United States
Environmental Protection
Agency
Health Effects
Research Laboratory
Research Triangle Park NC 27711
Research and Development
EPA/600/S1-87/010 Jan. 1988
oEPA Project Summary
Effects of 4,4'-Methylene-bis-
(2-Chloroaniline) and
Benzotrichloride in
Human and/or Animal Tissues
Gary D. Stoner, Herman A. J. Schut, Narayan Shivapurkar, and
In-Chang Hsu
'/
*f-
4.4'-Methylene-bis-(2-chloroaniline)
(MOCA) is an aromatic amine with
structural similarity to known human
bladder carcinogens, and induces
urothelial tumors in dogs. In this study
the binding to DNA and DNA-adduct
formation of MOCA in explant cultures
of human and dog bladder was
compared. The ability of MOCA to bind
to DNA in both human and dog bladder
explants was related to the dose of
MOCA. In both species, there appeared
to be a population with high DNA
binding activity and another with low
DNA binding activity. Preliminary
studies using the 32P-postlabeling
technique showed that several MOCA-
DNA adducts were formed in both
human and dog bladder with at least
three adducts in common between the
two species. These results indicate the
potential of MOCA to induce genetic
damage in human bladder and suggest
caution in the occupational exposure of
humans to this chemical.
Benzotrichloride (BTC) is used in the
synthesis of benzoyl chloride and
benzoyl peroxide, and epidemioJogical
data suggest that BTC is a human lung
carcinogen. In this project, BTC was
evaluated for its ability to induce lung
adenomas in strain A/J mice. Four
groups of male and female A/J mice
were injected i.p. with either tricaprylin
or BTC in tricaprylin three times a week
for 8 weeks. BTC groups received
doses totaling 1440 mg/kg, 719
mg/kg, or 287 mg/kg. The mean
number of lung tumors per mouse was
127.87 ± 5.81, 43 ± 2.44, and 17.73
± 1.09 in the groups treated with either
1440 mg/kg, 719 mg/kg, or 287 mg/
kg, respectively. Tricaprylin-vehicle
controls had a mean number of 0.46
± 0.15 lung tumors per mouse. There-
fore, BTC produced a significant
(p<0.001) and dose-related increase in
the lung tumor response when com-
pared to tricaprylin controls, and is a
potent carcinogen in the strain A mouse
lung tumor bioassay. These results
suggest caution in the exposure of
humans to BTC.
This Project Summary was devel-
oped by EPA's Health Effects Research
Laboratory, Research Triangle Park.
NC, to announce key findings of the
research project that is fully
documented in a separate report of the
same title (see Project Report ordering
information at back).
Introduction
4,4 '-Methylene-bis-(2-
Chloroaniline) (MOCA)
MOCA, a commerically important
curing agent in the manufacture of
polyurethane, is an aromatic amine with
structural similarity to known human
bladder carcinogens. It induces lung and
liver tumors in mice and rats and bladder
tumors in dogs. In addition, MOCA is
mutagenic in the Salmonella typhimu-
rium mutagenesis (Ames) assay, geno-
toxic in mouse and reta hepatocytes, and
positive in mammalian cell transforma-
tion assays. Human exposure to MOCA
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occurs during its manufacture and use.
Also, families of workers involved in the
manufacture of MOCA and residents in
the area of manufacturing plants are
exposed to MOCA as indicated by the
presence of MOCA in their urine. Evi-
dence of its mutagenic and carcinogenic
activity in experimental systems and of
human exposure to MOCA suggests the
importance of determining its genotoxic
potential in humans and especially in the
bladder.
Tissues from several human and
animal organs, including the bladder, can
be maintained as explants in serum-free
medium. Explant cultures of human and
animal tissues have been used exten-
sively for comparisons of the metabolism,
DMA-binding and DNA-adduct formation
of a wide variety of chemical carcino-
gens, including certain aromatic amines.
In general, these studies have demon-
strated that the metabolism of carcino-
gens and the profile of carcinogen-DNA
adducts in human tissues is similar to
that in animal model tissues in which
the carcinogen has been shown to induce
cancer. Most differences in metabolism
between humans and animals have been
quantitative rather than qualitative.
In this study, explant cultures were
used to compare the ability of MOCA to
induce genetic damage in cultured
explants of human and dog bladder. The
results indicate that MOCA induces
levels of genetic damage in Jiuman
bladder that are similar to those in the
dog, a species in which MOCA has been
shown to induce urothelial cancer.
Therefore, MOCA could represent a
significant risk to exposed individuals.
Benzotrichloride (BTC)
BTC is used exclusively as a chemical
intermediate. The most important deriv-
ative of BTC is benzoyl chloride, an
intermediate in the manufacture of
several dyes, pigments, herbicides,
Pharmaceuticals, and antimicrobial
agents. BTC does not occur naturally in
the environment; however, it has been
found in surface waters. BTC is unstable
in the environment and hydrolyzes to
benzoic acid and hydrochloric acid in the
presence of moisture.
Epidemiological studies indicate an
increase in the incidence of lung cancer
among workers in Japanese chemical
plants using BTC and other chemicals in
the manufacture of benzoyl chloride and
benzoyl peroxide.
Toxicity studies indicate that BTC is a
skin, eye and respiratory irritant. In
addition, BTC is mutagenic in \beBacillus
subtilus-rec assay, and in both the
Escherichia coli and Salmonella typhi-
murium (Ames test) reversion assays
Benzotrichloride has induced tumors
in experimental animals. Intragastric
doses of BTC produced forestomach
cancers (squamous cell carcinomas) and
lung cancers (adenomas and carcino-
mas) in female ICR mice. In a skin
painting study in female ICR mice, BTC
induced skin tumors, lymphomas, lung
tumors, and tumors of the lips, tongue
and forestomach.
The strain A mouse lung tumor bioas-
say is a short-term quantitative bioassay
for carcinogens The bioassay has been
utilized for the testing of more than 300
environmental chemicals for carcino-
genic activity. In this study, BTC was
tested for its ability to induce lung
adenomas in strain A mice and its
potency was compared to that of other
carcinogens. The data indicate that BTC
is a strong carcinogen in the strain A
mouse lung tumor bioassay, and could
represent a significant risk to exposed
individuals.
Discussion
MOCA Studies
The aromatic amines, lilke many
organic carcinogens, are not direct-
acting genotoxins. It is thought that they
are metabolized and conjugated predom-
inately in the liver and excreted in the
urine, where under mildly acidic condi-
tions, hydrolysis of the conjugates
occurs. The resulting free active metab-
olites may then decompose to form
electrophiles that react with DNA in the
bladder epithelium.
Recent studies have shown however,
that the bladder epithelium itself is
capable of metabolizing carcinogens,
including 2-acetylaminofluorene, to
genotoxic electrophiles. Moreover, in the
present study, it was shown that both
human and dog bladder urothelium are
capable of metabolizing MOCA mto
forms that induce DNA damage. The data
indicate that the binding of MOCA to
human bladder DNA is higher than in the
dog bladder. This result is of particular
importance since MOCA has been shown
to induce urothelial cancer in dogs. The
data also show a wide variation among
both humans and dogs in the levels of
binding of MOCA metabolites to bladder
DNA. This observation is consistent with
previous results in which there was a
wide variation in the binding of carcin-
ogens to human and animal bladder
DNA. It has been suggested that differ-
ences among individuals in their ability
to metabolize carcinogens into forms that
bind to DNA might be associated with
carcinogenic risk. It this were the case,
then some individuals exposed to MOCA
may be more likely to develop bladder
cancer than others
Although the studies on the detection
of MOCA-DNA adducts using 32P-
postlabeling assay are quite preliminary,
the data show formation of several
adducts in the dog as well as in the
human. There appear to be at least two
adducts in common between the dog and
the human. The observed qualitative and
quantitative differences in the number of
adducts detected in different samples
could be due to the low levels of adducts
formed. These studies need to be
expanded to include more samples
before any conclusions can be drawn. In
another set of preliminary studies, when
deoxynucleoside hydrolysates of DNA
from two different [3H]MOCA-treated dog
bladder samples and human bladder
samples were subjected to high perfor-
mance liquid chromatography (h.p I c )
analysis, there was evidence for several
adducts with at least four adducts in
common between the dog and the
human.
In this project, the metabolites of
MOCA produced mto the culture medium
were not identified. Recent studies by
others suggest that MOCA is metabolized
by both N-hydroxylation and C-
hydroxylation pathways. In addition, the
precise chemical nature of the adducts
formed between MOCA and bladder DNA
was not determined. Studies are in
progress to identify the MOCA metab-
olites and MOCA-DNA adducts in order
to develop a complete understanding of
MOCA metabolism and the mechanism
of MOCA-induced DNA damage Never-
theless, our results clearly indicate the
potential of MOCA to induce genetic
damage in the human bladder and
suggest caution in the occupational
exposure of humans to this chemical.
Benzotrichloride Studies
Although BTC has been shown to
induce tumors in experimental animals,
it was decided to evaluate the potential
carcinogenic activity of BTC m the strain
A mouse lung tumor bioassay and to
compare its tumongenic potency with
that of other known carcinogens. Results
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ndicate that BTC induces a high occur-
rence of lung adenomas in A/J mice
within 6 months after initial i.p. adminis-
tration. The tumorigenic response to BTC
is dose-related. The tumors exhibited the
morphology of the mouse lung adenoma
as previously described. Since lung
carcinomas occur rarely in strain A mice,
and usually are not seen until 1-1.5 years
after carcinogen administration, it was
not surprising that carcinomas were not
observed within the 6-month period of
the BTC bioassay.
At the MTD, approximately 58 jumol/kg
of BTC was required to induce an average
of one lung tumor per mouse. Therefore,
BTC is approximately one-half as active
as benzo(a)pyrene and seven times more
active than urethan as an inducer of lung
tumors in strain A mice. However, these
comparisons of carcinogenic potency
could be misleading since several of the
carcinogens were administered in a
single dose and BTC was given in
multiple doses. Nevertheless, in the
authors' experience with urethan, the
latency period for lung tumor develop-
ment is shorter and the number of
induced tumors is higher when the
compound is administered in a single
high dose injection than when that same
dose is fractionated in 24 injections
administered during an 8-week period.
If this is true for the other carcinogens
that were given in a single injection, then
the relative potency of BTC could actually
be underestimated.
Results from the strain A mouse lung
tumor bioassay, comparing BTC with
other known carcinogens, coupled with
the epidemiological, mutagenicity and
other carcinogenicity data, suggest that
exposure to BTC may represent a signif-
icant carcinogenic risk to man.
Conclusions
1.
MOCA induces genetic damage in
explant cultures of human uroth-
elium and could present a risk for
bladder carcinogenesis in exposed
individuals.
2.
BTC is a carcinogen in the strain
A/J mouse lung tumor bioassay
and could present a risk for lung
cancer in exposed individuals.
Recommendations
On the basis of previous epidemiolog-
ical and experimental investigations, and
on the results of the present studies, the
authors recommend caution in the
occupational exposure of humans to both
4,4'-methylene-bis-(2-chloroaniline)and
benzotrichloride.
G. D. Stoner, H. A. J. Schut, and N. Shivapurkar are with the Medical College
of Ohio, Toledo, OH 43614; and I-C. Hsu is with the University of Maryland,
Baltimore, MD212O1.
MarcJ. Mass is the EPA Project Officer (see below).
The complete report, entitled "Effects of 4,4'-Methylene-bis-(2-Chloroanilinel
and Benzotrichloride in Human and/or Animal Tissues," (Order No. PB 88-
117 882/AS; Cost: $12.95, subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
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