Environmental Health Perspectives Volume
102, Supplement 2, June 1994
[Citation
in PubMed] [Related
Articles]
Summary of the National Toxicology Program Benzidine Dye Initiative
Daniel L. Morgan,1 June K. Dunnick,1 Thomas
Goehl,1 Michael P. Jokinen,1 H. B. Matthews,1
Errol Zeiger,1 and John H. Mennear2
1National Institute of Environmental Health Sciences, Research
Triangle Park, North Carolina; 2Campbell University, Buies Creek,
North Carolina
Abstract
The benzidine dye initiative is a research program established by the
National Toxicology Program to generate an integrated body of scientific
information regarding the potential health risks associated with exposure
to benzidine- and benzidine-congener-derived dyes. Because an in-depth evaluation
of each of the hundreds of benzidine-congener-derived dyes was considered
impractical, the research program was designed to study the metabolism and
disposition, genetic toxicity, and in vivo toxicity and carcinogenicity
of two primary benzidine congeners, 3,3'-dimethylbenzidine and 3,3'-dimethoxybenzidine,
and a select group of prototypical dyes derived from those amines. It was
anticipated that by applying the basic information generated in these extensive
studies, it would be possible to make regulatory decisions about other dyes
after conducting only a minimal number of experiments such as studies of
disposition and metabolism, and in vitro mutagenicity. This paper
summarizes the results of studies conducted to evaluate the metabolism,
disposition, mutagenicity, toxicity, and carcinogenicity of representative
benzidine congeners and derived dyes. -- Environ Health Perspect 102(Suppl
2):63-78 (1994).
Key words: benzidine, 3,3'-dimethylbenzidine, 3,3'-dimethoxybenzidine,
C.I. Direct Blue 15, C.I. Direct Blue 218, C.I. Acid Red 114, chemistry,
metabolism, carcinogenesis, mutagenicity
The authors thank Drs. G. Boorman, R. Maronpot, and R.
Tennant for critIcally reviewing this manuscript.
Address correspondence to Dr. Daniel L. Morgan, National
Institute of Environmental Health Sciences, MD IF-00, P.O. Box 12233, Research
Triangle Park, NC 27709. Telephone (919) 541-2264. Fax (919) 541-0356.
Introduction
Haley (1) reviewed the early literature on the toxicologic and
carcinogenic effects of benzidine and a new review is currently in progress
by the Agency for Toxic Substances and Disease Registry. Since the intent
of our communication is to present a body of experimental results, only
a brief review of clinical and experimental highlights will be presented.
An increased incidence of cancer of the urinary bladder was first associated
with human occupational exposure to dyes and dye chemicals in 1895 (1).
These tumors, initially referred to as aniline cancers because of the extensive
use of aniline in dye chemistry, were attributed to exposure to either the
starting materials or to the finished dyes per se. Over the intervening
years several aromatic amines, used as starting materials in the dye manufacturing
process, were shown to be animal carcinogens and benzidine was identified
as a carcinogen for the human urinary bladder in 1973 (1). Simultaneously
the results of cancer epidemiology studies in Japanese Kimono painters implicated
benzidine-derived dyes as human carcinogens. In addition, two congeners
of benzidine, 3,3'-dimethylbenzidine and 3,3'-dimethoxybenzidine, also employed
in the dye industry, were found to increase tumor incidence in rats (1).
The results of these early studies on 3,3'-dimethylbenzidine and 3,3'-dimethoxybenzidine
were difficult to interpret because of the small groups of animals used
and reduced survival.
Although certain benzidine-derived dyes (e.g., Direct Blue 6, Direct
Brown 95, and Direct Black 38) were considered to be carcinogenic in rats
(2), it remained to be established whether the carcinogenic action
was attributable to the dyes per se, trace levels of benzidine present in
the dyes as a contaminant, or metabolites of the dyes. Further, in addition
to equivocal data concerning the carcinogenicity of 3,3'-dimethylbenzidine
and 3,3'-dimethoxybenzidine, dyes derived from either of these benzidine
congeners had not been adequately studied for carcinogenic activity.
These questions concerning possible carcinogenicity, combined with the
potential for exposure of humans to the benzidine congeners during dye manufacture
and to the dyes during their use, prompted the design and conduct of a series
of experiments to explore the carcinogenic potential of several benzidine-congener-derived
dyes in rats. The US Environmental Protection Agency, the Consumer Protection
and Safety Commission, and the Occupational Safety and Health Agency nominated
several benzidine dyes for carcinogenicity testing by the National Toxicology
Program (NTP). During the planning stages it was recognized that a large
number of dyes existed and that should one or more be found to possess undesirable
biological activity an alternate, most likely of ill defined toxicologic
potential, would be readily available as a substitute. The substitute would
then require testing before regulatory decisions relative to its use could
be made. Such a circuitous path would have placed an enormous burden on
the resources of the NTP.
To circumvent this potential dilemma the collaborating agencies agreed
to implement a research program on the benzidine congeners and dyes derived
from them, the results of which could be used to predict the carcinogenic
potential of other benzidine-congener-derived dyes. The broad objective
of the initiative was to generate an integrated body of scientific information
regarding the potential health risks likely to be associated with exposure
to any benzidine-congener-derived dye. To this end, experiments were designed
to study the metabolism and disposition, genetic toxicity, and in vivo
toxicity and carcinogenicity of 3,3'-dimethylbenzidine and 3,3'-dimethoxybenzidine,
and a select group of prototypical dyes derived from those amines. It was
anticipated that by applying the basic information generated in these extensive
studies it would be possible to make regulatory decisions about other dyes
after conducting only a minimal number of experiments such as studies of
disposition and metabolism, and in vitro mutagenicity.
Chemistry
Nomenclature and Structure
Nomenclature of colorants can be confusing. The difference between dyes
and pigments is in the method used to apply the coloring agent. Dyes are
applied as a solution or a vapor while pigments are applied without losing
their crystalline structure. The Colour Index (C.I.) (3) names dyes
by reference to application class and shade, and by using a sequential number.
The dyes used in the current studies belong to the direct and acid application
classes which are water-soluble anionic dyes (4). Benzidine- and
benzidine-congener- (i.e., chemically related to benzidine) based dyes include
those derived from 3,3'-dimethylbenzidine and 3,3'-dimethoxybenzidine, as
well as benzidine itself. Synonyms for 3,3'-dimethylbenzidine and 3,3'-dimethoxybenzidine,
the benzidine congeners used in the studies reported here, are o-toluidine
and o-dianisidine, respectively. Chemical structures, CAS numbers,
and C.I. numbers for benzidine, the benzidine congeners, and the related
dyes that were studied are presented in Table 1.
Synthesis
Benzidine and its congeners are dye intermediates, i.e., precursors of
dyes. A common method used to prepare benzidine and congeners involves the
reduction of nitrobenzenes followed by the acid catalyzed intramolecular
rearrangement of the resulting hydrazobenzenes. Subsequently, the dyes are
produced by the diazotization of the amino groups on the benzidines and
azo coupling to reactive aromatic ring systems (other dye intermediates).
A generalized synthetic scheme is shown in Figure 1. Metallizing of dyes
sometimes is carried out to improve the stability of the azo groups; however,
the mechanism by which the added metal chelates with the dye is not always
well understood. For the one metallized dye used in our carcinogenesis studies,
C.I. Direct Blue 218, the structure has not been completely defined. However,
the synthetic route consists of coupling 1 mole of o-dianisidine
(3,3'-dimethoxybenzidine) to 2 moles of 4-amino-5-hydroxy-2,7-napthalene
disulfonic acid under alkaline pH conditions followed by metallizing and
elimination of methyl groups from the methoxides to form the copper complex
(5). Depending on the synthesis methods employed and the needs of
the end user, the dyes may be impure "presscakes" or highly pure
crystalline products. The most important property of the dyes to the end
user is the color, while purity is of secondary concern. Because humans
are exposed to these impure commercial products, commercial sources for
the test materials were selected. As was found in our chemical characterization
studies, the purity of the dyes varies greatly.
Figure 1. Generalized
synthetic scheme for benzidine (R=H), the benzidine congeners 3,3'- dimethylbenzidine
(R=CH3), and 3,3'-dimethoxybenzidine (R=OCH3), and derived azo dyes (R'and
R"= various aromatic intermediates).
Chemical Analyses
Comprehensive purity, identity, and stability studies were performed
on the benzidine congeners and dyes used in the 2-year chronic studies.
Reports on the analyses performed are on file at the National Institute
of Environmental Health Sciences. The study chemicals were identified as
3,3'-dimethylbenzidine dihydrochloride, 3,3'-dimethoxybenzidine dihydrochloride,
C.I. Direct Blue 15, C.I. Acid Red 114, or C.I. Direct Blue 218 by infrared,
ultraviolet/visible, and nuclear magnetic resonance spectroscopy. The purities
of 3,3'-dimethylbenzidine dihydrochloride and 3,3'-dimethoxybenzidine dihydrochloride
were determined to be 99 and 98%, respectively, by elemental analysis, Karl
Fischer water analysis, titrations (nonaqueous amine and neutralization
titrations), thin-layer chromatography, and high-performance liquid chromatography
(HPLC). Because of its high salt content, Direct Blue 15 was desalted by
dialysis, which reduced the salt content from approximately 25 to about
3%. The purity of the desalted dye was determined to be about 50%. Approximately
35 impurities were detected by HPLC analysis accounting for about 50% of
the chromatographic peak area. No attempt was made to identify the chromatographic
peaks. However, the chemical was assayed for free benzidine and 3,3'-dimethoxybenzidine
content. Benzidine could not be detected at levels greater than 1 ppm in
either of the two lots used in the study, whereas 3,3'-dimethoxybenzidine
was found at 826 and 1310 ppm in two lots that were sampled.
The dye, Acid Red 114, was desalted by dialysis, and the salt content
reduced from approximately 14.9 to about 0.9%. The purity of the desalted
dye was estimated at 82 to 85%. There were approximately 15 organic impurities
observed by HPLC analysis; these impurities were similar in structure to
the major component, with the two largest components estimated at 3% each.
In addition, there was approximately 1 to 4% water present in the dye. The
level of benzidine detected by HPLC did not exceed 1 ppm; whereas, 3,3'-dimethylbenzidine
was detected at a concentration of approximately 5 ppm.
Direct Blue 218 contained about 2% salt after desalting by dialysis.
The purity of the desalted chemical was determined to be approximately 60%.
Over a dozen impurities were detected by HPLC analysis, accounting for approximately
40% of the chromatographic peak area. No attempt was made to identify the
chromatographic peaks, however, the concentrations of benzidine and 3,3'-dimethoxybenzidine
were determined. Benzidine could not be detected in either lot at levels
greater than 1 ppm. 3,3'-Dimethoxybezidine was found at levels less than
or equal to 7 ppm.
Dose Formulation
Dose formulations were characterized and the concentrations confirmed
during the toxicology studies. In all cases, except C.I. Direct Blue 218,
reverse-phase HPLC-UV systems were used. Detector wavelengths were 280 nm
for 3,3'-dimethylbenzidine and 3,3'-dimethoxybenzidine; 546 nm for C.I.
Direct Blue 15 and 512 nm for C.I. Acid Red 114. For C.I. Direct Blue 218,
visible spectroscopy at 622 nm was employed. Reports on the dose formulation
studies are on file at the National Institute of Environmental Health Sciences.
Initially, attempts were made to formulate 3,3'-dimethylbenzidine dihydrochloride
and 3,3'-dimethoxybenzidine dihydrochloride in feed. However, feed formulations
were unstable when stored in the dark in sealed containers at room temperature,
5°C, or 22°C. Drinking water was then investigated as a vehicle
for chemical administration. Solutions of 3,3'-dimethylbenzidine dihydrochloride
or 3,3'-dimethoxybenzidine dihydrochloride in water remained stable for
at least 14 days when stored at either room temperature or 5oC.
Solutions were also stable for up to 48 hr under simulated animal dosing
conditions. During the studies drinking water formulations were prepared
two times/week and were used immediately or stored at room temperature for
up to 7 days before being used. The drinking water solutions were analyzed
at approximately 4-week intervals. Of the dose formulations analyzed, 99%
were determined to be within 10% of the target concentration. Results of
periodic independent analyses agreed with the results of the toxicology
study laboratory.
The drinking water formulations of Direct Blue 15 and Acid Red 114 remained
stable for at least 21 days when stored at room temperature. Solutions were
stable for at least 3 days under simulated dosing conditions including exposure
to normal room light and air. Dose formulations were prepared twice weekly
and made available to the study animals on the day of mixing. Dose formulations
were analyzed at least once every 4 weeks for the duration of the studies;
all were determined to be within + 10% of the target concentrations.
Direct Blue 218 was insoluble in water at the required concentrations
for the 2-year studies. Because of the solubility limitations, the dose
formulations were prepared by mixing Direct Blue 218 with feed. Stability
tests showed that the formulations were homogeneous and stable for at least
21 days when stored at room temperature, and for at least 3 days under simulated
animal dosing conditions. Dose formulations were prepared once every 2 weeks.
The formulations used for dosing were analyzed at least once every 4 weeks;
97% were determined to be within 10% of the target concentration.
Metabolism and Mechanisms of Toxicity
As part of the initiative to characterize the toxicity and carcinogenicity
of benzidine and benzidine-congener based dyes, the NTP conducted and sponsored
studies of the metabolism of both the dyes and parent compounds. These studies
were conducted at the National Center for Toxicological Research (NCTR)
and through contractual mechanisms. This section presents a summary of that
work, and includes relevant work done by others since that time, to more
completely describe the fate and mechanisms of toxicity of these compounds.
Metabolism/Disposition of Dyes
The objectives of metabolism and disposition studies conducted or supported
by the NTP were to investigate the degree to which the dyes or parent amines
were absorbed from the gastrointestinal tract, and to determine the amount
of parent amine eliminated in urine following exposure to the dyes (Figure
2). The presence of the parent amine in urine was taken as evidence of exposure
of the target tissue for humans, the urinary bladder. It was further assumed
that results obtained with representatives of each dye class would be representative
of the respective classes. That is, the absence of the release of benzidine,
or the respective congeners, or metabolites of these compounds, would be
taken as evidence that the dyes were not metabolized to the parent compounds,
and that human risks associated with exposure to the dyes may not be related
to exposure to the respective parent amine. Similarly, release of the respective
parent amine following exposure to most or all of the dyes would indicate
that exposure to the dyes would entail risks similar to those associated
with the parent compound. Mixed results would be taken as evidence that
more dyes should be tested prior to judging the risks associated with using
this class of compounds.
Figure 2. Metabolism
of the benzidine congener and benzidine dyes (e.g., C.I. Direct Blue 6)
initially involves azo reduction in the gastrointestinal tract producing
free benzidine or benzidine congener, and other aromatic amine by-products.
The biological reduction of azo compounds to release aromatic amines
had been previously demonstrated by a number of investigators (6-8)
prior to the NTP studies of benzidine and related dyes. Further, the metabolism
of four benzidine- based dyes by rhesus monkeys to benzidine was described
in a brief report by Rinde and Troll (9) in 1975. However, metabolite
identification in the studies of Rinde and Troll were limited to thin-layer
chromatography versus authentic standards of benzidine and N-acetylbenzidine.
Therefore the NTP-sponsored studies were designed to confirm and extend
those observations by studying seven additional benzidine dyes, two dimethoxybenzidine-
based and four dimethylbenzidine-based dyes. The NTP studies also more fully
characterized the metabolism of these compounds and confirmed metabolite
structures by gas chromatography and mass spectral analysis.
Dyes representative of those in use at the time were selected for metabolism
and disposition studies. Benzidine-based dyes selected included Direct Orange
1, Direct Green 1, Direct Orange 8, Direct Black 4, Direct Brown 2, Direct
Blue 2 and Direct Red 28. The 3,3'-dimethoxybenzidine-based dyes studied
were Direct Blue 15 and Direct Blue 1. In addition, Direct Blue 25, Direct
Red 2, Direct Red 39 and Acid Red 114, all 3,3'-dimethylbenzidine-based
dyes, were studied. In addition to the dyes, these studies also included
the parent compounds (the structures of these compounds are presented in
Table 1).
The dog was chosen as a test species because, other than humans, it was
the only species known to develop bladder tumors as a result of benzidine
exposure (1). The rat was chosen for comparative purposes, and because
this species was to be used in chronic studies of several of these dyes.
The objective of the initial studies was to confirm the metabolism of
benzidine and benzidine-based dyes to the parent amines. In these initial
studies the test chemicals were administered to dogs at 100 mg/kg in meat
balls, and urine was collected at 24 hr intervals for 3 days. No benzidine
or related compounds were detectable in urine by the third day. Rats received
similar doses of the dyes by oral gavage in water daily for 10 days. Urine
from both species was collected over ice and stored at approximately 20°C.
Concentrations of benzidine, the congeners, and known metabolites of these
compounds in urine were determined by gas chromatography. Concentrations
of benzidine, 3,3'-dimethylbenzidine or 3,3'-dimethoxybenzidine in the starting
dyes also were determined by gas chromatography. In analyses of both the
urine and the dyes, the identities of the chromatographic peaks were confirmed
by mass spectrometry (10).
Results of studies with dogs established that the respective free parent
amines were excreted in urine following administration of each of the benzidine
and 3,3'-dimethoxybenzidine dyes studied, and following administration of
Direct Blue 25 and Acid Red 114, two of the four 3,3'-dimethylbenzidine-based
dyes. Administration of two other 3,3'-dimethyl- benzidine-based dyes, Direct
Reds 2 and 39, did not result in 3,3'-dimethylbenzidine in the urine of
dogs. After administration of benzidine-based dyes, the amounts of benzidine
excreted were small (about 0.1% of the dose), but they were at least nine
times greater than the amounts of benzidine administered as contaminants
in the dyes, and were equivalent to the amount excreted following administration
of neat benzidine. Since dogs do not readily acetylate aromatic amines such
as benzidine, free amines were excreted in the urine. On the other hand,
rats readily acetylate these compounds and excreted both the free amines
and significant amounts of the N-acetyl metabolites of benzidine
and its congeners (10).
Conclusions that could be drawn from these studies were that the azo
linkages of most dyes studied were reduced to release free benzidine or
the respective congeners, which were subsequently excreted in urine. Azo
reduction probably occurred in the intestine and, less probably, in liver.
Reduction of dyes to the parent amines by anaerobic intestinal bacteria
from humans, monkeys, and rats, was confirmed in a later report (11).
More important, based on the amount of benzidine or its congeners excreted
in urine, it appeared that most dyes were reduced quantitatively to release
the parent amines to result in exposure of the target tissue, the urinary
bladder.
As a result of their quantitative reduction to the parent amine, exposure
to benzidine-, or benzidine-congener-based dyes appears to result in exposure
to concentrations of benzidine (or congener) that are comparable to those
that occur following exposure to equivalent amounts of free benzidine (or
congener). Furthermore, the consistency of results obtained with benzidine
and related dyes implies that their reduction to release the respective
parent amine is probably a general phenomenon. That is, similar results
and risks associated with exposure to benzidine should be anticipated with
most dyes based on benzidine and its congeners (10). These results
also imply that benzidine detected in urine from workers exposed to benzidine-based
dyes was probably derived from the dyes, rather than from traces of benzidine
present in the dyes as a contaminant (12). The significance of this
finding is that the production and use of benzidine and related dyes carries
inherent risks which cannot be overcome by producing and using dyes which
contain less of the respective parent amine as a contaminant.
Benzidine Metabolism
Neither benzidine nor its congeners are thought to be carcinogenic prior
to metabolic activation. The presence of benzidine or its congeners in urine
was considered significant only because it demonstrated that the target
tissue in humans, urinary bladder, was exposed to a potential carcinogen
following exposure to the dyes. The speculated necessity for metabolic activation
was consistent with the report that benzidine is a bacterial mutagen only
when activated by a liver enzyme preparation (13). The first in
vitro study of benzidine metabolism demonstrated that benzidine is acetylated
to form N-acetylbenzidine, N,N´-diacetylbenzidine, and
N-hydroxy-N,N´-diacetylbenzidine (14). These
studies further implied that the latter metabolite was somehow involved
in the alkylation of DNA.
In an early study of comparative metabolism of benzidine in several species
Clayson et al. (15) demonstrated that the dog was the only species
studied that did not form N-acetylbenzidine when administered the
parent compound. In another early study, Sciarini and Meigs (16)
isolated the N- and O-glucuronides as well as the N-
and O-sulfate metabolites from dogs and other species administered
benzidine. A more thorough study of benzidine disposition and metabolism
in dogs and rats by Lynn et al. (17) demonstrated that benzidine
is readily absorbed from the gastrointestinal tract, metabolized extensively,
and rapidly excreted in urine and feces. Excretion in feces exceeded that
in urine by 2- to 3-fold, and less than 10% of the dose remained in the
tissues, primarily liver, after 3 days.
The use of radiolabeled benzidine and HPLC analysis permitted Lynn et
al. (17) to detect at least 17 distinct metabolite peaks in urine
following oral administration to rats. Benzidine accounted for less than
2% of the total radiolabel excreted in urine. The major metabolites excreted
in bile and urine were isolated and identified as glucuronide conjugates
of benzidine, N-acetylbenzidine, N,N´-diacetylbenzidine,
3-hydroxy-N,N´-diacetylbenzidine, and free N-acetyl-
and N,N´-diacetylbenzidine. 3-Hydroxy-N,N´-diacetylbenzidine
was the major metabolite excreted in urine and bile. A study of the mutagenicity
of these metabolites in the Salmonella/liver S9 system showed that both
the mono- and diacetylated metabolites were approximately 10 times as mutagenic
as benzidine. The most mutagenic metabolite isolated in this study, N-hydroxy-N,N´-diacetylbenzidine
glucuronide, was approximately 100 times more potent a mutagen than the
parent compound when incubated with ß-glucuronidase to release the
hydroxylated diacetylamine (18).
Lynn et al. (17) measured concentrations of benzidine-derived
radioactivity in all the major tissues at 0.5, 1, 2, 4, 8, 24, and 72 hr
after dosing. Radioactivity was concentrated only in liver, which contained
the highest concentrations at every time point. Extraction and analysis
of radiolabel from liver determined that very little of this radioactivity
was free benzidine, but approximately half was in the form of two metabolites,
N-acetyl- and N,N´-diacetylbenzidine. The remainder
was in the form of unidentified polar metabolites. N,N´-Diacetylbenzidine
accounted for approximately 80% of the benzidine-derived radioactivity in
blood at the early time points; however, concentrations at later time points
were too low to permit accurate analysis. Studies with isolated, perfused
rat livers demonstrated that all of the metabolites isolated and identified
from whole animals could be formed by the liver. Further, some of these
metabolites were themselves subject to additional metabolism, including
the rapid reduction of the most mutagenic metabolite, N-hydroxy-N,N´-diacetylbenzidine,
to the less mutagenic N,N´- diacetylbenzidine (18).
Metabolism and disposition of 3,3'-dimethoxybenzidine in intact rats
was similar to that reported for benzidine, except that the 3-methoxy groups
provided additional sites for metabolic attack and resulted in more rapid
and more extensive metabolism (19). The most mutagenic metabolite
isolated in this study was N-acetyldimethoxybenzidine.
Benzidine Interaction with DNA
As mentioned above, benzidine is believed to be carcinogenic only following
metabolic activation. Like other carcinogenic aromatic amines, such as 2-naphthylamine
and 2-acetylaminofluorene, benzidine and its congeners were presumed to
be metabolized to intermediates which reacted with DNA to result in mutations
and carcinogenicity. Evidence for the increased mutagenicity of benzidine
following metabolic activation was provided by the studies of Morton et
al. (14), who reported nucleic acid binding of benzidine; and Lynn
et al. (17), who reported that the glucuronide of N-hydroxy-N,N´-diacetylbenzidine
was as much as 100 times more mutagenic in the Salmonella/liver S9 assay
than benzidine. Martin et al. (20) studied the metabolism and covalent
binding of benzidine and a benzidine metabolite, N-acetylbenzidine,
in vitro and in vivo in the rat. They isolated and identified
a product of DNA alkylation, N-(deoxyguanosin-8-yl)-N´-acetylbenzidine,
which could have been formed from either N-acetylbenzidine or N,N´-diacetylbenzidine,
both of which are formed by rat liver (18) and excreted in urine
(17).
Following their isolation of a product of benzidine acetylation of DNA,
Martin et al. (21) demonstrated that this adduct is readily formed
in vivo by rats dosed with benzidine, N-acetylbenzidine or
a benzidine-based dye, Direct Blue 6. Only a trace of binding was detected
following administration of N,N´-diacetylbenzidine which implied
that this metabolite may not be the precursor to N-hydroxy-N,N´-diacetylbenzidine,
previously reported to be a potent bacterial mutagen (17). Subsequent
work using enzyme preparations from rat and mouse liver provided data to
support the hypothesis that N-hydroxy-N,N´-diacetylbenzidine
is the proximate carcinogen in these species, because of its capacity to
alkylate DNA. This work also demonstrated that this metabolite is formed
from N´-hydroxy-N-acetylbenzidine, rather than N,N´-diacetylbenzidine
(22).
The target organ for benzidine carcinogenesis in humans is the urinary
bladder, and the best experimental animal to model this lesion is thought
to be the dog. Unlike rats and mice, dogs do not acetylate benzidine or
other arylamines known to induce bladder cancer (23). Therefore,
the causative agent in benzidine carcinogenesis in the dog was not thought
to be the hydroxyacetylamine metabolite speculated to account for carcinogenesis
in rodent studies.
Beland et al. (24) studied the formation of DNA adducts in urinary
bladder of dogs following oral administration of a number of known carcinogenic
aromatic amines, including benzidine and N-acetylbenzidine. Their
studies demonstrated a positive correlation of the degree of binding of
carcinogenic aromatic amines to bladder DNA with the potency of the respective
compounds. Further, of the known carcinogens studied, binding to DNA in
the dog bladder following administration of benzidine and N-acetylbenzidine
was equivalent to that observed following administration of two other potent
bladder carcinogens, 2-naphthylamine and 4-nitrobiphenyl. These investigators
proposed that N-hydroxylamines account for the carcinogenicity of
each of these compounds in the urinary bladder of dogs, and speculated that
these reactive intermediates are formed in the liver and transported to
the bladder as glucuronide conjugates which hydrolyze in the weakly acidic
urine to release the reactive intermediate (24).
A more recent publication has demonstrated that, in the dog, benzidine
may also be transported to the bladder as a glucuronide conjugate which
could be hydrolyzed to release free benzidine (25). Later work by
members of this same group, however, demonstrated that benzidine is also
metabolized to a reactive intermediate by arachidonic acid-dependent prostaglandin
H synthase and that prostaglandin H synthase is present in high concentrations
in dog bladder (26,27). The reactive intermediate formed by prostaglandin
H synthase, benzidine diimine, was proposed to react directly with DNA to
form an adduct, N-(deoxyguanosin-8-yl)benzidine, which was later
isolated and identified (28). In studies of a series of human tissues
the urinary bladder was shown to be rich in prostaglandin H synthase, and
among the aromatic amines studied, benzidine was shown to be the optimum
substrate for this enzyme (29). Thus, the presence of prostaglandin
H synthase in dog and human urinary bladder probably accounts for the activation
and carcinogenicity of benzidine in this tissue.
Summary
Studies of the metabolism and disposition of dyes derived from benzidine
and benzidine congeners established that, following ingestion, the azo linkages
of these compounds are reduced, probably by bacteria in the intestines,
to release the parent amines. Reduction of the dyes to the respective free
amines appears to be nearly complete, therefore, ingestion of most benzidine
and related dyes is thought to be comparable to ingestion of an equivalent
amount of the respective free amine. The free amines (benzidine and its
congeners) are absorbed readily from the gastrointestinal tract, rapidly
and extensively metabolized and excreted in both urine and feces. The degree
of benzidine excretion in urine by experimental animals exposed to dyes,
and the actual detection of benzidine in urine of workers in the dye industry,
strongly support the potential for exposure of human urinary bladder to
this known bladder carcinogen following exposure to the respective dyes.
The probable reactive intermediate which accounts for the carcinogenicity
of benzidine in rodents is N-hydroxy-N,N´-diacetylbenzidine.
This reactive intermediate is formed in liver and is probably transported
to target tissues in blood or urine. Dogs do not acetylate benzidine and
humans acetylate benzidine less readily than rodents; therefore, the reactive
intermediates of benzidine vary with species. Metabolism to a reactive intermediate
by dogs and humans may also occur in liver, but urinary bladder carcinogenicity
is more probably mediated by the action of prostaglandin H synthase in the
target tissue. Prostaglandin H synthase forms a diimine metabolite which
reacts directly with DNA in the bladder.
Mutagenicity
Salmonella mutagenicity studies were prompted by the fact that benzidine
congener dyes were not uniformly and reliably positive in oxidative in
vitro mutagenicity assays. It was believed that the dyes required reductive
cleavage to yield the parent amine in order to be mutagenic. The objectives
of the mutagenicity portions of the initiative were to establish an optimal
protocol for the reductive cleavage of the dyes, and evaluate the mutagenic
effects of benzidine-congener-derived dyes. A part of this latter activity
was to determine if the mutagenicity of the dyes was attributable to the
parent amine metabolites, or if activity was either quantitatively or qualitatively
influenced by the chromophore portions of the molecules. In addition, the
mutagenicity of the benzidine and benzidine-congener metabolites found in
the urine of dosed rats was investigated. It was reasoned that the demonstration
of a genotoxic potential for these urinary metabolites would increase the
confidence in predictions of carcinogenicity after short-term oral administration
of these dyes in laboratory animals. These studies would also provide additional
information regarding the metabolic activation of the dyes.
Three Salmonella mutagenesis testing protocols were used. All dyes were
first tested (under code) using a standard, aerobic preincubation procedure
in Salmonella strains TA98, TA100, TA1535, and TA1537, with and without
metabolic activation. S9 fractions derived from the livers of Aroclor 1254-treated
male Sprague-Dawley rats and Syrian hamsters were used. Some dyes were also
tested using strain TA97. The detailed test procedure and criteria for a
positive response are in Mortelmans et al. (30) and Zeiger et al.
(31,32).
The mutagenic and nonmutagenic dyes were also tested following treatment
under reducing conditions to liberate the parent amine. Equimolar concentrations
of the parent benzidine congeners were tested in parallel with the dyes.
The FMN reduction protocol of Prival and Mitchell (33) and the cecal
flora reduction procedure of Reid et al. (34,35) were used. In the
Prival procedure, the dyes were preincubated using the standard aerobic
procedure except that the liver S9 and cofactors were supplemented with
NADH and FMN to provide reducing conditions (33,36). The cecal flora
reduction procedure employed a washed suspension of rat cecal flora. The
dye was added to this culture and incubated overnight under anoxic conditions.
The incubation mixture was extracted with ethyl acetate, dissolved in DMSO,
and tested for mutagenicity using the standard aerobic preincubation procedure
(34). All reduction experiments were performed using strains TA98
and TA1538, because these strains are more responsive to the mutagenic effects
of benzidine, 3,3'-dimethoxybenzidine, and 3,3'-dimethylbenzidine than the
other tester strains.
Mutagenicity of the Dyes
With the exception of Direct Blue 218 (30,34) and Pigment Yellow
12 (31,34), all dyes tested were mutagenic in Salmonella,
and produced frameshift mutations in strains TA98 or TA1538 when tested
under conditions that fostered reduction of the azo bonds (Table 2) (30-34).
Only Direct Black 38 (30,34) and Direct Blue 2 (30) were mutagenic
without activation. The dyes that were mutagenic with the standard aerobic
S9 preparation generally gave weaker responses than they did following azoreduction.
The positive responses with the standard S9 mix alone showed that this metabolic
activation preparation was able to induce low levels of azoreduction or
that the dye sample contained impurities that could be activated to mutagens
without azoreduction.
Many of the dyes tested had high levels of impurities, although the specific
impurities were not identified. It was not determined to what extent any
mutagenicity seen under aerobic conditions was due to bacterial reduction
of the dye, and what extent was due to mutagenic impurities.
Mutagenic Potency of Reduced Dyes and Congeners
In the azoreduction protocols, the various dyes were tested in parallel
with equimolar concentrations of the parent benzidine congeners (34).
This was done to compare the level of mutagenicity induced by the parent
amine with the mutagenicity induced by an equimolar concentration of amine
released from the dye. Benzidine was more mutagenic with hamster than rat
S9; 3,3'-dimethoxybenzidine was more mutagenic when tested with rat than
hamster S9; and 3,3'-dimethylbenzidine gave equivalent results with both
S9s. 3,3'-Dihydroxybenzidine, a putative metabolite of Direct Blue 218,
was more mutagenic with hamster than with rat liver S9 in Salmonella
strain TA98 (E Zeiger et al. unpublished data). 3,3'-Dichlorobenzidine was
the most potent mutagen, and the only congener that was mutagenic without
S9 activation. There did not appear to be a correlation between the mutagenic
responses induced by the parent amines and those induced by the reduced
dyes. For some dyes, the reduction products were more mutagenic than an
equimolar quantity of the amine; for other dyes the opposite was seen. These
results may be related to the possible mutagenic or inhibitory effects of
the other moieties released from the dye during azoreduction, to mutagenic
impurities in the dye preparation, or to incomplete reduction of the dye
(34).
Mutagenicity of Metabolites
The N-acetylated and the N,N-diacetylated urinary metabolites
of benzidine, 3,3'-dimethylbenzidine and 3,3'-dimethoxybenzidine were mutagenic
in Salmonella strains TA98 and TA1538; the N-acetylated metabolites
were also mutagenic in TA100 (35). These results show that, with
the exception of Direct Blue 218 and Pigment Yellow 12, all dyes tested
that can be reduced to their benzidine congeners, are mutagenic in Salmonella.
The mutagenic responses obtained with the rat cecal preparations show that
the dyes can be reduced to their constituent benzidine congeners by the
gut flora as effectively as by liver preparations under reduced conditions.
In addition, the results show that these cecal preparations do not further
metabolize the resulting aromatic amines to products that will not be mutagenic
upon subsequent aerobic metabolism by liver enzymes.
The azo linkages in Direct Blue 218 also appeared to be reduced by the
NADP/FMN-fortified rodent liver preparations and by the cecal extract, as
judged by the change in visible color intensity, but the resulting product
was not mutagenic. This may indicate that the reduction of this dye does
not release the free amine, but a copper complex of this amine with the
chromophore or to 3,3'-dihydroxybenzidine. In the Reid et al. study (34),
approximately 50% of the Direct Blue 218 was reduced by rat cecal bacteria
as determined by spectrophotometric analysis. However, comparison of the
mutagenic responses of this congener to the test responses of Direct Blue
218 showed that the highest dose tested of Direct Blue 218 was 0.5 µmole/plate,
whereas the mutagenic response of 3,3'-dihydroxybenzidine were seen only
at doses above 1.5 µmole/plate. Pigment Yellow 12 did not appear to
be reduced, probably as a consequence of its insolubility, and was not mutagenic.
The question of the mutagenicity of the chromophore moieties of the dyes
was not addressed. In a number of instances, however, the mutagenic responses
following reduction of the dye and subsequent oxidative metabolism of the
reduced products was greater than that obtained following similar treatment
of the parent congener amine (33,36). This suggests that the chromophore
was also mutagenic.
Summary
Mutagenicity studies provided evidence that the benzidine- and benzidine
congener-derived dyes require reductive metabolism before they are mutagenic.
All soluble dyes tested, with the exception of Direct Blue 218 (copper chelated),
produced frameshift mutations in Salmonella when tested under conditions
that fostered reduction of the azo bonds. Only Direct Black 38 and Direct
Blue 2 were mutagenic without activation, possibly due to the presence of
mutagenic impurities in these dye preparations. This information supports
the results of metabolism studies relative to the mechanisms of genotoxic
action, and increases confidence in predicting the carcinogenicity of these
chemicals.
Carcinogenesis
Chronic Carcinogenicity Testing
The objectives of the in vivo carcinogenicity studies were to:
conduct definitive studies of the carcinogenicity of orally administered
3,3'-dimethylbenzidine and 3,3'-dimethoxybenzidine in rats; assess the carcinogenicity
of orally administered prototypical benzidine congener derived dyes; develop
dose-response relationships for the carcinogenicity of the dyes and the
parent amines; determine similarities in the qualitative nature of the responses
to the parent amines and the derived dyes; and assess the carcinogenicity
of a metallized benzidine congener dye (Direct Blue 218).
Five chemicals were evaluated in the 2-year carcinogenicity studies:
3,3'-dimethoxybenzidine dihydrochloride, and 3,3'-dimethylbenzidine dihydrochloride
(benzidine congeners), Direct Blue 15 (a representative 3,3'-dimethoxybenzidine-based
dye), Acid Red 114 (a representative 3,3'-dimethylbenzidine-based dye) and
Direct Blue 218 a metallized 3,3'-dimethoxybenzidine-based dye). The oral
route of administration was selected to maximize the chances of detecting
systemic effects associated with chemical administration. 3,3'-Dimethylbenzidine,
Acid Red 114, 3,3'-dimethoxybenzidine, and Direct Blue 15 were all studied
using the same experimental design. Because of the instability of 3,3'-dimethylbenzidine
and 3,3'-dimethoxybenzidine in feed, all chemicals, except Direct Blue 218,
were administered in drinking water. Direct Blue 218 was not soluble in
drinking water, so it was administered in feed. Because long-term studies
of 3,3'-dimethylbenzidine and 3,3'-dimethoxybenzidine in mice were performed
at the National Center for Toxicology Research (37,38), only male
and female rats were used in the studies described here. Rats and mice of
both sexes were used in the Direct Blue 218 study.
All studies utilized untreated controls and three dosed groups of each
sex. These experimental groups consisted of "core" animals intended
for 24 months of treatment plus additional animals designated for interim
evaluations at 9 and 15 months. For all, except the Direct Blue 218 study,
the allocation of animals in each group followed a procedure recommended
by Portier and Hoel (39). The 9 month interim evaluations were performed
in each study; however, because of excessive early cancer-related mortality
in the 3,3'-dimethoxybenzidine and 3,3'-dimethylbenzidine studies, the 15
month evaluations were not performed and the animals originally designated
for interim evaluation were incorporated into the core group. The Direct
Blue 218 studies in both rats and mice followed a standard design with 50
core animals per group and 10 additional animals per group designated for
interim evaluation at 15 months; there was no 9 month interim evaluation.
Dose selections for the carcinogenicity studies were based on the results
of experiments in which groups of 10 male and 10 female rats received test
compound in their drinking water or food for from 2 to 13 weeks. Evaluative
criteria used to assess the toxicologic effects of the chemicals during
the prechronic studies included body weight gain, food and water intake,
grossly observable signs, mortality, and microscopic evidence of organ toxicity.
Experimental animals had free access to food and water throughout the
duration of the experiment. In experiments in which the drinking water was
used as the vehicle for the test material, water consumption was measured
weekly; weekly food consumption was measured when food was the dosing vehicle.
Animals were observed twice daily for morbidity or mortality. Clinical signs
and body weights were recorded at regular intervals. All animals were subjected
to a complete necropsy. A complete histologic evaluation, consisting of
microscopic examination of approximately 40 tissues and all gross lesions,
was performed on all animals except the low- and mid-dose animals from the
15 month interim evaluations of Acid Red 114, Direct Blue 15, and Direct
Blue 218. Only gross lesions and selected tissues were examined in these
groups.
Incidences of neoplasms in dosed and control groups were compared statistically
using survival adjusted analyses. The life table test, a procedure appropriate
for rapidly lethal tumors, was used for mononuclear cell leukemia and Zymbal's
gland tumors, both neoplasms that are rapidly fatal. Incidences of other
neoplasms in all but the 3,3'-dimethylbenzidine and 3,3'-dimethoxybenzidine
studies were analyzed by using the logistic regression test. Significantly
reduced survival in the 3,3'-dimethylbenzidine and 3,3'-dimethoxybenzidine
studies compromised the power of logistic regression so the Fisher exact
test utilizing the effective number of animals was employed for these two
studies. The effective number of animals is the number of animals that survived
until the appearance of the initial tumor.
Two- and Thirteen-Week Studies
In general, pronounced effects were not seen in the 2-week or 13-week
studies. Dose-related reductions in water consumption were observed in the
dosed water studies and reduced feed consumption was seen in the dosed feed
study. Both effects presumably were due to poor palatability caused by the
presence of the test compound. Reduced body weight gains were observed at
the higher dose levels in all studies. This effect was presumed to be secondary
to reduced water or food consumption. A few chemical related deaths occurred
in rats in the highest dose groups during the 2-week studies of 3,3'-dimethylbenzidine
and during the 13-week studies of 3,3'-dimethylbenzidine and Direct Blue
15.
The most common chemical-related lesions observed in rats treated with
each of the compounds for 13 weeks were a mild degree of hepatocyte degeneration
and necrosis, and degeneration of renal tubules. Hepatocyte hypertrophy
(enlargement) and necrosis were seen in the livers of mice from the 13-week
Direct Blue 218 study. The results of these studies have been reported in
detail elsewhere (40-46).
Two-Year Studies
Concentrations of 3,3'-dimethylbenzidine, 3,3'-dimethoxybenzidine, Acid
Red 114, and Direct Blue 15 added to the drinking water during the chronic
study are shown in Table 3. The estimated amounts of chemicals consumed,
based on measured water consumption, are also shown in Table 3. In general,
doses were highest for Direct Blue 15 followed by 3,3'-dimethoxybenzidine,
and with lower consumptions of Acid Red 114 and 3,3'-dimethylbenzidine.
Females typically consumed somewhat higher doses of chemicals than did males.
The survivals of male and female rats dosed with either 3,3'-dimethylbenzidine,
3,3'-dimethoxybenzidine, Acid Red 114, Direct Blue 15, or Direct Blue 218
are summarized in Tables 4 and 5. The administration of each of these compounds
significantly reduced the survival of both sexes of rats. These significant
increases in mortality were considered to be due to increased incidences
of chemical-induced, lethal neoplasms. Because of the high incidence of
tumors and decreased survivals, the 3,3'-dimethylbenzidine and 3,3'-dimethoxybenzidine
studies were terminated at 15 and 21 months, respectively, and the Direct
Blue 15 study was terminated at 22 months. Despite significantly reduced
survivals in both sexes, the Acid Red 114 study was continued until the
scheduled termination (24 months).
Nonneoplastic Lesions
Chemical-related nonneoplastic lesions were observed in the livers and
kidneys in some studies. The livers of male and female rats treated with
all the benzidine compounds except Direct Blue 218 had increased incidences
of foci of cellular alteration, cystic degeneration, hepatocyte degeneration,
necrosis, and regeneration. Increased incidences of foci of cellular alteration
were observed in mice treated with Direct Blue 218. The severity of nephropathy,
a common degenerative change of the kidney in aging F344 rats, was increased
in treated males and females from the 3,3'-dimethylbenzidine study, and
in treated females from the Acid Red 114 study. In addition, foci of hyperplasia,
presumably preneoplastic changes, were seen in the preputial, clitoral,
and Zymbal's glands in the 3,3'-dimethylbenzidine, 3,3'-dimethoxybenzidine,
Acid Red 114, and Direct Blue 15 studies, and in the lung in the 3,3'-dimethylbenzidine
and Acid Red 114 studies.
Neoplastic Lesions
3,3'-Dimethylbenzidine, 3,3'-dimethoxybenzidine, Acid Red 114, and Direct
Blue 15 all caused a similar spectrum of neoplastic lesions (Tables 6 to
8). The primary chemical related neoplasms were tumors of the skin, Zymbal's
gland, oral cavity epithelium, liver, preputial/clitoral glands, and intestines
of both males and females. A few of these neoplasms were seen as early as
the 9-month interim sacrifices, and the numbers of tumors at the various
sites increased as the studies progressed. The microscopic appearance of
the chemical-related neoplasms was similar in all studies.
While we found a wide variety of tumors in our studies, Robens et al.
(2) reported only liver involvement after administration of the benzidine-derived
dyes. It is unlikely that the 13-week studies reported by Robens et al.
were long enough for a full expression of toxicity, and therefore a comparison
of the studies is difficult to make. By way of contrast, however, we found
no evidence of hepatocarcinogenicity during the 13-week studies on the congeners
or dyes.
All four chemicals significantly increased the incidence of a variety
of epithelial neoplasms of the skin in both sexes of rats. These skin tumors
included adenomas and carcinomas of the basal cells and sebaceous glands,
squamous cell papillomas and carcinomas, and keratoacanthomas. Neoplasms
of the Zymbal's glands included adenomas and carcinomas, with carcinomas
being by far the more common of the two. Carcinomas were highly invasive
and occasionally metastasized. Chemical related neoplasms of the oral cavity
consisted of squamous cell papillomas and carcinomas originating from the
stratified squamous epithelium. Microscopically, these neoplasms resembled
the squamous cell neoplasms of the skin. Neoplastic nodules and hepatocellular
carcinomas occurred as a chemical related effect in the livers of treated
male and female rats ("neoplastic nodule" was the term used previously
for neoplasms now classified as hepatocellular adenomas). Adenomatous polyps
and adenocarcinomas of the small and large intestine, neoplasms rarely seen
in untreated F344 rats, occurred in treated male and female rats.
Adenomas and carcinomas of the preputial glands and clitoral glands (female
homologue of the preputial glands) occurred frequently in treated rats.
Because preputial gland neoplasms are usually not overtly aggressive or
invasive and rarely metastasize (47,48), classification of these
neoplasms as benign or malignant is difficult (49). The transplantability
of preputial gland neoplasms induced by 3,3'-dimethylbenzidine, 3,3'-dimethoxybenzidine
and Acid Red 114 was investigated to provide information on the biologic
behavior of these neoplasms (49,50). All neoplasms selected for transplantation
were retrospectively diagnosed as carcinomas and therefore, comparable information
was not obtained for preputial gland adenomas. The transplanted neoplasms
did not become anaplastic or less differentiated over four serial passages;
however, the transplants behaved biologically as malignant neoplasms in
spite of their well differentiated morphology. The latency period was short
and transplants grew rapidly. The results of these studies confirmed the
malignant nature of these preputial gland neoplasms from rats exposed to
the benzidine congeners and dyes.
In rats treated with 3,3'-dimethylbenzidine 3,3'-dimethoxybenzidine,
Direct Blue 15, and Acid Red 114, the total numbers of neoplasms tended
to be greater in males than in females. In addition, tumors of the skin,
liver, and oral cavity epithelium were more common in males than females.
Some, but not all, of the four chemicals tested caused marginal increases
in mononuclear cell leukemia, mesotheliomas, and tumors of the brain, mammary
gland, lung, or adrenal gland, which may been related to treatment.
The numbers of animals with primary malignant neoplasms in the 3,3'-dimethylbenzidine,
3,3'-dimethoxybenzidine, Acid Red 114, and Direct Blue 15 studies are shown
in Table 9. Malignant tumors were observed in 83 to 98% of all high dose
animals; 64 to 91% of mid-dose animals, and 13 to 80% of all low-dose animals.
Tumor multiplicity was calculated as the number of primary malignant tumors
per animal. Tumor multiplicity was greater than one for all dosed animals
except those treated with low and mid doses of 3,3'-dimethylbenzidine, and
females in the low-dose group of Direct Blue 15. In male rats, malignant
neoplasms were observed earliest in 3,3'-dimethylbenzidine-dosed rats, followed
by rats dosed with Direct Blue 15, 3,3'-dimethoxybenzidine, and Acid Red
114. In females the order in which tumors were first detected was: 3,3'-dimethylbenzidine,
3,3'-dimethoxybenzidine, Acid Red 114, and Direct Blue 15. The results of
the Direct Blue 218 study showed a spectrum of neoplastic lesions that was
different from that observed for the other four benzidine compounds. Incidences
of chemical-related neoplasms were not as dramatic, and were observed primarily
in the oral cavity epithelium of rats, and in the livers of mice (Table
10).
Dose-response relationships could not be established for the carcinogenicity
of the benzidine congener dyes and the parent amines because of the high
incidences of tumors at all doses. These high incidences of tumors after
chronic exposure were unexpected because higher doses of the benzidine congeners
and dyes produced little or no toxicity in prechronic studies. In early
studies of 3,3'-dimethoxybenzidine and 3,3'-dimethylbenzidine, only relatively
low incidences of tumors were observed in dosed animals (51-54).
However, in those studies, the administration of large, toxic doses of chemical
decreased survival and may have resulted in a severe underestimation of
the carcinogenic potency of these chemicals.
Quantitative comparisons of the carcinogenic potencies of the four chemicals
is difficult because of the lack of dose- response relationships, as well
as differences in doses and durations of treatments. Slight differences
in the carcinogenic potencies of the two benzidine congener dyes are in
part a result of the rate at which they are metabolized and in part by the
carcinogenicity of the benzidine congener metabolite. Quantitative differences
were observed in the liver and skin of males and females; males in all four
studies had a higher tumor incidence at these sites than did females. However,
chemical-induced clitoral gland tumors of females occurred more commonly
than preputial gland tumors in males.
3,3'-Dimethylbenzidine caused a neoplastic response at an earlier time
than did 3,3'-dimethoxybenzidine, suggesting that 3,3'-dimethylbenzidine
may be a more potent carcinogen, possibly because it is absorbed to a greater
extent, or metabolized to the ultimate carcinogen more efficiently than
3,3'-dimethoxybenzidine. In addition, Acid Red 114, the 3,3'-dimethylbenzidine-derived
dye, caused tumors at lower doses than Direct Blue 15, the 3,3'-dimethoxybenzidine
derived dye, also suggesting that 3,3'-dimethylbenzidine may be a more potent
carcinogen.
Qualitatively, 3,3'-dimethoxybenzidine, 3,3'-dimethylbenzidine, Direct
Blue 15, and Acid Red 114 caused a similar spectrum of neoplasms in the
Zymbal's gland, skin, liver, oral cavity, clitoral and preputial gland,
and to a lesser extent in the intestine of the Fischer F344/N rat. Tumors
occur infrequently at these sites, and the high incidences of tumors at
these sites in all four studies strongly indicates a common mechanism.
Two uncommon neoplasms that appeared at relatively high incidences in
treated rats were Zymbal's gland tumors (males and females) and skin basal
cell tumors (males). Most of the chemicals tested by the NTP that induced
either Zymbal's gland or skin tumors in rats also caused tumors in other
sites. These tumor inducing chemicals (e.g., 4-aminobiphenyl, 4,4´-thiodianiline)
all have in common an aromatic amine functional group that is considered
to be a "structural alert" for genotoxic activity, and all are
mutagenic in Salmonella. A number of aromatic amines, or chemicals that
are metabolized to aromatic amines, cause neoplasms in the Zymbal's gland
of rats (40). Benzidine, the parent compound of this series of chemicals,
also causes Zymbal's gland tumors in rats (55), implicating the free
amine group of benzidine as a common site for metabolic activation for this
related group of chemicals. Although, for the most part, the target sites
were similar for the four chemicals, some differences were observed. Acid
Red 114 and its parent congener, 3,3'-dimethylbenzidine, caused a higher
incidence of liver tumors than did Direct Blue 15 and its parent congener,
3,3'-dimethoxybenzidine. In addition, significantly increased incidences
of preputial gland tumors were seen in male rats treated with 3,3'-dimethoxybenzidine,
3,3'-dimethylbenzidine, and Direct Blue 15, but not with Acid Red 114. Some,
but not all, of the four chemicals tested caused variable increases in mononuclear
cell leukemia, mesotheliomas, and tumors of the brain, mammary gland, lung,
and adrenal medulla, which may have been related to chemical treatment.
These slight differences in sites of tumor origin among studies are likely
a result of differences in dose, the extent of absorption and metabolism
of the different chemicals, and possibly the effects of impurities in the
dyes. Direct Blue 218, a copper-chelated 3,3'-dimethoxybenzidine-derived
dye, was considerably less carcinogenic than the benzidine congeners and
nonchelated dyes; it was also nonmutagenic.
Chelation with copper of the hydroxy groups of the benzidine congener
is believed to prevent metabolism of the dye to the ultimate carcinogen.
The relatively low carcinogenic response to Direct Blue 218 supports the
concept that copper chelation inhibited metabolic activation of the dye
to a large extent; however, the presence of oral cavity tumors in rats and
liver neoplasms in mice suggests that copper chelation does not completely
eliminate the carcinogenic effects of the dye. These neoplasms may be a
result of incomplete inhibition of metabolism of the chelated dye, and possibly
a result of carcinogenic impurities in the dye. The Direct Blue 218 used
in these studies was only 60% pure. In addition to organic impurities, the
dye also contained free copper which may also be toxic. It is also possible
that the oral cavity tumors were a direct effect of the chemical in dosed
feed.
The different target sites of Direct Blue 218 in rats and mice suggests
potential species differences in metabolism of this dye. Further support
for species differences in metabolism of benzidine compounds is provided
by studies of 3,3'-dimethoxybenzidine and 3,3'- dimethylbenzidine administered
in drinking water to BALB/c mice. Although these compounds are potent, multisite
carcinogens in rats, 3,3'-dimethoxybenzidine had no apparent carcinogenic
effect (37),and 3,3'-dimethylbenzidine caused only a low incidence
of lung tumors in treated mice (38). Metabolic differences may also
exist between mouse strains, since liver neoplasms were observed in B6C3F1
mice and lung tumors were observed in BALB/c mice.
Oncogene Activation
DNA from neoplasms of skin, preputial and clitoral gland, mammary gland,
and intestines of rats exposed to 3,3'-dimethoxybenzidine, 3,3'-dimethylbenzidine,
Direct Blue 15, or Acid Red 114, was analyzed for dominant transforming
genes using the NIH 3T3 plate transfection assay (56). Oncogenes
detectable by DNA transfection analysis were present in 35 of 59 skin, clitoral
gland or preputial gland neoplasms that had been induced in F344 rats by
the benzidine congeners or derived dyes. DNA from both benign and malignant
neoplasms was capable of inducing morphologically transformed foci in NIH
3T3 fibroblasts.
A high percentage of the induced rat neoplasms contained activated alleles
of either H- or N-ras. Those neoplasms with activated H-ras
contained point mutations in codon 12, 13, or 61. Point mutations at codon
61 accounted for approximately 50% of the H-ras gene activations
in both 3,3'-dimethylbenzidine and 3,3'-dimethoxybenzidine induced tumors,
with C
A transversions (or GT transversions
in the antisense strand) being the predominant mutation at codon 61 in both
groups. The mutations detected at codon 12 were exclusively GA transitions
whereas the mutations at codon 13 were predominantly GC transversions.
The detection of a high percentage of activated oncogenes in these chemical-induced
neoplasms is in sharp contrast to the relatively low frequency of activated
oncogenes found in spontaneously occurring neoplasms of F344 rats (57).
These observations suggest that activation of cellular ras genes by point
mutation may be an important step in induction of tumors in rats treated
with the benzidine congeners and derived dyes.
A number of epidemiologic studies support the causal relationship between
exposure to benzidine and the occurrence of bladder cancer in humans (1).
The finding that H-ras gene activation occurs in a portion of human
bladder tumors (58,59) and the above results suggesting that ras
gene activation is an important step in the induction of tumors in rats
treated with the benzidine congeners and derived dyes, suggests that ras
activation can be involved in one pathway of human urothelial cell transformation.
Summary
Consumption of drinking water containing the benzidine congeners 3,3'-dimethylbenzidine
and 3,3'-dimethoxybenzidine, and the prototypical benzidine congener dyes,
Acid Red 114 and Direct Blue 15 led to a similar spectrum of neoplasms.
The high incidences of tumors at the same sites in all four studies strongly
indicates a common mechanism. The detection of a high percentage of activated
oncogenes in these chemical-induced neoplasms suggests that activation of
cellular ras genes by point mutation may be an important step in induction
of tumors in rats treated with benzidine congeners and derived dyes.
Direct Blue 218, a metallized 3,3'-dimethoxybenzidine-derived dye, was
considerably less carcinogenic than the benzidine congeners and nonchelated
dyes and was also nonmutagenic. However, the presence of oral cavity tumors
in rats and liver neoplasms in mice suggests that copper chelation does
not completely eliminate the carcinogenic effects of metallized dyes.
Conclusions
The broad objective of the benzidine initiative was to generate a body
of scientific information that would facilitate regulatory decision making
regarding the toxicologic and carcinogenic risk associated with exposure
to benzidine and benzidine congener derived dye materials. The carcinogenicity
studies provided clear evidence that the benzidine congeners and prototypical
dyes derived from these parent amines are potent carcinogens in animals.
Based upon the results of metabolism and mutagenicity studies of other benzidine-
and benzidine- congener-derived dyes, one would predict that most, if not
all, dyes based on these chemicals are carcinogens.
The metabolism, disposition, mutagenicity, and carcinogenicity data from
these studies provide sufficient evidence that the benzidine and benzidine-congener-derived
dyes should be regulated as probable human carcinogens.
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