria normally found in natural bodies of water would have the same capa-
bility. Benzidine dyes are used in a wide variety of commercial products,
but as in the case of benzidine, there is very little information on the
amounts being released into the environment.
8.6 POTENTIAL ENVIRONMENTAL HAZARDS
It has been shown that benzidine is potentially toxic to microorganisms,
a species of blue-green algae, one species of freshwater fish, and various
laboratory animals. Although the data are not very extensive, they lead to
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135
the conclusion that sufficiently high concentrations of benzidine may prove
to be disruptive to the environment. In view of the fact that the most
likely route of entry into the environment would be through industrial
effluents, aquatic systems may be the most susceptible to damage. There is,
however, no evidence that toxic levels of benzidine or any benzidine compound
are present in any natural bodies of water in the United States. This lack
of evidence may be due to an insufficient amount of environmental monitoring
data resulting from the general assumption that these substances are not
environmental hazards. More refined analytical techniques are currently
available to detect levels of contamination as low as 0.2 yg/liter and these
should be used to determine whether any significant environmental contamina-
tion is occurring.
Although it would appear that benzidine lacks the physiochemical charac-
teristics — such as resistance to chemical and biological degradation — to
make it a major long-range environmental problem, its toxicity and carcino-
genicity are sufficient to require that a continuing effort be made to
evaluate its effects on the environment.
8.7 REGULATIONS AND STANDARDS
The industrial standards instituted by OSHA in 1974 excluded from
regulation any compounds containing less than 0.1% benzidine. These stan-
dards did not recognize a safe level of air or water contamination, nor were
any provisions made for environmental monitoring.
In 1973 the Environmental Protection Agency proposed a toxic pollutant
standard for benzidine which was based primarily on an extrapolation of
animal test data (Federal Register, 1973). The standard proposed limited
the amount of benzidine discharged into navigable waters to 1.8 yg/liter
provided there was an immediate 1:10 rate of diffusion or to 0.18 yg/liter
in situations where there was a slower rate of diffusion. Because of the
limited data base, these standards were not promulgated.
In 1976 EPA proposed new standards for benzidine discharges (Federal
Register, 1976). These standards were supported by evidence presented in
Criteria Document: Benzidine (U.S. Environmental Protection Agency, 1976),
which contained information on toxicological and environmental effects and
fate of benzidine. These standards, which were promulgated in 1977 (Fed-
eral Register, 1977) , establish an ambient water criterion for benzidine of
0.1 yg/liter. Effluent standards were set at 10 yg/liter (daily average)
with a maximum for any single day of 50 yg/liter. Based on a monthly
average, daily loading was limited to 0.130 kg per 1000 kg of benzidine
produced. The standards set for users of benzidine-based dyes were the
same except that the maximum daily effluent concentration of benzidine was
limited to 25 yg/liter.
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SECTION 8
REFERENCES
1. Ames, B. N., W. E. Durston, E. Yamasaki, and F. D. Lee. 1973. Car-
cinogens Are Mutagens: A Simple Test System Combining Liver Homog-
enates for Activation and Bacteria for Detection. Proc. Natl. Acad.
Sci. U.S.A. 70(8):2281-2285.
2. Bowman, M. C., J. R. King, and C. L. Holder. 1976. Benzidine and
Congeners: Analytical Chemical Properties and Trace Analysis in Five
Substrates. Int. J. Environ. Anal. Chem. (Great Britain) 4:205-223.
3. Collier, H. B. 1974. Are Orthotolidine and Dianisidine Health Hazards
to Laboratory Workers? Clin. Biochem. (Canada) 7:3-4.
4. Federal Register. 1973. Proposed Toxic Pollutant Effluent Standards.
30(247):35388-35392.
5. Federal Register. 1974. Occupational Health and Safety Standards,
Carcinogens. 39(20):3756-3781.
6. Federal Register. 1976. Benzidine: Proposed Toxic Pollutant
Effluent Standards. 41(127) :27012-27017.
7. Federal Register. 1977. Toxic Pollutant Effluent Standards.
42(8):2617-2621.
8. Fitzgerald, G. P-, G. C. Gerloff, and F. Skoog. 1952. Studies on
Chemicals with Selective Toxicity to Blue-Green Algae. Sewage Ind.
Wastes 24(7) :888-896.
9. Gerarde, H. W., and D. F. Gerarde. 1974. Industrial Experience with
3, 3'-Dichlorobenzidine: An Epidemiological Study of a Chemical
Manufacturing Plant. J. Occup. Med. 16:322-344.
10. Jenkins, R. L., and R. B. Baird. 1975. The Determination of Benzidine
in Wastewaters. Bull. Environ. Contam. Toxicol. 13(4):436-442.
11. Lutin, P. A., J. J. Cibulka, and G. W. Malaney. 1965. Oxidation of
Selected Carcinogenic Compounds by Activated Sludge. Proc. Ind. Waste
Conf. 20:131-145.
12. Malaney, G. W., P- A. Lutin, J. J. Cibulka, and L. H. Hickerson. 1967.
Resistance of Carcinogenic Organic Compounds to Oxidation by Activated
Sludge. J. Water Pollut. Control Fed. 39(12):2020-2029.
13. Meigs, J. W., L. J. Sciarini, and W. A. Van Sandt. 1954. Skin Penetra-
tion by Diamines of the Benzidine Group. Arch. Ind. Hyg. Occup. Med.
9:122-132.
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14. Noto, T. 1967. The Effects of Some Carcinogens on the Morphogenesis
and Differentiation in the Early Chick Embryo. Sci. Rep. Tohoku Univ.
Ser. 4 (Japan) 33:65-69.
15. Pliss, G. B., and V- V- Khudoley. 1975. Tumor Induction by Carcino-
genic Agents in Aquarium Fish. J. Natl. Cancer Inst. 55(1):129-136.
16. Radding, S. B., B. R. Holt, J. L. Jones, D. H. Liu, T. Mill, and
D. G. Hendry. 1975. Review of the Environmental Fate of Selected
Chemicals. EPA 560/5-75-001, U.S. Environmental Protection Agency,
Washington, D.C. 37 pp.
17. Rinde, E., and W. Troll. 1975. Metabolic Reduction of Benzidine
Azo Dyes to Benzidine in the Rhesus Monkey. J. Natl. Cancer Inst.
55(1):181-182.
18. Schwartz, L., L. Tulipan, and S. M. Peck. 1947. Dermatitis in Synthetic
Dye Manufacture. In: Occupational Diseases of the Skin. Lea and
Febiger, Philadelphia. pp. 268-280.
19. Takemura, N., T. Akiyama, and C. Nakajima. 1965. A Survey of the
Pollution of the Sumida River, Especially on the Aromatic Amines in
the Water. Int. J. Air Water Pollut. (Great Britain) 9:665-670.
20. U.S. Environmental Protection Agency. 1976. Criteria Document:
Benzidine. EPA-440/9-76-017, Washington, D.C. 74 pp.
21. Walker, R. 1970. The Metabolism of Azo Compounds: A Review of the
Literature. Food Cosmet. Toxicol. (Great Britain) 8:659-676.
22. Wood, J. M., and R. Spencer. 1972. Carcinogenic Hazards in the
Microbiology Laboratory. In: Safety in Microbiology, D. A. Shapton
and R. G. Board, eds. Academic Press, London, pp. 185-189.
23. Veys, C. A. 1972. Aromatic Amines: The Present Status of the
Problem. Ann. Occup. Hyg. (Great Britain) 15:11-15.
24. Yoshida, 0., and M. Miyakawa. 1973. Etiology of Bladder Cancer:
"Metabolic" Aspects. In: Analytic and Experimental Epidemiology
of Cancer, W. Nakahara, T. Hirayama, K. Nishioka, and H. Sugano, eds.
University Park Press, Baltimore, pp. 31-39.
25. Zavon, M. R., U. Hoegg, and E. Bingham. 1973. Benzidine Exposure
As a Cause of Bladder Tumors. Arch. Environ. Health 27:1-7-
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ORNL/EIS-86
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29-30. Laboratory Records
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Theodore M. Albert, Office of Technology Impacts, Division of
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-------
.Suggested Format for the Human Effects Section
of Criteria Documents
I. Exposure (Route of Entry)
A. Ingestion
1. Water \
2. Food (including food chain magnification)
B. Inhalation
C. Dermal
1. Some statement as to relative contributions to total dose.
'II. Pharnacokinetics Human Data. Supplemented by Other Mammalian '
Species Where Pertinent. This section should be short but briefly
address each of the following and include citations of important papers.
A. Absorption
B. Distribution
C. Metabolism
D. Excretion (including body burden and half-life data)
III. Effects (including clinical, occupational, epideiniologic and pertinent
animal studies). The emphasis is to be upon E. Animal and human data
on cancer is the major item and should be as complete as possible.
A. Acute, Subacute, and Chronic Toxicity (Target Organs)
B. Synergistic or Antagonistic Compounds
C* Teratogenicity
D. Mutagenicity
E. Carcinogenicity
1. Animal data. This data, should where possible, be presented in
tabular form, i.e. describe species, route of administration,
sex, affected organ(s), time to tumor, reference, etc. as
well as dose response data. See Example.
2. Human data, case reports, etc.
IV- Criterion Rational
A. Existing Standards
B. Current Levels of Human Exposure
C. Special Groups at Risk
D. Basis for the Standard
1. Extrapolation - use the linear, non-threshold model. We will
supply method and assistance.
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138-286. Jerry F. Stara, Office of Program Operations, Health Effects
Research Laboratory, U.S. Environmental Protection Agency,
Cincinnati, Ohio 45268.
287. D. G. Stephan, Director, U.S. Environmental Protection Agency,
Industrial Environmental Research Laboratory, Cincinnati, Ohio
45268.
288-289. E. Stokes, U.S. Environmental Protection Agency, Region VI
Library, 345 Courtland Street NE, Atlanta, Georgia 30308.
290. Lester Sutton, U.S. Environmental Protection Agency, Region 1,
J. F. Kennedy Federal Building, Room 2304, Boston, Massachusetts
02203.
291. H. Sykes, Office of Pesticide Programs, Technical Information
Center, Room 42-B, U.S. Environmental Protection Agency,
Washington, D.C. 20460.
292. V. Tenney, R&D Representative, U.S. Environmental Protection
Agency, Region IX, 100 California Street, San Francisco,
California 94111.
293. L. Tilley, U.S. Environmental Protection Agency, Region V
Library, 230 South Dearborn, Chicago, Illinois 60604.
294. U.S. Environmental Protection Agency, Office of Research and
Development, Health Effects Division (RD-683), Washington,
D.C. 20460.
295. U.S. Environmental Protection Agency, Region 1, New England
Regional Laboratory, 60 Westview Street, Lexington, Massachusetts
02173.
296. U.S. Geological Survey Library, National Center (mail stop 950),
12201 Sunrise Valley Drive, Reston, Virginia 22092.
297. A. Weir, U.S. Environmental Protection Agency, Region X Library,
1200 Sixth Avenue, Seattle, Washington 98101.
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144
298. Wheeling Field Office, U.S. Environmental Protection Labora-
tory Director, 303 Methodist Building, Wheeling, West Virginia
26003.
299. H. Wiser, Principal Science Advisor (RD-676), U.S. Environmental
Protection Agency, Washington, D.C. 20460.
300. E. A. Young, U.S. Environmental Protection Agency, Field Studies
Section, P.O. Box 219, Wenatchee, Washington 98801.
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145
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
5. REPORT DATE
Reviews of the Environmental Effects of Pollutants:
II. Benzidine
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Carole R. Shriner, John S. Drury, Anna S. Hammons,
Leigh E. Towill, Eric B. Lewis, and Dennis M. Opresko
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Information Center Complex, Information Division
Oak Ridge National Laboratory
Oak Ridge, Tennessee 37830
10. PROGRAM ELEMENT NO.
1HA616
11. CONTRACT/GRANT NO.
IAG D5-0403
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory, Cin-OH
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
13. TYPE OF REPORT AND PERIOD COVERED
Final October 1977
14. SPONSORING AGENCY CODE
EPA/600/10
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This report is a review of the scientific literature on the biological
and environmental effects of benzidine. Included in the review are a general
summary and a comprehensive discussion of the following topics as related to
benzidine and specific benzidine compounds: physical and chemical properties;
occurrence; synthesis and use; analytical methodology; biological aspects in
microorganisms, plants, wild and domestic animals, and humans; distribution,
mobility, and persistence in the environment; assessment of present and
potential health and environmental hazards; and review of standards and
governmental regulations. More than 200 references are cited.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDEDTERMS
c. COSATI Field/Group
^Pollutants
Benzidine
Toxicology
Health Effects
06F
06T
13. DISTRIBUTION STATEMENT
Release to public
19. SECURITY CLASS (This Report)
Unclassified
!1. NO. OF PAGES
160
20. SECURITY CLASS (This page)
Unclassified
22. PRICE
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