5601180019
PRELIMINARY RISK ASSESSMENT: PHASE I
BENZIDINE, ITS CONGENERS, AND THEIR
DERIVATIVE DYES AND PIGMENTS
Dr. Theodore C. Jones
Project Manager
Assessment Division
Office of Pesticides and Toxic Substances
OFFICE OF TESTING AND EVALUATION
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
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This document has been reviewed and approved for
publication by the Office of Testing and Evaluation,
Office of Pesticides and Toxic Substances, U.S.
Environmental Protection Agency. Since this is a
preliminary assessment, it should not be construed
to present a full or final evaluation by the Agency
of the topics included. The mention of trade names
or commercial products does not constitute
endorsement or recommendation for use by the
Environmental Protection Agency.
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Foreword
Evaluations of chemical substances prepared by scientists in EPA's
Office of Testing and Evaluation, Office of Pesticides and Toxic Substances
(OPTS), to implement provisions in the Toxic Substances Control Act (TSCA),
will be published periodically and made available to the public in the TSCA
Chemical Assessment' Series. Some of the volumes in the series are reports
on single chemicals; others are compendiums of information received and
evaluated by the Agency about many chemicals. (The anticipated frequency
of publication will vary among titles: some will be published annually,
some semiannually, and others quarterly.)
Because the chemical assessments published in this series often will
reflect initial or intermediate steps in EPA's evaluation of a chemical
under TSCA, the Agency welcomes the submission of additional information
for or comments on its evaluations. Such submissions will be considered
either at a subsequent step in the assessment of the subject chemical or in
the decision not to proceed with further evaluation.
All information for or comments on volumes in the TSCA Chemical
Assessment Series should be submitted to:
Document Control Officer (TS-793)
Office of Pesticides and Toxic Substances
U.S. Environmental Protection Agency
401 M Street SW
Washington, D.C. 20460
Comments on this volume in the Series should bear the identifying
docket number OPTS 10003.
The TSCA Chemical Assessment Series is being distributed through the
Industry Assistance Office (IAO) in OPTS. IAO is maintaining two mailing
lists: a subscription list of persons who want to receive all volumes in
the series and a notification list of persons who want to receive announce-
ments of individual volumes as they become available. Requests for a place
on either list can be made by telephoning IAO (tollfree 800-424-9065 or, in
Washington, D.C., 554-1404) or writing to:
Document Control Officer (TS-793)
Office of Pesticides and Toxic Substances
U.S. Environmental Protection Agency
401 M Street SW
Washington, D.C. 20460
Generally, five thousand copies of each volume will be printed. When
this supply has been exhausted, copies can be purchased from the National
Technical Information Service (NTIS), whose "PB" reference number can be
found in the OPTS "Comprehensive List of Scientific and Technical Reports,"
also available from IAO.
iii
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Contents
Foreword ill
Introduction 1
Executive Summary 3
Disposition 9
I. Production and Uses 10
A. Production Methods 10
Benzidine and Congeners 10
Dyes and Pigments 10
B. Major Uses 14
C. Production and Import Values 15
Benzidine and Congeners 15
Dyes and Pigments 16
II. Health Effects 21
A. Benzidine and Benzidine-Based Dyes 21
Mutagenicity 21
Carcinogenicity 21
Metabolism and Bacterial Degradation 22
B. Dichlorobenzidine and Dichlorobenzidine-
Based Pigments 25
Mutagenicity 25
Carcinogenicity 25
Metabolism 27
C. o-Tolidine 28
Mutagenicity 28
Carcinogenicity and Teratogenicity 31
Metabolism 32
D. Dianisidine 32
Mutagenicity 32
Carcinogenicity . ..... 32
III. Ecological Effects 33
IV. Environmental Fate 35
A. Water 35
B. Air and Soil 36
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Contents ( cont . )
V. Exposure Analysis .... ................... 37
A. Distribution in the Environment .............. 37
B. Sources of Release to the Environment . .......... 37
Benzidine and Its Congeners ............... 37
Dyes and Pigments .................... 38
C. Population Exposed .................... 38
Industrial Workers ................... 38
Laboratory Workers ................... 41
General Population ......... . ......... 41
VI. Summary of Issues Including Validation
and Information Needs ..................... 44
A. Benzidine and Benzidine-Based Dyes ... ......... 44
Validation Needs .................... 45
B. p_-Tolidine, Dianisidine, and Their
Derivative Dyes and Pigments ............... 45
C. Dichlorobenzidine and Its
Derivative Pigments .................... 46
Identified Issues ........ . ........... 46
Validation Needs .................... 46
D. General Issues . ............... ...... 46
Information Needs .................... 47
References ............................. 48
Appendix A ............................. 54
Technical Report Data Sheet ..................... 55
vi
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Introduction
In the Office of Pesticides and Toxic Substances (OPTS), the
assessment of chemical substances is sequential. A decision to perform a
Preliminary Risk Assessment (Phase I assessment) of a chemical is based on
a Chemical Hazard Information Profile (CHIP)* or its equivalent.
Following validation of the key information in the preliminary risk
assessment, a detailed risk assessment (Risk Assessment in Support of
Regulatory Decision Making, Phase II) is prepared if the Phase I
disposition indicates that comprehensive assessment should continue.
The Preliminary Risk Assessment report is a summary and analysis of
information on the known sources and effects of exposure to a given
chemical, identifying the specific problems it is most likely to create.
The report arrives at preliminary conclusions about which effects present
significant hazards; whether existing or anticipated exposure levels may
pose a threat to human health or the environment; whether the information
available on a chemical is sufficient to complete all or part of the
Preliminary Risk Assessment; and the key technical and policy issues that
the information in the report has raised.
The structure of the Preliminary Risk Assessment report has evolved
over time to include an executive summary, an introduction to the chemical,
information about production and use, health and environmental effects,
principal sources of exposure, and a preliminary analysis of risk. As a
result of the evolution of the structure and content of the report, early
Phase I reports do not share a common outline and have not gone through the
same process of development as those now being prepared. However, in all
Phase I reports, to the extent that it is possible to do so, potential
sources of the chemical and its potential effects are ranked on the basis
of relative risk. This ranking considers any differences in absorption
route, frequency, or duration of exposure that may cause ranking by
relative risk to differ from ranking by relative exposure level.
The literature search for a Preliminary Risk Assessment report is
targeted to retrieve 95 percent of all published information on a given
chemical for a 30- to 50-year period. This search should turn up all
relevant published articles, reviews of the literature, research
monographs, and monitoring and field study reports. The search does not
include unpublished studies, particularly those in industry files. To
obtain such information, a TSCA section 8(d) reporting requirement may be
*The Chemical Hazard Information Profile (CHIP) is a brief summary
addressing potential for adverse health or environmental effects or
significant exposure based primarily on secondary literature. For a more
extensive discussion of the CHIP, see the Introduction in "Chemical Hazard
Information Profiles (CHIPs), August 1976-August 1978," Volume 1, April
1980, EPA 560/11-80-011.
1
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invoked. If essential use and production data are not available from the
section 8(b) Inventory or any other internal Agency source, a section 8 (a)
reporting requirement may be invoked. However, when a substantial amount
of data is available in the published literature, the literature search
generally will suffice.
A Preliminary Risk Assessment report is accompanied by a disposition
summary documenting the next course of action planned on the chemical. The
three major possible dispositions are to keep the chemical in the
assessment process, to suspend further assessment based on existing
information, and to initiate information-gathering efforts (such as testing
requirements under section 4 of TSCA) to obtain information critical to
further assessment.
At this stage in the process the Preliminary Risk Assessment report is
published in the TSCA Chemical Assessment Series, with a request for public
comment on the report's accuracy and completeness. All comments received
are available for inspection and copying in the OPTS reading room,* unless
specifically claimed as confidential in accordance with applicable EPA
rules and procedures (see 40 CFR Part 2 [41 FR 36902, Sept. 1, 1976]).
Public comments received on a Phase I report are considered in the
validation stage, which will be described subsequently, or in reconsidering
a decision not to proceed to validation.
The second stage of the preliminary risk assessment is the validation
of the Phase I assessment report and the preparation of a Control Options
paper. The validation is an in-depth staff review of the adequacy of the
key studies and the conclusions drawn about exposure and effects. To date,
not all of the Phase I reports written in OPTS have been validated. The
Control Options paper analyzes the feasibility of using various regulatory
controls to reduce the most serious of the risks presented by a chemical.
Once a Preliminary Risk Assessment report has been validated, the
report and its corresponding Control Options paper are submitted to the
Toxic Substances Priority Committee (TSPC). The TSPC considers whether the
chemical should proceed to initiation of rulemaking under TSCA or other
authorities, based on the Committee's evaluation of the relative importance
of the sources of environmental release and exposure, the severity and
probability of potential effects, the feasibility of the proposed controls,
and the mechanism to effect the needed controls.
Validated Preliminary Risk Assessment reports will be published if
they differ substantially from the unvalidated reports.
*Room E447 at EPA Headquarters, 401 M Street SW, Washington, D.C. 20460
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Executive Summary
Benzidine and its congeners, 3,3'-dichlorobenzidine, o-tolidine
(3,3'-dimethylbenzidine), and dianisidine (3,3'-dimethoxybenzidine)
constitute a family of similar synthetic aromatic compounds (see Figure 1)
NHt
3,3'-Dichlorobenzidine
O-Tolidin*
CH,0 OCH,
HiN
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Table 1. Dyes and Pigments Derived from Benzidine and Its Congeners
Currently on the U.S. Market, 1979
Derived from benzidine:
Direct Black #4, 38
Direct Blue #2, 6
Direct Brown #1A, 2, 6, 31, 59, 74, 95, 154
Direct Green #1, 6, 8
Direct Orange 8
Direct Red #1, 28, 37
Direct Violet #1, 22
Acid Red 85
Derived from o-tolidine:
Acid Red #114, 167
Acid Black 209
Azoic Coupling Component 5
Azoic Yellow Composition #1, 2, :
Azoic Orange Composition 3
Direct Blue #14, 25, 26
Direct Orange 6
Direct Red #2, 39
Direct Brown 230
Direct Yellow 95
(Dyes without C.I, generic name)
Diphenyl Green
Direct Fast Brown BCW-NB
Padazoic Yellow G
Padazoic Orange GR
Penetrating Black AM-NB
Sandolan Red N-3B
Derived from dianisidine:
Direct Black #94, 114, 118
Direct Blue #1, 8, 15, 22, 76, 80, 80(S),
90, 98, 100, 151, 160, 191(S),
218, 218/224 (S), 244,
269 (o-anisidine)
Direct Brown 200
Direct Yellow 68
Direct Violet 93
Azoic Blue Composition #2, 3, 6
Azoic Diazo Component 48
Azoic Coupling Component 3
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Table 1. Dyes and Pigments Derived from Benzidine and Its Congeners
Currently on the U.S. Market, 1979 (cont.)
(Dyes without C.I, generic names)
Atlantic Printing Black 2B P
Atlantic Resin Fast Blue ARL
Padazoic Brilliant Indigo 3B
Atlantic Printing Brown GGN
Atlantic Resin Fast Grey LVL
Superlitefast Rubine WLKS
Derived from dianisidine:
Pigment Orange 16
Pigment Blue 25
Derived from dichlorobenzidine:
Pigment Orange #13, 34
Pigment Yellow #12, 13, 14
Source: Information supplied by Dyes Environmental and Toxicology
Organization, Inc. (DETO), and Dry Color Manufacturer's Association
Benzidine and its three congeners all have been shown to be mutagenic
in the Ames Salmonella assay. Benzidine also is a demonstrated animal
carcinogen and has been shown to cause bladder cancer in humans. Positive
carcinogenieity studies (some of questionable validity by current
standards) also have been reported for the three benzidine congeners.
Three benzidine-based dyes (Direct Blue 6, Direct Black 38, and Direct
Brown 95) were reported to be carcinogenic in rats. These dyes, as well as
Direct Red 28, have been shown to metabolize to benzidine in rhesus
monkeys. Preliminary results suggest that Direct Red 28, and five
additional benzidine-based dyes not previously tested, are also metabolized
to benzidine in dogs.
The 2~tolidine-based component of Trypan Blue is a proven teratogen
and mutagen; the commercial dye, which is of mixed composition, is known to
be a carcinogen. No studies investigating the carcinogenic potential of
dianisidine-based dyes have been found in the available literature.
Metabolism tests in dogs are currently under way to determine whether
o-tolidine- and dianisidine-based dyes are metabolized to the parent
compounds.
Pigments Yellow 12, 13, and 83 (dichlorobenzidine-based) and Pigment
Yellow 16 (£-tolidine-based) are not carcinogenic in rats or mice.
Moreover, metabolism tests performed in the same species have failed to
demonstrate the release of free dichlorobenzidine from Pigments Yellow 12
and 13. Pigments Yellow 12 and 13 differ structurally from the above
benzidine-based dyes for which there is positive evidence of
carcinogenicity, accompanied by proof of metabolic breakdown to release the
parent benzidine compound. In addition to a difference in solubility
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(pigments are insoluble, whereas the dyes are water soluble),
dichlorobenzidine is a benzidine derivative with chlorine substituents
ortho to the amines; a keto-enol tautomeric group adjacent to the azo
linkage in the pigments may shield this linkage from enzymatic cleavage.
There currently is insufficient information, however, to draw firm
conclusions about the mutagenic, teratogenic, or carcinogenic potential of
other pigments derived from dichlorobenzidine or the other benzidine
congeners. Tables 2 and 3 summarize the evidence regarding the
careinogenicity, teratogenicity, and mutagenicity of benzidine, its three
congeners, and the dyes and pigments derived from them.
There is little published information on the environmental fate of
benzidine, its congeners, and the dyes and pigments derived from them.
Benzidine is thought to be oxidized readily in clay soils (with a half-life
of less than 1 day) and air (a half-life of 1 day) but less readily in
water (a half-life of 100 days, with organic peroxide ions). One field
study suggests that benzidine-derived dyes and pigments (in waste waters
from manufacturing plants) are reduced to the parent compound by hydrogen
sulfide or sulfur dioxide in the receiving waters. Adequate measures exist
for processing wastewaters to reduce the level of free benzidine to below
10 ppb, a level at which benzidine can be biodegraded by unacclimated
activated sludge in subsequent sewage treatment works. However, the
biological effects of the by-products of this process have not been
assessed. Regardless of the potential biodegradation and restricted
environmental release of benzidine, recent studies suggest that, if
available, benzidine is bioaccumulated in cesrtain fish. Dichlorobenzidine
also has been shown to bioaccumulate (135-fold) in bluegill sunfish. The
potential for bioaccumulation and possible contamination of the food chain
by benzidine (and dichlorobenzidine) may be enhanced by the lack of
restriction on release of dichlorobenzidine into the environment. Recent
studies suggest that the latter compound is rapidly degraded in aqueous
solutions (by natural sunlight), forming benzidine and 3-chlorobenzidine.
Direct exposures, both in terms of the number of people exposed cind in
exposure levels, to benzidine, its congeners, and their derivative dyes and
pigments occur primarily in occupational settings. Since 1976, occupa-
tional exposure to benzidine and benzidine-based dyes has been greatly
reduced as a result of a general reduction in the level of production and
of the imposition by the Environmental Protection Agency (EPA) of standards
for controlling benzidine levels in effluents from manufacturing plants and
dyeing operations. Currently there is only one major manufacturer of ben-
zidine-based dyes in the United States; only a few U.S. manufacturers still
produce dyes and pigments based on the three benzidine congeners. The
reduction in U.S. production, however, has been accompanied by an increase
in the importation of benzidine-based dyes, in which the level of free
benzidine is not controlled. In some imported dyes the level of free
benzidine has been reported to be as high as 500 ppm (25 times higher than
normally found in U.S. dyes). Thus, workers in plants using imported ben-
zidine-based dyes may constitute a new high-risk group.
The general public is exposed to benzidine, its congeners, and their
derivative dyes and pigments mainly in some paper, textile, leather, and
other substrates. Once applied to the substrate the dyes or pigments are
generally regarded as fast—i.e., they do not readily leach from the
material when used (e.g., when laundered) according to label directions.
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The possible extent of dye leaching under normal use conditions, however,
has not been determined for any of the dyes or pigments considered in this
report.
Other sources of exposure affect relatively small segments of the
general population but may be of greater concern. These include the
packaged dyes for home use (which may contain benzidine or benzidine-
congener derivative dyes) and those artist-craftsman products that contain
benzidine-congener derivative dyes and pigments—i.e., spray paints,
enamels, and lacquers.
Although additional information on exposure, release, and
environmental fate is needed to complete a detailed risk assessment on
benzidine, its congeners, and their derivative dyes and pigments, several
potential risks have been identified through a preliminary analysis of the
exposure and hazards associated with these compounds. The risks are (a) to
workers exposed to imported benzidine-based dyes that contain high
concentrations of free benzidine; (b) to workers using domestically
produced benzidine-based dyes (there are no occupational exposure standards
for either benzidine or its derivative dyes); (c) to the general
population, which may result from exposure to benzidine-based dyes in a
variety of products including home dyes and textiles; and (d) to the
environment from the release of dichlorobenzidine.
An assessment of the health risks that may be attributed to
o-tolidine, dianisidine, and their derivative dyes and pigments depends on
the validity of the carcinogenicity studies on the congeners and the
potential for metabolic conversion of the dyes/pigments to the parent
compound(s).
Disposition
Benzidine is one of 15 chemicals identified for assessment by Douglas
Costle, Administrator, U.S. Environmental Protection Agency. It is now in
the validation stage of preliminary risk assessment.
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I. PRODUCTION AND USES
A. Production Methods
1. Benzidine and Congeners
Benzidine is produced from nitrobenzene in two steps. First,
nitrobenzene in alkaline solution is treated with a mild reducing agent to
form what is mainly hydrazobenzene and lesser amounts of azobenzene and
azoxybenzene. Subsequent treatment of hydrazobenzene with mineral acid
results in the formation of benzidine by the well-known benzidine
rearrangement (Lurie 1964). The reaction scheme for the synthesis of
benzidine is shown in Figure 2.
At present, benzidine is produced domestically solely as a precursor
of azo dyes, by a single dye manufacturer, Fabricolor, using hydrazobenzene
as the starting material (Boeniger 1979). The synthesis of benzidine
congeners (Figure 3) is similar to benzidine synthesis, with nitrobenzene
derivatives being used as the starting materials.
2. Dyes and Pigments
The distinction between the terms "dye" and "pigment" as stated by the
dye industry (and as used in this report) is that dyes are soluble and
pigments are insoluble in the medium in which they are used. Where
solubility is not an important consideration, the word "dye" is sometimes
used to refer to dyes and pigments collectively, as in Lurie (1964).
Production of dyes and pigments from benzidine and its congeners
proceeds via tetrazotization to form the tetrazonium salt, which is
followed by coupling of the tetrazonium compound with a relative compound
(e.g., aromatic hydroxy compounds or arylamines) to form a colored product
(Lurie 1964). Tetrazotization is accomplished by reacting benzidine or a
congener with nitrous acid (sodium nitrite in hydrochloric acid) in a water
solution at 0-5°C. This produces the coupling agent, a tetrazonium
hydrochloride (Figure 4).
The final step in the production of a dye solution is the coupling of
the tetrazonium salt with a phenol, aromatic amine, or other reactive
compound. The coupled products have azo groups that are linked to
sp -hybridized carbon atoms; hence, they are azo dyes. A typical azo dye
made from benzidine (Congo Red, the first azo dye produced from benzidine)
is shown in Figure 5.
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Sodium nitrite + hydrochloric acid
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Benzidine
Benzidine tetrazonium salt
Figure 4. Tetrazotization of Benzidine.
NH2
N =
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Figure 5. Structure of Congo Red (Direct Red 28).
In the coupling reaction the first diazo group of the tetrazonium salt
of benzidine or a congener couples readily to give the diazomonoazo
compound. The activity of the second diazonium group is diminished so that
formation of unsymmetrical disazo dyes also can be accomplished (Lurie
1964). By proper choice of coupling agents, the resulting diazo compound
can be diazotized or used itself as a coupling component with another
diazonium compound to give rise to bisazo, trisazo, tetrakisazo, or polyazo
dyes (Lurie 1964). A wide variety of dyes spanning the color spectrum is
made possible by (1) a large number of possible coupling agents, (2) the
choice of symmetrical or unsymmetrical coupling, and (3) the possibility of
forming monoazo or polyazo derivatives. Table 4 shows the coupling agents
and their arrangements in some typical benzidine-based azo dyes.
13
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Table 4. Azo Dyes Derived from Coupling Benzidine
Reaction (-*-Indicates diazo coupling) Color
Salicylic acid-< Benzidine ^-Salicylic 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-Phenyldiamine •* Benzidine >-H-acid-*»-Aniline Black
Source: Adapted from Lurie (1964)
B. Major Uses
By far the most important and largest use of benzidine in the United
States is the production of the 22 benzidine dyes currently in commercial
production. These dyes are among the class known as direct dyes because
they can be applied directly to cellulosic substrates (e.g., cotton and
paper) without use of mordant. Relatively small amounts of benzidine also
have been used in analytical reagents to determine the presence of certain
inorganic ions (Welcher 1947); in thin-layer chromatography (Adamovic
1966); in analytical reactions to determine the presence of a number of
organic compounds (IARC 1972a); and in security printing, because it reacts
with ink erasers (IARC 1972a).
Dichlorobenzidine can be used for coupling to produce the 95 tetrazo
dyes listed in the Colour Index (1971); however, of these, only five
pigments are currently produced in the United States. Dyes and pigments
produced from dichlorobenzidine can be used for coloring plastic resins,
lacquers, rubbers, printing inks, metal finishes (Martens 1968), textile
and wallpaper prints (Colour Index 1971), interior grade "lead-free"
finishes (paints and toy enamels), and floor coverings (Colour Index 1957).
A few hundred metric tons of dichlorobenzidine also were used in 1969 as a
curing agent for liquid castable polyurethane elastomers (Woolrich and Rye
1969).
£-Tolidine is used domestically to produce 22 azo dyes. These dyes
are used for coloring products similar to those listed for benzidine and
dichlorobenzidine.
Dianisidine is used domestically to produce 36 azo dyes. These are
used for coloring leather, paper, plastics, rubber, and textiles. A small
quantity of dianisidine also has been used to manufacture 3,3'-dimethoxy-
4,4'-diphenylene diisocyanate, an ingredient in isocyanate adhesives and a
component of polyurethane elastomers (Woolrich and Rye 1969).
14
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C. Production and Import Volumes
1. Benzidine and Congeners
With the recognition of the potential health hazards of benzidine, its
manufacture and use have come under regulation in the United States and in
some foreign countries (Ferber 1978). By 1975, a number of U.S. producers
had ceased manufacturing benzidine/ and by 1978 its use was reported to be
diminishing rapidly (Ferber 1978). Although a complete compilation of
information on production and import volumes of benzidine and its congeners
could not be derived from available sources, the fragmentary data compiled
in Table 5 support the conclusion that benzidine production in the United
States has been greatly reduced since 1974. The general lack of
information on imports precludes an assessment of a trend in benzidine
importation; there were no imports reported by the International Trade
Commission in 1978. Production of dianisidine in 1978 was greatly reduced
from 1967, the only previous year for which production figures are
available; imports show no obvious long-term trend. Production of
dichlorobenzidine rose threefold between 1962 and 1977, while imports
fluctuated greatly; in general, however, imports seem to have decreased
since 1970. Since 1974, production of o-tolidine has remained stable;
imports have fluctuated since 1971.
Table 5. Production and Imports of Benzidine and Its Congeners
Chemical
Production (Ib)
Imports (Ib)
1978
1977
1976
1975
1974
1978
1977
1976
1975
1972
1970
1968
1962
Benzidine*
Small amount for research
only (a)
1,100,000 (b)
1,540,000 (b)
1,327,000 (c)
3,3*-Dichlorobenzidine
Estimated at several
million (a)
5,500,000
estimated in (a)
4,500,000
estimated in (a)
3,000,000
estimated in (a)
6,424,000 (b)
3,656,000 (d)
2,940,000 (d)
1,702,000 (c)
None (a)
9,500 (a)
None (a)
No data
No data
261,705 (e)
None (a)
2,002
None (a)
No data
979,812 (d)
928,786 (d)
No data
15
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Table 5. Production and Imports of Benzidine and Its Congeners (cont.)
Chemical
Production (Ib) Imports (Ib)
Dianisidine
1978 "Very small quantities 554,012 (e)
once or twice a year" (a)
1977
1976
1975
1972
1967
1960
1978
1977
1976
1975
1974
1971
1962
t
No data
t
No data
367,400 (f)
360,000 (c)
o-Tolidine
200,000 est. (g)
200,000 (a)
200,000 (a)
t
220,000
estimated in (b)
No data
243,000 (c)
428,007
751,784
400,780
272,800
No data
No data
330,547
353,281
472,794
312,351
600,000
97,680
No data
(a)
(a)
(a)
(f)
(e)
(a)
(a)
(a)
(b)
(f)
Source: Figures include base compounds and salts, where reported
separately. Data sources: (a) Powell et al. (1979, pp. 4-2, 4-3), (b)
Ferber (1978), (c) Lurie (1964), (d) Gerarde and Gerarde (1974), (e) USITC
(1979), (f) IARC (1972b), and (g) NIOSH (1978b).
*Production volume may not include benzidine produced in situ for dye
manufacture.
tThe U.S. International Trade Commission does not give production
statistics when there are fewer than three producers or when one producer
is dominant.
2. Dyes and Pigments
The available production and import data for specific dyes and
pigments derived from benzidine and its congeners for the period 1975-1978
are presented in Table 6. Dyes and pigments listed are those for which
figures were reported for more than one year. Most notable is the drop in
production of benzidine-based dyes; no significant trends can be discerned
for dyes and pigments based on o-tolidine, dianisidine, and dichloro-
benzidine.
Table 7 summarizes the available total sales and import data for dyes
and pigments based on benzidine and its congeners. Over the 4-year period,
a significant drop in U.S. sales of benzidine-based dyes can be seen, as
16
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well as a significant increase in imports (although total consumption
decreased slightly). An increase in U.S. sales of dichlorobenzidine-based
pigments (50 percent increase over a 2-year period) can also be discerned.
There appear to have been no significant changes in total consumption of
dyes and pigments based on o_-tolidine and dianisidine in the past few
years.
Table 7. Estimated Quantities of Dyes Consumed in U.S.
Type of dye
1975
Quantity (10 Ib)
1976 1977
1978
Benzidine-based:
U.S. sales
Imports
o-Tolidine-based:
U.S. sales
Imports
4.2
0.9
2.1
0.1
6.6
0.6
2.3
0.1
4.6
1.3
1.9
0.1
1.9
1.6
2.8
0.1
Diani s idine-based:
U.S. sales
Imports
Dichlorobenzidine-based:
U.S. sales
Imports
0.5
0.1
8.4
0.1
0.5
0.1
11.6
0.1
0.4
0.1
12.8
0.05
0.4
0.1
0.03
Source: Data on benzidine-, o-tolidine-, and dianisidine-based dyes are
from Powell et al. (1979). Data on dichlorobenzidine-based pigments are
from USEPA (1979a).
20
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II. HEALTH EFFECTS
A. Benzidine and Benzidine-Based Dyes
1. Mutagenicity
Benzidine was found to be mutagenic in two studies using the
Salmonella/mammalian microsome system developed by Ames (Ames et al. 1973,
Ferretti et al. 1977). In the Ames study, the maximum mutagenic effect was
obtained by the addition of 50 ug benzidine to Salmonella typhimurium TA
1538, in the presence of S-9 mix (a mixture of rat liver homogenate and
TPNH-generating system). This treatment produced 265 revertant colonies,
compared with 16 and 36 colonies on plates lacking S-9 mix and benzidine,
respectively. In a survey of compounds currently used in clinical
hemoglobin determinations, Ferretti et al. (1977) confirmed that benzidine
is mutagenic when activated by rat liver homogenate.
Benzidine also has been used as a positive control in testing the
bacteriocide Grotan BK, a cutting oil preservative (Urwin et al. 1976).
The test system was the micronucleus test in rats, in which treated and
untreated animals are examined for the presence of micronucleated
erythrocytes in bone marrow. Rats administered a total dose of 410 mg/kg
benzidine by either dermal or subcutaneous routes developed a content of
micronucleated erythrocytes nearly 100-fold higher than controls.
The benzidine-based dyes Direct Red 28 (Congo Red) and Direct Violet 1
(Chlorazol Violet N) were found to be mutagenic when tested by a
modification of the Salmonella/microsome system (Sugimura et al. 1977).
The dyes were mixed with bacterial cells and microsomes in DMSO and
incubated for 20 minutes at 37° C before plating. It was stated that the
preincubation was performed because it was a necessary step for obtaining
mutagenic activity with potential carcinogens such as dimethylamino-
azobenzene and dimethylnitrosamine.
2. Carcinogenicity
The evidence implicating benzidine as a carcinogen in animals and in
man has been reviewed by the International Agency for Research on Cancer
(IARC 1972a). The number of animal carcinogen studies is extensive; the
results of these studies led the IARC to conclude that "benzidine is
carcinogenic in the mouse, rat and hamster, and possibly the dog. Given
orally, it has produced bladder carcinoma in the dog after a long latent
period and liver tumors in the rat and hamster" (IARC 1972a, p. 84).
On the basis of epidemiological studies, the IARC (1972a) concluded
that there is a strong correlation between occupational exposure to
benzidine and bladder cancer in humans. This conclusion is supported by
the study of Zavon et al. (1973) showing that, over a 13-year period, 13 of
25 production workers exposed to benzidine at a dye manufacturing plant
subsequently developed bladder cancer. This study was initiated in 1958
when the authors were alerted by the manufacturer that a worker had
developed hematuria. At this time, it was not generally known that
21
-------
benzidine is a human carcinogen, and the plant was still manufacturing
benzidine as it had been since 1929, with only a few minor modifications.
A detailed investigation was immediately undertaken of the exposure levels
of benzidine, the work practices at the plant, and the medical condition of
production workers potentially exposed to benzidine. Shortly after this
initial investigation, production of benzidine was discontinued. A
follow-up study of 25 exposed workers 13 years later disclosed that in the
interval 13 had developed bladder tumors. Although multiple exposures to
other suspect chemicals did occur, benzidine was the only chemical to which
all were exposed, and benzidine thus was judged to be the most likely
carcinogen. The time of exposure to benzidine in those who developed
tumors ranged from 6-28 years (average 13.6 years), and concentrations of
benzidine at various work locations at the time the study was initiated
(1958), ranged from 0.005 mg/m to 0.415 mg/m (at six sites). A value of
17.6 mg/m was found at one location, where benzidine was hand shoveled
into drums. Benzidine also was found in the urine of 33 workers (including
the 25 production workers), before and after work shifts. Average values
were about 0.010 mg/1 before work shifts and 0.030 mg/1 after work shifts.
The EPA Office of Water Planning and Standards has used these values in
estimating that the total accumulated dose of benzidine required to produce
a 50 percent incidence of cancer (13/25 observed incidence) is 200 mg/kg
(USEPA 1976b).
There is less information available about the carcinogenicity of
benzidine-based dyes than there is for benzidine. Following the finding
that four benzidine-based dyes (Direct Black 38, Direct Blue 6, Direct
Brown 95, and Direct Red 28) are metabolized to free benzidine in rhesus
monkeys (Rinde and Troll 1975), the National. Cancer Institute (NCI)
performed carcinogen bioassays on Direct Black 38, Direct Blue 6, and
Direct Brown 95 (NCI 1978a). All three dyes produced tumors in Fischer 344
rats in 13 weeks when included in the diet, but results differed in males
and females. Males fed Direct Blue 6 or Direct Black 38 at 1,500 ppm
showed a highly significant incidence of hepatocellular carcinomas and
neoplastic nodules of the liver. Doses of 11,500 ppm or 3,000 ppm Direct
Brown 95 were lethal to males at 5 weeks; lower doses did not produce
neoplasms. Females given 3,000 ppm and 1,500 ppm Direct Brown 95 developed
hepatocellular tumors and neoplastic nodules of the liver; females given
similar concentrations of Direct Black 38 showed no hepatocellular
carcinomas but had a significant incidence of neoplastic nodules of the
liver. The time-to-tumor interval was 5 weeks for each of the dyes. (For
purposes of comparison, it can be noted that rats fed benzidine as 0.017
percent of the diet for 424 days were reported to show an increased
incidence of tumors of the liver and bile ducts [Boyland et al. 1954]). In
this study, no carcinomas were produced in B6C3F1 mice fed the same dyes at
doses ranging from 750 ppnr to 12,500 ppm.
3. Metabolism and Bacterial Degradation
The metabolism of benzidine, the benzidine congeners, and their
derivative dyes and pigments is of interest because of the reported
metabolic conversion of some benzidine-based dyes to the demonstrated
carcinogen, benzidine. Haley (1975) presents a comprehensive review of the
biotransformation of benzidine and its congeners, in which it was shown
that the pattern of metabolites excreted in urine differs among mammalian
22
-------
species. A summary of the patterns of metabolites excreted in urine by
various species following administration of benzidine (route not specified)
is shown in Table 8.
The azo linkage in benzidine-based dyes is susceptible to anaerobic
enzymatic attack in mammalian species; azo-reductase activity is associated
with cytochrome P-450, which occurs predominantly in the liver but also is
present in most other organs of the body (Walker 1970). Under anaerobic
conditions, this enzyme can catalyze the cleavage of benzidine-based dyes
to release free benzidine. Once released, benzidine is converted to other
metabolites that may be the active species in carcinogenesis and
mutagenesis. The ability of intestinal bacteria to reduce azo-containing
dyes also is well established (Walker 1970, Chung et al. 1978).
Table 8, Urinary Metabolites of Benzidine
Compound
Dog Rat Mouse Rabbit Guinea pig Man*
Benzidine
4-Acetamido-4-aminodiphenyl
3-Hydroxybenzidine
(ether extracts)
4,4'-diamino-3-diphenylyl
hydrogen sulfate
4'-Acetamido-4-amino-3-
diphenylyl hydrogen sulfate
4'-Amino-4-diphenylyl sulfamic
acid
N-Glucuronides
Acid-stable unknowns
i
+
1
+
3
0
•f
3
+
+
3
+
0
Source: Adapted from IARC (1972a), Haley (1975). Metabolites were
detected and identified by paper chromatography. Although not specified in
the references, symbols are interpreted as + = substance detected with
certainty? +_ = substance presumed to be detected, but not with certainty;
and 0 = substance sought but not detected, within limits of technique.
*Also found in man are 3,3'-dihydroxybenzidine, mono- and diacetylben-
zidine, and N-hydroxy acetylaminobenzidine.
Four benzidine-based dyes (Direct Black 38, Direct Red 28, Direct Blue
6, and Direct Brown 95) have been subjected to in vivo metabolism studies
in rhesus monkeys (Rinde and Troll 1975); the metabolic pathways in this
species closely resemble those in humans. Each dye was administered in a
23
-------
single dose by stomach intubation, and after 72 hours urine was collected
and analyzed for free benzidine and monoacetylbenzidine. All four dyes
gave similar results, namely, about 1.25 percent of the administered dye
was excreted as benzidine (versus 1.45 percent benzidine excreted when a
comparable dose of benzidine was administered). The amount of free
benzidine recovered was much greater than was present as impurities in the
dyes administered. NCI performed similar metabolism tests on rodents with
three of these dyes—Direct Black 38, Direct Brown 95, and Direct Blue
6—and also found free benzidine and monoacetylbenzidine excreted in the
urine (NCI 1978a).*
Feeding studies with additional dyes are under way in dogs at the
National Institute of Environmental Health Sciences (NIEHS); dyes are
administered orally at a dose of 100 mg/kg body weight and urine is
collected for 72 hours and examined for free benzidine using
chromatography-mass spectrometry (Matthews 1979). In preliminary results
obtained for six benzidine-based dyes (Direct Blue 2, Direct Black 4,
Direct Brown 2, Direct Red 28, Direct Orange 8, and Direct Green 1)
benzidine was excreted in urine in amounts that exceeded by at least
25-fold the free benzidine present as contaminants in the administered
dyes. It appears, therefore, that conversion of benzidine-based dyes to
benzidine may be a generalized phenomenon in the dog. If a similar
conversion occurs in humans, these dyes may have the potential to induce
human cancer.
With regard to the latter statement, a recently completed study
conducted by the National Institute for Occupational Safety and Health
(NIOSH) suggests that benzidine-based dyes can be metabolized to free
benzidine in humans (Boeniger 1979). Workers at a textile plant using
benzidine-based dyes were found to excrete benzidine in their urine at
concentrations as high as 0.039 mg/1 (39 ppb), much higher than could be
attributed to the free benzidine present as a contaminant in the dyes. The
finding that benzidine-based dyes can be metabolized to a known carcinogen
in humans is cause for concern; the fact that the concentration of
benzidine detected was higher than the average values of 0.010-0.030 mg/1
(10-30 ppb) found in 13 of 25 dye production workers who subsequently
developed bladder cancer (Zavon et al. 1973) is especially alarming.
Although multiple exposure routes may be postulated for workers
exposed to benzidine-based dyes (i.e., inhalation, dermal, ingestion
through hand contamination), a recent report from industry suggests that
dermal absorption may be a specific problem. A preliminary report, filed
*Highly polar compounds, however, are not well absorbed from the gut and
thus water-soluble sulphonated dyes such as the majority of commercially
available benzidine-based dyes would not be expected to be well absorbed by
mammals (Walker, 1970). Therefore, one can postulate that reductive
cleavage of benzidine-based dyes would be expected to occur mainly by the
gut flora. Oral administration of benzidine-based dyes in animals with
subsequent testing of urine for free benzidine would not distinguish among
the two mechanisms for reductive cleavage of the azo linkages.
24
-------
under section 8(e) of the Toxic Substances Control Act* suggests that the
benzidine-based dye Direct Black 38 is absorbed through the skin in rabbits
(International Business Machines.. 1979). Rabbits were dosed dermally with
dye (radioactively labeled with C in the biphenyl moiety) in a
proprietary solution. Over a period of 144 hours, 91 percent of the
applied radioactivity was recovered in the urine. Because a detailed test
protocol was not submitted, this report can only be considered suggestive
at present; however, it is noteworthy as the first report of the absorption
of a benzidine-based dye through the intact skin of a mammal.
B. Dichlorobenzidine and Dichlorobenzidine-Based dyes
1. Mutagenicity
Dichlorobenzidine (50 yg/plate) was weakly mutagenic in the Ames
Salmonella assay without S-9 mix (114 revertant colonies versus 8 colonies
on control plate, Garner et al. 1975). In the presence of S-9 mix, the
mutagenic activity was about thirtyfold higher (3,360 revertant colonies).
2. Carcinogenicity
Dichlorobenzidine has been reviewed by the International Agency for
Research on Cancer (IARC 1973a) and found to be a carcinogen in the rat and
hamster. Studies that support the lARC's conclusion are described below.
In a 12-month study of rats fed 10-20 mg dichlorobenzidine in the diet
six times per week (total dose 4.5 g/rat), a high incidence of tumors of
the Zymbal gland and other organs was found (Pliss 1959). Subcutaneous
administration of 15-60 mg dichlorobenzidine in sunflower seed oil or
glycerol and water (at unspecified intervals) to rats for 10-13 months gave
rise to tumors in about 75 percent of the animals. Tumors of the skin,
mammary glands, and sebaceous glands were most numerous; intestinal,
urinary bladder, and bone tumors also were observed. One of 25 control
rats, injected with the vehicle alone, developed a sarcoma.
Stula et al. (1971, 1975) fed 50 male and 50 female ChRCD rats 1,000
ppm dichlorobenzidine in a standard diet for up to 16 months and observed
malignant tumors of the mammary glands, skin, and acoustic ducts in both
sexes. Treated males also had an increased incidence of haemopoietic
tumors, compared to an equal number of controls.
Dietary levels of 0.1 percent dichlorobenzidine in lifetime feeding
studies, however, did not induce tumors in 30 male and 30 female Syrian
golden hamsters, when compared with a similar number of controls. Dietary
levels of 0.3 percent dichlorobenzidine, however, produced four
transitional cell carcinomas of the bladder and some liver cell tumors
among 60 test animals; no such tumors were observed in an equal number of
control animals (Saffiotti et al. 1967, Sellakumar et al. 1969).
*Under section 8(e) of TSCA information that a chemical may present
substantial risk must be submitted to the Administrator of the U.S.
Environmental Protection Agency.
25
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Finally, a study by Golub and Kolesnichenko (1974) is of theoretical
interest. Dichlorobenzidine was administered to mice during the last week
of pregnancy; the progeny were observed for 10-20 months. A significant
increase in tumors occurred in the progeny, suggesting that dichloro-
benzidine may be a transplacental carcinogen.
At the time of the review of dichlorobenzidine by the IARC,
epidemiological studies in humans had not been performed. Because
benzidine and dichlorobenzidine were commonly produced at the same plant,
the possibility could not be excluded that dichlorobenzidine had
contributed to the incidence of bladder cancer commonly attributed to
benzidine. In 1974, Gerarde and Gerarde presented an epidemiological study
of 175 out of 207 persons exposed to dichlorobenzidine over a period of 35
years; no cases of bladder tumors were found. However, this study was
published without review, owing to the untimely deaths of the authors, and
a number of questions concerning the reliability of the data and its
significance (considering that no medical information was available for 32
workers) necessarily remain unanswered (see Commentary section appended to
Gerarde and Gerarde 1974). Another serious limitation of the work is the
fact that data on bladder tumors only were reported, although dichloro-
benzidine is known to produce tumors of other organs in animals (Pliss
1959, 1963; Sellakumar et al. 1969).
Maclntyre (1975) also reported the lack of incidence of bladder tumors
among 225 British dye workers exposed to dichlorobenzidine for up to 30
years. Since 1965 it has been the practice at this plant to give a medical
examination to exposed workers regularly and to do Papanicolau smears every
6 months. In this particular study, most workers were first exposed fewer
than 20 years ago, and most have been exposed for fewer than 16 years (the
latency period for benzidine-induced urinary bladder cancer is about 16
years). Furthermore, work practices at the plant since the late 1950s have
greatly minimized the extent of actual exposure. All of these factors tend
to diminish the force of the negative findings with respect to bladder
tumors.
In addition to the above epidemiological study, Maclntyre (1975),
citing unpublished information presented at a scientific meeting in 1974,
noted that occupational physicians in Europe had found no bladder cancer
among some 1,000 persons exposed to dichlorobenzidine. Thus, the only
conclusion that can be drawn from the accumulated weight of negative
findings is that if dichlorobenzidine is a bladder carcinogen in humans, it
is probably much less potent than benzidine.
Animal studies also have been performed on some dichlorobenzidine-
based pigments. In a study at NCI, a technical grade of Pigment Yellow 12
was fed to Fischer 344 rats and B6C3F1 mice for 78 weeks, followed by
observation for 18-22 weeks (NCI 1978b). High-dose animals received the
pigment as 5 percent of their diet; low-dose animals received it as 2.5
percent of the diet. Treated rats showed no increase in the incidence of
neoplasms; however, a statistically insignificant increase in the following
neoplasms were observed: metastatic chordoma in 1/49 low-dose males and an
osteogenic sarcoma in 1/49 low-dose females. Treated mice also showed no
statistically significant increase in the incidence of neoplasms, although
three specific findings were noted: squamous cell carcinomas of the ear in
26
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1/49 low-dose males, an infiltrating duct carcinoma of the mammary gland in
1/50 low-dose females, and a mastocytoma of the subcutaneous tissue in 1/50
high-dose females. These tumors were not found among the controls,
although a comparable incidence of other tumors was found. The study
concluded that the results did not provide evidence for the carcinogenicity
of Pigment Yellow 12 in Fischer 344 rats or B6C3F1 mice.
In another study, mice (NMRI, Ivanovas) and rats (Sprague-Dawley,
Ivanovas) were fed Pigment Yellow 12 (containing 2 ppm dichlorobenzidine)
in the diet at concentrations of 0.1, 0.3, and 0.9 percent for 104 weeks
(Leuschner 1978). Test groups and controls consisted of 50 males and 50
females. No significant incidence of tumors was found in treated animals,
compared with controls. Similar results were obtained in this study
(following a similar protocol) with Pigment Yellow 83, another
dichlorobenzidine-based pigment.
3. Metabolism
Sciarini and Meigs (1961) followed the biotransformation of
dichlorobenzidine in a mongrel dog injected intraperitoneally with 1 gm
dichlorobenzidine suspended in gum tragacanth. The only recovery product
identified in urine and feces was dichlorobenzidine; about 2 percent of the
administered dose was excreted over a 15-day period, primarily (90 percent)
in feces. As the chemical tests applied should have detected metabolites
similar to those produced by benzidine, it was concluded that dichloro-
benzidine is metabolized by a different route, or not at all, in the dog.
Kellner et al. (1973) compared the distribution and elimination of
dichlorobenzidine and benzidine in rats, dogs, and monkeys after
intravenous injection. In all cases, dichlorobenzidine was excreted much
more slowly than was benzidine. In monkeys, unchanged dichlorobenzidine
was found in the urine a few hours after injection, whereas nearly all the
injected benzidine found in the urine was present as metabolites rather
than unchanged benzidine. These results support the view of Sciarini and
Meigs (1961) that dichlorobenzidine is metabolized quite differently from
benzidine.
A number of studies on the metabolic fate of dichlorobenzidine-based
pigments fail to provide any evidence that these pigments are broken down
to release free dichlorobenzidine. These included an NCI study (unpub-
lished) in which rats were fed Pigment Yellow 12 by stomach intubation and
the study by Leuschner (1978) in which rabbits were fed a single dose of
Pigment Yellow 12. (The protocol of Leuschner"s study duplicated that of
an earlier study by Akiyama [1970], which reported that metabolic release
of dichlorobenzidine from Pigment Yellow 13 did occur.) The preceding
studies are supported by the work of Stavenuiter (1977), who detected
practically no free dichlorobenzidine in urine following injection of
C-labeled Pigment Yellow 13 to rats and rabbits. Thus, the weight of the
evidence supports the view that dichlorobenzidine-based pigments are only
poorly (if at all) metabolized to dichlorobenzidine in mammals. Leuschner
(1978) attributed the poor metabolism of dichlorobenzidine-based pigments
(Yellows 12, 13, and 83) and one o-tolidine-based pigment (Yellow 16) to
poor absorption of the pigment particles (0.1 = 1.0 ]_im in size). Other
27
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explanations, however, may be based on the structural differences between
benzidine-based dyes and these pigments. Dichlorobenzidine obviously
differs from benzidine in that chlorines are substituted for hydrogens in
the 3 and 3'-positions of the molecule. A more significant structural
difference, however, may be the substituent group in the pigments adjacent
to the azo linkage. For comparison, Figure 6 shows the structures of the
eight benzidine-based dyes known to be metabolized to benzidine and the
three dichlorobenzidine-based pigments found not to be metabolized to
dichlorobenzidine. Also shown is the o-tolidine-based pigment. Pigment
Yellow 16, which was found not to be metabolized to o-tolidine in mice and
rats (Leuschner 1978). As can be seen, all of the pigments, but none of
the dyes, contain the substituent R-C=COH-CH adjacent to the azo linkage.
This group is potentially capable of keto-enol tautomerism and of forming a
resonant ring structure, as shown in Figure 7. If the keto form
predominates or exists exclusively, it is probable that these pigments?,
lacking an azo linkage, would be impervious to enzymatic reduction. A
similar explanation was advanced by Jones et al. (1963) to account for the
stability of several 4-arylazo-5-pyrazolones to enzymatic azo-reduction.
In the latter study, infrared and N.M.R. spectroscopy revealed that the
pyrazolones exist exclusively in a keto form that is stabilized by
intramolecular hydrogen bonding and therefore lack an azo linkage (Figure
8). If correct, this hypothesis (with respect to Pigments Yellow 12, 13,
and 83) may also account for the lack of carcinogenicity of the
dichlorobenzidine-based pigments.*
C. o_-Tolidine
1• Mutagenicity
£-Tolidine was found to be weakly mutagenic in the Ames Salmonella
assay system with S-9 activation (Feretti et al. 1977). Under these
conditions, addition of 100 yg c--tolidine produced 80 revertants/plate,
compared with 6 revertants/plate for the control lacking S-9 mix.
Trypan Blue, an o-tolidine-based dye, was found to be mutagenic to
Aspergillus (Cooke et al. 1970, Roper 1971) and Salmonella (Hartman et al.
1978). The commercial grade of Trypan Blue contains a significant amount
of monoazo dyes (containing substituted diphenyl groups other than
o-tolidine) as impurities (Field et al. 1977); the samples of dye used in
the mutagenicity studies reported here were of unspecified purity. In
order to increase the sensitivity of the Ames Salmonella system to Trypan
Blue, Hartman et al. (1978) determined the experimental conditions
necessary for obtaining azo dye reduction in cell-free extracts of the
intestinal anaerobe, Fusobacterium sp. 1. Maximum azoreductase activity
required FMN, glucose-6-P, and anaerobic conditions. When 500 yg Trypan
Blue was subjected to the azoreductase regimen, then tested in the Ames
*The possibility that dichlorobenzidine or pigments based on it can be
converted to benzidine in vivo is considered remote. The loss of halogens
from the benzene ring in vivo has never been reliably demonstrated (Gerarde
and Gerarde 1974).
28
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DYES
C.I. Direct Red 28 (Yellowish red)
Classical name Congo Red
NHj
SO,Na
NaO,S
C.I. Direct Black 38 (Black)
NH, H2N OH
H2N Q-N = N —£}~O"N = N"
*HO
C.I. Direct Brown 95 1 Reddish brown)
Copper complex derived from
NaOOC OH
HO Q- N = N -£}-Q-. N = N
HO
C.I. Direct Blue 6 (Blue)
H2N OH HO NH,
nry N = N-OO-N = N
^*^ CD.Na MaO.c_ ™' u c'
H,C ^_/NH-OC-C-N = N\J~{J-N = N-OCO-HN-^ CH, Cl (_}-N = N-C-CO-HN-^-^-NH-CO-C-N = N-£) Cl
CH, H,C
Figure 6. Comparison of structures of dyes and pigments.
29
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R—C
— R
R-C
(-keto)
Figure 7. Proposed keto-enol tautomerism of dichlorobenzidine-based
pigments, with presumed ring structures.
Figure 8. Structure of hydrazone form of arylazopyrazolones, as determined
by Jones et al. (1963).
30
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Salmonella system with S-9 activation, it was found to be clearly mutagenic
(118 revertant colonies on treated plates versus 39 revertant colonies on
control plates). A similar amount of Trypan Blue, not subjected to the
azoreductase pretreatment, was not mutagenic, with or without S-9
activation.
2. Carcinogenicity and Teratogenicity
o-Tolidine was reviewed by the IARC (1972b) and was judged to be a
carcinogen in rats when injected subcutaneously. In one study, Spitz et
al. (1950) injected 105 Sherman rats with commercial p_-tolidine in olive
oil (at weekly intervals) in doses of 60 mg/rat per week (total dose 5.5
g). Only 48 rats survived more than 300 days; these were kept for the
remainder of their life span. Five of the 48 surviving rats developed
cancer of the auditory canal. Although no concurrent controls were run,
none of the 578 untreated rats of the same colony were found to have
similar cancers (the control group had a total of 56 other tumors).
In a second study, random-bred rats were given weekly subcutaneous
injections of purified jD-tolidine in sunflower seed oil (20 mg per rat per
week) for 13 months. Tumors first appeared at 8 months; among 50 treated
animals 30 developed a total of 41 tumors, including 2 carcinomas of
Zymbal's gland (Pliss and Zabezhinsky 1970). Only one of 50 control rats
injected subcutaneously with sunflower seed oil alone developed a tumor, a
sarcoma associated with a parasitic cyst. Another group of 88 rats (equal
numbers of males and females) was given weekly subcutaneous implants of 20
mg purified o-tolidine in 10 mg glycerol for 13 weeks. In about half the
animals (equal numbers of males and females) the o_-tolidine was subjected
to ultraviolet irradiation prior to injection; no significant effect of
this treatment was observed. First tumors appeared at 11-12 months, and 48
of the 68 surviving animals developed a total of 60 tumors, including 27
Zymbal's gland carcinomas. No concurrent controls were run in this study,
but it was reported that the strain of rats used had a low incidence of
spontaneous tumors (Pliss 1965).
No human epidemiological studies are available for o_-tolidine (IARC
1972b). Several case studies of cancer in workers manufacturing o-tolidine
and benzidine are summarized in a report published by NIOSH (1978a). No
cases of cancer in workers exposed solely to c>-tolidine have been reported,
possibly because both chemicals historically have been handled at the same
factory sites.
One carcinogenicity study was found in the available literature on an
o-tolidine-based pigment, Pigment Yellow 16 (Leuschner 1978). Rats fed the
pigment (containing less than 1 ppm o-tolidine) as part of the diet (in
concentrations of up to 0.9 percent) for 104 weeks had no higher incidence
of tumors than did controls.
Trypan Blue, an o-tolidine-based dye, also has been tested for
carcinogenicity in rats (Field et al. 1977). Inbred Wistar rats were
injected at biweekly intervals with dye in water solution (5 or 10 mg per
injection) for up to 40 weeks. The commercial grade dye was tumorigenic (5
tumors among 17 rats); however, the crude dye contained a significant
proportion of monoazo dyes containing substituted diphenyl groups other
31
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than o_-tolidine. When the p_-tolidine component was purified and tested no
tumors were observed, but precancerous changes were reported in 11 of 33
rats (Field et al. 1977). From these results it is not possible to
determine the oncogenic potential of the pure c^-tolidine-based dye
component.
Both crude and purified Trypan Blue were found to be potential
teratogens in mice in the preceding carcinogen study. Specific pathogen-
free CFLP mice were injected with dye at about 7.5 days of pregnancy and
were killed and examined on day 18 of pregncincy. Mice dosed with crude or
pure Trypan Blue (60 or 120 mg/kg body weight) gave a significantly higher
incidence of resorptions and major malformations than did controls injected
with saline.
3. Metabolism
The metabolism of o>-tolidine has been reviewed in a report by NIOSH
(1978b). The available evidence indicates that in humans £-tolidine is
metabolized by a pathway similar to that of benzidine, with the first steps
being the acetylation of the amino groups and introduction of the phenolic
group on the aromatic ring. Urine of workers occupationally exposed to
o-tolidine has been reported to contain o-tolidine, N,N'-diacetyl-o-
tolidine, and 5-hydroxyl-o-tolidine and its conjugates (NIOSH 1978b).
Leuschner (1978) examined the urine of rats fed the o-tolidine-based
pigment, Pigment Yellow 16, for evidence of reductive metabolism; no
c>-tolidine was found (detectable limit was 0.3 ppm). Currently,
o-tolidine-based dyes are being tested for reductive metabolism in dogs
(Matthews 1979). Although no evidence is currently available from these
mammalian metabolism studies, the reader should note that intestinal
bacteria can release _o-tolidine from c3-tolidine-based dyes (Hartman et al.
1978).
D. Dianisidine
1. Mutagenicity
Dianisidine has been found to be weakly mutagenic in the Ames
Salmonella assay, but only with microsomal activation (Garner et al. 1975).
2. Carcinogenicity
Dianisidine has been reviewed by the IARC (1973b) and was judged to be
a carcinogen in rats when administered orally. In one experiment, rats
were administered 30 mg dianisidine in sunflower seed oil 3 times per week
for 13 months (Pliss 1965). Of the 18 rats surviving this treatment, 4
developed tumors not found in the controls. The tumors included two Zymbal
gland tumors, one ovarian tumor, and one fibroadenoma of the mammary gland.
None of the 50 control rats developed tumors at these sites.
In another study, Fischer rats were administered various doses (up to
30 mg/animal) of dianisidine by stomach tube 5 days a week for 52 weeks
(Hadidian et al. 1968). A significant number of the 30 male and 30 female
rats treated in this manner developed tumors that were not found among the
32
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360 control animals given the vehicle alone. Tumors occurred at various
sites including the bladder (two papillomas), the intestine (three
carcinomas)/ the skin (five carcinomas) and the Zymbal gland (three
carcinomas). No differences between sexes were noted.
No epidemiological data on the occurrence of cancer in workers exposed
to dianisidine in the absence of other suspect carcinogens were found in
the available literature.
III. ECOLOGICAL EFFECTS
Little information was found concerning the possible ecological
effects of benzidine. No reports on the effects of benzidine or its
congeners in the natural environment were found. In a letter to Dr. Donald
I. Mount, Director, National Water Quality Laboratory (EPA), Duluth,
Minnesota, November 20, 1973, A. E. Lemke described laboratory studies in
which it was determined that benzidine, at concentrations of a few ppm, is
acutely toxic to six species of fish (see Table 9). A study by the
Synthetic Organic Chemical Manufacturers Association (SOCMA) reported the
acute toxicity of benzidine to three additional fish species (SOCMA 1975).
This study is reviewed in the Criteria Document on benzidine, issued by the
U.S. EPA Office of Water Planning and Standards (USEPA 1976). The TL
values reported in this document are listed in Table 9.
14
In an uptake and elimination study using C -labeled benzidine,
bluegill sunfish suffered 8 percent mortality from exposure to 98 ppb
benzidine for 42 days (EG and G Bionomics 1975). Benzidine, at this
concentration, was found bioaccumulated 44-fold in muscle tissue at 21
days; this level of bioaccumulation remained constant throughout the
remainder of the 42 days of exposure.
A recent study indicates that dichlorobenzidine bioconcentrates into
both the edible and nonedible portions of bluegill sunfish (Sikka et al.
1978). Mortality occurred before equilibrium between water and fish was
attained; the bioconcentration factors reached at mortality were 135-fold
to 554-fold. These values, as well as the 44-fold bioaccumulation found
with benzidine, are well below the 5,000-fold bioaccumulation factor
currently considered by EPA to constitute substantial risk to the
environment.
No information was found on the potential ecological effects of other
benzidine congeners (dianisidine and p^-tolidine) or any of the dyes or
pigments derived from benzidine and its congeners.
33
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Table 9. TL Values, Benzidine (ppm)
(Static bioassay)
Species 24 hr 48 hr 72 hr 96 hr
Flagfish* >50 32.5 25.0 16.2
(Jordanella floridae)
Fathead Minnow* >20 >20 >20 >20
(Pimephales promelas)
Fathead Minnowt - -
Red Shiner* >20 10 2.5 2.5
(Notropis lutrensis)
Lake Trout* 8.7 5. 4.35 4.35
(Salvelinus namaycush)
Rainbow Trout* >20 114.1 10 7.4
(Salmo gairdneri)
Scud* >20 >20 >20 >20
(Gammarus pseudolimaeus)
Emerald Shinerst - - 5
(Notropis atherinoides)
Bluegill Sunfisht - - - 15
(Lepomis macrochirus)
*A. E. Lemke in a letter to Dr. Donald I. Mount, Director, National Water
Quality Laboratory (EPA), Duluth, Minnesota, November 20, 1973.
tUSEPA (1976)
34
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IV. ENVIRONMENTAL FATE
A. Water
The most important source of environmental release of benzidine and
benzidine congeners is probably the wastewaters from dye producer plants.
In the absence of any reported studies of persistence in water, Radding et
al. (1975) estimated the half-life of benzidine in water to be about 100
days. This estimate assumed that degradation would proceed mainly through
reaction with peroxy radicals formed by the action of sunlight on water.
Because the rate constants used for this calculation were only estimates,
the actual half-life of benzidine may be much greater or smaller.
Dichlorobenzidine is very rapidly degraded in aqueous solutions by the
action of natural or simulated sunlight (Sikka et al. 1978). Benzidine and
3-chlorobenzidine are intermediates in this process. Although the
mechanism of dechlorination is unknown, it is known not to involve carbon-
chlorine bond homolysis. In organic solvents the dechlorination reaction
is considerably slower (Sikka et al. 1978).
Benzidine is resistant to biooxidation by unacclimated microorganisms
in activated sludge (Taback and Barth 1978). Keinath (1976) reported that,
in an unacclimated activated sludge system, benzidine is biodegradable at
0.05 mg/1 but not at 1 mg/1. To determine whether microorganisms in sludge
can become acclimated to benzidine and can adapt enzymes to metabolize it
aerobically, Taback and Barth (1978) subjected sludge microorganisms to
benzidine in aerobic growth chambers for up to 9 weeks. A reservoir of
wastewater containing sludge was seeded with benzidine and refrigerated,
then fed through the aerobic growth chamber at a rate set to attain a
hydraulic retention time of 24 hours in the chamber. The results indicate
that acclimation to up to 5 mg/1 benzidine was readily attained; the
concentration of residual benzidine in effluent from the reactor was
reduced to 2.2 mg/1 after 1 week and to 0.3 mg/1 after 7 weeks. At 1 mg/1
input concentration, residual benzidine dropped to 0.09 mg/1 after 1 week,
and thereafter was undetectable (0.001 mg/1, or 0.1 percent of starting
concentration, could have been detected by the test method used).
A 1976 study of wastewater treatment methods disclosed that
housekeeping measures available to benzidine manufacturers (e.g., oxidation
with ozone or nitrous acid) are adequate to prevent the discharge of
benzidine directly into sewer lines (Keinath 1976). However, the effects
of possible by-products of these processes have not been assessed.
Benzidine concentrations can be reduced by 1-10 ppb by adsorption on
granulated charcoal. Analysis for benzidine in the exhausted dye liquids
from a textile industry making heavy use of benzidine-based dyes gave an
average concentration of 3.5 ppb. Similar mass balance analyses conducted
for "heavy benzidine-dye use" leather and manufacturing concerns showed
calculated residual benzidine concentrations of 0.25 and 3.5 ppb,
respectively (Keinath 1976). All of these values are well below the 50 ppb
limit for biodegradation by unacclimated activated sludge (Keinath 1976).
35
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Dichlorobenzidine adsorbs extensively to a variety of aquatic
sediments and becomes more tightly bound with time (Sikka et al. 1978).
Both benzidine and dichlorobenzidine are more soluble in water than is DDT
(a well-known bioaccumulator), and all three compounds are also quite
soluble in organic solvents. Thus, it is suspected that bioaccumulation
occurs when either of these benzidine compounds is present in large bodies
of water and that the compounds can move within the food chain. Recent
studies indicate that both benzidine and dichlorobenzidine are rapidly
bioconcentrated in bluegill sunfish (see section III, Ecological Effects).
Benzidine-based or congener-based dyes and pigments discharged into
waters from manufacturing facilities are believed to be chemically reduced
back to the parent compound if either hydrogen sulfide or sulfur dioxide is
present in the receiving waters. This was believed to be the case
downstream of a dye plant on the Sumida River in Japan, where benzidine
concentrations of up to 233 ppb were found (Takemura et al. 1965). Hitz et
al. (1978) carried out laboratory studies on the adsorption of dyes to
activated sludge. Dyes (in concentrations found in effluents received at
treatment plants) were mixed with activated sludge at a concentration that
is comparable to the concentration in the aeration stage of sewage plants
in parts of Britain and the rest of Europe. After 30 minutes the sludge
was removed by centrifugation, and the dye remaining in solution or
suspension was determined. Average adsorption of seven basic dyes ranged
from 50-92 percent; the two dianisidine-based dyes used, Direct Blues 1 and
15, showed 92 and 90 percent adsorption, respectively (no other dyes
derived from benzidine or its congeners were tested).
B* Air and Soil
No data were found on the distribution of benzidine in air. Radding
et al. (1975) considered the probable principal chemical reactions of
benzidine in air to be photolysis and oxidation by ozone; however, lacking
any reaction data, they could only estimate crudely the half-life of
benzidine in air as approximately one day. In soil, benzidine is probably
immobilized rapidly by adsorption to humic material (Radding et al, 1975)
and clays (Furukawa and Brindley 1973). The Fe , Al , and Cu ions,
which are readily available in some clays soils, are believed to oxidize
benzidine very rapidly (Furukawa and Brindley 1973).
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V. EXPOSURE ANALYSIS
A. Distribution in the Environment
Benzidine and its congeners are not known to occur naturally in the
environment, and there is little published information bearing on this
point. A review of the literature available in 1978 (Shriner et al. 1978)
identified only one study on the distribution of benzidine or its congeners
in the natural environment, and in this case only water was examined. In
1973-1975 a field survey was conducted by the Synthetic Organic Chemical
Manufacturers (SOCMA) Task Force on Benzidine to determine whether
benzidine, suspected of being released by upriver plants, could be detected
in the Buffalo, New York, and Niagara River areas (reviewed in Howard and
Saxena 1976). Samples of sediment and water were gathered from seven sites
upstream and seven sites downstream from a plant producing benzidine-based
dyes. No benzidine or benzidine-based salts were detected in any of the
samples (detectable limit was 0.2 ppm).
B. Sources of Release to the Environment
1. Benzidine and Its Congeners
Although few actual measurements of release of benzidine compounds
into the environment have been reported, it is thought that manufacturing
and processing plants for dyes and pigments derived form benzidine and its
congeners are the major sources of their release (Shriner et al. 1978). It
would be useful to know
(a) the total amount of each dye produced by each manufacturer,
(b) the total number of sites of production for each dye, and
(c) the amount of benzidine or congener released at each site;
however, this information could not be gleaned from available sources.
In the dye-manufacturing process, the major potential routes for
release of dye-base compounds are air-borne dust produced during weighing,
loading, and mixing operations and wastewaters. Boeniger (1979) made
cotton swipes at the following six locations inside a plant that produced
benzidine dyes: inside a benzidine azo reactor; at the opening of an azo
charging chute; in the bottom area of a diazo tank wall; inside the bottom
outlet spigot of a diazo tank; on the doorknob of a changing room used for
decontaminating workers; and at the charging chute for hydrazobenzene
dumped into a reactor vessel. No benzidine was detected at any of the
sites. Because dust would be expected to be present on most of these
surfaces, it is unlikely that airborne release is a significant source of
benzidine at dye manufacturing sites using procedures similar to the plant
in Boeniger's study. No information is available for estimating the extent
of release of benzidine congeners by this route.
EPA standards for benzidine and its salts in wastewaters from dye
manufacturers were established in January 1977 in the following rule:
37
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"(3)Discharges from a manufacturer shall not contain benzidine
concentrations exceeding an average per working day of 10 yg/1 calculated
over any calendar month, and shall not exceed a monthly average daily
loading of 0.130 kg/kkg of benzidine produced, and shall not exceed 50 ug/1
in a sample(s) representing any working day" (USEPA 1977). Standards set
for dye applicators are similar except that maximum daily concentrations of
benzidine in effluents were limited to 25 yg/1, one half the maximum
allowed benzidine-dye manufacturers. All standards apply only to wastes
discharged directly into streams or rivers; EPA is developing standards for
discharge into secondary treatment plants. The only current benzidine-
based dye producer (Fabricolor) discharges its wastewaters into municipal
wastewater systems; it is believed that the final wastewaters, after
biological treatment, probably show no detectable benzidine (Archer et al.
1977).
2. Dyes and Pigments
The Dyes Ecological and Toxicology Organization (DETO) identified the
following three major sources for environmental release of dyes and
pigments derived from benzidine and its congeners.
(a) Process wastewaters. Although this seems a likely source of
release of dyes and pigments, no information was obtained
regarding the amounts of dyes and pigments derived from the
benzidine and its congeners released into wastewaters, either
during the manufacturing process or in dyeing operations. It is
known that most manufacturers and dye applicators subject their
wastes to secondary treatment plants (memo from DETO to Dr., Fred
Clayton, TSCA/ITC vice-chairperson, June 27, 1979) but the
effectiveness of these treatments is not known.
(b) Atmospheric release. Release of dyes in the atmosphere as
particulate matter can occur during dye manufacturing processes
such as drying and grinding, as well as during handling of dyes
in application industries (e.g., in drug rooms of textile mills
where dyes are weighed out for dye liquor bath preparation).
Except in situations in which dusting is considered a serious
problem requiring special means of collection, dust particles
(including dyes) are collected by ventilators and exhausted into
the air outside the plant.
(c) Disposal and dyed articles. Clothing, colored paper, leather,
and other dyed goods are commonly disposed into landfill sites as
solid wastes. Dyes could be released from these objects into the
environment, but—due to the high affinity of the dyes for the
substrate on which they are applied—it is thought that any such
release from manufactured articles would be a slow process.
C. Population Exposed
1. Industrial Workers
The people most exposed are the workers who manufacture, handle, or
use the dyes and pigments derived from benzidine and its congeners. Data
38
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concerning the numbers of such workers and the extent of their exposure ar,
generally unavailable but are being compiled by DETO.
Occupational standards instituted by OSHA in 1974 identified benzidine
(including its salts) as a carcinogen and, therefore, subject to regulation
as a health hazard in the workplace. The standards require that any area
in which benzidine is manufactured, processed, used, repackaged, released,
handled, or stored be designated a regulated area, with entrance and exit
restricted and controlled. Detailed work rules aimed at minimizing
exposure are prescribed (e.g., washing and change of clothes required on
entering and leaving a regulated area; use of a half-face, filter-type
respirator required in regulated areas; use of a hood or an isolated system
such as a glovebox when handling benzidine). The standards, however, do
not apply to any compounds that contain less than 0.1 percent free
benzidine (1,000 ppm) by weight or volume. No safe levels of benzidine in
the workplace were established, and environmental monitoring was not
required. Presumably these regulations have reduced worker exposure to
benzidine, but the actual extent of exposure cannot be assessed at present.
Occupational exposure to benzidine-based dyes is presently under the
scrutiny of NIOSH. Based on a national survey from 1972-1974, NIOSH
estimated that no fewer than 6,500 workers are potentially exposed to
benzidine-based dyes (NIOSH 1979). Boeniger (1979) investigated worker
exposure to azo dyes, with special attention directed to those derived from
benzidine. This comprehensive study monitored a variety of workers and job
sites in two dye-manufacturing plants, two textile plants, a tanning and
leather finishing plant, and a paper producing plant. Dye concentrations
in the air were determined from particulate matter trapped in filters (both
stationary filters and filters attached to workers were used), with
subsequent spectroscopic analysis of soluble matter removed from filters.
Urine samples over varying lengths of time were collected from workers and
analyzed for the presence of total aromatic amines, benzidine, and
monoacetylbenzidine (MAB); the presence of these compounds is indicative of
metabolism of the dyes. In all, 38 workers potentially exposed to
benzidine (representing all 6 sites) were monitored. Highly significant
was the finding of benzidine or MAB in quantities ranging from about 1 ppb
up to 112 ppb benzidine and 590 ppb MAB in 8 of the 38 workers monitored; 6
of the 8 worked at one of the dye manufacturing plants, and 2 worked at a
textile finishing plant. The concentrations of benzidine in urine were
found to be higher than could be accounted for by traces of free benzidine
measured in the dyes used; thus, by inference, the benzidine and MAB
resulted from metabolic conversion of the dyes. The major conclusion of
this study is that the number of workers previously thought to have
potential exposure to benzidine may be greatly underestimated (Boeniger
1979).
A recent report states that the growing use of imported benzidine dyes
may result in increased exposure of dye workers to residual free benzidine,
reported to be as high as 500 ppm in some imported dyes, compared to 20 ppm
or less in domestically produced dyes, although no actual data were
presented (Castleman 1979). To investigate this question, Boeniger (1979)
examined 26 randomly chosen imported dyes and an equal number of domestic
dyes for residual benzidine content. Results are shown in Tables 10 and
11. Although the highest residual benzidine found was in a domestic dye,
39
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this was the only domestic sample containing more than 20 ppm benzidine,
whereas six of the imported dyes exceeded this value. Thus, it seems
likely that increased use of imported dyes will lead to an increase in
exposure to residual benzidine. Although the amount of increased exposure
cannot be determined from the information at hand, it is probably much less
than that implied by the value of 500 ppm residual benzidine cited by
Castleman (1979).
Table 10. Residual Benzidine in Direct Dyes from Domestic Sources
Residual benzidine
Company and dyes in ppm (w/w)
GAF Corporation
Black JXA (Blk. 38) 13
Black ER-200 (Blk. 38) 4
Black EA (Blk. 38) 2
Brown BRL (Brn. 95) 2
Blue 2B (Blue 6) 4
Scarlet 4BGP (Red 39) <1
Fabricolor
Brown 3GN (Brn. 95) 270
Black GX (Blk. 38) 20
Brown BRL 200% (Brn. 95) 19
Brown 3GN (Brn. 154) 15
Green WS 133% (Grn. 1) 12
Fast Blue 2B 250% (Blue 6) 12
Grown Brown B 125 (Brn. 31) 10
Black GX 200% (Blk. 38) 10
Gatechine 3G (Grn. 74) 4
Brown 3GN (Brn. 154) 4
Fast Brown B 125% (Brn. 31) 3
Fast Green BX 100% (Grn. 6) 3
Phenamine Black E-200 (Blk. 38) 2
Congo Red 4B (Red 28) 2
Brown M 100% (Brn. 2) <1
Green WS 100% (Grn. 1) <1
Diazo Black BH (Blue 2) <1
Brown 3GN (Brn. 154) <1
Allied Chemical
Niagara Blue <1
Erie Green GPD <1
Source: Boeniger (1979)
40
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Table 11. Residual Benzidine in Import Dye Samples
Dye
C.I
C.I
C.I
C.I
C.I
C.I
C.I
C.I
C.I
C.I
C.I
C.I
C.I
C.I
C.I
C.I
C.I
C.I
C.I
C.I
C.I
C.I
C.I
C.I
C.I
C.I
name
. Direct Red 1
. Direct Orange 8
. Direct Green 6
Direct Black 38
. Direct Black 38
. Direct Black 38
Direct Blue 6
Direct Black 38
. Direct Blue 2
. Direct Blue 2
. Direct Blue 2
. Direct Red 28
. Direct Red 28
. Direct Green 1
Direct Black 38
Direct Red 1
Direct Red 28
. Direct Red 28
Direct Blue 2
. Direct Red 28
Direct Blue 2
. Direct Red 37
Direct Brown/54
. Direct Brown 1:A
Direct Red 28
Direct Blue 2
Exporting
. country
Belgium
India
Holland
Egypt
Poland
Poland
India
India
?
Romania
India
Romania
India
Poland
Holland
Poland
Poland
Belgium
Poland
Korea
Poland
Holland
Poland
Poland
Poland
Poland
Concentration of
residual benzidine
in ppm (w/w)
224
143
70
53
40
38
ID
9
8
8
7
7
6
3
3
3
2
2
2
2
1
1
1
1
<1
0.4
Source: Boeniger (1979)
2. Laboratory Workers
Benzidine and its congeners are commonly used as analytical reagents
in chemical, biochemical, and clinical laboratories, and the potential
dangers of these compounds have been pointed out (Shriner et al. 1978).
Levels of contamination and exposure probably vary widely; neither the size
of the potentially exposed population nor the levels of actual exposure is
known.
3. General Population
The general public is exposed mainly to finished dyes and pigments
after they have been applied to textiles, leather, and other products;
however, direct exposure to benzidine or its congeners, present as residual
unreacted starting materials, or as breakdown products, is conceivable.
The amounts of free benzidine in finished products, however, have not been
reported (USCPSC 1977). The dyes and pigments in finished products are
considered to be essentially "fast" (they do not migrate or wash out);
major retailers' quality standards require colors to be durable through at
41
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least 20 washings. One report on chemicals in wearing apparel, prepared
for the U.S. Consumer Product Safety Commission (CPSC), stated that "there
is little chance of dyes coming off in perspiration, saliva, or washings if
labeling instructions are followed" (USCPSC 1977, p. 111-14); however, no
actual measurements of possible leaching were reported.
A report compiled by the Center for Occupational Hazards of the
Artist-Craftsmen of New York, Inc., calls attention to the potential
hazards to the general public—and artists and craftsmen in
particular—presented by exposure to dyes and pigments specially packaged
for craft use and the all-purpose dyes for general household use (Jenkins
1978). According to this report, direct dyes are the most common among
those used in the crafts, and many of these dyes, as well as general
purpose dyes, are thought to be derived from benzidine or its congeners.
To investigate this question, Boeniger (1979) analyzed 15 consumer retail
dyes purchased in arts and crafts shops in New York City. The dyes were
chemically reduced and the relative amounts of aniline, benzidine,
£-tolidine, and dianisidine were determined (Table 12). Nine of these dyes
appear to be wholly or predominantly benzidine based and five jo-tolidine
based; only one of the 15 dyes contained no detectable benzidine or
benzidine congener. Recent studies performed for the CPSC (1979), however,
indicate that benzidine-based dyes are no longer being used for formulation
of home dyes such as KIT. jo-Tolidine- and dianisidine-based dyes are being
substituted for the benzidine-based dyes. However, because the available
inventory of benzidine-based dyes was not recalled and removed from the;
consumer market, such dyes could be available for several years.
Two other points raised by Jenkins (1978) are pertinent to an exposure
analysis:
(a) The craftsman or home user is probably unaware of any potential
hazard and does not use special precautions (as are expected to
prevail in commercial dye plants) when using these dyes. Use of
a cooking vessel with insufficient cleaning, for example, might
lead to a. significant amount of ingestion of dye.
(b) Some home or craft dyeing operations proceed in boiling Welter, at
which temperature dyes based on benzidine substrates have been
reported to decompose (Jenkins 1978). Presumably benzidine or
other parent compounds are among the degradation products,.
42
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Table 12. Determination of Aniline, Benzidine, o-Tolidine, and Dianisidine
in Retail Dyes
Reduced products (relative %)
Company
FBS, Inc.
"
"
Tintex
"
"
11
"
Aljo
II
II
II
RIT
n
ii
Dye name
Fazan Brown
#4029
Black #1628
Dark Blue
#4025
Black-
Powder
Brown-
Powder
Navy Blue
#25
Royal Blue-
Powder
Chocolate
Brown-Powder
Black -Cotton
Dark Brown
Imperial Blue
Royal Blue
Chestnut
Brown #43
Navy Blue 30
Black 15
Aniline
1
2
0
2
1
1
0
0
12
0
0
0
2
2
5
Benzidine
99
98
0
98
99
74
1
99
88
100
96
2
6
0
2
o-Tolidine
0
0
0
0
0
0
98
1
0
0
4
96
87
93
92
Dianisidine
0
0
0
0
0
25
1
0
0
0
0
2
5
5
1
Source: Boeniger (1979)
43
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VI. SUMMARY OF ISSUES INCLUDING VALIDATION AND INFORMATION NEEDS
A. Benzidine and Benzidine-Based Dyes
Direct exposures to benzidine, a human and animal carcinogen, probably
occur primarily in occupational settings. Although direct exposure to
benzidine during the manufacture of dyes and pigments appears to be
controlled to a technologically feasible degree, several exposure problems
result from the application of dyes contaminated with benzidine.
1. Workers may be exposed to as much as 500 ppm free benzidine in
imported dyes; domestically produced dyes normally contain about
20 ppm free benzidine. Both the domestically produced and
imported dyes are excluded from consideration in implementation
of work practices prescribed by OSHA for dyes containing more
than 0.1 percent (1,000 ppm) free benzidine. In addition, no
occupational exposure limit has been established for either
benzidine or benzidine-based dyes.
2. At least 85 percent of the dyes used are imported. Assuming that
U.S. technology is not unique, one might question why these dyes
have approximately twenty-five times the free benzidine that
normally occurs in U.S. dyes. The lack of a system for tracking
the flow of benzidine dyes in commerce, including imports,
further complicates an assessment of the risks that may be
associated with these dyes. For example, what are the individual
dyes used for? Are exposures adequately controlled during use?
Are some of the dyes used in uncontrolled circumstances, such as
home craft uses?
Although one problem associated with the use of benzidine-based dyes
is the level of free benzidine in the dyes, the exposure potential to
benzidine may be greatly enhanced if one considers that a number of the
benzidine-based dyes have been found to be metabolized to benzidine in
mammalian systems. The following basic issues need to be considered:
(a) Are the metabolism studies on eight benzidine-based dyes and the
demonstrated carcinogenicity of three dyes sufficient to make
predictions about the dyes curresntly in commercial production or
the larger number of dyes that can be synthesized by existing
technology?
(b) Can all the dyes known to metabolize to benzidine be assumed to
exhibit the same effects (i.e., carcinogenicity) as benzidine?
(c) Considering that the time-to-tumor interval is 5 weeks in rats
orally dosed with benzidine-based dyes and somewhat longer in
rats orally dosed with benzidine (the dose of benzidine was much
lower than the dose of dyes) and that the pattern of tumors
observed with the dyes is different from that observed in rats
fed benzidine, can benzidine (or a benzidine metabolite) be
44
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assumed to be the carcinogenic agent derived from the dyes? Can
an adequate scientific basis be developed to explain differences
in the time-to-tumor interval if metabolism of dyes to benzidine
is used as the basis for a detailed risk assessment?
1. Validation Needs
Key review and validation needs for benzidine and benzidine-based
dyes appear in the following areas:
(a) benzidine epidemiological studies;
(b) key animal carcinogenicity studies on benzidine and
benzidine-based dyes (Direct Blue 6, Direct Black 38, and Direct
Brown 95); and
(c) metabolism studies on the benzidine-based dyes.
B. o-Tolidine, Dianisidine, and Their Derivative Dyes and Pigments
Positive animal carcinogenicity studies have been reported for both
o-tolidine and dianisidine; however, these studies may not be of sufficient
strength to support a detailed risk assessment. The validity of the
reported animal carcinogenicity studies, therefore, is a key issue in
considering this subset of the category. Metabolic studies that clearly
indicate that the derivative dyes and pigments are metabolized to the
parent compounds are not available. One commercial ^-tolidine-based dye
(Trypan Blue) has been found to be carcinogenic, mutagenic, and
teratogenic, although the carcinogenicity may be the result of components
of the commercial product other than the primary o-tolidine-based dye. One
o_-tolidine-based pigment (Pigment Yellow 16) has been found negative in a
carcinogenicity study and found not to be metabolized to ca-tolidine by
rats. Metabolism studies on several o-tolidine-based dyes (pigments) are
currently in progress through the National Toxicology Program. The
following questions should be addressed:
(a) Is there a need for additional carcinogenicity testing of
jD-tolidine and dianisidine?
(b) Is there enough structural similarity between benzidine- and
o-tolidine- or dianisidine-based dyes to make basic conclusions
about the risk associated with o-tolidine- and dianisidine-based
dyes?
(c) Should the o-tolidine and dianisidine-based pigments be addressed
separately from the dyes derived from these two benzidine
congeners?
(d) Are the carcinogenicity, mutagenicity, and/or teratogenicity of
Trypan Blue adequately characterized to permit a detailed risk
assessment for that product?
45
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C. Dichlorobenzidine and Its Derivative Pigments
1. Identified Issues
Dichlorobenzidine is reported to be an animal carcinogen, and at least
one study in mice suggests that dichlorobenzidine may have some potential
as a transplacental carcinogen. Reportedly this chemical also is
photolyzed in the aquatic environment to benzidine and other products.
Both benzidine and dichlorobenzidine have some potential for
bioaccumulation. Exposure to and release of dichlorobenzidine into the
environment currently are not controlled by Federal or state legislation.
The three dichlorobenzidine-based pigments thus far tested have not been
demonstrated to be carcinogenic; they have not been shown to metabolize to
free dichlorobenzidine. The following questions may be asked with respect
to these compounds:
(a) Does the weight of the accumulated evidence suggest that
dichlorobenzidine is a carcinogen whose exposure and
environmental release should be controlled?
(b) Are the existing metabolism and negative carcinogenicity studies
sufficient to dispell concern that dichlorobenzidine-based
pigments may be carcinogens?
(c) Is the hypothesis that structural features make dichlorobenzidine
pigments resistant to enzymatic reduction (to dichlorobenzidine)
sufficient to explain the lack of carcinogenicity observed with
these pigments (also see General Issues [c]).
2. Validation Needs
The key review and validation needs for dichlorobenzidine and
dichlorobenzidine-based pigments follow:
(a) A review of the animal carcinogenicity studies on
dichlorobenzidine and Pigments 5fellow 12, 13, and 83;
(b) Review and validation of the metabolism studies on
dichlorobenzidine-based pigments;
(c) Review and validation of the study that suggests that
dichlorobenzidine may be a transplacental carcinogen;
(d) Review and validation of the studies that show that
dichlorobenzidine is photolyzed to benzidine in aqueous
media; and
(e) Review and validation of bioaccumulation studies for
both benzidine and dichlorobenzidine.
D. General Issues
One difficulty encountered in assessing the dyes and pigments
available in the U.S. is obtaining current and specific production and
importation information. The difficulty is created by the rapidly shifting
46
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pattern of consumption. This is reflected in the differences between the
list of dyes reported here as currently available and a list prepared by
the Midwest Research Institute as a part of a materials balance (see
Appendix A).
Because any future information-gathering studies will be seriously
handicapped by these problems, the following questions must be addressed:
(a) Can the risk assessment be used for decision making on all dyes
and pigments included in the category (i.e., all that can be
synthesized by existing technology), or can conclusions be drawn
for only a smaller subset of the category (i.e., those for which
we have certain types of information)?
(b) Although this report addresses the primary benzidine congeners,
other congeners (e.g., 3,3'-disulfo-, 2,2'-dichloro-, and
3-nitrobenzidine) exist. There is little, if any, available
information on these congeners. Should these congeners be
included in an assessment of the benzidine-based dyes and
pigments or treated separately on an as-needed basis?
(c) Do factors such as the particulate nature of pigments, particle
size, and possible differences between the transport and storage
of particulate (pigment) and soluble (dye) materials suggest that
dyes and pigments should be treated as separate issues?
1. Information Needs
Information needs, in part, will reflect the extent of the category
and the thoroughness of additional assessment efforts. The information
needed to evaluate the risk(s) associated with exposures to dyes and
pigments that result from contact with textiles, for example, will differ
from that required for determining the risk of worker exposures to dyes and
pigments. Regardless of the decision concerning the type and extent of the
assessment, the following information needs are anticipated:
(a) Level-Ill literature searches on specific topics;
(b) More in-depth materials balances for individual congeners and
dyes and pigments (either individually or as a category);
(c) Information gathering under TSCA, section 8; and
(d) Monitoring studies to determine the quantity of dye or congener
(e.g., dichlorobenzidine) that is released and distributed in the
environment.
47
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51
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53
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TECHNICAL REPORT DATA
(Please read Instruction! on the reverse before completing)
1. REPORT NO.
560/11-80-019
2.
[3. RECIPIENT'S ACCESSION NO.
|4. TITUS AND SUBTITLE-
5.
Benzidine, its congeners/ and their
derivative dyes and pigments
6. PERFORMING ORGANIZATION COOS
7. AUTHOR(S)
Theodore -C. Jones
3. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAMS AND AOORE5S
U.S. Environmental Protection Agency
• 401 M St. S.W.
Washington/ DC 20460
10. PROGRAM 6C=M£NT NO.
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NA.VIE AND AOORESS
U.S. Environmental Protection Agency
401 M St. S.W.
Washington/ DC 20460
13. TYPE Of REPORT AND PERIOD COVERED
Final -
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. AflSTHACT
This report assesses the risk to health and the environment
presented by benzidine and three of its congeners (o-tolidine,
dianisidins1/ and dichlorobenzidine) and by dyes and pigments derived
from these compounds. Benzidine/ cs-tolldine/ dianisidine and
dichlorobenzidine are used almost entirely in the production of
dyes and pigments used to color textiles/ paper/ leather, rubber,
plastic products/ printing inks, paints and lacquers.
Several potential risks have been identified through a
preliminary analysis of the exposure and hazards associated with
these compounds. These include: (1) the oncogenic risks to workers
exposed to imported benzidine-based dyes that contain high
concentrations of" free benzidine; (2) a similar'risk to workers using
domestically produced benzidine-based dyes (because there are no
occupational exposure standards for either benzidine or its
derivative dyes); (3) the risk to the general population that may
result from exposure to benzidine-based dyes in such products as
textiles and home dyes; and (4) risks -of toxicity to aquatic life
that may result from release of dichlorobenzidine into the
17. KSY WORDS ANO OOt-UMSNT ANALYSIS
4. DESCRIPTORS
benzidine; ai-tolidine; dia-
nisidine; dichlorobenzidine;
bisazobiphenyl; dyes; pigments;
carcinogens; mutagens; hazards;
assessment
18. DISTRIBUTION STATEMENT
b.lOENTIFISaS/OPEN 5NOSD TERMS
preliminary risk
assessment;
phase I assessment
19. SECURITY CLASS (This Aeport)
unclassified
2O.S6C*08«TY CLASS iThi* pagcl
unclassified
c. COSATI Field/Group
21. NO. OP PAGES
22. PRICE
CFA Fvttn USO'l (3-73)
55
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