AROMATIC AMINES:
An Assessment of the Biological
and Environmental Effects
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AROMATIC AMINES:
An Assessment of the Biological
and Environmental Effects
Committee on Amines
Board on Toxicology and Environmental Health Hazards
Assembly of Life Sciences
National Research Council
NATIONAL ACADEMY PRESS
Washington, D.C. 1981
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NOTICE: The project that is the subject of this report was approved
by the Governing Board of the National Research Council, whose
members are drawn from the Councils of the National Academy of
Sciences, the National Academy of Engineering, and the Institute of
Medicine. The members of the Committee responsible for the report
were chosen for their competences and with regard for appropriate
balance.
This report has been reviewed by a group other than the authors
according to procedures approved by a Report Review Committee
consisting of members of the National Academy of Sciences, the
National Academy of Engineering, and the Institute of Medicine.
The National Research Council was established by the National
Academy of Sciences in 1916 to associate the broad community of
science and technology with the Academy’s purposes of furthering
knowledge and of advertising the federal government. The Council
operates in accordance with general policies determined by the
Academy under the authority of its Congressional charter of 1863,
which establishes the Academy as a private, non—profit,
self—governing membership corporation. The Council has become the
principal operating agency of both the Academy of Sciences and the
National Academy of Engineering in the conduct of their services to
the government, the public, and the scientific and engineering
communities. It is administered jointly by both Academies and the
Institute of Medicine. The Academy of Engineering and the Institute
of Medicine were established in 1964 and 1970, respectively, under
the charter of the Academy of Sciences.
At the request of and funded by the
U.S. Environmental Protection Agency,
Contract No. 68—01—4655
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List of participants
COMZ4ITTEE ON AMINES
DAVID B. CLAYSON, University of Nebraska Medical Center, Omaha,
Nebraska, Chairman
GEORGE T. BRYAN, University of Wisconsin, Center for Health Sciences,
Madison, Wisconsin
DAVID H. FINE, New England Institute for Life Sciences, Waltham,
Massachusetts
CHARLES C. IRVING, Veterens Administration, Center for Health
Sciences, Memphis, Tennessee
CHARLES M. KING, Michigan Cancer Foundation, Detroit, Michigan
RICHARD MONSON, Harvard School of Public Health, Boston,
Massachusetts
JACK L. RADOMSKI, University of Miami, Miami, Florida
DONALD H. STEDMAN, University of Michigan, Ann Arbor, Michigan
STEVEN R. TANNENBAUM, Massachusetts Institute of Technology,
Cambridge, Massachusetts
SNORRI S. THORGEIRSSON, National Cancer Institute, Bethesda, Maryland
JOHN H. WEISBUI ER, Naylor Dana Institute, Valhalla, New York
ERROL ZEIGER, National Institute of Environmental Health Sciences,
Research Triangle Park, North Carolina
Consultants
EMERICH FIALA, Naylor Dana Institute, Valhalla, New York
MALCOLM C. BOWMAN, National Center for Toxicological Research,
Jefferson, Arkansas
National Research Council Staff
ROBERT J. GOLDEN, Project Director
FRENCES M. PETER, Editor
EPA Project Officer
ALAN CARLIN, Office of Research and Development, U.S. Environmental
Protection Agency, Washington, D.C.
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BOARD ON TOXICOLOGY AND ENVIRONMENTAL HEALTH HAZARDS
RONALD W. ESTABROOK, University of Texas Medical School
(Southwestern) , Dallas, Texas, Chairman
THEODORE CAIRNS, DuPont Chemical Co. (retired), Greenville, Delaware
VICTOR COHN, George Washington University Medical Center,
Washington, D.C.
JOHN W. DRAKE, National Institute of Environmental Health Sciences,
Research Triangle Park, North Carolina
ALBERT M. FREEMAN, Bowdoin College, Brunswick, Maine
RICHARD HALL, McCormick & Company, Hunt Valley, Maryland
RONALD W. HART, National Center for Toxicological Research,
Jefferson, Arkansas
PHILIP LANDRIGAN, National Institute for Occupational Safety and
Health, Cincinnati, Ohio
MICHAEL LIEBERMAN, Washington University School of Medicine, St.
Louis, Missouri
BRIAN MacMAHON, Harvard School of Public Health, Boston,
Massachusetts
RICHARD MERRILL, University of Virginia, Charlottesville, Virginia
ROBERT A. NEAL, Chemical Industry Institute of Toxicology,
Research Triangle Park, North Carolina
IAN NISBET, Massachusetts Audubon Society, Lincoln, Massachusetts
CHARLES R. SCHUSTER, JR., University of Chicago, Chicago, Illinois
GERALD WOGAN, Massachusetts Institute of Technology, Cambridge,
Massachusetts
ROBERT G. TARDIFF, National Research Coincil, Washington, D.C.,
Executive Director
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CONTENTS
PAGE
Executive Sununary 1
CHAPTER 1 — Control, Occurrence, and Identification 23
CHAPTER 2—MetabolismOfArOmatiCAiflifleS....... .... 40
CHAPTER 3 — Structure—Activity Relationships Among The
Carcinogenic AromaticAmineS......... 60
CHAPTER 4 — Carcinogenic Potency and Risk Estimation 86
CHAPTER 5 — Epidemiologic Aspects of Exposure to Aromatic
Amines •.... ..o . .s . . . .o .• . 102
CHAPTER 6 — An ii in e 12 3
CHPATER 7 — 4,4’—Methylene—Bis(2—ChlOrOaflilifle).............. 168
CHAPTER 8—2,4—Diaminotoluene.. . 198
CHAPTER 9—Trifluralin and Oryzalin . 228
CHAPTER 10 — 2 —Cresidine...................................... 274
CHAPTER 11 — Fur azoiLdone • • 288
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EXECUTIVE SUMMARY
Aromatic amines and compounds related through metabolism are
used widely in industry. As a result of such applications, they are
also dispersed into the environment, thereby creating a potential
for human exposure. Four aromatic amines are known to lead to
urinary tract cancer in exposed humans, and some are also
responsible for the induction of methemoglobinemia. Some other
structurally similiar amines are carcinogenic to one or more tissues
in laboratory animals.
This report summarizes the key information concerning the
occurrence, analysis, and toxicology of the aromatic aniines and then
considers six specific aniines in detail.
H ISTORICAL PERSPECTIVE
Aromatic amines comprise one of the major groups of
carcinogens. In 1895 Rehn reported that four workmen, three of whom
were employed at a single plant manufacturing magenta (fusChin) from
crude, commercial aniline, appeared at his clinic with bladder
cancer. He correctly deduced that these comparatively rare tumors
were associated with the workers’ occupation. Hueper (1942)
suggested that a limited number of aromatic amines were human
bladder carcinogens in humans. Case and his colleagues (1954)
confirmed this finding in their classic investigation of the British
chemical industry. They demonstrated that 2—naphthylamine,
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benzidine, and 1—naphthylami.ne, contaminated with between 4% and 10%
of the 2—isomer, were carcinogenic in the human bladder. Melick and
his associates (1955, 1971) also showed that 4—aminobiphenyl
(xenylamifle) was a potent carcinogen in the bladders of those
occupationally exposed and of users.
The only other aromatic amine for which there is significant
evidence of probable carcinogenicity in humans is
4—ethoxyacetaflhlide (phenacetin), which was a major component of the
analgesics consumed in excessively large quantities by certain
well—defined populations. These exposed populations developed renal
papillary necrosis and renal pelvic and bladder cancer (Bengsston et
al., 1968; Rathert et al., 1973)
Two consequences followed the identification of bladder
carcinogens in occupationally exposed humans. First, the
manufacture and use of 4—aminobiphenyl and 2—naphthylamine have, for
all practical purposes, been phased out, although benzidine
continues to be manufactured and used, even in the United States.
Traces of a variety of aromatic amines, including 2—naphthylamifle,
still occur in the environment as the result of combustion of
organic materials, including cigarettes. Second, there has been a
major research effort to determine which aromatic amines are
carcinogenic in laboratory experimental animals (Clayson and Garner,
1976), how they may be measured in the environment, and how their
mechanism of action operates (Miller and M1ll L, 197’). Results
from this effort demonstrate that many aromatic amines or their
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derivatives are indeed potent carcinogens in animals and that they
are converted to derivatives of aryihydroxylamines during metabolic
activation. These intermediates probably dissociate to
electrophilic nitrenium ions which interact with nucleophilic
centers in major cell macromolecules (e.g., DNA, RNA, and protein)
in exerting their carcinogenic and other toxic effects.
CHARGE TO THE COMMITTEE
The committee was convened by the National Research Council at
the request of the Environmental Protection Agency (EPA) to assess
the health and environmental effects of amines. The committee
decided that the topic could be addressed best by dividing the
subject into two parts: (1) aromatic amines and related compounds
and (2) aliphatic amines. This report on aromatic amines consists
of chapters on the general characteristics of the class followed by
chapters concerned with specific chemicals that illustrate problem
areas.
The committee reviewed the background information on aromatic
amines and related compounds, stressing, for example, general
analytic methods, toxicity, mutagenic and carcinogenic properties,
and the utility of these substances to industry. It decided that it
would exclude chemicals such as 2—naphthylamine,. 4—aminobipheny] . and
benzidine since they have been extensively and repeatedly reviewed
in the past. Benzidine and its cogeners were especially well
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covered in a recent report by Shriner et al. (1978)
Discussions with the EPA led to the selection of the following
compounds for intensive review: aniline,
4,4’—methylenebis(2—chloroaniline) , 2,4—diaminotoluene, trifluralin
and oryzalin, £—cresidine, and furazolidone. Both trifluralin and
furazolidone are nitro compounds, but were considered relevant to
this study because of the relatively easy biological conversion of
the nitro groups to the amino group and the fact that
aryihydroxylamine derivatives, in which the nitrogen is in an
intermediate oxygenated state between the amine and nitro compound
forms, is now generally recognized as the proximate biologically
active form.
The committee did not address the environmental aspects of
exposure after an intensive search of the literature revealed a lack
of information on this subject for the compounds selected.
OCCURRENCE, CONTROL, AND ANALYTIC METHODS
Aromatic amines are used in dyes, antioxidants, polymers,
explosives, pesticides, and pharmacologic agents. Workers in plants
producing these products can be exposed to a health hazard. An
evaluation of the data indicating that aromatic amines are
potentially toxic in humans indicates that it is prudent to suggest
that exposure be held to a minimum both for the worker and others
who may be exposed to inadequately controlled wastes associated with
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manufacture of the products or who are users of the products. LOW
levels of exposure to aromatic amines may result from the products
of destructive distillation of materials containing carbon,
hydrogen, and nitrogen, as in smoke from tobacco and the gases
resulting from the combustion of fossil fuels. Because of the
anticipated changes in the consumption pattern of fossil
fuels——those implicit in the promotion of diesel engines and the use
of coal——occupational and environmental exposure to the aromatic
amines may increase (National Academy of Sciences, 1981). The
committee recommends that both the qualitative and quantitative
factors that attend these changes be evaluated.
Techniques such as colorimetry, high—pressure liquid
chromatography (HPLC), gas chromatography (GC) and mass spectrometry
(P15) have been used to analyze aromatic amines. Modification of
sampling and clean—up procedures is required for certain substrates,
and techniques must be instituted to ensure good recoveries of the
more volatile compounds such as aniline. The use of
electron—capture derivatives, HPLC, and MS are attractive
prospects. Derivatives such as trifluralin and furazolidone require
the development of special recovery techniques.
METABOLISM
N—Hydroxylation of the aromatic amine or N—acetyl derivative,
followed by conjugation of the aryihydroxylamine or arylbydroxamic
acid, appears to be the key to the activation of aromatic amines.
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Other metabolic reactions, such as acetylation and ring hydroxylation
followed by conjugation, are detoxifying pathways.
Understanding of the metabolism of aromatic amines in laboratory
animals has advanced to the point that research workers are better
able to understand the process in humans. To further this objective,
it is first necessary to know which animal most closely approximates
the metabolic responses of humans. Assuming that most chemical
carcinogens need to be metabolized in the host to active forms in
order to exert a carcinogenic effect, it becomes necessary to know
how specific carcinogens are activated in animals. If the metabolism
of a specific chemical carcinogen in human tissues is qualitatively
similiar to that observed in studies of tissues from a susceptible
test animal, then a potential carcinogenic effect might also be
observed in humans exposed to the chemical. Although the use of
humans for testing is difficult or impossible and laboratory animals
tests are expensive, it should be feasible to make inferences
concerning responses in humans based on in vitro tests with human
cell lines, mutagenicity testing with human liver S—9 fractions, and
careful monitoring of blood and urine of humans accidently exposed to
compounds of interest. It would be easier to assess the risk to
humans from many aromatic amines if additional biological data on
occupationally or otherwise exposed humans were obtained.
STRUCTURE-ACTIVITY RELATIONSHIPS
Many aromatic amine and nitro compounds exhibit the ability to
induce cancer in animals. Unless polar groups, such as sulfonic or
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carboxylic acid substituents, are present in the molecule, these
chemicals should be regarded as potentially carcinogenic. Fortunately,
however, the most potent carcinogens among the aromatic amines appear
to possess certain structural identifying characteristics, such as:
o one, two, or three conjugated aromatic ring systems,
o an aromatic amino group substituted in the position para to the
conjugated aromatic system; or
o groups, such as methyl, methoxyl, or fluorine, substituted in
relative positions to the amino group.
However, carcinogenicity has been associated with aromatic amines that
lack one or more of these characteristics.
A thorough understanding of these relationships may make it
possible to predict the potential toxicity of aromatic amines before
they are adopted for widespread use. Furthermore, this knowledge and
the anticipated increases in the understanding of structure—activity
characteristics of aromatic amines and of other chemicals may allow for
the selective development of desirable chemical species without the
accompanying toxicity.
CARCINOGENIC POTENCY AND RISK ESTIMATION
It is not yet possible to predict the potency of a carcinogen in
any species. One prudent approach is to assume that humans are at
least as sensitive to these carcinogens as are the most sensitive
species. For example, 2—naphthylarnine should be assumed to be as
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potent in humans as it is in dogs.
The potency of a carcinogen depends on three factors: the dose of
carcinogen required to induce tumors, the time to tumor induction,
and the percentage tumor response. A method for expressing relative
potency is described in Chapter 4. Whatever tumor incidence—dose
model is used to describe a biologic event, the suggested means of
expressing potency values discussed herein may have a considerable
advantage in that these values may be derived without excessive
effort or data extrapolation. Ways of predicting carcinogenic
potency based on the present knowledge of the mechanisms of
carcinogenesis urgently need to be improved.
The use of statistical models to estimate possible risk to humans
exposed to very low concentrations is filled with uncertainty.
Studies of animals usually involve exposure to a high level of a
single carcinogen and, sometimes, just one modifying agent. On the
other hand, humans are exposed to a wide range of carcinogens and
carcinogenesis—modifying agents that may enhance or inhibit the
development of cancer especially if the carcinogen exposure is low.
The recently completed ED 01 experiment conducted by the
National Center for Toxicological Research has forced a rethinking of
some aspects of dose—response modeling for carcinogeriesis. More
attention may need to be directed toward the concepts of initiation
and promotion and the inherent abilities of chemicals to act in one
or the other capacity as well as in both.
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EPIDEMIOLOGY
Occupational exposure to 4—aminobiphenyl, 2—naphthylamine, and
benzidine has been clearly associated with an increased rate of bladder
cancer in workers. The use of 2—naphthylamine—containing ingredients
in the British rubber industry was associated with bladder cancer in
workers; however, U.S. rubber workers have not exhibited an increased
incidence of bladder cancer as a result of exposure to that compound.
There is no epidemiologic evidence from which to assess the effects on
humans from exposure to the specific chemicals examined in this report.
With the exception of trifluralin, the chemicals assessed in this
report have induced cancer at various sites in one or more species of
animals. Most toxicologists accept the concept that a demonstration of
carcinogenicity in laboratory animals implies that the causative agent
is a potential carcinogen in humans. Of the chemicals discussed in
this report, 2,4—diaminotoluene (a component of some hair dyes) and
4,4’—methylene—bis(2—chloroaniline) (MOCA) are of most interest because
they have been shown to be carcinogenic in animals and because humans
are frequently exposed to products containing them. Case—control
studies have raised suspicions, while not providing conclusive proof,
that the use of widely available hair dyes containing
2,4—diaminotoluene may be associated with cancer of the breast and of
other 8ites. Because of faulty industrial waste disposal methods, many
people in Adrian, Michigan have been exposed to MOCA, which is known to
be a potent carcinogen in animals. However, there is little evidence
upon which to judge its carcinogenicity or other effects to humans.
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Retrospective studies are not as likely to provide definitive
information on carcinogenicity in humans. In case—control studies of
cancer, the recollection of, for example, hair dye use is subject to a
high degree of recall bias. In retrospective cohort studies of persons
exposed to other chemicals, there is a very imprecise measure of
exposure.
The prospective follow—up study is the only realistic study design
to evaluate the carcinogenicity of these substances in humans. To
determine the effects of hair dyes, it would be necessary to interview
women (and possibly men) about their lifetime use of hair dyes and
follow them for 5 to 20 years to measure the rate of cancer occurrence.
To determine MOCA’s health effects, persons exposed occupationally to
relatively high levels of MOCA, their families, and preschool children
living near the plant would have to be identified, categorized as to
level of current (and future) exposure, and followed for 20 to 40 years.
The epidemiologic evaluation of the possible health effects from
exposure to low levels of aromatic amines as well as to other substances
may be costly and time consuming. To the extent that disease among an
exposed group is increased relatively little above background, perhaps
less than 50%, the excess may not be detectable against the background
variability. One of the best ways to minimize this variability is to
conduct prospective follow—up studies, so that the measure of exposure
is as precise as possible and does not, for example, depend on
memory—based recall of hair dye use. If this strategy is adopted, long
term and costly follow—up is the price that must be paid.
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ANILINE
In the United States, more than 300 x 1O 3 metric tons of
aniline is produced per year. It is the parent compound for more
than 300 chemical products. A metabolite of aniline,
phenyihydroxylainine, is known to cause methemoglobinemia in exposed
workers. Although the mechanism is fairly well understood, it now
appears that some people are more susceptible to this condition than
others. The mechanism of this increased sensitivity needs further
investigation. The National Cancer Institute recently reported that
exposure to the maximum tolerated dose of aniline led to
hemangiosarcomas and other sarcomas of the spleen in rats, but not
in mice. Mutagenicity tests produced negative results except in the
presence of a comutagen. These findings indicate the need for
further research, including carcinogenic studies, possibly on dogs.
There is no evidence indicating that aniline causes cancer in
humans, but further epidemiologic studies are required. There is
also a need to explore the mechanism by which aniline induces
splenic tumors in rats and to determine why it is not carcinogenic
in mice.
4,4 ‘-METHYLENE-BIS ( 2—CHLOROANILINE )
Studies in laboratory animals have demonstrated conclusively
that 4,4’—methylene—bis(2—chloroaniline) (MOCA) is a carcinogen.
Such activity is expected because of the chemical’s structural
similarity to other carcinogenic aromatic amines. Widespread
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environmental contamination by MOCA is attributed to one point source
in Adrian, Michigan, thus increasing the need for epidemiologic
investigations of the exposed population. There is also a need to
study the metabolic fate of MOCA so that those who have been highly
exposed can be identified by these metabolic indicators and
prospective epidemiologic investigations can be facilitated.
2, 4-DIAMINOTOLUENE
2,4—Diaminotoluene (2,4—DT) has been used in some hair dye
formulations. It is also used as an antioxidant and antiozonant in
some rubber products.
When administered orally, 2,4—DT is carcinogenic in rats and
mice, leading to liver and mammary gland tumors. This compound is
also a potent microbial mutagen, and induces mutations in Drosophila
melanogaster . There is no information on the mechanism by which
2,4—DT is activated in susceptible species, including rats and mice.
Given the positive demonstration of carcinogenicity in two animal
species and the data on the genotoxic effects of 2,4—DT in in vitro
systems, it is prudent to assume that humans may be under some
increased risk from exposure to 2,4—DT.
TRIFLURALIN
At the request of the Environmental Protection Agency, tLa.. Ô
report assesses only the potential mutagenic and genotoxic
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properties of trifluralin, a widely used herbicide. During its
commercial preparation, an impurity, dipropylnitrosamine(NDPA), is
produced. Since NDPA is a mutagen, it is difficult to assess this
property in trifluralin. Most mutagenicity studies of trifluralin
have produced negative results. Those that are positive for
chromosomal damage and aneuploidy may be due to the presence of
NDPA. Parallel studies with NDPA have not been reported. NDPA—free
trifluralin and pure NDPA need to be tested, in tandem, for their
abilities to induce chromosomal damage and aneuploidy. There is an
inadequate data base from which to evaluate the potential hazards of
trifluraliri and NDPA, to DNA and cell spindles in laboratory animals
or humans. Should NDPA be a germinal mutagen, exposures are
expected to occur at such low levels that its mutagenic potential
should be correspondingly low. Current manufacturing practice has
considerably reduced the level of NDPA in trifluralin and its
formulations.
Positive mutagenicity test results obtained with trifluralin
have subsequently been attributed to a 177—ppm NDPA impurity
contained in the trifluralin rather than to the test chemical
itself. There is a need for additional mutagenicity testing on
NDPA—free trifluralin for comparison with the existing studies.
Until these studies are performed, pure (NDPA—free) trifluralin
should not be considered mutagenic.
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p—CRESIDINE
E—Cresidine is used almost exclusively as a chemical
intermediate in the manufacture of dyes. Other than a cancer
bioassay conducted by the National Cancer Institute, there is
virtually no other existing biologic data on this compound. Chronic
oral exposures to —cresidine produced bladder cancer in both male
and female rats and mice as well as hepatocelluar carcinomas in male
rats and female mice. In a preliminary investigation, the compound
showed dose—response mutagenicity without metabolic activation in
the Salmonella assay.
Because of the lack of data, it is difficult to evaluate the
potential health effects of this compound. Nonetheless, it must be
considered as a potential carcinogen in humans on the basis of the
carcinogenicity demonstrated in rats and mice. The preliminary
mutagenicity data appear to show a positive response for
E_cresidines but confirmation is needed. Additional data are also
needed on metabolism, metabolic activation, mutagenicity, and
genetic toxicity in both animal and human in vitro test systems.
FURAZOLI DONE
Furazolidone is one of the 5—nitrofurans currently approved for
use as a systemic veterinary medicine in the United States, thereby
finding its way into some edible tissues. It has also been used to
treat bacillary dysentery, typhoid, and other infectious diseases in
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4.0 ppb with current analytical methods. Various spectrophotOmetriC
and thin layer chromotography procedures have failed to yield
satisfactory and reproducible recoveries at these levels.
Furazolidine has exhibited carcinogenic effects in male and
female rats and mice just as other 5—nitrofurans have done. A
variety of different tissues in each species have displayed these
effects. The compound is highly mutagenic in both microbial
( Escherichia coli ) and insect ( Drosophila Inelanogaster ) test
systems, produces chromosomal damage (breakage, sister—chromatid
exchange, mitotic suppression) in human lymphocytes, and forms
interstrand cross—linking in bacterial ( Vibrio cholera ) DNA.
For the reasons discussed above, the use of furazolidone is now
being reviewed by the Food and Drug Administration. Resolution of
this matter awaits the development of a sufficiently sensitive and
reliable analytical method. Any identified problem most certainly
would be associated with the veterinary use of furazolidone. A
“solution” may involve the substitution of an efficacious product
known not to have the mutagenic and carcinogenic potential of
furazolidone.
RESEARCH RECOMMENDATIONS
The following recommendations have been excerpted from the
various chapters to highlight and focus attention on specific topics
the committee felt deserved further consideration.
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Occurrence in the Environment
The anticipated changes in the use of fossil fuels, implicit in
the promotion of diesel engines and coal, may raise existing
environmental levels of some aromatic amines. Accordingly, it is
recommended that steps be taken to evaluate both the qualitative and
quantitative factors that attend these changes in practice.
General Analysis
Techniques must be developed to analyze for the presence of the
more volatile compounds such as aniline. Furthermore, methods must
be developed to determine the kinds and amounts of metabolites
present in persons exposed to these compounds.
Metabolism
understanding of the metabolism of aromatic amines in laboratory
animals has advanced to the point that it can be extended to improve
the understanding of this process in humans. First it is necessary
to know which animal most closely approximates the metabolic
responses of humans. Assuming that most chemical carcinogens need
to be metabolized in the host to active forms in order to exert a
carcinogenic effect, it becomes necessary to know how specific
carcinogens are activated in animals. If the metabolism of a
specific chemical carcinogen in human tissues is qualitatively
similiar to that observed in studies of tissues from a susceptible
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test animal, then a potential carcinogenic effect might also be
observed in humans exposed to the chemical. Although the use of
humans for testing is difficult or impossible, it should be feasible
to make inferences based on in vitro tests with human cell lines,
mutagenicity testing with human liver S—9 fractions, and careful
monitoring of blood and urine of humans accidently exposed to
compounds of interest. It would be easier to assess the potential
risk to humans from many aromatic amines if additional biological
data on humans were obtained.
Carcinogenic Potency and Risk Estimation
More attention needs to be focused on the concepts of initiation
and promotion and the inherent abilities of chemicals to act in one
or the other capacity as well as in both. Also, ways of predicting
carcinogenic potency based on the present knowledge of the
mechanisms of carcinogenesis urgently need to be expanded. In the
absence of contrary evidence, aromatic amines which lack polar
groups should be regarded as carcinogens.
Epidemiology
Epidemiologic methodology needs to be improved so small
differences between exposed and control groups can be detected above
the background variability. One of the best ways to minimize this
variability is to conduct prospective follow—up studies so that the
measure of exposure is at least as precise as possible. Of all the
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compounds discussed in this report, the widespread exposure to
4,4’—methylene—bis(2—chloroaniline) (MOCA) represents the best
opportunity for this approach.
Aniline
Although there has been considerable research on aniline, there is
still much to be learned about its possible health effects. A compond
of such industrial importance deserves to be studied more thoroughly.
The hemangiosarcomas and sarcomas of the spleen and other organs
observed at the maximally tolerated dose (MTD) in the National Cancer
Institute bioassay need to be examined further in another lifetime
feeding study at three or four dose levels in a different strain of rat
to interpret the significance of previous observations.
A carcinogenicity study using Syrian golden hamsters may also be
useful since these animals to develop bladder tumors after exposure to
other aromatic amines. In addition, a long term (preferably 8—10
years) dog-feeding study at the MTD should allay any suspicions
concerning the possible role of aniline in the causation of human
bladder cancer. The only test on dogs was conducted many years ago on
a few animals for too short a duration. Further studies on the
metabolites of aniline in urine should be directed toward explaining
the failure of this compound to induce bladder cancer in dogs (if this
failure is confirmed). Special attention should be paid to
N—hydroxylated urinary metabolites. Moreover, studies should be
conducted on its potential for teratogenicity and reproductive toxicity.
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Epidemiological investigations on workers exposed to aniline
should also be undertaken. Monitoring of urine for aniline
metabolites to confirm and quantitate exposures should be considered.
4,4 ‘—Methylene—bis(2—chloroaniline) (MOCA )
Individuals exposed to MOCA in Adrian, Michigan as a result of
faulty industrial waste methods and others exposed to the compound
should be studied further to learn whether or not the compound is
carcinogenic in humans. First, the metabolic disposition of MOCA
should be explored so that methods for evaluating exposure can be
developed. Then these methods should be applied, to the population
at risk, including an evaluation of necropsy specimens from any
member of this population who dies during the course of this
investigation. Such studies would clarify the potential risk to
individuals, and aid in monitoring the effects of the cleanup
efforts. The final step is the prospective surveillance of this
population to determine whether exposure to MOCA increases their
tendency to develop cancer. In addition, studies on the potential
for teratogenicity and reproductive toxicity should be conducted.
2,4—Diaminotoluene (2 ,4—DT )
Recommendations for future research on 2,4—DT include studies of
the mechanism by which the chemical is activated in rats, mice, and
humans, testing for carcinogenicity in other species to obtain more
data on the relationship between metabolism and carcinogenicity, and
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examination of the in vitro metabolism of 2,4—D’r in human tissues.
The resulting data would facilitate estimatation of risk to humans
exposed to 2,4—DT. Studies on the potential for teratOgenicity and
reproductive toxicity should also be conducted.
Tr ifluralin
Tandem mutagenicity studies on pure trifluralin and its
contaminant, N—nitrosodipropylamine should be conducted to determine
which compound is responsible for chromosonial damage and aneuploidy
noted in previous studies.
p—Cresidine
In vitro studies of human tissues and tests with animals should
be conducted to gather data on the metabolism, metabolic activation,
mutagenicity, and genetic toxicity of p—cresidine. Furthermore,
studies on the potential for teratogenicity and reproductive
toxicity need to be performed.
Furazol idone
A sufficiently sensitive and reliable analytical method for
furazolidone is needed. Moreover, studies on that compound’s
potential for teratogenicity and reproductive toxicity should be
conducted.
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Re ferences
Bengtsson, ri.,, L. Angervall, H. Ekman, and L. Lehman. 1968.
Transitional cell. tumours of the renal pelvis in analgesic
abusers. Scand. 3. Urol. Nephrol. 2:145—150.
Case, R.A.M., M.E. Hosker, D.B. McDonald, and J.T. Pearson. 1954.
Turnouts of the urinary bladder in workmen engaged in the
manufacture and use of certain dyestuff intermediates in the
British chemical industry. Part I: The role of aniline,
benzidine, alpha—naphthylamine and beta—naphthylamine. Br. 3.
md. Med. 11:75—104.
Clayson, D.B., and R.C. Garner. 1976. Carcinogenic aromatic amines
and related compounds. Pp. 366—461 in C.E. Searle, ed. Chemical
Carcinogens. ACS Monograph 173. American Chemical Society,
Washington, D.C.
Hueper, W.C. 1942. Occupational Tumors and Allied Diseases. Thomas,
Springfield, Ill.
Melick, W.F., H.M. Escue, J.J. Naryka, LA. Mezera, and E.P. Wheeler.
1955. The first reported cases of human bladder tumors due to a
new carcinogen——xenylamine. 3. Urol. 74:760—766.
Melick, W.F., H.M. Escue, J.J. Naryka, and R.E. Kelly. 1971.
Bladder cancer due to exposure to para—aminobiphenyl: A 17—year
followup. 7. Urol .. 106:220—226.
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Miller, E.C., and J.A. Miller. 1976. The metabolism of chemical
carcinogens to reactive electrophiles and their possible
mechanisms of action in carcinogenesis. Pp. 737—762 in C.E.
Searle, ed. Chemical Carcinogens. ACS Monograph 173. American
Chemical Society, Washington, D.C.
National Academy of Sciences. 1981. Health Effects of Exposure
to Diesel Exhaust. Report of the Health Effects Panel. Diesel
Impacts Study Committee. National Research Council, Washington,
D.C. 197 pp.
Rathert, P., H. Meichior, and W. Lutzeyer. 1975. Phenacetin: A
carcinogen for the urinary tract. J. Urol. 113:653—657.
Rehn, L. 1895. Ueber Blasentumoren bei Fuchsinarbeitern. Arch.
Klin. Chir. 50:588—600.
Shriner, C.R.,J.S. Drury, A.S. Hammons, L.E. Towill, E.B. Lewis,
and D.M. Opresko. 1978. Reviews of the environmental effects of
pollutants: II. Benzidine. Information Center Complex,
Information Division, Oak Ridge National Laboratory, prepared for
Health Effects Research Laboratory, Office of Research and
Development, U.S. Environmental Protection Agency, Cincinnati,
Ohio, EPA—600/l—78—024, 139 pp.
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Chapter 1
CONTROL, OCCURRENCE, AND IDENTIFICATION
Although concern for the health effects of aromatic amines has
focused primarily on industrial use of these substances, it is
becoming increasingly evident that there are many sources of
exposure to these compounds and to their precursors. The facile
biochemical reduction of arylnitro compounds by both mammalian and
microbial organisms necessitates that their precursors be identified
so that the distribution of potentially hazardous aromatic amines
can be surveyed. Similarly, azo dyes are readily reduced to free
amines by a variety of enzymes. Given the metabolic capacities
likely to be involved, it is prudent to regard any N—substituted
aromatic compound as a potential aromatic amine.
In general, aromatic amine derivatives to which humans might be
exposed are either synthesized intentionally for some specific
commercial use or produced by enzymic reduction of aromatic nitro or
azo compounds or are formed inadvertently as byproducts in processes
a parently directly or indirectly related to combustion.
Commercial. Products
The development of the synthetic dye industry in Europe during
the latter half of the 19th century led to the first recognition of
arylamine—induced bladder cancer in humans. Since that time,
industrial organic chemistry has become more sophisticated and
23
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it is not surprising that aromatic amines are now widely marketed in
dyes and as compounds to be used in various manufacturing
processes. The synthesis of these substances can cause occupational
health hazards, and inadequate control of wastes associated with
their production can result in contamination of the environment.
Furthermore, the practical use of the products can expose both
workers and consumers to their dangers.
Because of the various uses intended for the aromatic amines and
the ingenuity of the chemist, commercial products have various and
ever—changing compositions. Ainines are integral to the following
compounds:
o dyes
o antioxidants
o polymers
o explosives
o pesticides
o pharmacologic agents
Byproducts and Combustion Processes
In contrast to the often large—scale industrial synthesis,
aromatic amines are also produced inadvertently in low
concentrations as byproducts of processes that expose organic
materials to elevated atmospheric temperatures. The combustion of
organic materials can generate aromatic amine derivatives by two
different mechanisms. The partial combustion or pyrolysis of
nitrogen—containing organic material can produce both
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azoheterocyclics and arylamine compounds, as exemplified by the
detection of 22 pg of 2—naphthylaxnine in the smoke of 100 cigarettes
(Hoffmann and Wynder, 1976), amino—substituted carbolines in amino
acid pyrolysates (Kosuge et al., 1978), and indirect mutagens
believed to be primary aromatic amines in synthetic fuels (Epler et
al., 1980).
The presentation by Guerin (1980) and subsequent discussion by
several authors agreed that most of the observed mutagenic activity
resulting from many different samples derived from coal,
shale—derived oil, and petroleum crudes, could be attributed to an
alkaline isolate fraction constituting only a fraction of a percent
of the sample mass. These samples contained (among other things)
identifiable polynuclear aromatic amines.
A second, more indirect, mechanism that produces amines is seen
in the formation of nitroaromatic compounds as a consequence of
combustion processes. Polycyclic aromatic hydrocarbons can be
nitrated by nitrogen oxides formed at high temperatures from
atmospheric nitrogen. These compounds may be formed during or
immediately after the combustion process, but the possibility of
their subsequent photochemical formation has not been excluded.
Indirect evidence for the formation of nitrated aromatics as
byproducts of the combustion process comes from two sources. The
model studies of Pitts and his collaborators (1980) demonstrated
that polycyclic aromatic hydrocarbons were readily nitrated by
levels of nitrogen oxides found in the atmosphere. The second line
of evidence comes from analysis of the mutagenicity of particulatea
25
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collected from internal combustion engines (Claxton and Huisingh,
1980) and from the atmosphere (Wang etal., 1980). The mutagenicity
of these materials in Salmonella typhimurium are decreased if tested
in strains that are deficient in the ability to be reverted to
prototrophy by arylnitro compounds.
Thus, the population may be exposed to arylamine precursors from
several noncommercial sources (LofrOth, 1978):
o tobacco smoke,
o food pyrolysates,
o synthetic fuels,
o internal combustion engines,
o atmospheric particulates, and
o fossil fuel—fired power plants.
The last three sources contain direct—acting mutagens to bacteria
that contain nitroreductase which suggests the presence of
nitroaromatics.
Unlike the long—recognized potential hazards of the industrially
produced aromatic amines, the widespread distribution of these
compounds in prepared food and the environment has only recently
been demonstrated. Thus, the analytic methodology required for
their study is only now being developed. When the appropriate
techniques are available, it will be possible to evaluate the
compounds involved, the levels of exposure, and the biologic
consequences of contamination.
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RECOMMENDATION
The anticipated changes in the use of fossil fuel, implicit in
the promotion of diesel engines and coal, may raise existing
environmental levels of aromatic amines. Accordingly, the committee
recommends that steps be taken to evaluate both the qualitative and
quantitative factors that attend these changes in practice.
GENERAL ANALYTICAL PROCEDURES
Aniline, 2—cresidine, 2,4—diaminotoluene, and MOCA
(4,4’—methylene—bis(2—chloroaniline)) are primary aromatic amines
and should respond to the following general analytic procedures.
Trifluralin, a tertiary aromatic amine, and furazolidone, a
nitrofuran, do not respond to these procedures and must be analyzed
by other means as discussed at the end of this chapter.
General Procedures for Primary Aromatic Amines
Colorimetric Methods . A spectrophotometric method for amines,
amino acids, and a peptide using 2,4,6—trinitrobenzenesulfonic acid
(TNBS) as the chromogenic agent was reported by Satake et al.
(1960). Rinde and Troll (1975) modified this method to determine
free benzidine in the urine from monkeys dosed with a
benzidine—based dye. Urine samples were extracted with chloroform
and back—extracted with 0.01 M hydrochloric acid. The acid extract
(pH 5) was reacted with BS and the yellow color extracted into
chloroform for analysis. traction of the reaction products into
27
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an organic solvent was required to prevent excess reagent in the
aqueous phase from interfering with the analysis. TNBS was also
used as a spray to render the spots visible on thin—layer
chromatographic (TLC) plates. Although the procedure is sensitive
in the nanomole range, it lacks the specificity usually required of
analytic methods. Nevertheless, the procedure is currently used by
the National Institute for Occupational Safety and Health (NIOSH) to
monitor urine from industrial workers (Lowry et al., in press).
The reagent fluorescamine (Fluram) was introduced by Udenfriend
et al. (1972) to quantify aliphatic amines in a sensitive
fluorescent assay. Aromatic amines form fluorescent products just
as the aliphatics do, but the aromatic products are unstable.
However, the parent compounds do form stable yellow derivatives.
Rinde and Troll (1976) used Fluram to develop a colorimetric
procedure to identify several carcinogenic aromatic amines. Dry
residues of the amines were reacted with 50 p1 of Fluram solution (1
mg/mi in glacial acetic acid) for 10 minutes. The reaction was
stopped by adding 0.5 ml of methanol. Optical density readings of
the yellow color were made in 0.5 ml cuvettes in a spectrophotometer
set at the approximate wavelength for maximum absorption. Fluram is
said to react only with aromatic amines in a glacial acetic acid
medium. The reaction can be performed directly on TIC plates to add
specificity to the measurement, and the yellow product can be
quantitatively eluted from the TIC plates. Fluram is colorless, so
blanks appear as zero, a distinct advantage over procedures
employing a TNBS reagent. The sensitivity of the procedure ranges
from 2 to 10 nanomoles for compounds such as benzidine and
28
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2-naphthylamine.
Spot Tests . Strict procedural guidelines imposed by the
Occupational Safety and Health Administration (OSHA) concerning 14
potentially carcinogenic chemicals (including some aromatic amines)
prompted Weeks et a].. (1976) to investigate various spot—test
procedures for the compounds. Chromogenic agents, such as Ehrlich’s
reagent, —diethylaminobenzaldehyde, 2 —dimethylaminocinnamaldehyde,
chioranil, and chioramine T, and fluorogenic agents, such as Fluram
and isomeric phthaldehydes, were evaluated. The limit of detection
values in terms of grams of analyte per square centimeter of surface
being examined ranged from the low nanogram to the 5—pg level,
depending on the compound, sampling technique, and surface involved.
Thin—Layer Chromatography . There are numerous procedures for
separating and detecting aromatic amines. In recent years, reaction
chromatography has been found to identify amines most successfully.
In this technique, a derivative is prepared, and then both the
derivative and the parent compound are subjected to separation.
Some of the derivatives of aromatic amines that have been prepared
for this purpose are 3,5—dinitrobenzamides, 4—dimethylamino—
benzeneazo—4—benzamides, 2,4—dinitrophenylamines, 2—toluene—
sulfonamides, and arylazo—2—naphthols. Isomeric toluidines and
aniline, which are difficult to separate as salts, can be converted
into bromine derivatives; dansyl chloride has also been used. More
recently, Franc and Koudelkova (1979) investigated TLC separations
of 128 differently substituted aromatic amines and their
2,4—dinitrophenyl derivatives. Three solvent systems were used and
29
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in certain cases, assays were completed by paper electrophoresis.
The derivatives were prepared by reacting the amines with
l_fluoro—2,4—dinitrObeflzene. The three—solvent system used with
Silufol UV—254 were diethyl ether—cyclohexane (4:3) and ethyl
acetate—N—prOpaflOl—amfllOnia (5:4:1) and (2:1:2). The aromatic amines
were detected by spraying with a 1% solution of Ehrlich’S reagent in
1 M hydrochloric acid. The derivatives were detected by spraying
with 5% stannous chloride to reduce the nitro groups to amino
groups. After the chromatogram dried, Ehrlich’S reagent was used.
Lepri et al. (1978) in experiments with soap PLC studied the
behavior of 35 primary aromatic amines on layers of silanized silica
gel alone or impregnated with 2% or 4% triethanolamifle
dodecylbenzenesulfOnate solutions. Eluents such as a mixture of 1 M
acetic acid and 30% methanol were employed. Ehrlich’s reagent was
used to detect the amines. Several separations that could not be
made either b ion exchange or reverse—phase chromatography were
carried out.
Additonal TLC procedures for specific compounds are discussed
later in this chapter.
Gas ChrOmatograp . Many of the aromatic amines are amenable
to the conditions imposed by gas chromatography (GC) and can be
measured b using the nonspecific flame ionization detector (FID)
where extremely high sensitivity (e.g., low nanogram or picogram
range) is not required. Also, the rubidium—sensitized
thertnjonic—type nitrogen/phosphorus (N/P) detector responds to the
30
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compounds with various degrees of sensitivity and specificity.
Nevertheless, most of the work in CC methods development for the
aromatic amines has centered around the formation of halogenated
derivatives with subsequent analysis by electron—capture (EC) CC.
Such procedures greatly enhance sensitivity and often improve
stability and GC characteristics.
Francis et al. (1978) demonstrated that flophemesyl
(pentafluorophenyl—dimethylsilyl) derivatives of amines and other
classes of organic compounds could be prepared and analyzed with
high sensitivity by EC—GC. The amine (10 mg or less) dissolved in
toluene (30 pl) was reacted with 30 pl of flophemesylamine in a
sealed vial at 60°C for approximately 15 minutes. Aniline and
2—phenylethylamine were among the amines evaluated. The gas
chromatography was accomplished by using a 1.5—rn glass column packed
with 10% SE—30 on Chromosorb P AW DMCS and a 63 Ni EC detector;
sensitivities were reported in the picogram range.
Nony and Bowman (1978, 1980) adapted the method of Walle and
Ehrsson (1970) to assay several aromatic amines as their
pentafluoropropionyl (PFP) or heptafluorobutyryl (HFB) derivatives.
The amine (10 pg or less) in 1.5 ml of benzene is added to a tube
containing 0.5 ml of trimethylamine catalyst (0.05 M in benzene),
then 50 p1 of heptofluorobutyric acid anhydride is added. The
sealed system is heated at 50°C for 20 minutes, the reaction is
terminated, and the benzene phase is extracted by using 2 ml of
phosphate buffer (pH 6). The benzene phase, dried over sodium
sulfate, is analyzed on a 1.8—M glass column packed with 5% Dexail
31
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300 on Gas Chrom Q by EC-GC employing a 63 Ni detector.
Sensitivities are generally in the low picogram range; however, the
procedure requires modification to preclude low recoveries of
volatile compounds such as aniline. No cleanup procedures were used
in the assays. In another study using PFP derivatives, Bowman and
Rushing (1977) used both EC and N/P detectors to assay for trace
levels of 3,3’—dichlorobenzidine in animal feed, wastewater, and
human urine. The use of cleanup procedures using XAD—2, silica gel,
and/or liquid—liquid partitioning permitted the detection of low ppb
levels of the compound in feed and low ppt levels in wastewater and
human urine. These procedures could probably be adapted to assay
all four primary aromatic amines discussed in this report.
High Pressure Liquid Chromatography . Although few high—pressure
liquid chromatography (HPLC) methods for aromatic amines have been
described in the literature, the technique appears promising when
the compounds are not amenable to GC or derivatization techniques or
when optimum sensitivities are not required. Popi et al. (1978)
measured retention data for polar—substituted benzenes and
naphthalenes, including several aromatic amines, by using
reverse—phase, liquid-liquid chromatography on macroporous
polystyrene gel with methanol—water and acetonitrile—water as the
eluents. Riggin and Howard (1979) employed electrochemical
detection (thin—layer glassy carbon electrode) for HPLC assays of
benzidine and related compounds at ppb levels. Rice and Kissinger
(1979) described a specific method using HPLC with amperometric
detection (carbon paste electrode vs silver/silver chloride
reference electrode) for benzidine and its mono— and diacetyl
32
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metabolites in human urine. A detection limit of 10 ppb based on
twice noise was reported for benzidine. Nony and Bowman (1980)
reported HPr C methods for a variety of aromatic amines and related
compounds in hamster and human urine U8ing a Bondapak C 18 column,
mixtures of methanol—phosphate buffer (pH 6) as the mobile phase,
and a variable ultravoilet absorption detector set at the
appropriate wavelength for maximum response. The detection limit
for benzidine in the urine by this procedure was approximately 180
ppb, based on twice background.
Where applicable, the use of a fluorescence detector generally
enhances the sensitivity and specificity of assays by HPLC.
Other Aromatic Amines
Considerable research has been conducted on methodologies for
detecting trifluralin because of its widespread use as a herbicide.
Because the compound captures electrons well and is amenable to CC,
the EC—GC procedures (with minor modifications) should meet most of
the requirements. Alternative methods include TLC, HPLC, and mass
spectrometry (MS).
There are adequate procedures (e.g., colorimetry, TLC, and HPLC)
for analyzing relatively high concentrations of furazolidone.
However, assays for the compound at low ppb levels in various
substrates appear almost intractable. Although HPLC is probably the
best procedure available, deficiencies in cleanup prior to injection
and in the inherent sensitivity limits of the system may preclude
33
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its use at low ppb levels. Research in the areas of sample cleanup
and mass spectrometry methodology is the next step to improve the
stateofthear t.
CONC LUS IONS
Techniques such as colorimetry, TLC, GC, HPLC, and MS have been
used to analyze primary aromatic amines. Modifications of sampling
and cleanup procedures may be required for different substrates, and
techniques must be instituted to ensure adequate measurements of the
more volatile compounds, such as aniline. Nevertheless, existing
procedures with appropriate modifications should satisfy most
analytic requirements. The use of EC derivatives, HPLC, and MS are
attractive prospects.
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REFERENCES
Control
Claxton, Ii., and J. Huisingh. 1980. Characterization of the
mutagens associated with diesel particle emissions. Environ.
Mutagenesis 2:239 (Abstract).
Epler, J.L., T.K. Rao, and F.W. Larimer. 1980. Isolation and
identification of mutagenic polycyclic aromatic amines in
synthetic crude oils. Environ. Mutagenesis 2:238 (Abstract).
Guerin, M.R. 1980. Bioassay chemistry and the characterization of
polycyclic aromatic organonitrogen compounds — New environmental
analytical problems. Second Symposium on Environmental Analytical
Chemistry, Provo, Utah, June 1980.
Hoffinann, D., and E.L. Wynder. 1976. Environmental respiratory
carcinogenesis. Pp. 324—365 in C. E. Searle, ed. Chemical
Carcinogens. ACS Monograph 173. American Chemical Society,
Washington, D.C.
Kosuge, T., K. Tsuji, K. Wakabayashi, P. Okamoto, K. Shudo,
Y.Iitaka, A. Itai, T. Sugimura, P. Kawachi, M. Nagao, P. Yahagi,
and Y. Seino. 1978. Isolation and structure studies of mutagenic
principles in amino acid pyrolysates. Chem. Pharm. Bull.
26:611—619.
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Lofroth, G. 1978. Mutagenicity assay of combustion emissions.
Chemosphere 7:791—798.
Pitts, J.N., Jr., LA. Van Cauwenberghe, D. Grosjean, J.P. Schmid,
D.R. Fitz, W.L. Belser, Jr., G.B. Knudson, and P.M. Hynds. 1978.
Atmospheric reactions of polycyclic aromatic hydrocarbons: Facile
formation of mutagenic nitro derivatives. Science 202:515—519.
Wang, C.Y., M.—S. Lee, C.M. King, and P.O. Warner. 1980. Evidence
for nitroaromatics as direct—acting mutagens of airborne
particulates. Chemosphere 9:83—87.
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General Analytic Procedures
Bowman, M.C., and L.G. Rushing. 1977. Trace analysis of
3,3’—dichlorobenzidine in animal chow, wastewater and human urine
by three gas chromatographic procedures. Arch. Environ. Contam.
Toxicol. 6:471—482.
Franc, J., and V. Koude1kova ’. 1979. Thin—layer chromatography of
aromatic amines and their derivatives after reactions with
1—fluoro—2 ,4—dinitrobenzene. J. Chromatogr. 170:89—97.
Francis, A.J., E,D. Morgan, and C.F. Poole. 1978. Flophemesyl
derivatives of alcohols, phenols, amines and carboxylic acids and
their use in gas chromatography with electron—capture detection.
J. Chromatogr. 161:111—117.
Lepri, L., P.G. Desideri, and D. Heimler. 1978. Soap thin—layer
chromatography of primary aromatic amines. J. Chromatogr.
155:119—127.
Lowry, L.K., W.P. Tolos, M.F. Boeniger, C.R. Nony, and M.C. Bowman.
In press. chemical monitoring of urine from workers potentially
exposed to benzidine—derived azo dyes. Toxicol. Lett. 7:29—36.
37
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Nony, C.R,, and M.C. Bowman. 1978. Carcinogens and analogs: Trace
analysis of thirteen compounds in admixture in wastewater and
human urine. mt. J. Environ. Anal. Chem. 5:203—220.
Nony, C.R., and M.C. Bowman. 1980. Trace analysis of potentially
carcinogenic metabolites of an azo dye and pigment in hamster and
human urine as determined by two chromatographic procedures. J.
Chromatogr. Sci. 18:64—74.
Popi, M., V. Do1ansky and J. FYhnrich. 1978. Reversed—phase
liquid—liquid chromatography of aromatics on macroporous
polystyrene gel. J. Chromatogr. 148:195—201.
Rice, J.R., and P.T. Kissinger. 1979. Determination of benzidine
and its acetylated metabolites in urine by liquid chromatography.
J. Anal. ‘1\ xico1. 3:64—66.
Riggin, R.M., and C.C. Howard. 1979. Determination of benzidine,
dichlorobenzidine, and diphenyihydrazine in aqueous media by high
performance liquid chromatography. Anal. Chem. 51:210—214.
Rinde, E., and W.Troll. 1975. Metabolic reduction of benzidine azo
dyes to benzidine in the rhesus monkey. J. Natl. Cancer Inst.
55:181—182.
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Rinde, E., and W. Troll. 1976. Colorimetric assay for aromatic
amines. Anal. Chem. 48:542—544.
Satake, K., T. Okuyama, M. Ohashi, and T. Shinoda. 1960. The
spectrophotometric determination of amine, amino acid and a
peptide with 2,4,6—trinitrobenzene 1—sulfonic acid. 1. Biochem.
47:654—660.
Udenfriend, S., S. Stein, P. B hlen, W. Dairinan, W. Leimgruber, and
M. weigele. 1972. Fluorescamine: A reagent for assay of amino
acids, peptides, proteins and primary amines in the picomole
range. Science 178:871—872.
Walls, T., and H. Ehrsson. 1970. Quantitative gas chromatographic
determination of picogram quantities of amino and alcoholic
compounds by electron capture detection. Part I. Preparation and
properties of the heptafluorobutyryl derivatives. Acta Pharina.
Suec. 7:389—406.
Weeks, R.W., 8.1. Dean, and S.K. Yasuda. 1976. Detection limits of
chemical spot tests toward certain carcinogens on metal, painted,
and concrete surfaces. Anal. Chew. 48:2227—2233.
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Chapter 2
METABOLISM OF AROMATIC AMINES
The presence of an amine group has a strong tendency to activate
the aromatic ring, resulting in a complex pattern of metabolism and
a multiplicity of metabolites. For more than 40 years, it was
assumed that 2—naphthylamine was simply metabolized to
2—amino—1—naphthylsulfate in dogs (Wiley, 1938). Subsequent studies
with 14 c— labeled 2—naphthylamine indicated that although 90% of
the radioactivity could be accounted for by this metabolite, seven
other metabolites were also present. In addition, metabolism in the
dog is much simpler than in other species. For example, acetyl
metabolites are not formed in dogs.
These results illustrate the difficulty of conducting studies of
metabolism to delineate the best test species for evaluating a new
compound when there is little or no information concerning the
identity of the metabolite responsible for toxic or pharmacologic
effects. The active metabolite could well be quantitatively
insignificant. In addition, despite the difference in acetylating
ability between humans and dogs, the dog does appear to be a good
test species for induction of bladder cancer because it develops
bladder cancer from the same amines that humans do, which is a more
important consideration (Radomski, 1979b). However, the high cost
of maintenance and long lifespan of dogs may preclude their routine
use.
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Both aromatic axnines and amides are extensively metabolized by
enzyme systems, principally located in the liver. These enzyme
systems are usually divided into two groups: phase I and phase II
(Williams, 1959). During phase I metabolism, one or more polar
groups (such as hydroxyl) are introduced into the hydrophobic parent
molecule, thus allowing a “handle” or position for the phase II
conjugating enzymes (such as uridine diphosphoglucuronyl
transferase) to attack. The conjugated products are sufficiently
polar that these “detoxified” chemicals can be more efficiently
excreted from the cell and from the body.
One of the more interesting phase I enzyme systems is a group of
enzymes known collectively as the cytochrome P—450—mediated
monooxygenases (Cooper et al., 1975; Gillette et al., 1972; Haugen
et al., 1975; Johnson, 1979; Lu and Levin, 1974: Neims et al., 1976;
Thorgeirsson and Nebert, 1977). This enzyme system is involved in a
wide range of biologic activities, as it mediates the metabolism of
numerous, structurally diverse substrates. It catalyzes the
metabolism of many drugs, the transformation of steroids,
cholesterol, and bile acids, and the activation and detoxification
of a large number of carcinogens. Among the carcinogens extensively
metabolized by the cytochrome P—450—mediated monooxygenases are the
aromatic amines and amides. This metabolism involves both
activation (e.g., N—hydroxylation) and detoxification (e.g.,
C—hydroxylation). Therefore, a balance exists in each tissue
between the enzymes that potentiate and those that detoxify
xenobiotics. This balance is known to vary with species, sex, age,
tissue, hormonal status, and exposure of the animal to certain
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xenobiotics (Cooper et al., 1974; Gillette et al., 1972; Lu and
Levin, 1974; Neims et al., 1976; Thorgeirsson and Nebert, 1974).
The terminal oxidase of these monooxygenase systems is a group
of heineproteins collectively termed cytochrome P—450. The
cytochromes are characterized by their spectral absorption maximum,
which occurs at 450 nm when they are associated with carbon monoxide
in their reduced state. Recent efforts have successfully resolved
and characterized multiple forms of the cytochroine, and the study of
these isolated forms of cytochrome P—450 provides information
regarding the properties of individual cytochromes and aids
investigations concerned with regulating the occurrence of each
cytochrome.
The term “multiple forms of cytochrome P—450” refers to
experimentally distinguishable forms of the cytochrome that occur
naturally in a single species. The significance of multiplicity to
the many biologic processes in which the cytochrome has been
implicated will depend largely on the difference in regulatory and
functional properties of the individual forms. The most extensively
purified and characterized forms of the cytochromes have been those
that are induced by compounds such as phenobarbital or the group of
compounds designated as arylhydrocarbon(Ah)iflduCers which include
3—methylcholanthrene, —naphthoflavone, and 2,3,7,8—tetra—
chlorodibenzo— —dioxin (TCDD).
The role of the different forms of cytochroine P—450 in the
metabolic processing of aromatic arnines and amides has not been
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extensively studied. The existing data, however, indicate that the
first step in the metabolic activation of this class of chemicals,
namely N—hydroxylation, may be, at least in some species, catalyzed
by a single form of cytochrome P—450. The clearest demonstration of
this selectivity in the oxidative metabolism of aromatic amines and
amides involves the metabolism of 2—acetylaminofluorene (AAF) by
four purified forms of rabbit cytochrome P—450 (Johnson et al.,
1980) . Of these four forms, two (forms 3 and 6) are exclusively
involved in C—hydroxylation (i.e., detoxification), one (form 4) is
solely responsible the N—hydroxylatiori (i.e., metabolic activation),
and one (form 2) does not catalyze either C— or N—hydroxylation of
the AAF molecule. Also, although the evidence is indirect, genetic
studies in mice on the regulation of N—hydroxylation of AAF indicate
that one or very few genes are responsible for the induction of the
cytochrome P—450 form that catalyzes N—hydroxylation of AAF
(Thorgeirsson et al., 1977).
The occurrence of each cytochrome is dependent on many factors,
and the relative role of each cytochrome must be integrated with
other processes occurring during metabolism and carcinogenesis.
Thus, it is difficult to predict the effect of these metabolic
differences on carcinogenesis. Despite these uncertainties,
metabolism of aromatic ainines and amides plays an important role in
understanding the carcinogenic potential of these compounds by
either lifetime animal or short—term in—vitro tests. The outcome of
this testing has major consequences for health, society, and the
economy. Thus, there is a need for a more clearly defined
43
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relationship between the elements of metabolism of aromatic amines
and carcinogenicity.
OXIDATION
The primary metabolic attack on aromatic amines is usually
oxidation. Two types of oxidation may occur: oxidation of the
nitrogen atom (N—oxidation) and oxidation of carbon of the aromatic
ring (C—oxidation). Primary aromatic amines may be oxidized through
the following stages:
-NH 2 -NOH — N O -NO
amine hdroxylaflhine nitrosO nitro
There is little evidence that aromatic amines are oxidized to
nitro compounds. On the other hand, the nitro compound is reducible
through all the above steps to the amine. Secondary (N—alkyl
aromatic amines) and tertiary amines are also N—oxidized. Tertiary
amines are oxidized to the N—oxide only. Tertiary amines may also
be dealkylated to secondary amines (e.g., dimethylaminoazObeflZefle to
monomethylaminOaZObeflZefle). Secondary amines may be partially
N—dealkylated with the formation of hydroxylamineS (Zeigler et a]..,
1969). Acetamides are N—hydroxylated with the formation of
hydroxamic acids (Miller et a].., 1.960). Hydroxamic acids are quite
stable, in contrast to the notorious instability of
aryihydroxylamines. At present, aryihydroxylamines are believed to
be the proximal carcinogens in the induction of bladder cancer by
44
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some aromatic amines. The esters of hydroxamic acids are believed
to be the proximal carcinogenic metabolites responsible for the
induction of liver cancer by the acetamides (Radomski, 1979a).
Activation of the free amine group of an aromatic amine results
in the metabolic hydroxylation of the aromatic ring
(C—hydroxylation). The positions attacked generally correspond to
the regions of highest electron density (Weisburger and Weisburger,
1958). Thus, aniline and l—naphthylamine are primarily hydroxylated
in the 3 position and secondarily in the 2 position.
2—Naphthylamine and 4—aminobiphenyl, on the other hand, are
primarily hydroxylated in the 1 position and the 3 position,
respectively (Radomski, 1979a). 2—Aminofluorene either has been
more extensively studied or more positions on the aromatic nucleus
are fairly equivalent in electronic density, since metabolites
hydroxylated in the 1, 3, 5, 7, 8, and recently the 9 positions have
been detected (Weisburger and Weisburger, 1958).
GLUCURONI DATI ON
Perhaps the most important detoxification process is conjugation
of rnetabolites of aromatic amines with glucuronic acid. As far as
is known, all species are capable of this metabolic reaction. This
conjugation is generally regarded as a phase II process in which the
hydroxyl groups introduced by the liver mixed —function oxidase
system in phase I are further modified. Conjugation with glucuronic
acid results in highly polar metabolites that are rapidly excreted
45
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by the kidney through filtration without reabsorption or sometimes
through active secretion. Glucuronides are also excreted in the
bile (Mandel, 1971>.
The conjugation does not occur directly with glucuronic acid but
requires an activated intermediate, uridine diphosphoglucuronic acid
(UDPGA). The reaction is catalyzed by the enzyme glucuronyl
transferase, which occurs in liver microsomes according to the
following scheme (Mandel, 1971).
pvrophosphorylase
glucose—i—phosphate + UTP ——- -, UDP—glucose + pyrophosphate
UDP—glucose + 2NA& ÷ H 2 0 UDP—glucuronic acid + 2NADH + 2H+
UDP—glucuronic acid + Ar—OR ____ 2 _4 Ar-O-glucuronic acid + UDP
transf erase
Ar—OH = hydroxylated aromatic amine
UT? uridine triphosphate
Glucuronidation occurs primarily on hydroxyl groups, but may
also occur with carboxyl and amine groups. The formation of highly
acidic, labile N—glucuronides may or may not be enzymatic. In a
newly discovered metabolic reaction, hydroxylamines formed by the
N—hydroxy].ation of aromatic amines are conjugated with glucuronic
acid with the formation of an N—C conjugate (Kadlubar et al., 1977;
Radomakietal., 1973, 1977). For thearomatic amines that induce
bladder cancer, these conjugates may represent the carrier form,
which transports the carcinogenic metabolite (hydroxy]amjne) as an
aglycone from the site of N—hydroxylation in the liver to the
46
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bladder. In the bladder, the acid pH of the urine and/or the
presence of —glucuronidase in the mucosa liberates the
hydroxylamine to produce its carcinogenic action (Radomski et al.,
1977).
SULFATI ON
A perhaps less important phase II synthetic mechanism carried
out by all species of test animals is conjugation of hydroxyl groups
with sulfuric acid (sulfate). Thus, the phenolic hydroxyl groups
introduced on the aromatic nuclei (phase I) are used to increase the
polarity of the original, relatively hydrophobic amine. The primary
amine groups may also be directly conjugated, producing N—sulfate
conjugates (sulfamates) that are readily hydrolyzed in weakly acidic
Solutions and by ubiquitous hydrolytic enzymes (sulfatases). Of
Course, these enzymes also hydrolyze 0—sulfates. As with the
formation of glucuronides, sulfate (S04_) must first be activated
according to the following scheme (Mandf1, 1971).
SO +AT? ___AT • adenosine-5’-phosphosuifate(AP3) 4 roohof ’ te
suit las e
APS + A? _ :2 9 + p p
kinase
PAPS + RZ I R-Z-SO 3 H + 3-phosphoadenosine-5’-phosphste
transferase
whereZjsOo N1j
The key enzymes in the process are sulfotranferaeea (su1fokinases).
Several distinct enzymes that exbibitconside ab1e substrate
Specificity are known to exist.
47
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Because the total sulfate pool is quite limited, it is readily
exhausted when large amounts of exogenous chemicals are
administered. Thus, sulfate conjugation becomes quantitatively less
important with increasing doses.
Conjugation of hydroxamic acids with sulfate may also occur.
This reaction has been postulated as being responsible for the final
activation of N—hydroxy—N,2—fluorenylaCetamifle in the induction of
liver cancer (Irving, 1979).
ACETYLATI ON
Primary amines are acetylated to an appreciable extent by many
animal species. Secondary and tertiary amines are never acetylated
(Mandel, 1971). The metabolic reaction apparently occurs in the
reticuloendothelial cells of the liver, but not in the parenchymal
cells (Govier, 1965). Mucosa of the spleen and intestines are also
capable of acetylation (Mandel, 1971). For exogenous compounds,
acetylation occurs because of the presence in tissues of
acetyl—coenzyme A, a prominent component in the Krebs cycle.
Most species also contain,a hydrolytic enzyme, a deacetylase,
which is capable of removing the acetyl group from acetamides
However, the ability to deacetylate is apparently not absolute;
trace amounts of 2—naphthylamifle were detected in the urine of a man
who had ingested 2—naphthylacetalflide (Conzelman, personal
communication). The failure of the dog to acetylate aromatic amines
-------
(Marshal, 1954) may be due to the dog’s powerful deacetylating
ability, the deficiency of an enzyme, or the presence of an
inhibitor (Leiberman and Anaclerio, 1962). in the rat, aromatic
amines and their acetylated products appear to exist in biologic
eguilibra with each other (Krebs eta].., 1947; Peters and Gutmann,
1955). This finding is also evident from the observation that
2—amjnof]uorene and N,2—fluorenylacetalllide yield very similar
patterns of acetylated and nonacetylated metabolites.
Almost all metabolic alterations of exogenous compounds result
in increased polarity of the metabolized compounds. This property
is very important to survival: in this manner, the animal is able to
dispose of potentially noxious substances efficiently. Acetylation,
however, usually results in compounds having decreased polarity.
Acetylation of aromatic amines is carried out with the aid of the
enzyme N—acetyltransferaee , according to the following scheme:
N-acetyltransferase 1i Q
CH 3 — —CoA + RNH 2 —--—— —-——--—9’ Ru N— ..CH 3 + CoA
—acety1transferase appears to be present in twodifferent
genetically determined forma, oneof whiCh is moçe, efficient than
the other, as observed by Mandel(1971) in studies with4eoniazid.
The;activemetabolite of thecarcinOgeniC srOmattC aminesfor the
induction of bladder cancer appearLtObe the: bYdrOXYlamine; tl.ue,
acetylation appears to be proteCttVe. BU *sUVeeV deflCLto
Support this finding baø:alSO been obtained tn humans (Lower, 1979).
49
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METABOLIC ACTIVATION
Aromatic amines are thought to initiate tumor formation by
modifying tissue macromolecules (Clayson and Garner ,1976; Miller,
1978). These amines can be transformed to metabolites that can
react with proteins and nucleic acids by an initial N—oxidation,
followed by a second activation step. The reactivity of the
activated metabo].ites renders them unstable and limits the distance
through which they are likely to exert their carcinogenic
properties. Conversely, it is likely that all or only the final
metabolic activation step takes place in the tissue in which tumors
are induced. Consequently, the capacity of tissues to carry out
these metabolic events is an important determinant of the
susceptability of that tissue to a carcinogen. Furthermore, at a
higher level of resolution, it is probable that the intracellular
location of the metabolic activation event can be equally crucial.
The induction of liver tumors in rats by primary aromatic amines
has been associated with their conversion to reactive, toxic sulfate
conjugates of their hydroxamic acid derivatives (Irving, 1977).
This pathway is apparently restricted to rat liver and is
ineffective in the livers of female rats of some strains. The
hepatocarcinogenicity of the secondary amine
N—methyl—4—aminoazobenzene in rats appears to result from an initial
N—oxidation and a secondary conjugation with sulfate to yield a
reactive metabolite (ICadlubar et al., 1976).
50
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An alternative mechanism for the metabolic activation of
aryihydroxamic acids is by the formation of reactive
N—acetoxyarylamines as a consequence of N,O—acyltransfer&se (King
and Allaben, 1978). Cytosolic enzymes capable of activating
N—hydroxy—AAF have been demonstrated in a wide variety of tissues
from a number of species that are susceptible to the carcinogenic
effects of aromatic amines. Previous studies have shown that the
lactating mammary glands of the rat, like rat liver, possess an
N,O—acyltransferase and that RNA adducts formed in this tissue are
compatible with an acyltransferase—mediated mechanism of activation
(King etal., 1979). This metabolic pathway has been implicated in
tumor production in two ways. Malejka—Giganti and Gutmahn (1975)
demonstrated that the direct application of N—hydroxy—AAF was more
tumorigenic than either N—hydroxy—2—aminofluorene or’ N—2—AAF.
Allaben et al. (1978) reported that direct application of the
N—formyl,’ N—acetyl, or N-propionyl derivatives of
N—hydroxy—2—aminoflurOrefle resulted in tumor induction that was
greater than that of the N—acetyl derivative, which. was also the
most effective substrate with partially purified rat liver
aryihydroxamic acid N,O—acyltransferase.
A third type of esterification of —oxidize derivatives has
been reported in the activation of 4—hydroxyleininoquinoline—N—oxide
in incubations containing ATP—serine and seryl—adenosine—
monophosphate synthetase (Tada and Tada, 1976). The activity of
this system does not appear to be of general importance in the
activation of other’ aromatic amine derivatives.
31
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Another mechanism for activating aromatic amines is the
generation of reactive metabolites via pathways involving
peroxidation. Bartsch et al., (1972) described the oxidation of
N—hydroxy—N-2—AAF by peroxidase and hydrogen peroxide to yield the
reactive ester, N—acetoxy-N—2—AAF. Subsequent studies have
described the generation of radicals associated with this reaction
by preparations from rat mammary gland (Floyd et a].., 1978).
Although adduct formation with lipids has been demonstrated in
this system, reactions involving nucleic acids have apparently not
been considered. A more recent variation in this area has been the
generation of reactive benzidine derivatives by an arachidonic—
acid—dependent, prostaglandin synthetase system obtained from rat
kidney CT. Zenser, St. Louis University, personal communication).
One unique feature of these findings is that the prostaglandin
synthetase activation uses the free primary amines, prior
N—oxidation is not required. The relationship of this metabolic
activation pathway to the carcinogenicity of benzidine, however,
remains to be explored.
52
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Metabolism
Allaben, W.T., C.E. Weeks, N.C. Treep, S.C. Louie, EJ. Lazear, and
C.M. King. 1978. Mammary tumor induction in the rat by
N —acyl —N —2—flUoreflYlhYdrOXYlamifleS Structure—activity
relationship. Fed. Proc. Fed. Am. Soc. Exp. Biol. 37:1543
(Abstract No. 15030).
Bartech, H., J.A. Miller, and B.C. Miller. 1972.
N —Acetoxy—N—acetylamiflOarefle e and nitrosoarenes • One—electron
non—enzymatic and enzymatic oxidation products of various
carcinogenic aromatic acethydroxamic acids. Biochim. Biophys.
Acta 273:40—51.
Clayson, D.B., and R.C. Garner. 1976. Carcinogenic aromatic amines
and related compounds. pp. 366—461 in •C.E. Searle, ed Chemical
Carcinogens. ACS Monograph 173. American Chemical Society,
Washington, D.C.
Cooper, D.Y., 0. Rosenthal, R. Snyder, and C Witmer, eds. 1975.
Cytochromes p—450 and b 5 . Structure, Function, and
Interaction. Advancee in Experimental Medicine and Biology,
Volume 58. Plenum Publishing Corp., Navyork.
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Floyd, R.A., L.M. Soong, M.A. Stuart, and D.L. Reigh. 1978. Free
radicals and carcinogenesis: Some properties of the nitroxyl free
radical products by covalent binding of 2—nitrosofluorene to
unsaturated lipids of membranes. Arch. Blochem. Biophys.
185:450—457.
Gillette, JR., D.C. Davis, and }T.A. Sasame. 1972. Cytochrome P—450
and its role in drug metabolism. Annu. Rev. Pharmacol. 12:57—84.
Govier, W.C. 1965. Reticuloendothelial cells as the site of
sulfanilamide acetylation in the rabbit. J. Pharmacol. Exp. Ther.
150:305—308.
Haugen, D.A., T.A. van der Hoeven, and M.J. Coon. 1975. Purified
liver microsomal cytochrome P—450: Separation and characterization
of multiple forms. J. Biol. Chew. 250:3567—3570.
Irving, C.C. 1977. Influence of the aryl group on the reaction of
glucuronides of N—arylacethydroxaxnic acids with polynucleotides.
Cancer Res. 37:524—528.
Irving, C.C. 1979. Species and tissue variations in the metabolic
activation of aromatic amines. Pp. 211—227 in A.C. Griffin and C.R.
Shaw, eds. Carcinogens: Identification and Mechanisms of Action.
31st Annual Symposium on Fundamental Cancer Research, M.D. Anderson
Hospital and Tumor Institute, Universities of Texas Cancer Center,
Houston, 1978. Raven Press, New York.
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Johnson, E.F. 1979. Multiple forms of cytochrome P—450: Criteria and
significance. Rev. Biochem. Toxicol. 1:1—26.
Johnson, E.F., D.S. Levitt, U. Muel].er—Eberhard, and -
S.S. Thorgeirsson. 1980. Catalysis of divergent pathways of
2—acety].aminofluOrefle metabolism by multiple forms of cytochrome
P—450. Cancer Res. 40:4456—4459.
Kadlubar, F.F., J.A. Miller, and E.C. Miller. 1976. Hepatic
metabolism of N_hydroxy_ —1flethY14aXfliflOaZObeflZefle and other
N—hydroxy arylamines to reactive sulfuric acid esters. Cancer Res.
36:2350—2359.
Kadlubar, F.F., JA. Miller, and E.C. Miller. 1977 Hepatic
microsomal N—g1ucuronidatiOfl and nucleic acid binding of N—hydroxy
arylamines in relation to urinary bladder carcinogenesis. Cancer
Res. 37:805—814.
Ring, C.M., andW.T. Allaben. 1978. The role of aryihydroxamic acid
N—O—acyltraneferase in the carcinogenicity of aromatic amines. Pp.
431—441 in A. Aitio ed. Conjugation Reactions in Drug
Biotransformation. Proceedings of the Symposium held in Turku,
Finland, 1978. B1sevier/NOrth HOl 3 *nd, New- York.
King, C.M., N.R. Traub, Z,M. Iottz, and M.R.. Thisser. l979.
Metabolic activation of aryihydroxalLic aO1 1.by) acyltransferaa.
of rat mammary gland. Cancer Res. 39:3369—3372.
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Krebs, H.A., W.O. Sykes, and W.C. Bartley. 1947. Acetylation and
deacetylation of the 2—amino group of suiphonamide drugs in animal
tissues. Biocheni. 3. 41:622—630.
Liebman, K.C., and A.M. Anaclerio. 1962. Comparative studies of
sulfanilamide acetylation; an inhibitor in dog liver. Pp. 91—96 in
B.B. Brodie and E.G. Erdos, eds. Proceedings of the First
International Pharmacology Meeting, Volume 6: Metabolic Factors
Controlling Duration of Drug Action. Macmillan Co., New York.
Lower, G.M. 1979. N—acetyltransferase phenotype and risk in
industrial urinary bladder cancer. Approaches to high risk groups.
Pp. 209—219 in Toxicology and Occupational Medicine, Proceedings of
the Tenth Inter—American Conference on Toxicology and Occupational
Medicine, Key Biscayne (Miami). Fla., October 22—25, 1978.
Developments in Toxicology and Environmental Science, Volume 4.
Elsevier/North—Holland, New York.
Lu, A.Y.H., and W. Levin. 1974. The resolution and reconstitution of
the liver microsomal hydroxylation system. Biochim. Biophys. Acta
344:205—240.
Malejka—Giganti, D., and H.R. Gutinann. 1975.
N—Flydroxy—2—fluorenylacetamide, an active intermediate of the
mammary carcinogen N—hydroxy—2—fluorenylbenzenesulfonamide. Proc.
Soc. Exp. Biol. Med. 150:92—97.
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Mandel, H.G. 1971. Pathway of drug biotransformation: Biochemical
conjugations. Pp. 149—186 in B.N. La Du, H.G. Mande]., and E.L. Way,
eds. Fundamentals of Drug Metabolism and Drug Disposition. Williams
and Wilkins, Baltimore.
Marshall, E.K. 1954. AcetylatiOn of sulfonamides in the dog. J.
Biol. Chem. 211:499—503.
Miller, J.A. , J.W. Cramer, E.C. Miller. 1960. The N— and ring—
hydroxylation of 2—acetylaminOflUOrene during carcinogenesie in the
rat. Cancer ReS. 20:950—962.
Miller, E.C. 1978. Some current perspectives on chemical
carcinogenesis in humans and experimental animals: Presidential
address. Cancer Res. 38:1479—1496.
Neims, A.H., M. Warner, P.M. Loughnan, and J.V. Aranda. 1976.
Developmental aspects of the hepatic cytochrome P 450 monooxygenase
System. Annu. Rev. PharmacOl. Toxicol. 16:427—445.
Peters, J.H., and H.R. Gutmafln. 1955.- The acetylation of
2—aminofluorene and the deacetYlatiofl and concurrent reacetylation
of 2_acetylaminofluorefle by rat liver slices. J. Biol. Chem.
216:713—726.
Radomeki, J. L. l979a. The primary aromatic aminea: Their biological
properties and structUre.aCtivity relationships. Annual Rev.
Pharmacol. !roxicol. 19:129157.
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Radoinski, 3,L. 1979b. Evaluating the role of environmental chemicals
in human cancer. Pp. 27—41 in M.A. Meh].man, RE. Shapiro, and H.
Blumenthal, eds. Advances in Modern Toxicology, Volume 1: New
Concepts in Safety Evaluation, Part 2. Hemisphere Publishing Corp ,
Washington, D.C. Distributed by John Wiley & Sons, New York.
Radomski, J.L., W.L. Hearn, T. Radomski, H. Moreno, and W.E. Scott.
1977. Isolation of the glucuronic acid conjugate on
N—hydroxy—4—aminobiphenyl from dog urine and its mutagenic
activity. Cancer Res. 37:1757—1762.
Radomski, J.L., A.A. Rey, E. Brill. 1973. Evidence for a glucuronic
acid conjugate of N—hydroxy—4—aminobiphenyl in the urine of dogs
given 4—aminobiphenyl. Cancer Res. 33:1284—1289.
Thorgeirsson, S.S., P.J. Wirth, W.L. Nelson, and G.H. Lambert. 1977.
Genetic regulation of metabolism and mutagenicity of
2—acety].aminofluorene and related compounds in mice. Pp. 869—886 in
H.H. Hiatt, J.D. Watson, and J.A. Winsten, eds. Origins of Human
Cancer, Book B: Mechanisms of Carcinogenesis. Cold Spring Harbor
Conferences on Cell. Proliferation, Volume 4. Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.
Thorgeirsson, S.S., and D.W. Nebert. 1977. The Ab locus and the
metabolism of chemical carcinogens and other foreign compounds.
Adv. Cancer Res. 25:149—193.
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Tada, M., and M. Tada. 1976. Metabolic activation of
4—nitroquinOlifle 1—oxide and its binding to nucleic acid. Pp.
217—228 in P.N. Magee, S. Takayama, T. Sugimura, and T. Matsushima,
eds. Fundamentals in Cancer Prevention: Proceedings of the 6th
International Symposium on The Princess Takamatsu Cancer Research
Fund, Tokyo, 1975. University Park Press, Baltimore.
Weisburger, E.K., and J.H. Weisburger. 1958. Chemistry,
carcinogenicity, and metabolism of 2—f luorenamine and related
compounds. Adv. Cancer Res. 331—431.
Wiley, F.H. 1938. The metabolism of —naphthylamine. J. Biol. Chem.
124:627—630.
Williams, R.T. 1959. Detoxification Mechaniins. 2d edition. John
Wiley & Sons, New York. 796 pp.
Zeigler, D.M., C.H. Mitchell, and D. Jollow. 1969. The properties of
a purified hepatic microsomal mixed function amine oxidase. Pp.
173—188 in JR. Gillette, A.H. Conney, G.J. Cosinides, R.W.
Estabrook, J.R. Pouts, and G. 3. Mannering, eds. Microsomes and Drug
Oxidations. Academic Press, New York.
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Chapter 3
STRUCTURE-ACTIVITY RELATIONSHIPS AMONG THE CARCINOGENIC
AROMATIC AMINES
In certain instances, aromatic amines are converted in the host
organism to aryihydroxamic acids or aryihydroxylamine derivatives,
which are believed to be the ultimate carcinogenic forms of those
amines which are carcinogens (Miller and Miller, 1976). These
substances induce tumors, usually in tissues distant from their
sites of administration (Clayson and Garner, 1976). The tumor site
varies with the chemical, species, and strain of test animal used.
So far, there is little understanding as to why one aromatic
amine attacks one tissue while another amine affects a different
one. Irving (1979) discussed species and tissue differences in
aromatic amine metabolism as one factor in determining the
distribution of induced tumors. Other factors, such as differing
tissue and species levels in prereplicative DNA repair, cell
proliferation, and hormonal responsiveness, also need to be
considered before a comprehensive picture of tumor distribution can
be formulated. Besides the structure—activity relationships of the
aromatic amines themselves, aromatic aroyl— and acylamides, aromatic
hydroxylantines and hydroxamic acids, and aromatic azo, hydrazo,
nitroso, and nitro compounds are also discussed in this chapter. In
this discussion, aromatic amines and their derivatives are regarded
as carcinogens if they significantly induce cancer in any tissue in
any species.
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Clayson (1953) suggested that, to be carcinogenic, an aromatic
amine had to:
o O8S5B5 two or three conjugated aromatic ring systems
(eg., biphenyl or naphthalene) and
o have the amino group substituted in the aromatic ring
in the para position to a conjugated aromatic system.
This suggestion has been refined more recently by the clear
demonstration that single—ring aromatic amines such as —to1udine
may induce cancer (weisburger et ., 1978), although structures of
the type suggested by Clayson (1953) represent some of the more
Potent carcinogens.
Another important discovery has been the demonstration of the
Considerable carcinogenic potency of certain amino and
nitroheterocyclic arouiatics, such as the derivatives of
5—nitrofuran, njtrothiazo]e, and nitroirnidazole. These compounds
also have considerable importance because of their use in medicines
for animals and humans, to treat parasitic infections. Because
these substances are structurally dissimilar to : - simpler aromatic
amines, they are discussed separatelY.
The information on which the conc1u iofle in thi. chapter are
based has been reviewed by Millet and Miller (1976), Clayson and
Garner (1976), and Weisburger •t ii. (1978).
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SUBSTITUENTS ON THE NITROGEN
Alkyla t ion
Alkylation of the aromatic amino group has been intensively
studied only with derivatives of _dimethylaminoazObeflZefle. In this
particular series, the presence of at least one methyl group on the
amino group is held to be essential to hepatocarcinogenicity in
rats; longer alkyl groups depress activity. Detailed examination
of the evidence supporting this statement reveals that few
nonalkylated derivatives (analogs of 2 —aminoazobenzene) have been
studied and that the evidence for the essential nature of the methyl
group is confined mainly to 4—dimethylaminoazobeflZefle itself. In
specific cases, such as 3—inethoxy—4-aminoaZobeflzefle or
4—(o—tolylazo)—o—toluidine, the N—methyl group is not essential, and
tumors are induced. Painting —aminoazobenzene or a range of
similar chemicals on rat skin leads to skin tumors (Fare and Orr,
1965; Fare, 1966).
There is very limited evidence for the importance of an alky].
group on the nitrogen to the carcinogenic potency of aromatic
amines. A methyl group may be essential to the activity of
4—dimethylaminoazObenzene and certain of its analogs in rat
hepatocarcinogenesis. Dimethyl derivatives are metabolically
monodernethylated to monomethylaminoazobenzene derivatives and then
to the unsubstituted amines. Contrary to earlier evidence, the
reverse process, arylamine methylation, is not a major metabolic
62
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pathway (Scribner etal., 1965). Higher alkyl groups generally
result in chemicals with lower carcinogenicity than do those
provided by the free amines. Monoalkylaromatic amines can be
converted to the corresponding nitrosamines in the presence of
nitrous acid at an acid pH.
Aryla tion
Arylation of the amino group to diarylamines or triary].amines is
believed to abolish carcinogenic activity, although the evidence for
this conclusion is tenuous. Phenyl—2—naphthylamine has been 8tudied
intensively because of its use as a substitute for 2—naphthylamine,
a rubber—compounding ingredient. The urine of humans and dogs
exposed to this apparently noncarcinogenic chemical contains low
levels of free 2—naphthylamine (Batten and Hathway, 1977; Kummer and
Tordoir, 1975;). At this time, there is no explanation for this
finding——whether the 2—naphthylamine is liberated within the host or
is an artifact of urine collection, or whether there is a similar
degradation of other diarylamines. The biologic significance of
this observation can be judged from the fact that the amount of
urinary 2—naphthylamine found after exposing humans to 10 mg of
phenyl—2—naphthylamine is equivalent to that in the smoke of 4—40
Cigarettes. Hoffmann et al. (1969) found 1 pg of 2—naphthylamine in
the smoke of 40 cigarettes.
Acyi.ation
Acetylation and deacetylation of aromatic amines are common
63
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metabolic reactions in most species, except dogs, which lack the
ability to acetylate these chemicals. Consequently, aromatic amines
and acetamidea generally possess similar carcinogenic potencies.
Higher homologs of the acetyl group do not appear to have been
investigated. N—2—fluorenylformamide is less potent than the acetyl
derivative (Miller et al., 1962).
Aroyl Derivatives
Aroyl derivatives have been examined, particularly in the
2—fluorenylamine series. Although N—2—f].uorenylbenzamide is
inactive, the corresponding N—hydroxy—2—fluorenylbenzamide is
carcinogenic. This finding suggests that aroylation blocks
N—hydroxylation, the essential activation route for this
carcinogen. The benzenesulfonamide derivatives of 2—fluorenylamine
are likewise inactive, but its N—hydroxy derivative is carcinogenic
(Gutjuann at al., 1967).
N—Hydroxy lation
N—Hydroxylation of aromatic amines or of their amides is
generally regarded as the first step toward their metabolic
activation. If active, aromatic hydroxylamines or hydroxamic acids
are more potently carcinogenic than are the non—N—hydroxyla ted
chemicals; however, the demonstration of this increase in potency
may require careful selection of a system (Miller at a].., 1964). In
specific instances, the N—hydroxylated compound demonstrates
activity, and the parent compound does not. 1—Naphthylamine, for
64
-------
example, is not demonstrably carcinogenic if free from the potently
carcinogenic 2—isomer; however, the corresponding N—hydroxy
derivative, is carcinogenic (Radoiflski ! .!.i • ’ 1971).
Esters of N—Hydroxy Derivatives
Esters of N—HydrOxy derivatives of aromatic amines have been
regarded as the ultimate carcinogenic forms of the aromatic amines.
Some of these derivatives are highly genotoxic, if they can be
delivered to the test system and the critical receptors before they
interact with other possible targets such as thiols. The less polar
model esters (such as N—acetoxy—2—fluorenylacetamide) are thus more
readily shown to be carcinogenic than are the more polar derivatives
(such as the sulfate ester of N—hydroxy—2—fluorenylacetamide).
Different esters appear to vary in their ability to act as
leaving groups in the production of the arylnitrenium ion. For
example, the Q—glucuronic acid ester of N—hydroxy—2—fluor
enylacetamide does not appear as capable of producing the nitrenium
ion as do either the acetoxy or sulfate esters (Irving and Wiseman,
1971).
Azo Compounds and Hydrazo Compounds
These compounds are reduced in the anaerobic portions of the
gastrointestinal tract, or by the tissue enzyme, azoreduatase, to
compounds, each of which contains an amino group. Several examples
of azo compounds, themselves carcinogenic and capable of reduction
65
-------
to carcinogenic aromatic amines have been reported. Bonser et al.
1954 and weisburger et al. (1978) reported that 1—(o—tolyl)azo—
2—naphthol is carcinogenic and may be reduced to o—toluidine, which
is also carcinogenic. The high carcinogenic potency of a series of
dyes (Direct Black 38, Direct Brown 95, and Direct Blue 6), which
are capable of reduction to benzidine, has been reported by the
National Cancer Institute (1978).
Aromatic C-Nitroso Compounds
These compounds are of interest because of their easy conversion
to arylbydroxylamines. Certain C—nitroso compounds are effective
nitrosating agents.
THE RING SYSTEM AND THE POSITION OF THE AROMATIC AMINO GROUP
Ring—substituted amino derivatives of most substances containing
one, two, three, and possibly four aromatic rings may be
carcinogenic. The placement of the amino group is the critical
factor. Thus, 2—naphthylamine, 2—acetamidofluorene, 2—anthramine,
and 2—phenanthrylacetamide are potent carcinogens; l—naphthy]amine,
3—acetamidofluorene, 9—anthramine, and 9—phenanthrylacetamide are
not. The presence of a large conjugated group para to the amino
group appears to enhance carcinogenic activity but is not a
requirement for this property, as is clearly demonstrated by the
fact that single—ring aromatic amines and derivatives of
niethylenedianiline are carcinogenic (International. Agency for
Research on Cancer, 1974a,b,c; Weisburger etal., 1978).
66
-------
The basic ring system for carcinogenic aromatic amines may be
entirely carbocyclic (2—naphthylamine, 4—biphenylamine), may show
limited numbers of heteroatoms (3—aminodipheny].ene oxide,
4—N—quinoline—l—oxide), or may be highly heterocyc].ic (nitridazole,
metronidazole). The heterocyclic chemicals are discussed separately
later in this chapter.
Ring substituents on the carcinogenic potential of aromatic
amines are subdivided into
Analogs of E —dimethylaminoazobenzene
Aromatic amines with single amino groups
Analogs of the phenylenediamines
Analogs of benzidine.
This subdivision is necessary because the various types of
carcinogenic aromatic amines appear to be affected differently by
substituents.
Analogs of p —DimethylaminoazObeflzefle
Analogs of 2 —dimethylaininoazobenzefle have been extensively
studied for their induction of rat liver tumors. There are five
different positions available for monosubstitution The effects of
the substituents on carcinogenic activity are presented in Tables
3—1, and 3—2.
67
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Table 3-1
Effect of Substituents on the Carcinoqenic Activit.y of
N , N
Subs t i ti jen t . 2 3 2’ 3’ 4’ _ ‘ 2,4’ 3’ , 5 2 , 4 ’
-CH - + -F + + +
- + + +
-CF 3 - - -
-F + + + -f + + +
-Cl + + + +
-Br -
-OH - - - - -
-OCH + + + +
-0C 2 H 5 +
-NO 2 + +
-NH 2
-SO 3 H
-CO 2 H 4 +
- From Clayson arid Garner, 1976, with permission.
Ear duct, skin, and intestinal tumors, but no hepatornas.
- Bladder papillomas; hepatomas possibly induced•
6
-------
Table 3-2
Liver Carcinogenicity of N, -Dimethyl-p-pheny1azoanilines in Rats
Poai we
N, V-DimethyI-4 (4’-benzimidazolylazo)aniline
N,N-Dimethyl-4 (6’-benzthiazolylazo)aniline
N,N-Dimethyl-4 (7’-benzthiazolylazo)aniline
N ,N-Dimethyl-4 14’(2’ ,6’-dimethyIpyridy -1 ‘-oxide)azoJaniline
N,N-Dimethyl-4 (6’-IH-indazylazo)aniline
N ,N-Diinethyl -4 (4’-i oquinoIiiiylazo)aniIiue
N,N-Dimethyl-4 (5’-iaoquinolinylazo)aniline
N,N-Dimethy1-4(7’-isoquinoIjny1 zo)&njJjne
N,AV-Diniethyl-4 (5’-i oquinoIyI-2’ .oxide)azoanj1ine
N ,N-Dimethy l -4 [ 4’-(2’,5’ - Iutidyl)azo]aniIjne
N,N-Dimethyl-4 [ 4’-(2’,6’-lutidyl-I ‘-oxide)azolaniline
N ,N-Dimethyl-4 [ 3’ ,5’-Iutidyl- 1 ‘—oxide)azo aniline
N,N-Dimethy l -4 [ 4’(2’_methylpyridy l)azolanjline
N ,W-Diznethyl-4 [ 2’-methylpyridyl- 1 ‘-oxide)az.olaniline
N.N-Dimethyl-4 [ 4’-(3’-niethylpyridyl-1 ‘-oxide)azojaniline
N ,N-Dimethyl-4 [ 4’ (3’-m&4hylpyridyl-1’-oxide)azolaniline
‘ 1 ,N-Dimethyl-4 [ 4’ (2’-niethylpyridyl-l ‘-oxide)azc)aniline
W,iV-Dimethyl-4 [ 4’ (2’-riiethylpyridyl-l ‘-oxide)azojo-t.o luidine
t ’ ,N-Dimethy1-4 [ 5’(3’-z icthylquino1y1)azoJaniIine
A ,N-Dimethy1-4 [ 5’(6’-rnt thy1quinoly1)a2o [ aniline
P ’ ,N-Dixnethyl-4 [ 5’ (7’-rncthylquinolyl)azo]aniline
N,N-Dimethyl-4 [ 5’(8’ -niethylquinolyl)azo [ aniLine
N, M-Dimethyt-4 (2’-naphthylazo)aniline
N,N-Dimethyl-4(3’-picolyl-l ‘-oxide)azo}-o-toluidine
N,N-Dimethyl-4 3’-picolyl-l’-oxide)azoj-m-toluuline
N,N-Dimethyl -4 [ 4’-pyridyl-l ‘-oxide)&zoj-2,3-xybdine
N,N-Dimethyl-4( (4’-pyridyl-l ‘ oxide)azoJ-2 ,5-xy1idine
N,N-Dimethyl-4 [ (4’-py ri Iy1-1’-oxide)aw1-3 ,5-xy1idine
N ,N-Dimethyl-4 (3’-pyri. iylazo)aniline
N ,N-DimethyL-4I4’ pyridyl-1’-oxide)azo [ s&niIine
N,N-Dimethyl-4(5’-quu uidy1azo)aniline
N,N-Dimethyl-4(3’-quinolylazo) aniline
N,N-Dimethyl-4 (4’-quinolylazo) aniline
N,N-Dimethy l-4(5’--quino ly l&zo)anitine
N,N-Dimethy l-4(6’..quino ly l.azo)ani line
N,N-Dimethyl-4( (4’-quinolyl-l’-oxide)azo [ axnline
N,N-Dunethyl-4 [ (5’-quino lyl-l’.-oxide)azo]anthne
N,N-Dimet.hyl-4 [ (6’.quinolyl-1’_oxide)azo &niIine
N,N-Dunethyl--4 (5’-quinolyLazo)-m-toluiduie
N,N-Dimethyl-4 (2’-quinoxaiylazo)anilme
N,N-Dimethy l -4(5’-quinoxa ly lazo)ani line
N,N-Dimethy l -4(6’ -quiaoxilylaao).-niline
2 -Dimethylamino-5(phenylazo)pyrjdine
N,N-I)imethyl-4 (5’-benzimidazolyla.z.o)aniline
N,N-Dimethy1-4(2’-dibenzofurany1a o) a njIjne
N,N-Dimethyl-4( 1’-dibenzothienylazo)aniline
N,N-Dimethyl-4 (2’-dibenzothienyla o)ani1ine
N,N-Dimethyl-4(3’dibenzothjenyIazo)a.ni 1j
N,N-I)imethyl-4(3-dibenzothieny lazo)anj ljne
N,N-1)imethyt-4 (4’-benzthiazylazo)anihne
N,N-Dimethyl-4 (5’-benzthiazy1azo) niIine
N,N-Dimethyl-4 (2-fluoreny lazo)aniline
N ,N-i)irnethyl-4 (3’-IH-indazylazo)analine
N ,N-Dimethy l-4 (4’-IH-indazylazo)aniline
N,N-Dimethyl-4 (5’-IH-indazylazo)amline
N,N-Dimethyl--4 (7’-IH-indazylazo)aniline
N,N-Djmethy1 _4 2’-(4’ -methyIpyrjdyI)azo [ .nj1jne
N,N-IJunet.hyl..4 [ 2’- (6’-methylpyridyl)az.oja.niline
N,N-Dmethy l-4-(7’-quinoly lazo)aniline
N,N-Dimethyl-4-(8’-quinolylazo)auiline
N,N-I1 imethy14 [ 2’-quuio1y1-1 ‘-oxide)az.ojaniline
N,N-Dimethyl -4 [ 3’. ’quinolyl-l ‘-oxide)azojaniline
N,N-Dimethyl-4f7’-quinolyl-l ‘-oxide)azolaniline
N,N-Dimethyl-4(8’-quinolyl- 1 ‘-oxide)azojaniline
N,N-Dimethyl-4(2’-(4’-methylpyridyi-1’-oxide)azo [ aniline
N,N-Dimethyl-4 [ 2’-(&’-wetby lpyridyt- l’-oxide)azo [ anihne
Ncg 4ive
- From Clayson and Garner, 1976, with permission.
-------
Aromatic Amines Containing Single Amino Groups
These aromatic amines have a number of similarities that lead to
structure—activity relationships.
Methyl Substitution . Especially when ortho to the amino group,
methyl substitution appears to enhance activity, as is demonstrated
by comparing the carcinogenic potential of 2’,3—dimethyl—4—
aminobiphenyl to 4—aminobiphenyl, or of 3—methyl—2—amino—
naphthylamine to 2—naphthylamine. The effect of methyl groups in
other positions in the 4—aminobiphenyl system was extensively
studied by Walpole and Williams (1958) . Methyl groups in both ortho
positions, as with 2,6—dimethy].aniline (2,6—xylidine), may be
deactivating (National Cancer Institute, in press)
Methoxyl Substitution . Ortho substitution of a methoxyl group
has a varied effect. Thus, 3—methoxy—4—aminobiphenyl and
l—methoxy—2—fluorenylacetamjde are just as, or even more, potent
than the parent carcinogen in rats, although
3 —methoxy—2—fluorenylacetamjde was found to be without activity.
Similar results were reported for the free amines. Other methoxyl
substitutions also make the molecule more carcinogenic as, for
example, in the case of £—cresidine as compared to aniline or
7—methoxy-2—fluorenylacetamide as compared to 2—fluorenylacetamide.
Halogen Substitution . This is best illustrated by using
fluorine substitution to block metabolic detoxification of aromatic
amines. Fluorine enhances carcinogenicity, as illustrated by the
70
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potent carcinogens 4 ‘—fluoro—4—biphenylamine and
7—fluoro—2-fluorenylacetamide. Most fluoro derivatives of
N—2—fluorenylacetamide have been tested (Miller et ., 1962).
Insufficient data are available to permit a uBeful statement on
other halogen—substituted aromatic amines.
Polar Group Substitution . It is generally held that
substitution with sulfonic acid, carboxylic acid, or phenolic groups
diminishes the carcinogenicity of aromatic amines. There are,
however, few published animal studies to support this viewpoint.
Epidemiology can do little to provide useful information because the
noxious parent aromatic amines are usually used in proximity to the
polar derivatives.
nologs of Phenylenediamines
More than 14 phenylenediamines or the corresponding nitro
compounds have been adequately tested for carcinogenicity (National
Cancer Institute, l978a,b,c,d,e,f; 1979a,b; Weisburger et al.,
1978). A limited number of these agents are effective carcinogens,
including 2,4—diaminotoluene, 5—nitro—o—anisidine, and
4—chloro—o—phenylenediamine; others, under the test conditions used,
exhibited more marginal carcinogenicity, including
2—N-. —phenylenediamine, 2, 5—diaminotoluene sulfate,
2,4—dinitrotoluene, and tetrafluorometaphenylenediamine. The
remainder were inactive.
71
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This evidence demonstrates that derivatives of each of the three
positional isomers of phenylenediainine or the corresponding nitro
compounds may exhibit carcinogenicity. So far as the limited data
permit a decision, it appears that the carcinogenicity of
phenylenediamine, is activated by the presence of a methyl or
methoxyl group ortho to one of the amino groups in the same way as
are the aromatic monoamines. Blocking metabolically important
positions for detoxification, as in tetrafluorometaphenylenediamine,
may also be important.
Analogs of Benzidine
Information obtained mainly from studies of rats indicates that
benzidine is more carcinogenic than o—tolidine, o—dianisidine, or
o—dichlorobenzjdjne. In a limited study, 3,5,3’,S’—tetramethyl—
benzidine appears inactive. Overall, benzidine derivatives appear
to behave differently in their structure—activity relationships than
do either the aromatic monoamines or derivatives of
methylene(bis—aniline), in which 0—methyl— or o-chloro—substitutj.on
appears to enhance carcinogenicity (Munn, 1967).
NITRO- AND AMINO-AROMATIC HETEROCYCLIIC COMPOUNDS
In the furan, thiophene, imidazole, and thiazole series, both of
the unsaturated bonds provide a pair of ir electrons, and one of the
hetero atoms provides a lone pair of electrons to form the aromatic
sextet. Thus, the amino and nitro derivatives of these heterocyclic
resemble the carbocyclic analogs in various ways, including cancer
72
-------
induction. The initial discovery by Price et al. (1966) and Stein
et al. (1966) has been followed by the bioassay of many
environmentally important substances of this type (Table 3—3). The
nitrofuran derivatives have been fully reviewed by Bryan (1978).
It is apparent that many of the nitroheterocyclic compounds are
potent carcinogens with demonstrable effects in many tissues. The
amino—substituted analogs have been much less intensively studied.
Structure—activity relationships are difficult to evaluate because
of the competing effects of the hetero atoms, the substitutents, and
the various conjugated aromatic systems. It does, however, appear
that compounds with two conjugated aromatic ring systems are, when
carcinogenic, more potent than are single—ring systems. Thus, the
low activity of the single—ring substance, metronidazole (FlagyI ),
can be compared to the considerable carcinogenic potency of
niridazole, which possesses two ring systems (Bulay et a]., 1977;
Rustia and Shubik, 1972).
CONCLUS IONS
Many aromatic amino and nitro compounds can induce cancer in
humans or animals. Unless polar groups, such as sulfonic acid or
carboxylic acid substituents, are present in the molecule, it is
possible that these chemicals are potentially carcinogenic.
Fortunately, however, the most potent aromatic amine carcinogens
73
-------
Foinuc *cid, 244-(5-nitro-2-furyl)-
2-tbiaaolytlhydraaide
ham.ter 0
mouse 0
2-Hydnl -(&-nitro -2-fur3rl)thiasole rat 0
mouse 0
3 Hydrzin4tropbenyl)t1 .i. nI. rat 0
N.44-45 -Nito.2-furyl).2-thiaaoly
fona&m de
5-Ac mmido-3-(5-nitro-2-f uryl)-
6ff- i ,2,4-oxadiuiae
5-Nitro-2-(uraldehyde eermicarbazone rat
N44 -(5 -Nitro-2-fury i)-24biasoly lJ rat
acatam
mouse
1-(5-Nitzo-2-(urylilcne)amino hydaxitoin rat
4-Met l vl-I-{5-nutzuIurylido)amiao)- rat
m mose
i -5-Morphohnomethyl.3-((5-nitzo- rat
24urylklene)wli oJ4-oxuolidine
1 -(2 -Hydioxyethyl) -3 -iutroturyhdsee) rat
aino}.2 - mI 4 tn liiWe
1{(5Nirofury lidor e)ammno -
u ooe
6-Nit o -2-f uranudoxime
4 .6-Diaseino-2-(5n itro-2-furyl) ’s-triuine
N,N ’*(5 -Nitro -2-furylñse in.-
2,4 -diyl)-biacetamide
Hezamethylme l amine
2-ifydraino-pbenylthiaso le
p4- )-Th(p-nitiop&iseyl)-
i-(2-Hydrozyethy l) -2-methy l-
5 -nitroãnidaao le
l,2 -Dimethyl-5-niu ’oimidaaol.
2-Amino -5-pbenyl-2-oxaao lia-
4-one + Mg(OH),
1-Aminotria sole
1-(2-Hydroxy)ethyl-2-metbyl-
5-nilinimidasok
S
B
S
8
B
o s
S
S
S
0
o s
o
o
o 8
— 7 7
- - +
- + -
— — 7
- ÷ -
- + +
- + -
— + -
Cn ,o .sp4 Spectra Ro.4. A&qiIaCIJ ’
2-(2,2-Dimethyibydraaino)-4-(5-rütro- rat 0 A
24uy1)thiaso l.
Table -3 a
Carcinogenicity of 2-Nitrofuryl Corirnounds and Related S 1 u arjces—
1*
mouse 0
rat 0
S
B
mouse 0 S
dog 0 B
rat 0 A
hamster S
mouse 0 8
rat 0 8
BLod- Kid- In- Eer
I4 1 .d _! 1L Lis.r gesMa J Breast ( * r
— — — + selivary gland,
various othse
- - - -_
7 — — + hing?skin7
- - - - omath?
- - - +hing?
— — — + 5 4 fl 11 ?
- - - + stomach?
- - - + am?
selivary gland?
— — — — stomach?
- - - + gallbladder
- - - + -
- - - - stomach
— — — — lung?
leukemia?
+ - - - lung, mearotely
(bp
wcomu)
— — — — — — 7 —
+ - - - + lung livary
+ - - -
- - - - leukemia,
- - - + -
- — ! + lymphosna
- - - + -
— - - + lymphoina
— - — - + —
- - - + -
— — 7 — — — 7 —
- — - ÷ -
- - - + -
— — — 7 —
+ - - - thyroid
+ - - - thyroid
- - - - lymphoma, lung
• +, tu oil lepoiled; - ‘ uioia aol reported; ?, cs .quivoorL
o s
o s
o 8
o s
o 8
o 8
rat
rat
rat
rat
rat
rat
rat
rat 0 8
rat 0 S
rat 0 8
rat 0 A
mouse 0 A
mouse 0 8
0, or.l.
• A. ts sd .oi, thee sea isstMiaI.; 8, e,idsem l thee oomvncInL.
- From Clayson and Garner, 1976, with permission.
-------
appear to possess certain specific structural characteristics, such
as
o one, two, or three conjugated aromatic ring Systems,
o an aromatic amino group substituted in the position
para to the conjugated aromatic system, or
o groups such as methyl, methoxyl, or fluorine
substituted in specific positions relative to the
amino group.
Aniline, as the simplest aromatic amine, might be considered the
reference chemical for structure—carcinogenic activity relationships
in this series. However, although it has induced cancer in rats
(National Cancer Institute, 1978g), but not in mice, it has shown
negative results in mutagenicity tests. If norharmon is present in
Salmonella tests the results are positive (Nagao et al., 1977).
Thus, it nay induce splenic hemangiosarcoma and other sarcomas by a
mechanism different from that by which other aromatic amines induce
their effects. For example, the methemoglobinemia induced by
aniline may stress the spleen, which removes debris in red blood
cells from the circulation, thereby setting up the conditions for
splenic tumorigenesis. £—Chloroani].ine induces similar cancers and,
likewise, induces high levels of metheinoglobinemia (National Cancer
Institute, 1979d).
p—Cresidine (2—methoxy—5—methylani line) induced bladder
carcinomas and olfactory neuroblastomas in rats and bladder
75
-------
carcinomas and hepatocellular carcinomas in mice (National Cancer
Institute, 1979c). E—Cresidine produced positive results in
microbial mutagenicity tests. There is no reason to doubt that
genotoxic factors play a role in inducing these tumors and, as they
occur at sites usual for aromatic amines, E—cresidine should be
regarded as a potent carcinogen. The ortho inethoxyl group is
probably responsible for enhancing the activity of this amine.
2,4—Diaminotoluene (National Cancer Institute, 1978h) induced
hepatocellular carcinomas in male and female rats and in female
mice. In female rats, mammary adenocarcinomas were induced. The
substance is mutagenic in microbial systems. It is a genotoxic
carcinogen, and its activity is enhanced by the methyl group ortho
to the amino group.
Methylene—bis(o—chloraniline) is clearly more carcinogenic than
is methylenedianiline (Munn, 1967). It provides an example of the
enhancing effects of ortho chloro substitution; ortho methyl
substitution also enhances carcinogenicity.
Furazolidine is a borderline carcinogen. This is to be expected
from its single—ring structure, as activity in the nitro
heterocyclic series is highest when two aromatic heterocyclic ring
systems exist in the molecule.
76
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24:2018—2031.
Miller, E.C., T.L. Fletcher, A. Margreth, and J.A. Miller. 1962. The
carcinogenicities of derivatives of fluorene and biphenyl: Fluoro
derivatives as probes for active sites in 2—acetylaminofluorene.
Cancer Res. 22:1002—1014.
Munn, A. 1967. Occupational bladder tumors and carcinogens: Recent
developments in Britain. Pp. 187—193 in K.F. Lampe, R.A. Penalver,
and A. Soto, eds. Bladder Cancer: A Symposium. Aesculapius
Publishing Co., Birmingham.
Nagao, M., T. Yahagi, T. Kawachi, T. Sugimura, T. Kosuge, K. Tsuji, K.
Wakabayashi, S. Mizusaki, and T. Matsumoto. 1977. Coniutagenic
action of norharman and harman. Proc. Jap. Acad. 53(2):95—98.
National Cancer Institute. 1978. 13—Week subchronic toxicity studies
of Direct Blue 6, Direct Black 38, Direct Brown 95 dyes. ITS
Carcinogenesjs Technical Report Series No. 108. DHEW Publication No.
(NIH) 78—1358. U.S. Dept. of Health, Education, and Welfare, Public
Health Service, National Institutes of Health, Bethesda, Md. 127 Pp.
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National Cancer Institute. 1978a. Bioassay of 2,4—dinitrotoluene for
possible carcinogenicity. CAS No. 121—14—2. ITS CarcinogeflesiS
Technical Report Series No. 54. DHEW Publication No. (NIH) 78—1360.
U.S. Dept. of Health, Education, and Welfare, Public Health Service,
National Institutes of Health, Bethesda, Md. [ 99) pp
National Cancer Institute. l978b. Bioassay of
4—chloro—o—phenylenediamine for possible carcinogenicity. CAS No.
95—83—0. ITS Carcinogenesis Technical Report Series No. 63. DHEW
Publication No. (NIH) 78—1313. U.S. Dept. of Health, Education, and
Welfare, Public Health Service, National Institutes of Health,
Bethesda, Md. [ 94) pp.
National Cancer Institute. 1978c. Bioassay of 5—nitro—o—toluidine for
possible carcinogenicity. CAS No. 99—55—8. ITS Carcinogenesis
Technical Report Series No. 107. DHEW Publication No. (NIH)
78—1357. U.S. Dept. of Health, Education, and Welfare, Public Health
Service, National Institutes of Health, Bethesda, Md. (95) pp.
National Cancer Institute. 1978d. Bioassay of 4—nitroanthranilic acid
for possible carcinogenicity. CAS No. 619—17—0. ITS Carcinogenesis
Technical Report Series No. 109. DHEW Publication No. (NIH)
78—1364. U.S. Dept. of Health, Education, and Welfare, Public Health
Service, National Institutes of Health, Bethesda, Md. (105) pp.
81
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National Cancer Institute. 1978e. Bioassay of
2—chloro—p—phenylenediamine sulfate for possible carcinogenicity.
CAS No. 61702—44—1. ITS Carcinogenesis Technical Report Series No.
113. DHEW Publication No. (NIH) 78—1368. U.S. Dept. of Health,
Education, and Welfare, Public Health Service, National Institutes of
Health, Bethesda, Md. [ 87) pp.
National Cancer Institute. l978f. Bioassay of 5—njtro—o—anisidine for
possible carcinogenicity. CAS No. 99—59—2. ITS Carcinogenesis
Technical Report Series No. 127. DHEW Publication No. (NIH)
78—1382. U.S. Dept. of Health, Education, and Welfare, Public Health
Service, National Institutes of Health, Bethesda, Md. (117) pp.
National Cancer Institute. 1978g. Bioassay of aniline hydrochloride
for possible carcinogenicity. CAS No. 142—04-1. ITS Carcinogenesis
Technical Report Series No. 130. DHEW Publication No. (NIH)
78—1385. U.S. Dept. of Health, Education, and Welfare, Public Health
Service, National Institutes of Health, Bethesda, Md. [ 96) pp.
National Cancer Institute. l978h. Bioassay of 2—5—toluenediamine
sulfate for possible carcinogenicity. CAS No. 6369—59—1. ITS
Carcinogenesis Technical Report Series No. 126. DHEW Publication No.
(NIH) 78—1381. U.S. Dept. of Health, Education, and Welfare, Public
Health Service, National Institutes of Health, Bethesda, Md. (101]
pp.
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National Cancer Institute. 1979a. Bioassay of
2—nitro—p—phenylenediamine for possible carcinogenicity. CAS No.
5307—14—2. ITS Carcinogenesis Technical Report Series No. 169.
DHEW Publication No. (NIH) 79—1725. U.S. Dept. of Health, Education,
arid Welfare, Public Health Service, National Institutes of Health.
Bethesda, Md. [ 84] pp.
National Cancer Institute. 1979b. Bioassay of 4—nitro—o—phenyldiamine
for possible carcinogenicity. CAS No. 99—56—9. ITS Carcinogenesis
Technical Report Series No. 180. DHEW Publication No. (NIH)
79—1736. U.s. Dept. of Health, Education, and Welfare, Public Health
Service, National Institutes of Health, Bethesda Md. (85) pp.
National Cancer Institute. 1979c. Bioassay of p—cresidine for
possible carcinogenicity. CAS No. 120—71—8. ITS Carcinogenesis
Technical Report Series No. 142. DHEW Publication No. (NIH)
79—1397. U.s. Dept. of Health, Education, and Welfare, Public Health
Service, National Institutes of Health, Bethesda, Md. [ 105) pp.
National Cancer Institute. 1979d. Bioassay of p—chloraniline for
possible carcinogenicity. CAS No. 106—47—8. ITS Carcinogenesis
Technical Report Series No. 189. DHEW Publication No. (NIH) 79—1745.
U.S. Dept. of Health, Education, and Welfare, Public Health Service,
National Institutes of Health, Bethesda, Md. (90) pp.
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National Cancer Institute. In press. Bioassay of 2,6—dimethylaniline
for possible carcinogenicity. National Cancer Institute
Carcinogenesis Technical Report Series. U.S Public Health Service,
National Institutes of Health, Washington, D.C.
Price, J.M., J.E. Morris, and J.J. Lalich. 1966. Evaluation of the
carcinogenic activity of 5—nitrofuran derivatives in the rat. Fed.
Proc. Fed. Am. Soc. Exp. Biol. 25:419. (Abstract No. 1297).
Radomski, J.L., E. Brill, W.B. Deichmann, and E.M. Glass. 1971.
Carcinogenicity testing of N—hydroxy and other oxidation and
decomposition products of 1— and 2—naphthylamine. Cancer Res.
31:1461—1467.
Rustia, M., and P. Shubik 1972. Induction of lung tumors and
malignant lymphomas in mice by metronidazole. .3. Natl. Cancer. Inst.
48:721—729.
Schribner, J.D., J.A. Miller, and E.C. Miller. 1965.
3—Methylmercapto—N—methyl—4—aminoazobenzene: An alkal me—degradation
product of a labile protein—bound dye in the livers of rats fed
N,N—dimethyl—4—aminoazobenzene. Biochem. Biophys. Res. Commun.
20:560—565.
Stein, R.J., D. Yost, F. Petroliunas, and A. von Esch. 1966.
Carcinogenic activity of nitrofurans: A histological evaluation.
Fed. Proc. Fed. Am. Soc. Exp. Biol. 25:291 (Abstract No. 578).
84
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Weisburger, E.K., A.B. Russfield, F. Homburger, J.H. WeisbL]rger,
E. Boger, C.G. Van Dongen, and K.C. Chu. 1978. Testing of
twenty—one environmental aromatic amines or derivatives for long—term
toxicity for carcinogenicity. J. Environ. Pathol. Toxicol. 2:325—356.
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Chapter 4
CARCINOGENIC POTENCY AND RISK ESTIMATION
In this chapter the committee uses information concerning the
carcinogenicity of aromatic amines to suggest a means for expressing
concepts of carcinogenic potency and for estimating risk.
Aromatic amines are excellent candidates for risk assessment. They
have been assayed for carcinogenicity in several animal species and
strains, and some members of this class (2—naphthylamine, benzidine,
4—aminobiphenyl, and probably phenacetin) are indeed bladder tumor
inducers in humans. l—Naphthylamine, as manufactured at one time, was
associated with bladder cancer in workers; this was probably due to a
high level (4—10%) of contamination with 2—naphthylamine. Of course,
there are no reliable data concerning the level of adventitious human
exposure in the workplace or elsewhere, although the amount of chemical
administered to laboratory animals is readily determined. Limited data
may possibly be obtained from studying iatrogenic carcinogens, e.g.,
2—naphthylamine mustard (Thiede and Christensen, 1975), but in such
cases, animal data are usually deficient.
CARCINOGENIC POTENCY
The potency of a carcinogen depends on three factors: the dose of
carcinogen required to induce tumors, the time to tumor induction, and
the percentage of tumor response. For purposes of the following
discussion the committee has defined potency as
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potency = 7 —log dE5O
where dE SO is the dose expressed in pmol/kg body weight/week
required to induce tumors in 50% of the animals in a lifetime
experiment. This dose is expressed as a logarithm in order to
compress the range of values to a readily understandable format and to
avoid undue emphasis on small differences in potency that lack
biologic significance. The negative of the logarithm is used because
potency is inversely related to the dose required to induce tumors,
and the number 7 is used to bring all values to a readily
comprehensible positive form.
As calculated according to the above criteria, typical potencies
of a number of carcinogens that induce liver or bladder tumors in rat
or mouse are shown in Table 4—1. The data illustrate the range of
potency from the most potent (aflatoxin Bl) to the least potent
(trichioroethylene and saccharin) carcinogens. The calculations used
to determine these values are admittedly approximate. For example, an
approximation has been made that in attaining a 50% dose response, the
dose experimentally shown to give a 25% tumor incidence has been
doubled; likewise, doses that lead to tumors in half the lifespan of
the animal have been halved to calculate dE S O. These assumptions
may well be adequate because the logarithmic scale compresses small
differences. The values for the lifespan of several test species and
their food and water consumption requirements are compared in Table
4—2. These are the values used for dE So calculations. Better
approximations would lead to more accurate potency values.
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Table 4—1
The Potency of a Range of Carcinogens to Rat or
Mouse Liver Following Continuous Feeding
Chemical Species log 10 dE5Q Potency
(mg/kg/week)
Aflatoxin Bi Rat 0.67 9.18
Michler’s ketone Rat 4.88 4.62
Dimethylnitrosamine Rat 4.90 4.00
Carbon tetrachioride Rat 5.27 3.87
2—Aininoanthraquine Rat 6.72 4.44
Trichloroethylene Mouse 7.03 2.12
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Table 4—2
Factors Used for Calculating dE5o in Animals
Factors(s) Mouse Rat Dog
Lifespan (years) 2.5 3.0 10
Food (g/day) 4—6 12—15 300—500
Drinking water (mi/day) 2.1 20.0 NI@.
Gestation (days) 21.0 21.0 63
Weight (g) 25—40 100—500 10,000
. NE = not known
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Certain features of this potency derivation need emphasis.
First, the dose rate ( imol/kg body weight/week) is used in
preference to the total dose to avoid giving the impression that
long—lived species are less sensitive to carcinogens than are
shorter—lived species. For example, it has been accepted that dogs
are sensitive to the carcinogenicity of 2—naphthylamine, and that
mice are less sensitive. This finding is shown in Table 4—3. Total
exposures (450—500 weeks in dogs compared to 100 weeks in mice)
indicates that dogs and mice are more similarly sensitive to this
chemical.
The potency as calculated here defines one point on the tumor
incidence—dose curve. If one accepts the linear one—hit model
(National Academy of Sciences, 1976) of carcinogenesis, which, is
probably not universally accurate, as shown later in this chapter,
the potency value effectively defines the slope of the dose—response
curve. Whatever tumor incidence—dose model is used, the potency
values discussed here have a considerable advantage in that only
they may be derived without excessive data extrapolation. In many
cases, where two or more doses of a carcinogen have been used,
interpolation rather than extrapolation may be required.
Tables 4—4 and 4—5 show the potency values for 4—aminobiphenyl
and methylene—bis(o—chloraniline) in different species. Although
the potency of a carcinogenic aromatic amine in different species
appears to lie within two orders of magnitude, this impression, to a
certain extent, is false; species that fail to respond to an aromatic
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Table 4-3
Potency of 2—Naphthylamine in Different Species
Followir Oral dministration
Species log 10 dE5O Potency
(mg/kg/week)
Dog 4.92 4.34
Mouse 5.23 3.93
Rat 6.72 2.74
Hamster 6.72 2.14
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Table 4—4
Potency of 4--Aminobiphenyl Following Administration
to Different Species
Route of
Species Administration Tissue Potency
Dog Oral Bladder 6.22
Mouse Gavage Liver 4.52
Rat Subcutaneous Intestine 4.37
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Table 4—5
Potency of Methylene—bis(o—chloranilifle) in
Various Species Following Oral Administration
log 10 dE5O
Species Tissues (mg/kg/week) Potency
Dog Bladder 4.57 4.86
Rat . (adequate diet) Lung 4.81 4.57
Breast 5.15 4.25
Zymbal’s gland 4.85 4.58
Liver 4.72 4.70
Rat . (low protein diet) Lung 5.08 4.35
Breast 5.48 3.95
Zymbal’s gland 5.18 4.25
Each value of dE5Q is calculated as life correction x tumor
yield correction x dose x 105 pg/kg/week. It is assumed that
rats live 95—104 weeks and consume an average 105 g/food/week
and that dogs live an average of 9 years, or 468 weeks.
Only male animals were used.
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amine are omitted from the tables. It is not possible to decide
whether these nonresponsive species are completely insensitive to
the particular agent or are sensitive at a level too low to be
detected in the animal bioassay. The cutoff point for the
sensitivity of a carcinogenesis bioassay is determined first by the
toxicity of the chemical being tested and, second, by the maximum
amount of test substance it is reasonable to give to an animal
during any one period. The use of the maximum—tolerated dose, as
recommended in the bioassay protocols of the National Cancer
Institute/National Toxicology Program (Sóntag et al., 1976) , ensures
that the highest feasible dose level is used, but that this level
may lead to abnormal results due to the intervention of the agent’s
toxic properties in the carcinogenic process. However, substances
with a very low level of toxicity may, if the maximum tolerated dose
is used, be administered at levels that interfere physiologically
with the host, for example, by inducing nutritional imbalance, and
similarly lead to difficulty in interpreting results.
At present, it is not possible to predict with confidence that
the demonstration of potency of a carcinogen in one animal species
means the carcinogen will be as potent in another species. Thus, it
may be prudent to assume (since it cannot be tested in humans) that
humans are at least as sensitive to a carcinogen as are the most
sensitive species. Consequently, 2—naphthylamine in humans could be
assumed to be as potent as it is in dogs (Table 4—3) and
methylene—bis—(o—chloraniline) to be as potent in humans as it is in
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rats (Table 4—5). Table 4—6 shows the carcinogenic potency of
N,2—fluorenylacetamide in different species and strains of test
animals. Ways of predicting carcinogenic potency based on the
present knowledge of the mechanisms of carcinogenesis urgently need
to be expanded. Crouch and Wilson (1979) discuss interspecies
differences in carcinogenic potency, including humans; however, they
do not describe how they calculate potency to humans in the absence
of reliable exposure data.
HIGH- TO LOW-DOSE EXTRAPOLATION
It is now generally regarded as prudent to assume, when
extrapolating data from high carcinogenic dose rates in animals to
low—dose exposure in humans, that the mathematically simple linear
one—hit model is appropriate even if the limited experimental
evidence does not necessarily support this conclusion. The
relevance of this model, which implies that a single
carcinogen—critical receptor interaction is involved, has not been
seriously questioned, although it may prove unsuitable in many cases.
Chemical carcinogenesis is a multifaceted process. It is
generally accepted that the procarcinogen has to be metabolically
activated to the ultimate or reactive form, and the ultimate
carcinogen then has to interact with its critical target. When the
critical target is DNA, this interaction is followed by DNA
replication to “lock in” genetic damage, or by DNA repair to restore
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Table 4—6
The Carcinogenic Potency of N,2-Fluorenylacetamide in
Different Species and Strains of Test Animals !
Species Route Site of Tumor Potency
Dog Diet Liver, bladder 4.5
Rabbit Gavage Bladder, ureter 4.46
Hamster Diet Gall bladder 4.29
Rat
Slonaker (M+F) Diet Bladder 4.40
Wistar (M) Diet Liver 5.14
Wistar (F) Diet Breast 5.03
Piebald (M+F) Diet Intestine 4.93
Mouse
BALB/c (F) Diet Liver 4.17
BALB/c (F) Diet Bladder 4.17
! Some evaluations based on early studies.
M = male; F = female.
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the cells to quasi-normality, and the damaged cells or tumor
progenitor cells then undergo various biologic interactions before
frank clinical neoplasia is observed. It is difficult to conceive
all these changes involving only a single interaction between the
carcinogen and its critical receptor(s)
The simplest biologic model of carcinogeneSis is the two—stage
hypothesis, proposed by Berenbium and Shubik (1947a,b; 1949) in
mouse skin, and now being shown applicable for many other tissues
such as liver, bladder, and pancreas. This model may not
effectively describe what may well be a more complex multi—stage
process. Initiation and promotion are separate processes induced b
different agents. Therefore, they are independent of each other. A
complete carcinogen, however, is capable of both initiation and
promotion. Since both processes are presumably dose dependent, an
exponential rather than linear relationship should exist between
dose of carcinogen and tumor response. A linear relationship be
applicable for pure initiators or pure promoters.
The validity of these suggestions is suggested by the large
scale (ED 01 ) experiment conducted by the National Center for
Toxicological Research (Staffa and Mehlman, 1979), in which low
levels of N,2—fluorenylacetamide were fed to BALB/c mice. The liver
tumor yield was apparently related linearly at low doses to the
carcinogen dose; the bladder tumor yield was not (Figure 4—1).
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0
1—
I
I —
0
I —
0
a-
0
a-
Figure 4—1
Comparison between liver and bladder tumor yield in
response to different doses of N,2—fluorenylacetamide
given over 33 months. These data are for BALB/c mice,
(Staffa and Mehiman, 1979).
1.
Liver
33 months
0
0
0
0
All
Bladder
I
0
0
20
40
60
DOSE (ppm
80
120
140
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Between 24 and 33 months, BALB/c mice developed a significant incidence
of naturally occurring liver tumors, which the carcinogen enhanced.
The naturally occurring incidence of bladder tumors was very low.
Thus, one explanation of the dose—response relationships exhibited is
that the carcinogen must act as both initiator and promoter in bladder
carcinogenicity to give a tumor incidence—dose curve that is very
differently shaped than that for the liver, where only promotion occurs.
These observations could mean that, although a linear dose—response
curve may be appropriate in specific cases (possibly with pure
initiators or with pure promoters acting on an appreciable spontaneous
tumor yield), it may be inappropriate in other cases. The use of
linear dose—response relationships may indicate a level of risk higher
than actually occurs. This result is possibly beneficial in that it
provides estimates at a time when methods for risk estimations are
little understood. As risk assessment techniques become more precise
it may become possible to make estimates with greater accuracy.
The use of these statistical models to estimate possible risk to
humans at very low exposures is filled with uncertainty. Studies with
animals usually involve exposure to a high level of a single carcinogen
and, sometimes, just one modifying agent. In the real, nonexperimental
world, humans are exposed to a wide range of carcinogens and
carcinogenesis—modifying agents, which may enhance or inhibit cancer
development due to low levels of a particular agent. The suggestion
that a given agent will induce, for example, one tumor in a population
of a million is, under these conditions, trite speculation.
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REFERENCE S
Carcinogenic Potency and Risk Estimation
Berenbium, I., and P. Shubik. 1947. The role of croton oil
applications, associated with a single painting of a carcinogen,
in tumour induction of the mouse’s skin. Br. J. Cancer 1:379—382.
Berenbium, I., and P. Shubik. 1947. A new, quantitative approach
to the study of the stages of chemical carcinogenesis in the
mouse’s skin. Br. J. Cancer 1:383—391.
Berenbium, I .,, and P. Shubik. 1949. The persistence of latent
tumour cells induced in the mouse’s skin by a single application
of 9 :l0—dimethyl—1;2—benzanthracene. Br. J. Cancer 3:384—386.
Crouch, E., and R. Wilson. 1979. Interspecies comparison of
carcinogenic potency. J. Toxicol. Environ. Health 5:1095—1118.
National Academy of Sciences. 1976. Drinking Water and Health.
Safe Drinking Water Committee, Assembly of Life Sciences, National
Research Council, Washington, D.C. 938 pp.
Sontag, J.M., N.P. Page, and U. Saffiotti. 1976. Guidelines for
Carcinogenic Bioassay in Small Rodents. NCI—CG—Tr—l. U.S.
Department of Health, Education, and Welfare, Public Health
Service, National Institutes of Health, National Cancer Institute,
Washington, D.C. 65 pp.
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Staffa, J.A., and r4.A. Mehlman, eds. 1980. Innovations in Cancer
Risk Assessment (ED 01 Study): proceedings of a Symposium
Sponsored by the National Center for Toxicological Research, U.S.
Food and Drug Administration, and the American College of
Toxicology. J. Environ. Pathol. ToxiCol. 3(3):l—246.
Thiede, T., and B.C. Christensen. 1975. Tumours of the bladder
induced by chiornaphazine treatment. Ugeskr. Laeg. 137:661—666
[ in Danish; English abstract).
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Chapter 5
EPIDEMIOLOGIC ASPECTS OF EXPOSURE TO AROMATIC AMINES
The studies of pase et al. (1954a,b) in Great Britain provided
the first systematic evidence of the carcinogenicity of specific
aromatic amines. More recently, Clayson (1976), Clayson and Garner
(1976), and the working group for the International Agency for
Research on Cancer (1972, 1974a, 1978a) have concluded that bladder
cancer is caused in workers who manufacture and use 2—naphthylamine,
benzidine, and 4—aminobiphenyl. It is difficult to identify persons
exposed only to l—naphthylamine, auramine, magenta, and
N—phenyl--2—naphthylamine (International Agency for Research on
Cancer, 1972, 1974a, l978a); thus, it has not been possible to
determine whether any one compound alone is carcinogenic in numans.
It is generally accepted that aniline has not been shown to cause
bladder cancer in humans (International Agency for Research on
Cancer, 1974a).
The data from the studies of Case et al. are presented in Table
5—1. The largest increase in fatalities occurred among men exposed
to a mixture of compounds. Increased mortality rate is
statistically significant and has been interpreted as causal for
2—naphthylamine and benzidine. The increased mortality rate among
workers exposed to l—naphthylamine is complicated by the fact that
l—naphthylamine contains 4—10% of 2—naphthylamine.
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Table 5—1
Observed and Expected Deaths from Bladder Cancer According to
Class of Exposure to Specific SubstancesL
Class Observed Expected
Aniline without benzidine, naphthylamifle, 1 0.83
magenta, or auramine contact
Aniline with magenta contact 3 0.13
Aniline with auramine contact 6 0.45
Benzidine 10 0.72
l—Naphthylamine 6 0.70
2—Naphthylamine 26 0.30
Mixed exposure 81 1.48
From Case et al., 1954a,b, with permission.
Expected numbers derived from age—time specific mortality rates for
British males.
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The excess mortality among persons having had contact with
magenta or auramine has been interpreted as indicating that bladder
cancer is associated with the manufacture of these chemicals, not
necessarily with the substances themselves (Case and Pearson,
1954b). Melick etal. (1971) concluded that 4—aminobiphenyl is
carcinogenic in humans from their study that 53 of 315 men exposed
to the substance developed bladder cancer.
Studies of persons in the chemical and rubber industries, who
were exposed during the manufacture and use of the compounds, have
provided evidence that aromatic amines are carcinogenic. Benzidine
and naphthylamines are used to manufacture dyes; naphthylamines and
4—aminobiphenyl are antioxidants used to manufacture rubber.
Persons who were exposed only to benzidine, 2—naphthylamine, or
4—aminobiphenyl, and who developed bladder cancer can be identified;
from this, a causal association between exposure to these chemicals
and bladder cancer has been derived.
Another reason the above aromatic amines are accepted as a cause
of bladder cancer in humans is that the rate of bladder cancer among
those exposed is many times greater than the rate among persons not
exposed. Not only is it relatively straightforward to detect the
increased cancer incidence among a comparatively small group of
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exposed persons, it is also clear that apart from the possible
effects due to cigarette smoking and certain drugs no other cause of
bladder cancer is responsible for the increase. This situation is
in Contrast to the usual one in epidemiology where, among a group of
exposed persons, the rate of the disease of interest is elevated by
perhaps only 50—100%. Also, it is often impossible to eliminate or
control for the possible effects of other causes of the disease.
Tables 5—2 through 5—5 contain data on mortality and incidence
of cancer in the rubber industry from 1940 to 1973. These data were
assembled by Monson (1978) from studies of rubber workers in the
United States and Great Britain (Andjelkovich et al., 1976; Fox and
Collier, 1976; McMichael et a]., 1976; Monson and Fine, 1978), who
died between 1964 and 1974.
In the United Kingdom, the rubber industry used as a
rubber—compounding ingredient a mixture containing a condensate of
acetaldehyde with l—naphthylamine and 2—naphthylamine (Nonox S). The
discovery that there was an increased incidence of bladder cancer
mortality in those who worked with these compounds (Case and Hosker,
1954) and an associated increased mortality rate in electric
cablemakers (Davies, 1965) using contaminated rubber led, in 1949,
to the abandonment of the use of Nonox S and other related bladder
carcinogens by the rubber industry. Fox and Collier (1976) reported
on a survey designed to determine whether the action taken in 1949
had removed the bladder cancer hazard from the industry, but
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Table 5—2
Characteristics of Four Groups of Male
Rubber Workers
Mi n irnum
Number of Initial Years Years
Group Location Ethnicity Workers Ages Employed Followed
A Akron All 6,678 40—84 10 1964—72
B Great Britain All 40,867 3565 1 1968—74
C Akron White 13,571 20—79 5 1940—74
D Akron White 8,418 40—84 10 1964—73
! From Monson, 1978, with permission.
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Table 5—3
General Mortality Among Four Groups of Male
Rubber Workers
Group A Group B Group C Group D
Cause of deaths Obs Obs Exp Obs Exp Obs
All causes 1873 1798.5 4079 4055.7 5079 6186.9 2373 2524.5
All cancers 351 336.9 1256 1106.0 980 1046.4 457 456.3
Circulatory disease 953 940.0 1999 2022.6 2938 3482.8 1311 1351.7
External causes 59 83.6 118 138.4 278 446.8 91 111.0
Residual 420 438.0 706 788.7 833 1210.9 514 605.5
! From Monson, 1978, with permission.
Expected numbers based on mortality rates for:
A - U.S males
B — English and Welsh males
C — U.S white males
D — U.S white males
Slight differences exist in classification of cause of death.
£ Observed.
Expected.
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Table 5—4
Mortality from Specific Types of Cancer Among
Four Groups of Male Rubber Workers!’
Group A
Type of Cancer Obs. .
Group
B
Group
C
Group
Obs.
D
Exp.
Obs.
.
Obs.
Stomach 39 20.9 153 122.3 98 93.9 34 27.6
Large Intestine 39 31.8 104 103.1 53 45.7
Lung 91 109.3 585 493.5 234 253.1 116 139.8
Prostate 49 34.4 82 89.0 50 45.9
Bladder 9 12.3 60 38.9 48 39.5 21 18.1
Leukemia 16 12.5 28 23.3 55 43.0 25 18.1
. From Monson, 1978, with permission.
Expected numbers based on mortality rates for:
A — U.S males
B — English and Welsh males
C — U.S. white males
D — U.S. white males
£ Obs. = Observed
Exp. = Expected
Slight differences exist in classification of type of cancer.
! Data not given.
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Table 5—5
Observed and Expected Numbers of Bladder
Cancers in Selected Work Areas
Group Work Area Observed Expected
A Receiving and shipping 2 0.7
Tire building 1 2.5
B Tires 20 15.3
C Warehouse/shipping 8 2.6
Tire building 9 4.8
D Product fabrication . 5 2.6
. From Monson, 1978, with permission.
Product fabrication is subset of tire building.
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they refrained from reaching a conclusion because the number of
tumors occurring in workers employed after 1949 still had not
reached a sufficient level for a statistically valid comparison.
Table 5—3 shows that there is little to suggest a substantial
increase in fatal cancer among rubber workers. The greatest
increase in bladder cancer has been observed among British rubber
workers (Table 5—4). However, compared to the data in Table 5—1,
the increment is relatively small. In the U.S. studies, it has not
been possible to obtain detailed exposure histories as did Case ,
a).. (1954a, l954b). It was only possible for the investigators to
group workers on the basis of where they worked within the factory.
The data in Table 5—5 show the observed and expected numbers among
men who make tires and who have close contact with uncured rubber.
There is minimal evidence that these men have an increased incidence
of bladder cancer. Also, among men who make rubber and who might be
expected to come into contact with antioxidants such as
2 —naphthylamjne or phenyl—2—naphthylamine, no increased incidence of
fatal bladder cancer was identified.
On balance, there is minimal evidence of an increased incidence
of bladder cancer among American rubber workers. Rather than using
1— or 2—naphthylamine as antioxidants, the American rubber industry
has used phenyl—2—naphthylamine. Ingestion of phenyl—2—
naphthylamine has been shown to be associated with the appearance of
minimal amounts of 2—naphthylamine in the urine
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(Kummer and Tordoir, 1975). Among an unknown number of men who
worked for at least 15 years with a phenyl—2—naphthylamine autoclave,
two developed bladder cancer (Monson and Fine, 1978).
HAIR DYES
A number of aromatic amines are used in the manufacture of hair
dyes. It has been speculated that users and appliers of hair dyes
might be at an increased risk of developing cancer; however, it has
not been possible to assess with certainty the effects on humans of
specific substances used in hair dye. These are not the only
substances to which humans are exposed, and therefore it is not
possible to identify persons with isolated exposure. Furthermore,
since the adverse effect most postulated is cancer, any substance in
hair dyes leading to cancer in either appliers or users will require
many years of observation before the effect can be detected. Unless
the adverse effect is very strong, it may not be possible to gather
sufficient data on which to base an association.
Volume 16 of the IARC Monographs of the Evaluation of
Carcinogenic Risk of Chemicals to Humans (International Agency for
Research on Cancer, l978b), presents a general review of the data
available on the exposures of humans to hair dyes. For users, the
results were judged equivocal. There was more evidence for an
increased risk of cancer at certain sites (bladder, lung, larnyx) for
persons with occupational exposure. However, further epidemiologic
studies were recommended before any firm conclusions are drawn.
i-li
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Reports since that review have not provided more conclusive
data. In a followup study of cosmetologists (Wairath, 1978), breast
cancer occurred less often than was expected, and leukemia occurred
more often than expected. Four recent case—control studies provided
minimal evidence for an excess incidence of breast cancer. Crude
relative risks between hair dye use and breast cancer were 0.83
(Shore et al., 1979), 1.06 (Hennekens et al., 1979) , 1.11 (Stavraky
et al., 1979), and 1.28 (Nasca et a]., 1980). Although stronger
associations were observed between subsets of each study group, all
authors cautioned against overinterpretation of the results of each
individual study.
In summary, the epidemiologic data relating hair dyes and cancer
are inconclusive. There is some suggestion that persons with
occupational exposure to hair—care products are at increased risk of
developing cancer and less evidence that users of hair dyes develop
such cancer. These inconclusive studies need to be balanced by the
positive results for carcinogenicity and mutagenicity found for a
number of the ingredients in hair dyes.
DRUGS
Phe n ace tin
Among a group of employed Swiss women, those who were regular
users of compounds containing phenacetin developed increased serum
creatinine levels and low urine specific gravity in comparision to
those of controls (Dubach et al., 1975; International Agency for
Research on Cancer, 1977). Abuse of analgesics has been reported to
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be associated with the development of renal papillary necrosis
(Bengtsson et al., 1978). In a number of clinical and epidemiologic
studies, heavy users of phenacetin—containing compounds have
reported an increased incidence of cancer of the kidney and of the
bladder (Fokkens, 1979; International Agency for Research on Cancer,
1977) . On the basis of these reports, it seems prudent to associate
heavy phenacetin use with kidney and bladder disease in humans.
Chloranaphaz me
Chiornaphazine (N,N—bis(2—ch lorOethyl)—2—flaPhthY lamifle), a
derivative of 2—naphthylamine, has been used to treat persons with
polycythemia and Hodgkin’s disease. It is generallly accepted that
this antineoplastic agent has led to the development of bladder
cancer in humans (Hoover and Fraumeni, 19767 International Agency
for Research on Cancer, 1974a; Lower and Bryan, 1979; Thiede and
Christensen, 1975; Thiede et al., 1964).
Tobacco
Cigarette smoking is the major cause of lung cancer and is
associated with the increased incidence of many types of cancer,
including bladder cancer (Hammond, 1975). Arylamines and
nitrosamines occur in tobacco smoke (Lower and Bryan, 1979).
Although the presence of these amines may cause bladder cancer in
smokers, the link at the moment is tenuous.
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Amitrole (3—amino—l,2,4—triazole )
Amitrole residues were found in cranberries in the United States
in 1959. Sale of cranberries and cranberry products from 1958 and
1959 crops were prohibited because amitrole produced thyroid tumors in
rats (International Agency for Research on Cancer 1974b).
In Sweden, amitrole was used as an herbicide from the 1950’s to
the 1970’s (Axelson et al., 1974). Increased cancer incidence and
mortality rates have been reported among Swedish railway workers who
sprayed amitrole and the chlorophenoxyacetic acids (2,4—D and 2,4,5—T)
(Axelson etal., 1974, 1979). Among 348 workers, 18 cases of cancer
occurred, in comparision to the 11.9 expected from the Swedish
incidence rates. Because of the difficulty in separating the possible
effects of amitrole and the phenoxy acids, and because many different
types of cancer occurred among those exposed, it is difficult to judge
whether this excess may be causally related to amitrole exposure.
RECOMMENDATIONS FOR EPIDEMIOLOGIC STUDY OF AROMATIC AMINES
Hair dyes and nitrosamines are the substances that are of most
current interest as to their potential carcinogenicity in humans.
Nitrosamines are discussed in more detail in a companion report on
aliphatic amines. Hair dyes are widely used, and case—control studies
have raised suspicions that they are associated with cancer of the
breast and other sites. Nitrosamines are recognized to be potent
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carcinogens in animals and are present in low concentrations in many
substances to which humans are exposed. However, there is currently
little evidence upon which to judge their carcinogenicity in humans.
The only realistic study design to evaluate the carcinogenicity
of these substances is the prospective follow—up study. To study
the effects of hair dyes, women should be interviewed to determine
their life—time use of hair dyes and followed for 5—20 years to
measure the rate of occurrence of cancer. For nitrosamines, persons
exposed to relatively high levels in the workplace should be
identified, categorized as to level of current (and future)
nitrosamine exposure, and followed from 20 to 40 years.
Retrospective studies are not as likely to provide definitive
information on carcinogenicity in humans. In case—control studies
of cancer, the recollection of hair dye use is subject to a high
degree of recall bias. In retrospective cohort studies of persons
exposed to nitrosamines, there is a very imprecise measurement of
exposure to nitrosamines.
Also, cross—sectional or short—term prospective cohort studies
can be conducted on workers exposed to nitrosamines. Here, the
health outcome would be either acute illness or physiologic
abnormality. These studies would provide an initial evaluation of
the association between exposure to nitrosamine and human health.
However, they would not be expected to address carcinogenicity.
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The epidemiologic evaluation of the possible health effects from
exposure to low levels of aromatic amines as well as to other
substances may not be possible. To the extent that disease among an
exposed group is increased relatively little above background,
perhaps less than 50%, the excess may not be detectable against the
background variability. One of the best ways to minimize this
variability is to conduct prospective follow—up studies, so that at
least the measure of exposure is as precise as possible. However,
if this strategy is adopted, long—term follow—up is the price that
must be paid.
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References
Epidemiology
Andjelkovich, D., J. Taulbee, and M. SymonS. 1976. Mortality
experience of a cohort of rubber workers, 1963—1973. 3. Occup.
Med. 18:387—394.
Axelson, 0., C. Edling, H. Kling, K. Anderson, C. Hogstedt, and L.
Sundell. 1979. tippdatering av mortaliteten hos
bekampningsmedelsexponerade banarbetare. Lakartidningen
76:3505—3507.
Axelson, 0., and L. Sundell. 1974. Herbicide exposure, mortality
and tumor incidence. An epidemiological investigation on Swedish
railroad workers. Work Environ. Health 11:21—28.
Bengtsson, U., S. Johansson, and L. Angervall. 1978. Malignancies
of the urinary tract and their relation to analgesic abuse.
Kidney mt. 13:107—113.
Case, R.A.M., and M.E. Hosker. 1954. Tumour of the urinary bladder
as an occupational disease in the rubber industry in England and
Wales. Br. J. Prey. Soc. Med. 8:39—50.
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Case, R.A.M., M.E. Hosker, D.B. McDonald, and J.T. Pearson. 1954a.
Tumours of the urinary bladder in workmen engaged in the
manufacture and use of certain dyestuff intermediates in the
British chemical industry. Part I. The role of aniline,
benzidine, alpha—naphthylamine, and beta—naphthylamine. Br. 3.
md. Med. 11:75—104.
Case, R.A.M., and J.T. Pearson. 1954b. Tumours of the urinary
bladder in workmen engaged in the manufacture and use of certain
dyestuff intermediates in the British chemical industry. Part
II. Further considerations of the role of aniline and of the
manufacture of auramine and magenta (fuchsine) as possible
causative agents. Br. 3. md. Med. 11:213—216.
Clayson, D.B. 1976. Occupational bladder cancer. Prey. Med.
5:228—244.
Clayson, D.B., and R.C. Garner. 1976. Carcinogenic aromatic amines
and related compounds. Pp.366—461 in C.E. Searle, ed. Chemical
Carcinogens. ACS Monograph 173. American Chemical Society,
Washington, D.C.
Davies, J.M. 1965. Bladder tumours in the electric—cable
industry. Lancet 2:143—146.
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Dubach, U.C., P.S. Levy, B. Rosner, H.R. Baumeler, A. Muller, A.
Peier, and P. Ehrensperger. 1975. Relation between regular
intake of phenacetin—containing analgesics and laboratory evidence
for urorenal disorders in a working female population of
Switzerland. Lancet 1:539—543.
Fokkens, W. 1979. Phenacetin abuse related to bladder cancer.
Environ. Res. 20:192—198.
Fox, A.J., and P.F. Collier. 1976. A survey of occupational cancer
in the rubber and cablemaking industries: Analysis of deaths
occurring in 1972—74. Br. 3. md. Med. 33:249—264.
Hammond, E.C. 1975. Tobacco. Pp. 131—138 in J.F. Fraumeni, Jr.,
ed. Persons at High Risk of Cancer; An Approach to Cancer
Etiology and Control. Academic Press, New York.
Hennekens, C.H., F.E. Speizer, B. Rosner, C.J. Dam, C. Belanger,
and R. Peto. 1979. Use of permanent hair dyes and cancer among
registered nurses. Lancet 1:1390—1393.
Hoover, R., and J.F. Fraumeni, Jr. 1975. Drugs. Pp. 185—199 in
J.F. Fraumeni, Jr., ed. Persons at High Risk of Cancer; An
Approach to Cancer Etiology and Control. Academic Press, New York.
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International Agency for Research on Cancer. 1972. IARC Monographs
on the Evaluation of Carcinogenic Risk of Chemicals to Man.
Volume 1. International Agency for Research on Cancer, Lyon. 184
pp.
International Agency for Research on Cancer. 1974a. IARC Monographs
on the Evaluation of Carcinogenic Risk of Chemical to Man. Volume
4. Some Aromatic Amines, Hydrazine and Related Substances,
N—Nitroso Compounds and Miscellaneous Alkylating Agents.
International Agency for Research on Cancer, Lyon. 286 pp.
International Agency for Research on Cancer. l974b. IARC Monographs
on the Evaluation of Carcinogenic Risk of Chemicals to Man.
Volume 7. Some Anti—Thyroid and Related Substances, Nitrofurans
and Industrial Chemicals. International Agency for Research on
Cancer, Lyon. 326 pp.
International Agency for Research on Cancer. 1977. IARC Monographs
on the Evaluation of Carcinogenic Risk of Chemicals to Man.
Volume 13. Some Miscellaneous Pharmaceutical Substances.
International Agency for Research on Cancer, Lyon. 255 pp.
International Agency for Research on Cancer. 1978a. IARC Monographs
on the Evaluation of the Carcinogenic Risk of Chemicals to Man.
Volume 16. Some Aromatic Axnines and Related Nitro Compounds——Hair
Dyes, Colouring Agents and Miscellaneous Industrial Chemicals.
International Agency for Research on Cancer, Lyon. 400 pp.
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International Agency for Research on Cancer. 1978b. IARC Monographs
on the Evaluation of the Carcinogenic Risk of Chemicals to
Humans. Volume 17. Some N—nitroso compounds. International
Agency for Research on Cancer, Lyon. 365 pp.
Kummer, R., and W.F. Tordoir. 1975. Phenyl—betanaphthylamine
(PENA), another carcinogenic agent? Tijdschr. Soc. Geneeskd.
53: 415—419.
Lower, G.M., and G.T. Bryan. 1979. Etiology and carcinogenesis:
Natural systems approaches to causality and control. Pp. 29—53 in
N. Javadpour, ed. Principles and Management of Urologic Cancer.
Williams and Wilkins, Baltimore.
McMichael, A.J., R. Spirtas, J.F. Gamble, and P.M. Tousey. 1976.
Mortality among rubber workers: Relationship to specific jobs. .3.
Occup. Med. 18:178—185
Melick, W.F., .3.3. Naryka, and R.E. Kelly. 1971. Bladder cancer
due to exposure to para—aminobipheny].: A 17—year follow—up. 3.
Urol. 106:220—226.
Monson, R.R. 1978. Effects of industrial environment on health.
Environ. Law 8:663—700.
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Monson, R.R., and L.J. Fine. 1978. Cancer mortality and morbidity
among rubber workers. J. Natl. Cancer Inst. 61:1047—1053.
Nasca, P.C., C.E. Lawrence, P. Greewald, S. Chorost, J.T. Arbuckle,
and A. Paulson. 1980. Relationship of hair dye use, benign
breast disease, and breast cancer. J. Nati. Cancer Inst. 64:23—28.
Shore, RE.,, B.S. Pasternack, E.U. Thiessen, M. Sadow, R. Forbes,
and R.E. Albert. 1979. A case—control study of hair dye use and
breast cancer. J. Nati. Cancer Inst. 62:277—283.
Stavraky, K.M. , E.A. Clarke, and A. Donner. 1979. Case—control
study of hair dye use by patients with breast cancer and
endometrja]. cancer. J. Nat].. Cancer Inst. 63:941—945.
Thiede, T., E. Chievitz, and B.C. Christensen. 1964.
Chlornaphazjne as a bladder carcinogen. Acta Med. Scand.
175: 721—725.
Thiede, T., and B.C. Christensen. 1975. Turnouts of the bladder
induced by chlornaphazjne treatment. Ugeskr. Laeg. 137:661—666
(in Danish; English Summary).
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Chapter 6
ANILINE
NH 2
Aniline (also called aminobenzene or benzenaxuine) is a
colorless, oily liquid that freezes at —6.2°C and boils at 184°C.
It is combustible and is moderately soluble in water. At 25°C,
aniline has a vapor pressure of 0.67 mm Hg.
Aniline is one of the most important organic bases and is the
parent compound for more than 300 chemical products. It is
typically produced by the catalytic hydrogenation of riitrobenzene.
The gas—phase reaction of hydrogen and nitrobenzene over a catalyst
at temperatures below 350°C yields more than 98% aniline.
Aniline as a free base is a relatively unstable compound, which
is rapidly oxidized in the presence of air and light to a complex
mixture of quinoneimines, quinones, and highly colored polymers of
unknown composition. It is a weak base that is readily converted to
a water—soluble, stable salt in acid solution (a hydrochloride)
(International Agency for Research on Cancer, 1974; Radomaki, 1979).
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PRODUCTION
Table 6—1 lists the current producers of aniline, their
locations, and their annual capacities.
Three of the companies are planning to increase production in
the near future. Rubicon Chemicals, Inc. plans to expand its
capacity at Geismer, La. by an additional 9,100 metric tons per year
during 1980 (Chemical Marketing Reporter, 1979) . American Cyanamid
Co. will increase the capacity of its Willow Island, W. Va. facility
to a total of 50,000 metric tons per year during 1980 (Chemical
Marketing Reporter, 1979). The Polyurethane Division of Mobay
Chemical Corp. in New Martinsville, W. Va., plans to begin recovery
of aniline from its iron oxide plant in the first quarter of 1981.
Capacity will be 12,000 metric tons. By 1985, Mobay’s polyurethane
capacity is expected to reach 18,000 metric tons (Chemical Economics
Handbook, 1978).
USE S
U.S. consumption patterns of aniline in 1979 are shown in Table
6—2.
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Table 6—1
ANILINE PRODUCERS AND CAPACITIES
Capacity
Producer and Location (103 metric tons)
Rubicon Chemicals, Inc.
Geismar, La. 127
E. I. du Pont de Nemours & Co., Inc.
Beaumont, Tex. 118
Gibbstown, N.J. 73
First Chemical Corp., subsidiary
of First Mississippi Corp.
Pascagoula, Miss. 114
American Cyanamid Co.
Bound Brook, N.J. 27
Organic Chemicals Division
Willow Island, W. Va. 23
Mobay Chemical Corp.,
Industrial Chemicals Division
New Martinsville, W. Va. 45
Total annual U. S. aniline production for recent years:
Thousands of metric tons
1975 1976 1977 1978
247.2 265.5 275.4 309.7
! SRI (Standard Research Institute), 1979.
U.S. International Trade Commission, 1976, 1977, 1978, 1979.
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Table 6—2
ANILINE CONSUMPTION PATTERNS ,
Percent of
Use total 1O . metric tons
Intermediate for monomeric 50 155
and polymeric isocyanates
Intermediate for rubber 27 84
chemicals
Dyes and dye intermediates 6 19
Hydroquinone 5 15
Intermediate for pharmaceuticalS 3 9
Miscellaneous 9 28
. Chemical Marketing Reporter, 1979.
Total U. S. consumption is considered equal to U.S
production; imports and exports are negligible.
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Aniline is used as an intermediate in the production of
, ‘—methylenediphenyl diisocyanate (MDI) and polymeric MDI, which
are used primarily in the manufacture of rigid polyurethane foam for
building insulation (Chemical Economics Handbook, 1978). The U.S.
producers of MDI include Mobay Chemical Corp. in Cedar Bayou, Tex.,
and New Martinsville, W. Va., Eubicon Chemicals, Inc. in Geismar,
La., and the Upjohn Co. in La Porte, Tex. (Stanford Research
Institute, 1979).
The chemicals derived from aniline are used in rubber
manufacture as vulcanization accelerators, antioxidants, and
antidegradants (Northcott, 1978). The most commercially significant
are 2—mercaptobenzothiazole and N—cyclohexyl—2—benzothiazole
(Chemical Economics Handbook, 1978), produced by American Cyanamid
Co. in Bound Brook, N. J. the B.F. Goodrich Co. in Henry, Ill.,
Monsanto Co. in Nitro, W. Va., Pennwalt Corp. in Wyandotte, Mich.,
and Uniroyal, Inc. in Geismar, La. 2—MercaptobenzothiazOle is also
produced by Eastman Kodak Co. in Rochester, N.Y., and the Goodyear
Tire and Rubber Co. in Niagara Falls, N.Y. (Stanford Research
Institute, 1979).
Dyes prepared from aniline and aniline derivatives are included
in the following four dye classes: azo, triphenylmethane,
anthraquinone, and safranines (International Agency for Research on
Cancer, 1974). The Colour Index (1971) lists 174 dyes that can be
prepared from aniline, and more than 700 dyes that can be prepared
from aniline derivatives. Because of the increased use of synthetic
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fibers and stricter controls imposed by the Food and Drug
Administration (FDA), very few of these dyes are currently produced
in commercially significant quantities (Northcott, 1978). One of
the more significant of the aniline—based dyes, from a commercial
standpoint is C.I. (Color Index) Vat Blue 1, used widely to dye
cotton fibers such as those used in denim. C. I. Vat Blue 1 (D&C
Blue No. 6) has also been used as a colorant for surgical sutures.
(Bauer, 1979; 21 CFR 74). This dye is produced by BASF Wyandotte
Corp. in Parsippany, N. J., and Buffalo Color Corp. in Buffalo, N.Y.
(Standford Research Institute, 1979).
Among the commercially more significant dye intermediates
derived from aniline are p—nitroaniline, which is produced by
Monsanto Co. in Sauget, Ill., American Color & Chemical Corp. in
Lock Haven Pa., and the Signal Companies Inc. in Shreveport, La.;
N,N—diethylaniline 1 - and N,N—dimethylaniline, both produced by
American Cyanamid Co. in Bound Brook, N.J., Buffalo Color Corp. in
Buffalo, N.Y., and E.I. du Pont de Nemours & Co. in Deepwater, N.Y.;
and o—, m—, and Q—chloroaniline, which is produced
1 Also used to make 2—chloro—2’,6’—diethyl-N—(methoxymethyl)
acetanilide, an herbicide marketed under the trade name Lasso
(Chemical Economics Handbook, 1978).
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by E. I. du Pont de Nemours & Co., Inc. in Deepwater, N. J. 2 and
2—Chloroaniline are also produced by Monsanto Co.in Luling, La.
(Stanford Research Institute, 1979; Colour Index, 1971).
Aniline is also involved in the production of hydroquinone,
which is used primarily as a developing agent for black—and—white
photography (Woodlief, 1973), and as a polymerization shortstop in
styrene—butadiene rubber production (Bauer, 1979). Hydroquinone is
produced by Eastman Kodak Co. in Kingsport, Tenn. and the Goodyear
Tire & Rubber Co. in Bayport, Tex. (Stanford Research Institute,
1979.
In the pharmaceutical industry, aniline is used in the
production of acetanilide, which was once widely included in
analgesic and antipyretic formulations: it is currently used as an
intermediate in the manufacture of most sulfanilamide drugs
(Northcott, 1978). Pharmaceutical aniline is produced by Eastman
Kodak Co. in Rochester, N.Y., Merck & Co., Inc. in Albany, Ga.,
Salisbury Laboratories in Charles City, Iowa, and Syntex Corp. in
Newport, Tenn. (Stanford Research Institute, 1979).
There are a number of miscellaneous applications of aniline. It
is used in the production of intermediates for herbicides,
fungicides, insecticides, animal repellants, and defoliants
(Northcott, 1978) and in the production of cyclohexylamine (formerly
an intermediate in the manufacture of cyclamate synthetic sweeteners
and presently an intermediate in the production of a
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variety of other chemicals and as a corrosion inhibitor).
Cyclohexylamine and its derivatives are produced by Abbott
Laboratories in Wichita, Kans., Monsanto Co. in Sauget, Ill., and
Virginia Chemicals Inc. in Bucks, Ala. and Portsmouth, Va. (Stanford
Research Institute, 1979). Aniline is also used in the production
of , ‘—methylenedianiline, an intermediate for the commercial
synthesis of a polyamide fiber marketed under the trade name
Quiana. The sole producer of Quiana is E.I. du Pont de Nemours &
Co., Inc. The monomer is produced at Belle, W. Va., and the polymer
is spun into yarn at the plant in Chattanooga, Tenn. (Chemical
Economics Handbook, 1977).
EXPOSURE
As demonstrated above, aniline is produced in large quantities
and has numerous applications. Although the potential for human
exposure is correspondingly large, there are no quantitative
estimates of environmental exposures of the general public.
Nonetheless, the National Institute on Occupational Safety and
Health (NIOSH), based on results of a National Occupational Hazards
Survey, has estimated that a potential 1.26 million workers could be
exposed to aniline.
Exposure to aniline in the workplace is regulated by the
Occupational Safety and Health Administration (OSHA). The health
standards for occupational exposure to air contaminants require that
an employee’s exposure to aniline shall not exceed
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S ppm or 19 mg/rn 3 air in any 8—hour workday of a 40—hour workweek
(Occupational Safety and Health Administration, 1980).
In 1979, the American Conference of Governmental Industrial
Hygienists (ACGIH) adopted a threshold limit value time weighted
average for dermal exposure to aniline and its homologs of 2 ppm or
10 mg/rn 3 air for any 8—hour workday or 40—hour workweek; and a
threshold limit value, short—term exposure limit of 5 ppm or 20
mg/rn 3 air for a period of up to 15 minutes, not to occur more than
4 times per day (American Conference of Governmental Industrial
Hygienists, 1979).
Because of aniline’s widespread use, it is generally considered
to be a likely component of many industrial wastewater discharges.
However, the committee found only one reference (Jungclaus et al.,
1978) in which aniline concentrations had actually been measured in
such a discharge; the aniline concentration in the wastewater
discharge. These investigators reported that of a specialty
chemicals plant was 0.02 ppm. The compound was not detected
downstream of the plant nor in the stream sediment.
Aniline is biodegradable. It is susceptible to treatment in
wastewater with activated sludge (Joel and Grady, 1977). In air, it
is subject to attack by the hydroxyl radical (Spicer et al., 1974),
but its overall half-life in air is not known. No information could
be found on the presence of aniline in consumer products, and no
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evidence was found that aniline is covered by Food and Drug
Administration regulations.
The Interagency Testing Committee, established under section
4 (e) of the Toxic Substance Control Act (TSCA), has added aniline to
its Priority List of Chemicals despite the previous National Cancer
Institute (Nd, 1978) test. Chemicals on this list are considered
for testing by the U.S. Environmental Protection Agency (EPA) in
accordance with section 4(a) of TSCA. Within 12 months of such a
recommendation, the EPA must initiate rulemaking to require testing
of chemical or publish its reasons for not doing so. The committee
recommended studies to determine the carcinogenicity, mutagenicity,
teratogenicity, chronic effects, environmental effects, and
epidemiology of aniline.
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ANALYTIC METHODS
In addition to the general analytic procedures for primary
aromatic amines, discussed in Chapter 1, the following additional
information from recent literature should help in methods selection.
lodination and Bromination
The sensitivity of aniline in electron capture—gas
chromatography (EC—GC) assays is greatly enhanced by iodination or
bromination of the molecule. Kofman et al. (1979), described the
following process: For iodination, the compound in 1 N hydrochloric
acid is treated with sodium nitrite at 0°C, iodinated with potassium
iodide at room temperature, and boiled at reflux. The iodine
derivative is extracted with hexane; iodination efficiency is 87%.
Bromination of aniline is carried out in 3. M sulfuric acid with
mesidine, potassium bromide, and potassium broinate. The reaction
product (2,4,6—tribromoaniline) is extracted with toluene after
alkalinjzatjon with 10 N sodium hydroxide. Efficiency of
bromination is 99.6%.
Cigarette Smoke
The amines from cigarette smoke were trapped in dilute
hydrochloric acid and enriched together with the basic portions,
derivatized to pentafluoropropionamides, and determined by EC—GC
with a nickel—63 electron—capture detector. The detection limit
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was approximately 50 pg of aniline per cigarette (Patrianakos and
Hoffmann, 1979).
Aniline and its Metabolites
Sternson and Dewitte (1977) reported a high—pressure liquid
chromatography (HPLC) method for determining nanomole quantities of
aniline and its metabolites, 0— and 2—aminophenol,
phenylhydroxylamine, nitrosoberizene, nitrobenzene, azobenzene and
azoxybenzene, which form nonenzymatically by condensation of
reactive metabolites. The compounds were separated by reverse—phase
HPLC on a Bondapak carbon-18 column and detected
spectrophotometrically. The eluent for the first four components
was methanol—water (15:85) containing 0.26 M ammonium acetate and
0.0l5—M nickel acetate. The remaining components were eluted with a
solution of methanol and water (50:50).
Air and Personal Sampling
Wood and Anderson (1975) described procedures for assaying
airborne vapors of aniline and related compounds. The vapors were
absorbed on silica gel, eluted from the gel with 95% ethyl alcohol
containing 0.1% heptanol, and separated and analyzed by gas
chromatography with a column of OV-25. Bovkun et al. (1974) devised
a simple but sensitive and selective method for determining aniline
vapors in air. The air sample was drawn through an indicator tube
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filled with porcelain powder treated with a mixture of alcohol
ammonium hexanitrocerate (IV) solution and aqueous potassium
persulfate solution. The concentration of aniline in the air was
determined by the length of the indicator mass, which had changed
colors. Grorniec and Adamlak—Ziemba (1974) determined vapors of
N—ethylaniline and aniline in admixture in air. Both compounds were
adsorbed in 2% ethyl alcohol, and the sum of the two amines was
determined colorimetrically by using an indophenol procedure.
Simultaneously, aniline was determined by diazotization and coupling
with N—l—naphthyiethylenediamine. The concentration of
N—ethylaniline was calculated by difference. The determinable limit
for N—ethylaniline and aniline was 1.0 and 0.37 mg/mi, respectively.
A rather unique method for personal monitoring was reported by
Schaffernicht and Schreinicke (1974). A personal sampler connected
to a telemetric system was placed directly on a workman, thereby
permitting continuous measurement of the toxic substances in his
breathing zone. The toxic substances were colorimetrically
determined by absorption in a tube of the personal sampler.
Electrolytic current was proportional to the concentration of the
toxic substance and was used as the basis for a frequency—modulated
telemetric signal. After demodulation on the receiving side, the
data were recorded by a strip recorder. The system is suitable for
measuring sulfur dioxide, hydrogen sulfide, hydrogen cyanide,
phenol, and aniline in the ranges likely to occur in industrial
situations. The person being monitored is free to move within a
radius of 150 meters around the receiver.
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Volumetric Analys is
Madraimov et a].. (1973) reported an iodometric method for
determining aniline or Anesthesin (ethylene aminobenzoate) using a
neutral iodine—bromide solution. The procedure is reported to be
sensitive to 5 pg of aniline in 0.0005% solutions and 40 pg of
Anesthesin in 0.0025% solutions. The preparations were treated with
5 to 6 ml of l—M hydrochloric acid and 50 ml of 0.l—M iodine—bromine
solution and heated at 40°c to 50°C for 3 minutes. After the
addition of both 10 ml of ethanol and 10% potassium iodide solution,
the released iodine was titrated with a sodium thiosulfate solution,
with starch as an indicator.
Other Analytic Methods for Aniline
Ascik et al. (1975) determined toxic compounds in pulp and paper
mills. They discussed methods of sampling and instrumental analysis
and tabulted maximum permissible concentrations for several
compounds, including aniline. zaugol’nikov et al. (1975) determined
several environmental contaminants, including aniline, and used
nomograms and equations to determine maximum permissible
concentrations of the compounds in industrial environments, city
air, and municipal water reservoirs. Hartstein and Forshey (1974)
reported on experiments performed by the Bureau of Mines to
investigate products formed on thermal oxidative degradation of
selected compounds under both dynamic and static conditions. Four
broad classes of materials were studied: polyvinyl chloride
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compounds, neoprene compounds, rigid urethane foams, and variously
treated woods. Thermogravimetric and differential thermal analyses
were performed to explore the feasibility of using these analyses to
identify materials. Sixteen toxic products including aniline were
detected and measured. Dutkiewicz and Szymanska (1973) employed
thin—layer chromatography (TLC) to analyze the urine from rats given
an oral dose of hydrazobenzene
(200—400 mg/kg). Aniline was one of the products detected in the
urine.
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HEALTH EFFECTS
The primary exposure of humans to aniline is occupational,
however, exposure to aniline in the environment may also occur.
Aniline is a volatile liquid at room temperature and is rapidly
absorbed when inhaled (Dutkiewicz and Piotrowski, 1961; Vasilenko et
a].., 1972). It is also rapidly and efficiently absorbed through the
skin and from the gastrointestinal tract following oral ingestion.
These properties led to the establishment of a threshold limit value
(TLV) of 5 ppm (19 ing/m 3 ).
Metabolism
The metabolism of aniline is complex and multifaceted. As with
many other compounds, the metabolic process seems to take place in
two stages. The first stage, which is mediated by microsomal
enzymes in the liver, consists of oxidation (hydroxylation) of the 2
and 4 positions on the aromatic ring and the nitrogen atom
(N-hydroxylation). Usually 4—hydroxylation predominates with the
formation of E—aminophenol, the principal metabolite (Parke, 1960;
Smith and Williams, 1949; Williams, 1959). N—hydroxylation results
in the formation of a possible biologically significant metabolite,
phenylhydroxylamine. However, phenyihydroxylamine does not appear
to be carcinogenic or mutagenic under test conditions where a series
of other aryihydroxylamines were positive for both effects (Belntan
et a].., 1968)
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The second stage consists of conjugation of these ring hydroxyl
groups with glucuronic and/or sulfuric acid. In addition, the
unoxidized amine group may be conjugated with g].ucuronic acid with
the formation of a N—glucuronide or with sulfuric acid with the
formation of an N—sulfate (sulfamate) (Boyland et al., 1957).
Phenyihydroxylamine may react with cysteine, leading eventually to
the formation of mercapturic acid conjugates (Boyland et al., 1963)
In all species except dogs, aniline is N—acetylated with the
subsequent formation of a second series of metabolites containing
the acetyl group (Williams, 1959). Since deacetylation also occurs,
these acetyl metabolites are usually present in small quantities in
urine. N—oxidation of the acetamide could result in the formation
of an hydroxamic acid, N—hydroxyacetanilide, but evidence for its
actual occurrence in tissues and urine is lacking. —Hydroxy1ation
forms N—acetyl—Q—aminophenol (Williams, 1959).
Not all of these metabolites occur in all species, and the
relative amounts formed vary considerably among species (Conney and
Levin, 1974). However, it appears that 2—aminophenol is excreted in
the urine of all species as a glucuronic acid or sulfate conjugate
(Gut and Becker, 1975; WIlliams, 1959). In rats, 42.3% of the
administered dose was recovered as 2—aminophenol in urine after acid
hydrolysis (BUS et al., 1978) Concentrations of 2 —aminophenol in
the urine of workers has been measured as a rough means of
estimating occupational exposure. An average of 39.44 mg/i was
associated with the occurrence of significant methemogiobinemia
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(Pacseri, 1961). Phenyihydroxylamine is apparently also an important
metabolite in most species including humans, but its presence seems
to be limited to the blood where it reacts with hemoglobin to form
methemoglobin, after which it is oxidized to nitrosobenzene. It has
never been detected in the urine of animals given aniline (Kiese,
1966).
Both N—hydroxylation and ring hydroxylation are carried out by
the mixed—function oxidase system of the liver microsomes and are
stimulated by pretreatment of rat with either phenobarbital or
aniline itself and inhibited by SKF 525A (Boobis and Powis, 1975;
Conney and Levin, 1974; Patterson and Roberts, 1971; Wisniewska—Knypl
and Jablonska, 1975 Wisniewska—Knypl et al., 1975;). Aniline is very
rapidly metabolized in rabbits and mice; its metabolic half—life in
these species is approximately 40 minutes. It is metabolized less
rapidly in rats and still less rapidly in dogs (Conney and Levin,
1974)
Acute Toxicity
Mechanism of Methemoglobin Induction . Because of the widespread use
of aniline in industry and its high vapor pressure, the occurrence of
methemoglobinemia in chemical workers is a rather common experience.
The mechanism of the induction of methemoglobinemia bY aniline has
been widely studied. The bulk of the available evidence indicates
that aniline itself is not directly responsible for the induction of
methernoglobinemia, but its metabolite, phenyihydroxylamine, is
140
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responsible for this effect (Kiese, 1966; Lin and Wu, 1973; McLean
et al., 1969;). The phenylhydroxylamine, in the presence of oxygen,
reacts with hemoglobin forming methemoglobin and nitrosobenzene.
Nitrosobenzene is in turn reduced by the diaphorase
nicotinamide—adenine dinucleotide phosphate (NADP)—methenioglobin
reductase (in the presence of NADP) back to phenyihydroxylamine,
which can in turn oxidize another molecule of hemoglobin. This
cyclic reaction goes on until as many as 50 i*1 of methemoglobin are
produced from a single millimole of hydroxylamine (Kiese, 1966). It
appears that a small amount of nitrosobenzene is reduced all the way
to the amine, and the reaction is then terminated. In addition,
nitrosobenzene appears to be inactivated by glutathione, which is
present in red blood cells (Aikawa eta].., 1978; Eyer,1979; ).
NADP—methemoglobin reductase is the enzyme mainly responsible
for the physiologic reduction of methemog].obin to hemoglobin.
However, NADP—methemoglobin reductase has a greater affinity for
nitrosobenzene than for niethemoglobin. This affinity inhibits the
reconversion of methemoglobin back to hemoglobin as long as the
nitroso compound is present.
Sensitivity to the induction of methemoglobin by aromatic amines
varies among species. On a mg/kg basis cats are the most sensitive,
and humans are approximately 60% as sensitive; dogs are about 30% as
sensitive, rats 5%, and rabbits and monkeys seem to be quite
resistant to aromatic—amine—induced methemoglobin (Hamblin, 1963).
Other evidence indicates that humans are roughly 70 times more
141
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sensitive than rats to aniline itself (Jenkins et al., 1972).
Although it seems clear that phenylhydroxylamine is the primary
metabolite of aniline responsible for methemoglobin induction, it is
not necessarily the only one. For years, —aminophenol was regarded
as the precursor of methemoglobin formation, until it was found that
phenylhydroxylanijne is approximately 20 times more potent. However,
it is possible that E—aminophenol, o—aminophenol, and even other
metabolites of aniline may be involved (Smith et al., 1967).
—Aininophenol also requires oxygen to oxidize hemoglobin to
methemoglobin, resulting in the formation of the p-guinoneimine,
which may go back to —aminopheno1 in a manner analogous to the
phenylhydroxylamine—nitrosobenzene cycle. However, only a few
equivalents of methemoglobin are produced by one equivalent of
—aniinophenol (Kiese, 1966).
Although NADP—methemoglobin reductase is normally responsible
for the reduction of methemoglobin to hemoglobin, some individuals
have a hereditary reduction or absence of this enzyme. Such
individuals are hypersensitive to the induction of methemoglobinemia
by nitrates. This genetic conditon is due to an autosomal recessive
allele and is manifested in homozygotes of both sexes (Goldstein et
al., 1968). Such individuals are presumed to be more sensitive to
aniline and related methemoglobin inducers, but there is no
conclusive evidence for this hypothesis at the present time.
Although lack of the reductase may retard the conversion of
methemoglobin back to hemoglobin, it may also prevent the reduction
of the nitroso compounds back to the hydroxylamine (Radomski,
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1979). In contrast, the action of nitrite is a direct one, not
involving NADP—methenoglobin reductase.
Although generally regarded as a highly toxic compound for
humans, the acute toxicity in laboratory animals is relatively low.
The oral LD5O’s in rats, mice, and cats are 440, 460, and 1,750
mg/kg, respectively. Derma]. LD5O’s are 1,400 mg/kg in rats and
1,290 mg/kg in guinea pigs (National Institute for Occupational
Safety and Health, 1977). Fatal poisoning in humans rarely occurs,
even following severe exposure. The unfavorable reputation of
aniline as an intoxicant is undoubtedly due to the rapidity and
efficiency of its absorption through both the skin and the
respiratory tract, resulting in the rapid induction of
methemoglobinemia. This condition results in symptoms such as
headache, nausea, and dizziness (Hamlin, 1963). Indeed, four cases
of methemoglobinemia, in which methemoglobin values reached 17—26
g%, were reported from the wearing of shoes dyed black with aniline
(Ghiringhelli and Molina, 1951).
Methemoglobinemia is a relatively benign and reversible
condition, however, at least in normal individuals. Conversion of
75% of the hemoglobin in the body to methemoglobin can occur without
life—threatening results (Hamlin, 1963). Experiments in dogs
indicates that 4—aminobiphenyl is 10 to 20 times more potent than is
aniline, even in the induction of methemoglobinemia. Experiments in
cats have shown that 2—nitrosobenzene is 50 to 80 times more potent
than aniline (Radomaki, 1979).
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Chronic Toxicity
Carcinogenicity
When Rehn first observed the induction of bladder cancer in
chemical workers, he believed these tumors to be due to exposure to
aniline and named them “aniline tumors”. This unjustified misnomer
persisted for many years until a careful epidemiologic investigation
by Case et al. (1954) and Case and Pearson (1954) convincingly
attributed these tumors to 2—naphthylamine and to benzidine, rather
than to aniline.
Following these observations, aniline has been regarded as a
noncarcinogenic substance. Unfortunately, aniline was never
adequately tested in dogs, a test species often used for the
evaluation of bladder carcinogens. In the only dog experiment
reported in the literature, in which aniline was administered daily
to three dogs for 4 years no tumors were observed (Gerhman et al.,
1948)
Aniline, as the hydrochloride, was given to rats in drinking
water in an amount calculated to provide a dose of 22 mg/day for 750
days. One—half of the rats survived longer than 425 days. No
tumors of the bladder, spleen, liver, or kidney were observed
(Druckrey, 1950). Aniline was also tested both as the free base in
lard (1 mg/mouse) or olive oil (8 x 5 mg/mouse) or as hydrochloride
in water (13 x 4 mg/mouse). These experiments indicated that
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aniline was not tumorigenic, when injected subcutaneously into mice
in tests ranging from 12 to 24 months (International Agency for
Research on Cancer, 1974).
A subacute pilot study in rats was conducted primarily to
determine the maximally tolerated dose (MTD) for a chronic feeding
experiment. In this experiment, concentrations of 30, 100, 300, and
1,000 mg/kg body weight per day were administered in the diet to
male and female Fisher—344 rats. Only the 1,000 mg/kg dose was
clearly toxic, causing death in many female rats. In addition, the
investigator observed depression of body weight gain, pathology of
the liver, kidney, and spleen, and elevated methemoglobin
concentrations (Gralla, 1977).
Until recently, these inadequate experiments plus the
publication of a series of epidemiologic studies that exonerated
aniline as a bladder carcinogen have led to the belief that aniline
is not a carcinogen. In 1978, the results of a carcinogenesis
bioassay in rats and mice given aniline hydrochloride were
released. The compound was fed in the diet of male and female rats
and mice for 103 weeks at two concentrations: 0.6% and 0.3% for rats
and 1.2% and 0.6% for mice.
Male rats had a significant number of hernangiosarcomas of the
spleen. A significant increase in the combined incidence of
fibrosarcomas and sarcomas of the spleen and other organs was also
observed in rats of both sexes. There was no evidence of
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aniline—induced carcinogenicity in mice of either sex (National
Cancer Institute, 1978) . This carcinogenesis bioassay was conducted
according to the usual National Cancer Institute (NCI) protocol,
which utilizes an MTD and one—half 4TD as the doses for the study.
Mutagenicity
The data from inutagenicity tests of aniline are summarized in
Table 6—3. Aniline and aniline—derivatives (hydroxylamine and the
riitroso derivatives) did not induce mutations in the Salmonella test
system in four out of five studies, and weakly inutagenic in one
(Mitchell, 1978). However, aniline was, in the presence of the
“comutagens” norharman and harman ( —carboline derivatives),
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Observations
Gene mutations
TABI. .E 6-3
Mutagenicity Tests of Aniline
Species/Strain Results
Salmonella , TA 1535, TA 1537,
TA 98, TA 100
Salmonella , TA 100, TA 98
Salmonella , TA 1538
Salmonella , TA 98, TA 100
Salmonella , TA 98
4 :-
References
neg! McCann et al., 1975
neg
neg!
post
pos/negE
Hecht et al., 1979
Garner et al., 1977
Mitchell, 1978
Nagao et 1., 1977
DNA damage
Mammalian cells
in culture Chinese hamster, V79 cells neg Swenberg et al., 1976
Negative, both with and without metabolic activation system (S—9, microsomes etc.)
b Weakly positive in the liquid—medium assay plus the metabolic activation system.
£ Positive only in the presence of harman or norharman and S—9 fraction.
The V79 cells were incubated with liver S—9 fraction from rat and mouse.
-------
strongly mutagenic in the Salmonella strain TA 98 (Nagao et al.,
1977). Aniline caused no DNA strand breakage in mammalian cells in
culture (Swenberg etal., 1976). The results are discussed below.
Bacterial Tests . In an extensive study of the mutagenicity of
chemical carcinogens in the Salmonella/microsome mutagenicity test,
McCann et a]. (1975) found aniline to be nonmutagenic in all four
tester strains (TA 1535, TA 1537, TA 98, TA 100). Similarly, Hecht
et a]. (1979), testing aniline as well as the hydroxylamine and
nitroso—derivatjves, and Garner and Nutman (1977) also obtained
negative results for aniline in the Salmonella test. However,
Mitchell (1978) obtained weakly positive data on aniline
mutagenicjty in the Salmonella test by using a liquid—medium method,
which involves a liquid incubation of the tester strain, metabolic
activation system (S—9 or microsomes) and the test compound before
plating the mixture on the agar. Under these conditions, the
highest rate of mutagenicity observed was twice the number of
spontaneous nutations. The significance of these results is highly
questionable no data were provided on the purity of the compound;
no dose—response relationship was observed for the mutagenicity of
aniline; the mutagenic effect was independent of metabolic
activation and the possibility that the aniline was oxidized under
the test conditions to form a mutagenic oxidation — coupling product
has not been ruled out.
The nonmutagenic 8—carboline derivative norharman has been shown
to enhance the mutagenic activity of benzo(a)pyrene,
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dimethylaminoazobenzene, and tryptophan pyrolysates on Salmonella
typhimurium strain TA 98 (Sugimura et al., 1977). The mutagenicity of
aniline and o—toluidjne was first demonstrated in the Salmonella system
in the presence of norharman and S—9 fraction from rat liver (Nagao et
al., 1977). The mutagenicity of aniline followed a clear dose response
pattern in strain TA 98, when coincubated with norharman (200 pg/plate).
The metabolic changes (activation) of aniline and/or norharman were
necessary to demonstrate the mutageni.city of aniline in the presence of
norharman, because no mutagenicity was observed without the S—9 fraction
(Nagao et al., 1977).
Mammalian Cell . Swenberg et al. (1976) evaluated the capacity of
aniline to Induce DNA strand breaks using an in—vitro/alkaline elution
assay using Chinese hamster V79 cells with and without a liver microsomal
activation system. No detectable chromosomal damage, measured as an
increase in the rate of DNA elution was observed with aniline.
Teratogen icit
No data were available to evaluate the potential teratogenicity or
reproductive toxicity of aniline.
CONCLUSIONS
Until the recent publication of the results of the NCI bioassay of
aniline, aniline had been considered noncarcinogenic. Concern
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about exposure to aromatic amines has always been focused on the
induction of bladder cancer because this is the only form of cancer
known to have been produced in humans by some of these substances.
There was no evidence of the induction of bladder tumors in either
the rats or mice at the MTD in the NCI bioassay experiment.
Whether the sp].enic tumors observed in rats, but not in mice, at
the large doses tested in this experiment indicate that aniline
represents a carcinogenic threat to humans at a site other than the
bladder cannot presently be ascertained. There is no evidence that
primary splenic tumors result from the exposure of humans to
aniline. On the other hand, there is evidence that a carcinogen may
induce tumors in different tissues in different species, and this
observation has been extended to the generalization that a substance
inducing cancer in any tissue of one species may induce tumors in a
different tissue in other species, including humans. Although this
may be true as a generalization, there are undoubtedly exceptions
since some types of tumors in test animals do not appear to be
correlated with carcinogenic potential in humans.
The present TLV for aniline in the United States is in line with
that of most other countries, except the Soviet Union, which has
lowered it to 0.1 mg/rn 3 . The basis for this action is unclear
(Bardodej, 1975; Vasilenko, 1972; Winell, 1975).
The potential mutagenic activity of aniline has been evaluated
extensively in the Salmonella test. The resulting data indicate
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strongly that aniline alone is without mutagenic effect, although
one study reports that aniline is weakly mutagenic (Mitchell,
1978). However, in the presence of the “comutagen” norharman,
aniline becomes highly mutagenic in the Saln nella test. However,
when aniline and norharman were given in the diet either alone or in
combination to male Wistar rats there was no carcinogenic effect to
the urinary bladder or other organs which could be treatment
related. Although this experiment was terminated after only 80
weeks it does strongly suggest that norharman does not enhance
aniline carcinogenicity as it does mutagenicity (Hagiwara et al.,
1980).
RECOMMENDATI ONS
Although considerable research has already been performed on
aniline, gaps in our knowledge of its possible health effects
continue to exist, and a compound of such industrial importance
deserves to be more thoroughly studied. The carcinogenic effects
(hemangiosarcomas of the spleen and sarcomas of the spleen and other
organs) observed at the MTD in the NCI bioassay need to be studied
further with another lifetime feeding study at 3 or 4 dose levels in
a different strain of rat. This will greatly assist in interpreting
the significance of the previously observed effects.
A carcinogenicity study using Syrian golden hamsters may also be
useful since these animals have been shown to develop bladder tumors
after exposure to other aromatic amines. In addition, a long—term
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(preferably 8 — 10 years), feeding study at the MTD should be
conducted on a large number of dogs. Such an experiment will allay
any suspicions concerning the possible role of aniline in the
causation of human bladder cancer. This test is desirable because
the only test with dogs was conducted many years ago on a few
animals for too short a time. Along with these studies, there
should be further studies on the occurrence of metabolites of
aniline in urine to explain the failure of this compound to induce
bladder cancer in dogs (if this failure is confirmed). Special
attention should be paid to N—hydroxylated urinary metabolites. In
addition, studies on the potential for teratogenicity and
reproductive toxicity need to be performed.
Further epidemiologic investigations on workers exposed to
aniline are also needed. Urine should be monitored and analyzed for
aniline metabolites to confirm and quantitate exposures.
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American Conference of Governmental Industrial Hygienists.
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and Physical Agents in the Workroom Environment with Intended
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Chemical Economics Handbook. 1978. Stanford Research
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Chemical Marketing Reporter. 1979. p. 4 September 3, 1979
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Code of Federal Regulations. 1980. Title 21, Part 74.
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Colour Index. 1971. The Society of Dyers and Colourists,
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International Agency for Research on Cancer. 1974. Aniline.
Pp. 27—29 in IARC Monographs on the Evaluation of Carcinogenic
Risk of Chemicals to Man. Volume 4. International Agency for
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Joel, A.R., and C.P.L. Grady, Jr. 1977. Inhibition of
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Jungclaus, G.A., V. Lopez—Avila, and R.A. Hites. 1978. Organic
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National Cancer Institute. 1978. Bioassay of aniline
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U.S. Occupational Safety and Health Administration. 1979.
Occupational safety and health standards. Subpart Z——Toxic
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1979. Office of the Federal Register, Washington, D.C.
Radomski, J.L. 1979. The primary aromatic amines: Their
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Annu. Rev. Pharmacol. Toxicol. 19:129—157.
Spicer, C.W., D.F. Miller, and A. Levy. 1974. Inhibition of
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Analytic Methods
Ascik, K., M. Glinska, and K. Szypruc. 1975. Determination of
toxic compounds in pulp and paper mills. Przegl. Papier.
31:232—233. (Chem. Abs. 84:34936j, 1976.]
Bovkun, E.K., R.K. Voronova, S.A. Psaltyra, and M.I.
Bukovskjj. 1974. Quantitative determination of aniline
vapors in air. U.S.S.R. Patent No. 443,311. [ Chem. Abs.
82 :l74810x, 1975.]
Dutkiewicz, T., and J. Szymanska. 1973. Chromatographic
determination of hydrazobenzene metabo].jtes in rats.
Bromato].. Chem. Toksyko].. 6:323—327. [ Chem. Abs. 80:116838k,
1974.)
Gromiec, 3., and J. Adamlak—zjemj a. 1974. Determination of
N—ethylanj].ine in air in the presence of aniline. Chem.
Anal. (Warsaw) 19:853—860. [ Chem. Abs. 82:34691j, 1975.]
Hartstejn, A.M., and D.R. Forshey. 1974. Coal mine combustion
products. Neoprenes, polyvinyl chloride compositions,
urethane foam and wood. Bureau of Mines, Pittsburgh, Pa.
Available fron National Technical Information Service,
Springfield, Virginia, as PB—240 211. 30 pp.
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Kofman, t.Sh., M.A. Klisenko, and V.1. Kofanov. 1979.
Gas—chromatographic determination of certain products of the
transformation of phenylamide—group herbicides. Fiziol.
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Chapter 7
4 , 4 ‘—METHYLENE—BIS ( 2—CHLOROANILINE )
H 2 N_(> _CH 2 _Q_ NH 2
4,4 ‘—Methylerle—bis(2—chloroanjline), commonly referred to as
MOCA (a registered trade name), is a nearly odorless, crystalline
solid that is yellow to tan in color (du Pont, 1977). Its molecular
weight is 267, its specific gravity (solid) is 1.44, its melting
point is 110°C, and its solubility (% by weight) at 24°C is as
follows: trichioroethylene, 4.2; toluene, 7.5; ethoxyethylacetate,
34.4; mesityloxide, 43.0; methylethylketone, 51.0; tetrahydrofuran,
55.5; dimethylformamide, 61.7; and dimethylsulfoxide, 75.0. Its
vapor pressure ranges from 1.3 x 10 mm Hg at 60°C to 5.4 x
l0 mm Hg at 120°C.
MOCA is also known by the following synomyms, acronyms, and
trade names: diamino—3—chlorophenylmethane, bisarnine,
di— ( 4 —amino—3—chlorophenyl)methane,
4,4 ‘—diamino—3,3 ‘—dichlorodiphenylmethane,
3,3 ‘—dich].oro—4—4 ‘—diaminodiphenylmethane,
methylene—bis(o—chloroani].jne ), , ‘—methy1ene—bis(o—chloroanj1jne),
DACPM, MBOCA, MCA, Curaline M, Curene 442, and Cyanaset.
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PRODUCTION
The sole U.S. producer of MaCA is Anderson Development Company in
Adrian, Mich. (Stanford Research Institute International, 1979), but
production has presumably been halted as a result of current litigation
(see below). du Pont had produced MOCA at its Deepwater, N.J. plant until
1978, when the company decided to phase out production (Chemical Week,
1978)
Because of the proprietary nature of the data, actual production levels
are not reported, and estimates of annual MOCA production have varied
substantially since 1974 (Table 7—1). As indicated, estimated annual
production rates have been as low as 500 kg to more than 4,500 kg. Because
estimated annual consumption levels are much higher than these figures, it
is likely that production levels are closer to 2 to 3 million kilograms.
Some unknown quantity of MOCA is probably imported.
USES
MOCA is applied principally as a curing agent for polyurethanes and
epoxy resins which are then used in the manufacture of specialized
products, particularly integral—skin polyurethane semirigid foam (used for
crash padding) and solid urethane rubber molding such as gear blanks and
industrial tires (National Institute for Occupational Safety and Health,
1978). MOCA is added to vary the hardness, flexibility, and impact
strength of these products. The most recent information on MOCA
consumption indicates that more than 99% is used to manufacture
polyurethanes.
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TABLE 7-1
Estimated! Production of MOCA, 1972—1978
Year
Estimated Gross
Production
( Thousands of Kg )
Data Source
3,300 approx.
0.5
1+
4.5
2,000—2 ,700
Bell, 1973
U.S. International Trade
U.S. International Trade
U.S. International Trade
Chemical and Engineering
U.S. International Trade
Commission, 1975
Commission, 1976
Commission, 1977
News, 1978
Commision, 1979
! Actual rates are not reported because of company
confidentiality.
U.S. consumption.
1972
1974
1975
1976
1977
1978 4.5
170
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Polyurethanes cured with MOCA have been used in hundreds of
applications (International Agency for Research on Cancer, 1974).
Military applications include ball seals on nuclear submarines,
positioning strips in Poseidon missiles, and in encapsulation of
electric components. In the automotive industry, they have been
used in dashboard padding and in numerous small parts. Other
reported uses include shoe soles; rolls for postage stamp machines;
cutting bars in plywood manufacture; rolls and belt drives on
cameras, computers, and reproducing equipment; and wheels and
pulleys for escalators and elevators.
Although systems to produce polyurethane elastomers without MOCA
have been developed in recent years, many manufacturers continue to
use MOCA—based methods because of the superior performance of the
resulting products (Ulrich, 1978)
EXPOSURE
The great potential for the distribution of aromatic amines
throughout the environment as a consequence of their production has
recently been documented (Williams, 1979). Beginning in 1970, a
small chemical plant began producing MOCA in the southeastern
Michigan town of Adrian. Between 1971 and 1978, production ranged
from 184,137 kg to 580,684 kg per year CHarger, 1979). Initially,
wastes from the plant were discharged into the Raisin River, which
serves as a water source for some downstream communities. After
1973, the wastes were channeled into a lagoon before they entered
Adrian’s wastewater treatment system. In the winter of 1978—1979,
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continuing problems at the treatment facility prompted a closer
examination of the mat.rials coming from this lagoon and the plant.
Local residents had long been disturbed by odors and dust that
periodically permeated the area. Consequently, when the state
recognized that MOCA was one of several products of this plant,
numerous samples were analyzed.
The results of these studies disclosed that nearly 18 km 2 is
contaminated with MOCA including approximately one—half of the town
of approximately 20,000 inhabitants. The compound appears to have
been spread by every possible mechanism. Airborne particulates were
the probable source of the material (up to 400 ppm) that collected
in the eavetroughs of adjacent houses (Michigan Department of
Natural Resources, 1979). Mechanical tracking from the plant was
suggested by the high levels of MOCA along the road that led from
the facility. Surface soil samples from public roads as far as 1.6
km from the plant contained up to 2 ppm MOCA; those adjacent to the
plant, up to 590 ppm; garden and yard samples from the local
residences contained, up to 55 ppm; and house dust from vacuum
cleaners, up to 18 ppm (Michigan Department of Natural Resources,
1979). According to Parris et al. (1980) and Walkington (1979),
transport via water was evident from the MOCA content of sludge from
the settling lagoon (1,600 ppm), the wastewater treatment plant (18
ppm) ,and the Raisin River (10 ppm). Secondary transport of the
compound also is believed to have occurred as a consequence of the
agricultural use of the sludge from the wastewater treatment plant.
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Evidence that humans have been exposed is provided by analyzing
urine for MOCA. There are no methods for anzlyzing urine for its
metabolites. In experiments with rats, only a small quantity
(usually less than 0.3%) of MOCA was excreted unchanged (Kominineni
et al. 1978). Thus, the actual exposure is far greater than is
implied by the concentration detected in the urine. Since
essentially nothing is known about the metabolic disposition of MOCA
in animals or humans, it is difficult to estimate exposure from
urine analyses. Furthermore, it is likely that there is significant
excretion of MOCA in bile. Available MaCA metabolism data indicate
that the substance behaves as a polymorphic substrate for the acetyl
CoA—dependent N—acetyltransferase of both rabbit and human liver
(Glowinski et al., 1978). The genetically determined levels of this
enzyme may, in fact, determine the rapidity with which MOCA is
excreted and/or converted to the reactive species involved in the
carcinogenic process. From previous knowledge of aromatic amine
metabolism in vivo , it is also expected that MOCA metabolites would
be excreted relatively rapidly. Thus far, only the 5—hydroxy
derivative has been reported as a urinary metabolite from an
observation made in a study of dogs, a species incapable of
N—acetylation (Barnes, 1964). The paucity of knowledge about MOCA
metabolism complicates attempts to evaluate exposure by analyses of
urinary excretion.
In spite of these complications, however, three categories of
individuals were shown to have been exposed: the workers, their
families (including spouses and children of all ages), and preschool
children (ages 2 to 5 years) living in the area of the plant
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(Williams, 1979). It is believed that the families were
contaminated by workers carrying MOCA home on their clothing since
direct contamination of clothing was demonstrated. The preschool
children were presumed to have been exposed while playing in
MOCA—contaminated soil; older neighborhood children did not have
detectable levels of MOCA (i.e., 0.3 ppb) in urine. The workers
were found to have up to 59 ppm MOCA in their urine; the members of
the workers’ families had urine MOCA levels of up to 15 ppb;
neighborhood children had up to 2 ppb. The detection of MOCA in the
urine of the workers’ families was not dependent on the location of
residence.
It is not known whether plant materials grown in the gardens of
the area represent a source of human exposure. Experiments to
resolve this question are in progress. Measurments suggest that the
level of MOCA in the soil has declined little if any since the
summer of 1979.
Analyses of urine specimens from workers at several facilities
that used the product from Adrian and other sources demonstrated
that some individuals excreted up to 0.7 ppm MOCA (Harger and
Saftlas, 1979). These observations confirm findings of earlier
studies (Hosein and Van Roosmalen, 1978). Environmental samples
obtained from sites that had not been exposed to MOCA for more than
4 years still contained detectable levels of the amine (Schleusener,
personal communication, 1980). As far as is known, the Adrian plant
has been the only MOCA production site in the United States for
several years. Since current litigation has presumably halted
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production in Adrian, MOCA is now believed to be available to American
users only from foreign sources. Conceivably, the use of MaCA—containing
products could be a hazard. It is possible, for example, that hydrolysis
of MOCA—containing polyurethanes could release very low levels free MOCA,
or unreacted MOCA might be leached from plastic under certain conditions
(Henning, 1974).
Occupational exposures to MOCA are of concern, as indicated by recent
federal actions. In 1969, the Food and Drug Administration disallowed
the use of MOCA as a component of certain food—contact articles (Federal
Register, 1969). The National Institute for Occupational Safety and
Health (NIOSH) recommended a standard of 3.0 pg/rn 3 in breathing zone
air determined as a time—weighted average. The Occupational Safety and
Health Administration (OSHA) is expected to set a standard for MOCA in
1980. The American Conference of Governmental Industrial Hygienists
(ACGIH) has adopted a threshold limit value, time—weighted average
concentration of 0.02 ppm for MOCA (American Conference of Governmental
Industrial Hygienists, 1979).
A national NIOSH survey (1978) indicated that in the early 1970’s
approximately 55,000 U.S. workers could have been exposed to MOCA. The
majority of these workers were employed in small— to medium—sized
establishments.
Concentrations in the workplace were sampled in one study in Italy.
As indicated in Table 7—2, concentrations ranged from 0.04 to 4.5
mg/m 3 ——much higher than the proposed OSHA standard. It is not possible
to judge the similarity between Italian and U.S. production conditions.
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Table 7—2
Sampled Concentrations of MaCA in the Workplace
at One Facility in Italy .
MOCA
Duration Concentration
Sample Location (minutes) mg/rn 3 )
Near the blending reactor 180 0.0400
where MOCA is mixed manu-
ally
Near another reactor 180 0.110
where MOCA is mixed
automatically
Above the oven in which the 140 0.283
container of MOCA is
reheated
Near the oven in which the 140 0.041
container of MOCA is
rehea ted
Above the exhaust of the 140 4.5
MOCA blending reactors
Source: Abstracted from Traina et al., 1978.
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ANALYTIC METHODS
In additon to the general procedures already described for
primary aromatic amines, most of the analytic methods for MOCA have
focused on the analysis of air or urine by using gas chromatography
(GC) or high—pressure liquid chromatography (HPLC) procedures.
Sawicki (1975) sampled air by drawing it through a tube of
Gas—chrom S. Sections of the tube packing were then extracted with
0.5 ml of acetone and analyzed by direct injection into a gas
chromatograph equipped with a 30—cm long, 2.3 nun (internal diameter)
stainless steel column packed with 10% Dexsil 300 GC coated on 80/90
mesh Anakrom ABS. The investigator used helium as the carrier gas
and a flame ionization detector. An injection of 1 pl of the
acetone extract permitted the detection of 2 ng of MOCA or
approximately 2 pg/rn 3 for a 500—1 air sample. In field trials, no
impurity was encountered that caused interference with the retention
time of MOCA. Isomers of chloroaniline commonly associated with
MOCA were completely resolved and did not interfere. The solvent
effect was pronounced in that the MOCA peak appeared on the tailing
edge of the acetone peak. It was therefore necessary to restrict
injection volumes to 2 p1 or less. At about the same time, Yasuda
(1975) reported a method essentially identical to that of Sawicki,
except that a 0.33 in long, 0.04 cm (internal diameter) stainless
steel column packed with the 10% Dexsil 300 GC was used.
Sensitivities of both methods were identical.
177
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Van Roosmalen et al. (1979) reported a procedure to determine
trace levels of MOCA in urine. The samples were partially cleaned
up by solvent extraction, followed by thin—layer chromatography
(TLC) on plates of silica gel G. The MOCA was then extracted from
the TLC plate, converted to its trifluoroacetyl derivative, and
analyzed by GC. The gas chromatograph was equipped with a 1.8 m
long, 0.32 cm (internal diameter) glass column packed with 3% OV—l
on Gas Chrom Q and a flame ionization detector (FID). A detection
limit of 1 pg/l was claimed. After the investigators prepared the
trifluoroacetyl derivative which is highly electron—capturing, they
chose to use the FID and did not mention the possibility of using
the more sensitive and specific electron capture—gas chromatograph
system.
The only HPLC procedure found in the literature for MOCA
was reported in 1979 by Rappaport and Morales (1979) for
determining airborne exposure of humans to MOCA. A personal sampler
consisted of a filter to remove the particulate MOCA, followed by a
bed of silica gel to remove the vapors. The compound was extracted
from the sampler stages with methanol, and a 10 pl aliquot was
injected into an HPLC instrument equipped with a reverse—phase
system and a 254 nm UV detector. Quantitation of 3 ng of MOCA
corresponded to 0.15 pg/sample. Precision levels were 9.2% and 14%
for 1.5 and 0.15 pg samples, respectively.
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HEALTH EFFECTS
Metabol isrn
Although little is known about the disposition of MOCA, it is
likely that it is metabolized via the pathways have been
demonstrated for other aromatic amines. These pathways include
N—oxidation, N—acetylation, C—oxidation, and conjugation with
glucuronate or sulfate as described elsewhere. Indirect evidence
for the N—oxidation of MOCA comes from its mutagenicity in a
Salmonella assay (see mutagenicity section). The genetically
polymorphic acetyl CoA—dependent acetylation of MOCA has been
demonstrated with preparations from both human and rabbit liver
(Glowinski et al., 1978). Evidence for the oxidation of the
aromatic ring is supported by the report that 5—hydroxy—MOCA is a
urinary metabolite of the parent amine in dogs (Barnes, 1964).
By analogy to other aromatic amines, transformation of MOCA to
derivatives capable of reacting with tissue macromolecules can be
expected to occur as a consequence of the production of an
N—oxidized derivative and a subsequent activation step. With other
compounds, this latter step has been shown to involve the formation
of reactive N—sulfoxy and/or N—acetoxy metabolites. An alternative
activation pathway can result from the peroxidation of the amine.
MOCA possesses one structural feature, the methylene bridge carbon,
not usually found in aromatic amines. This feature may make
possible an additional pathway for metabolic activation. Conjugates
179
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of metabolites oxidized at the methylene carbon may, for example, be
reactive as a consequence of this benzylic structure. If this type
of metabolite were to be responsible, in part, for the
carcinogenicity of MOCA, studies predicated solely on MOCA’S
aromatic amine structure might fail to detect the involvement of
this kind of metabolite.
Acute Toxicity
The LD 50 (single dose) of MOCA in rats (strain and sex
unspecified) is 750 mg/kg (Barnes, 1964). As with most other
aromatic amines, MOCA produces methemoglobin in rats and dogs.
Rabbits appear to be less susceptible to the acute toxic affects of
MOCA: the acute lethal dose by skin application was < 5 g/kg. The
protection afforded rabbits by their effective N—acetylatiOn of MOCA
may reduce methemoglobin formation as compared to its formation in
rats, which are less effective acetylators of aromatic amines, or
dogs, which are unable to acetylate aromatic amines. Although du
Pont has reported no acute toxicity in humans as a consequence of
its manufacture of MOCA (Linch etal., 1971), an increased urinary
frequency and hematuria (Mastromatteo, 1965) and a transitory
inability to reabsorb low molecular weight protein and concentrate
urine (Hosein and Van Roosmalen, 1978) have been related to MOCA
exposure.
180
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Chronic Toxicity
Carcinogenicity
MOCA is of primary concern because of its demonstrated
carcinogenicity in animals, and its structural relationship to
aromatic amines known to be capable of inducing bladder cancer in
humans. It is carcinogenic in mice, rats, and dogs (Table 7—3).The
evidence for tumor development is less strong in mice (Russfield et
al., 1975). Only hepatoma development in female CD—l mice was
significantly greater in the treated animals. Two dose levels,
1,000 and 2,000 ppm, were used, but the effective number of animals,
21 and 14 per group, respectively, were small. The length of the
experiment, 24 months, was appropriate, and the animal survival
adequate. There was a suggestion, however, that a greater number of
animals would have resulted in a significant increase of vascular
tumor induction.
Experiments proved that MOCA does induce bladder tumors in dogs
(Stula et al., 1977). Doses of 8 to 15 mg/kg/day were given 5 times
per week for up to 9 years. Five of six treated dogs developed
transitional cell carcinomas of the lower urinary tract. None of
the six control dogs developed these tumors. Thus, the
carcinogenicity of MOCA in the bladder of dogs is roughly comparable
to that of 2—acetylaminofluorene and 3,3—dichlorobenzidine, with
respect to dose and total test period; somewhat less carcinogenic
than 2—naphthylamine; much less carcinogenic than 4—aminobiphenyl;
and more carcinogenic than benzidine (Table 7—4).
181
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TABLE 7-3
Tumor Induction by MOCA
Spec leg
Tumor Mouse Rat Dog
Lung adenocarcinoina +
Mammary adenocarcinoma +
Zymbal gland +
adenocarc inoma
Hepatocellular carcinoma +
Hepatoma + +
Hemangiosarcoma +
Bladder +
182
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TABLE 7—4
Comparative Carcinogenicity of (Orally Administered) Aromatic Amines in the
Lower Urinary Tract of Dog .
Total Intake
Compound Per Dog (Mol)
Benzidine 1.77
MOCA 0.77 — 0.85
3,3’—Dichlorobenzidine 0.65 — 0.70
2—AcetylaThinofluorene 0.20 — 0.89
2—Naphthylaniine 0.24 — 0.28
4—Aminobiphenyl 0.03 - 0.04
! Adapted from Stula et al., 1977.
Treatment
Period (Yr)
5.0
8.3 — 9.0
6.6 — 7.1
2.6 — 3.9
2.6
2.8 — 3.].
Total
Period (Yr)
7.0 — 10.0
8.3 — 9.0
6.6 — 7.1
5.7 — 7.5
2.6
2.8 — 3.1
Tumor
Incidence
3/7
5/6
4/5
4/5
4/5
6/6
183
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The most detailed data on the carcinogenicity of MOCA are
available on male rats (Komniineni et al., 1978). Three dose levels
of MOCA were given to male CD—rats, fed either a protein—adequate or
protein—deficient diet. Sufficient numbers of animals were used
for each group (50 to 100 per group), and the animals were observed
for a long period. The mean survival time ranged from 65 to 89
weeks. A summary of the pertinent data are presented in Table 7—5.
Kominineni et al. have appropriately pointed out that the urine
levels of MOCA in these animals is actually less than in some of the
exposed workers. It seems very likely that, if a similar experiment
were to be conducted with female rats, the mammary incidence of
mammary gland tumors would be significantly higher than it was in
the male animals used in this experiment. Although Stula et al.
(1975) used female animals in the earlier study, the differences in
survival between control and treated animals clouded the
interpretation of the results.
The only information regarding the carcinogenicity of MOCA
in humans is du Pont’s report (Linch et al., 1971) of its limited
experience producing the compound. The authors indicated that they
had not observed malignancies attributable to MOCA in 31 men with
exposure lasting 6 months to 16 years.
Mutagenicity
MOCA has been teste’l fnr mutaqenicity in a large number of
systems, but the only results reported thus far are in the
184
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TABLE 7—5
Incidence (Percentage) of Predominant Neoplasms in Male Rats Fed MOCA!
Protein—Adequate Diet Protein—Deficient Diet
MOCA (ppm) 0 250 500 1,000 0 125 250
No. of rats 100 100 75 50 100 100 75 50
autopsied
Lung adenO 0 142 272 622 0 3 9 . 162
carciflOmaS
Mammary adeno— 1 ll 282 0 1 4 6!
carcinomas
zyinbal gland 1 8! 7 222 0 0 5! 122
carcinomas
HepatOCellU lar 0 3 4 3 C 0 o 0 182
carcinomas
emaflgiOSarc0maS 2 4 4 0 1 2 5 8 .
Mean survival 88.9 86.6 80.4 65.32 87.3 80.6 79.4 76.8!
(weeks)
Urinary MOCA <10 30 70 330 <10 <10 60 120
(ppb) at 26
weeks
!Abstracted from Koitimineni et al., 1979, with permission.
. Includes bronChiOlaralVeOlar cell carcinomas.
2 Difference from respective controls is significant (p < 0.001).
. Difference from respective controls is significant (p < 0.01 and >0.001).
! Difference from respective controls is significant (p < 0.05 and > 0.01).
185
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Salmonella/microsome test. These reports either include no data (Ho
et al.; 1979; Takemura and Shimizu, 1978) or have reported the
results obtained as bacterial revertants per single dose (Anderson
and Styles, 1978; McCann et al., 1975).
McCann et al. (1975) reported MOCA as positive in the TA 100
strain of Salmonella with Aroclor—induced rat liver S—9 (1,050
revertants at the 100 pg/plate dose, equivalent to 2.7
revertants/nmol). Anderson and Styles (1978) reported a fivefold
increase over background at 100 Pg/plate in the TA 100 strain using
rat liver S—9. Ho et al. (1979) also indicated that MOCA was
mutagenic in yeast, but reported no information as to strain, test
system, or treatment.
MOCA was also tested for mutagenicity as an unknown in 37
laboratories in a variety of test systems in the International
Program for Evaluation of Short Term Tests for Carcinogenicity
(IPESTTC). The data from this study have not yet been published.
However, MOCA was positive in bacterial DNA repair tests, phage
induction tests, the Salmonell.a/microgome tests, the Salmonella
fluctuation test, and in E. coli WP—2. Mixed results were observed
in the seven yeast systems used. MOCA induced unscheduled DNA
synthesis in HeLa cells in culture but did not induce sister
chromatjd exchanges (SCE’s) in culture. It produced transformation
in baby Syrian hamster kidney (BHK) cells in culture. In vivo , MOCA
produced sex—linked recessive lethal mu$ atirrns in Drosophila
melanogaster and mixed results in the micronucleus test in rodents.
Further analysis of these data must await the report’s publication.
186
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MOCA was also tested in a preincubation modification of the
Salmonel].a/microsome test in the National Institute of Environmental
Health Sciences (NIEHS) Environmental Mutagenesis Test Development
Program. S—9 from Aroclor 1254 induced male rat and Syrian hamster
liver was used with strains TA 98, TA 100, TA 1535, and TA 1537.
MOCA was mutagenic for strains TA 98 and TA 100 with S—9; hamster
S—9 produced a higher response than rat S-9 in the TA 100 strain,
and the responses in TA 98 were equivalent (K. Mortlemans, personal
communication).
Teratogenicity
No data were available to evaluate the potential teratogenicity
or reproductive toxicity of MaCA.
CONCLUSIONS
MOCA is a mutagen for Salmonella , requiring liver S—9 for its
activity. Results in press show it is also active in bacterial
repair tests, phage induction tests, . coli mutagenesis,
Drosophila , and cell transformation in vivo . Mixed results were
observed with yeast, chromosomal effects in cultured cells, and the
micronucleus test in vivo . A more complete evaluation awaits
publication of the IPESTTC study.
Studies in test animals have demonstrated conclusively that MOCA
is a carcinogen. This activity is to be expected from its
structure, which is similar to that of other aromatic amines that
187
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induce tumors in humans as well as in animals. Although the paucity
of epidemiologic evidence does not permit an evaluation of the
carcinogenic effects of MOCA, it is reasonable to assume that, given
a sufficiently high exposure, it may also be carcinogenic in humans.
RECOMMENDAT IONS
The MOCA—exposed population in and around Adrian, Mich. and
individuals exposed as a consequence of the use of MOCA should be
studied further to learn whether or not the compound is carcinogenic
in humans. This goal requires three types of effort. The first is
to explore the metabolic disposition of MOCA so that methods for
evaluating exposure to it can be developed. The second phase is to
apply these methods, including an evaluation of necropsy specimens
from any member of this population who dies during the course of
investigation. Such studies would help better define the potential
for risk to individuals, as well as aid in monitoring the effects of
the cleanup efforts. The final step is the prospective surveillance
of this population to determine whether exposure to MOCA increases
their tendency to develop cancer.
188
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RE FERENCES
Production, Uses, Exposure
American Conference of Governmental Industrial Hygienists. 1979.
TLVs: Threshold Limit Values for Chemical Substances and Physical
Agents in the Workroom Environment with Intended Changes for 1979.
American Conference of Governmental Industrial Hygienists,
Cincinnati, Ohio. 94 pp.
Barnes, J.R. 1964. Toxicity study on “MOCA TM ——4,4’methylene—bis
(2—chioroaniline). Study No. MR—652—2, Sep. 10. Dupont Haskell.
Laboratory, Wilmington, Del.
Bell, D.R. September 28, 1973. Final Environmental Impact
Statement Proposed Regulation (Administrative Action), Handling of
Certain Carcinogens. Occupational Safety and Health Administration,
Washington, D.C.
Chemical & Engineering News. 1978. Du Pont to halt MOCA curative
manufacture. 56(36) :7.
Chemical Week. 1978. Du Pont phasing out Moca production.
123(10) :13.
E. I. du Pont de Nemours & Co. 1977. ‘MBOCA’ and LD—8l3: Diamine
curing agents for isocyanate—containing polymers. Pub. No. AP—710.1.
Federal Register. 1969. 34(230): 19073. Tuesday, December 2, 1969.
189
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Glowinski, I.B., H.E. Radtke, and W.W. Weber. 1978. Genetic
variation in N—acetylation of carcinogenic arylainines b human and
rabbit liver. Mol. Pharmacol. 14:940—949.
Harger, J.R.E. 1979. Toxic Substance Control Commission, State of
Michigan. Additional information concerning curene, memo to P.S.
Cole, October 10, 1979 (curene production).
Harger, J.R.E., and P..F. Saftias. 1979. Toxic Substance Control
Commission. Analysis of 4,4 I_methylene ..biS(2 chloroaniline) user
data, memo to P.S. Cole. December 19,1979.
Henning, H.F. 1974. Precautions in the use of
methylene_biS_ —Ch1Ok:Oa ilifle (MEOCA). Ann. Occup. Hyg.
17:137—142.
Hoseiri, H.R., and P.B. Van Roosmalen. 1978. Acute exposure to
ntethylene_bis—orthO—ChlOroanilifle (MOCA). Am. md. Hyg. AsSOC. .3.
39:496—497.
International Agency for Research on Cancer. 1974. 4,4’—Methylene
bis (2—chioroaniline). Pp. 65—71 in IARC Monographs Ofl the
Evaluation of Carcinogenic Risk of Chemicals to Man. Volume 4.
International Agency for Research on Cancer, Lyon.
190
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Kommineni, C., D.H. Groth, I.J. Frockt, R.W. Voelker, and R.P.
Stanovick. 1979. Determination of the tumorigeniC potential of
methylene—bis—orthochloroaniline. J. Environ. Pathol. Toxicol.
2(5) :149—171.
Michigan Departments of Natural Resources. 1979. Curene
Contamination in Adrian, Summary of Investigations 1—9. Michigan
Department of Natural Resources, Air Quality Division, Lansing,
Michigan.
National Institute for Occupational Safety and Health. 1978.
Special Hazard Review with Control Recommendations for
4,4 ‘—Methylene-bis(2—chloroaniline). DHEW (NIOSH) Publication No.
78—188. Available from National Technical Information Service,
Springfield, Va., as PB—297 822. U.S. Dept. of Health, Education,
and Welfare, Public Health Service, Center for Disease Control,
Cincinnati, Ohio. 67 pp.
Parris, G.E., G.W. Diachenko, R.C. Entz, J.A. Poppiti, P. Lombardo,
T.K. Rohrer, and J.L. }iesse. 1980. Waterborne methylene bis
(2—obloroaniline) and 2—chioroaniline contamination around Adrian,
Michigan. Bull. Environ. Contam. Toxicol. 24:497—503.
Schleusener, P.L. 1980. Letter to C. M. King with enclosures.
Michigan Department of Natural Resources. April 28, 1980.
191
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SRI International. 1979. 1979 Directory of Chemical Producers:
United States of America. Stanford Research Institute
International, Menlo Park, Calif. 1122 pp.
Traina, G., C. Sala, F. Beretta, and G. Cortona. 1978.
Determinazone dell’inquinamento ambrentale da MBOCA in una
fabbrica di elastomeri polivretanici. Med. Lay. 69:530—536
Ulrich, H. 1978. Polyurethane. Modern Plastics 55(1OA);88, 90,
96—97.
U.S. International Trade Commission. 1976. Synthetic Organic
Chemicals. United States Production and Sales, 1975. USITC
Publication 804. U.S. Government Printing Office, Washington,
D.C. 246 pp.
U.S. International Trade Commission. 1977. Synthetic Organic
Chemicals. United States Production and Sales, 1976. rJSITC
Publication 833. U.S. Government Printing Office, Washington,
D.C. 357 pp.
U.S. International Trade Commission. 1979. Synthetic Organic
Chemicals. United States Production and Sales, 1978. USITC
Publication 1001. U.S. Government Printing Office, Washington,
D.C. 369 pp.
192
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U. S. International Trade Commission, 1975. Synthetic Organic
Chemicals. United States Production and Sales, 1974. USITC
Production 776. U.S. Government Printing Office, Washington, D.C.
256 pp.
Walkington, T. 1979. Michigan Department of Natural Resources,
meeting with the city (Adrian) on April 23, 1979, Adrian—Anderson
Development Co. File, May 7, 1979.
Williams, D.E. 1979. Curene 442 test results, Tables 1-9, October
5, 1979. Division of Environmental Epidemiology, Michigan
Department of Public Health, Lansing, Michigan.
193
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Analytic Methods
Rappaport, S.M., and Ft. Morales. 1979. Air sampling and analytical
method for 4,4’—methylenebis(2—chloroaniline). Anal. Chem.
51:19—23.
Sawicki, E. 1975. 3,3 ‘—Dichloro—4,4’—diaminodiphenylmethane (MOCA)
in air: Analytical method. Health Lab. Sci. 12:415—418.
Van Roosinalen, P.B., A.L. Klein, and I. Drummond. 1979. An
improved method for determination of 4,U—methylene
bis—(2—chloroaniline) (MOCA) in urine. Am. md. }Iyg. Assoc. J.
40: 66—69.
Yasuda, S.K. 1975. Determination of 3,3’—dichloro—4,4’—
diaminodiphenylmethane in air. J. Chromatogr. 104:283—290.
194
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Health Effects
Anderson, D., and J.A. Styles. 1978. Appendix II. The
bacterial mutation test. Br. 3. Cancer 37:924—930.
Barnes, J.R. 1964. Toxicity study on “MOCA” ——
4,4 ‘rnethylene—bis—(2—chloroaniline). Study NO. MR—652—2,
Sept. 10. Dupont Haskell Laboratory, Wilmington, Del.
Glowinski, I.B., H. E. Radtke, and W.W. Weber. 1978. Genetic
variation in N—acetylation of carcinogenic arylaniines by
human and rabbit liver. Mol. Pharmacol. 14:940—949.
Ho, P., A.A. Hardigree, F.W. Larimer, C.E. Nix, T.K. Rao, S.C.
Tipton, and J.L. Epler. 1979. Comparative mutagenicity
study of potentially carcinogenic industrial compounds.
Environ. Mutagen. 1:167—168 (Abstract No. Ea—10).
Hosein, H.R., and P.B. Van Roosmalen. 1978. Summary report:
Acute exposure to rnethylene—bis—o—chloroaniline (MOCA). Am.
md. Hyg. Assoc. 3. 39:496—497.
Kommineni, C., D.H. Groth, 1.3. Frockt, LW. Voelker, and R.P.
Stanovick. 1979. Determination of the tuinorigenic potential
of methylene—bis—orthochloroanjljne. 3. Environ. Pathol.
Toxicol. 2(5) :149—171.
195
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Linch, AL., G.B. O’Conner, J.R. Barnes, A.S. Killian, Jr., and W.E.
Neeld, Jr. 1971. Methylene—bis—ortho—chioroaniline (MOCA ):
Evaluation of hazards and exposure control. Am. md. Hyg. Assoc.
J. 32:802—819.
Mastromatteo, E. 1965. Recent occupational experiences in Ontario.
J. Occup. Med. 7:502—511.
McCann, J., E. Choi, E. Yamasaki, and B.N. Ames. 1975. Detection
of carcinogens as mutagens in the Salmonella/microsome test: Assay
of 300 chemicals. Proc. Nati. Acad. Sci. U.S.A. 72:5135—5139.
Russfield, A.B., F. Homburger, E. Boger, C.G. Van Dongen, E.K.
Weisburger, and J.H. Weisburger. 1975. The carcinogenic effect
of 4,4’—methylene--bis—(2—chloroaniline) in mice and rats.
Toxicol. Appl. Pharmacol. 31:47—54.
Stula, E.F., H. Sherman, J.A. Zapp, Jr., and J.W. Clayton, Jr.
1975. Experimental neoplasia in rats from oral administration of
3,3’—dichlorobenzidine, 4,4’—methylene—bis(2—chloroaniline) , and
4,4’—methylene—bis(2—methylaniline). Toxicol. P.ppl. Pharmacol.
31:159—176.
196
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Stula, E.F., J.R. Barnes, H. Sherman, C.F. Reinhardt, and J.A.
Zapp, Jr. 1977. Urinary bladder tumors in dogs from
4,4’—methylene—bis(2—chloroaniline) (MOCA ). J. Environ.
Pathol. Toxicol. 1:31 -50.
Takemura, N., and H. Shimizu. 1978. Mutagenicity of some
aromatic amino— and nitro—compounds. Mutat. Res. 54:256—257
(Abstract No. 35).
197
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Chapter 8
2, 4—DIAMINOTOLUENE
H 3 NH 2
2,4—Diaminotoluene (toluene—2,4—djamjne 2,4—DT) is a colorless
crystal that melts at 99°C. It is soluble in hot water.
2,4—rYi’ synthesis takes place in three steps, beginning with the
nitration of toluene in a mixture of nitric and sulfuric acids at 30°C
to 70°C. The resultant mononitrotoluene mixture is then nitrated again
in a somewhat stronger acid medium to a mixture of dinitrotoluene
isomers, of which the largest fraction is 2,4—dinitrotoluene (75.8%).
A number of processes can then be used to produce 2,4—DT from
2 ,4—dinitroto].uene. All of these methods involve catalytic
hydrogenation, followed by purification, to remove unwanted isomers and
byproducts.
PRODUCTION
Table 8—1 lists the current producers of 2,4—DT, their locations,
and (where available) their estimated annual capacities (Chemical
Economics Handbook, 1977—1978; Stanford Research Institute
International 1979; United States International Trade Commission, 1979).
198
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TABLE 8-1
Producers of 2,4-DT
Estimated Annual
Capacity as of
January 1, 1979g.
Company and Plant Location Thousands of metric tons
Air Products and Chemicals, Inc.
Industrial Gases Div.
Middlesex, N. J. NRP
Pasadena, Tex. 57 .
Allied Chemical Corporation
Specialty Chemicals Division
Moundsville, W. Va. 30
American Cyanamid Co.
Organic Chemicals Division
Bound Brook, N. J. NR .
RASF Wyandotte Corporation
Polymers Group
Urethane Division
Geismar, La. 36
E. I. du Pont de Nemours & Company, Inc.
Elastomer Chemicals Department
Deepwater, N. J. 25
Mobay Chemical Corporation
(owned by Bayer AG, Federal Rep. of Germany)
Polyurethane Division
Cedar Bayou, Tex. 47
New Martinsvilj.e, W. Va. 36
Olin Corporation
Olins Chemicals Group
Ashtabula, Ohio 11
Brandenburg, Ky. NR
Lake Charles, La. 36
Rochester, N. Y. NR
Rubicon Chemicals Inc.
(jointly owned by Uniroyal, Inc., and Imperial
Chemical Industries Limited, United Kingdom)
Geismar, La. 15
! Estimates of production capacities are based on an assumed 2,4 —
diaminotoluene capacity of 0.8 metric tons for each metric ton of
toluene diisocyanate capacity, except for Air Products and Chemicals,
Inc., which does not produce toluene diisocyanate.
Not reported.
199
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Total U.S. production of 2,4—DT has declined in recent years
(U.S. International Trade Commission, 1976—1978) as shown in Table
8—2.
USES
2,4—DT is used almost exclusively for the production of toluene
diisocyanate. Other U.S. consumption is estimated to amount to less
than 230 metric tons annually (Chemical Economics Handbook,
1977—1978)
Mixtures of isomers of 2,4—DT are normally used to produce
toluene diisocyanate. The most important mixture contains 80% of
the 2,4—isomer and 20% of the 2,6—isomer. Eight U.S. companies
produced 284,072.4 metric tons of the 80/20 toluene diisocyanate
mixture in 1978 (International Trade Commission, 1978). A mixture
containing 65% 2,4—isomer and 35% 2,6—isomer is also used in
significant quantities. Only a small quantity of 2,4—DT is isolated
for conversion to pure 2,4—toluene diisocyanate.
The Chemical Economics Handbook (1977—1978) estimated the U.S.
consumption pattern for toluene diisocyanate in 1978 as shown in
Table 8—3.
2,4—DT can be used to produce (approximately) 60 dyes, which are
used to color silk, wool, paper, boat fibers, cellulosic fibers, and
cotton. The following nine dyes are believed to have been produced
from 2,4—DT in the United States during 1978: Basic Brown 4, Basic
200
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Table 8—2
Annual Production of 2,4—DT .
Year Thousands of metric tons
1976 105.9
1977 101.1
1978 63.3
a Data from U.S. International Trade Commission, 1976—1978.
201
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Table 8—3
u.s. Consumption of Toluene Di—isocyanate in l978 .
Use Percent of Total
Flexible polyurethane foams 83%
Polyurethane surface coatings 6
Polyurethane elastomers 3
Other (including rigid polyurethane foam) 8
Total 100%
Data from Chemical Economics Handbook, 1977—1978.
202
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Orange 1, Direct Brown 2, Direct Brown 154, Direct Black 4, LeucO
Sulphur Orange 1, Leuco Sulphur Brown 10, Solvent Brown 12, and
Sulphur Black (Colour Index, 1975; U.S. International Trade
Commission, 1978). They are used in spirit varnishes and wood
stains as indicators, in the manufacture of pigments, and as
biologic stains.
2,4—DT is used as a developer for direct dyes, particularly to
obtain black, dark blue, and brown shades, and to obtain navy blue
and black colors on leather. It is also used to dye furs and was an
ingredient in hair dye formulations until banned in 1971
(International Agency for Research on Cancer, 1978).
EXPOSURE
More than 99% of the 2,4—DT produced in the United States is
used to produce toluene diisocyanate, generally at the site of
production. The single exception is the Air Products and Chemicals
Plant at Pasadena, Tex., which produces no toluene diisocyanate and,
conversely, a 45,000 metric ton/year capacity toluene diisocyanate
plant at Freeport, Tex. operated by Dow Chemical, which does not
produce its own 2,4—DT. Therefore, the main sources from which
humans are exposed will be the plants listed in Table 8—1. However,
there is no information on which to base an analysis of either
occupational or general exposures from plant emissions.
Because the substance is solid at ambient temperatures, air
emissions from 2,4—DT production do not appear to be significant.
203
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However, contamination of wastewater discharges from the plants is a
likely route of exposure. The hydrogenation of dinitrotoluene
yields 4 mol of water for every mol of 2,4—DT produced (600 1 of
water per metric ton of 2,4—DT). This water is separated in a
dehydration column at l00°C—150 0 C at atmospheric pressure (Milligan
and Gilbert, 1978). Because the vapor pressure of 2,4—DT is 11 mm
Hg at 150°C and it is soluble in hot water, the separated water is
undoubtedly highly contaminated. This wastewater is probably
treated before it is discharged.
Some 2,4—DT could find its way into consumer products as an
impurity in dyes. However, such exposure is nearly impossible to
quantify.
Under the U.S. Food and Drug Administration (FDA) regulation
concerning 2,4—DT, (21 CFR 177) is listed under the category of
antioxidants and antiozonants. The total of these components is not
to exceed 5% by weight of rubber product.
The U.S. Occupational Safety and Health Administration does not
have an occupational standard covering the exposure of workers to
2,4—DT.
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ANALYTIC METHODS
Jones et al. (1978) reported separations of nondye components in
the commercial food color preparation Brown FK The dye, which is
manufactured by the reaction of diazotized sulfanilic acid with a
mixture of rn—phenylenediamine and 2,4—diaminotoluene, has been shown
to consist of six major colored components. High—pressure liquid
chromatography (HPLC) and thin—layer chromatography (TLC) procedures
were evaluated for their ability to separate a mixture of the six
dye components as well as the starting compounds (e.g., 2,4—DT) used
in the synthesis. Two different columns and solvent systems were
used in the HPLC investigations with a UV absorption detector set at
254 nm. One of the systems, which consisted of a column of Partisil
5 loaded with 7% aminopropyl phase, was subjected to a 30—minute
linear gradient from acetonitrile—water (2:3) to acetonitrile—water
(2:3 containing sodium biphosphate (2 g/l). Only partial resolution
of the components was obtained. The other HPLC system, which
consisted of a column of Chromosorb Si 100 loaded with a 21%
octadecyl phase subjected to a 17—minute linear gradient from 5% to
40% acetonitrile in water (containing 1.2 and 2.4 g/l of sodium
biphosphate and sodium hypophosphate, respectively) was more
successful: all nine components of the mixture were separated with
essentially baseline resolution.
rJnger and Friedman (1979) developed an HPLC procedure to assay
2,6— and 2,4—DT. Their technique was adaptable to biomonitoring and
metabolic studies with samples of urine and plasma from rats. A
normal—phase silica column was used with a mobile phase consisting
205
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of acetonitri].e—water saturated chloroform (8:2) and a 250 nm UV
absorption detector. The two compounds were resolved as sharp peaks
in 3 minutes, and from 1 ng to 2 ng of each substance was
quantitated. Dichloromethane extraction of urine and plasma spiked
with 2,4—Dr (10 to 200 ppm), yielded recoveries of about 90% or more.
TLC tests were performed by using unactivated silica gel G
plates (layer thickness 0.25 mm) with a solvent system of
phenol—water (4:1) . The starting materials of the manufacturing
process (e.g., 2,4—DT) were detected by spraying with 1% Ehrlich’s
reagent in 50% acetic acid. Although the TLC system resolved for
the six dye components, it was not suitable for the other compounds
since sulfanilic acid was not resolved from one of the dye
components. The two aromatic amines (2,6— and 2,4—DT) were not well
resolved and ran close to the solvent front.
Spectrophotofluorimetry (SPF) was the basis for a method
described by Guthrie and Mckinney (1977) to analyze 2,4— and 2,6—Dr
in flexible polyurethane foams at levels as low as 1 ppm. The
amines were extracted with methanol, separated by TLC, and assayed
by SPF after reaction with Fluram reagent. The SPF assay was
accomplished by uniformly spraying the developed and dried TLC plate
with an 0.015% solution of Fluram in acetone and measuring the
fluorescent spots (AEX = 390, AEm = 500 nm) with a thin—film
chromatographic scanner. The instability of the Fluram derivative
required that quantitative measurements be completed within 1 hour.
The nonuniform characteristic of foams was determined by assaying
sample extracts in duplicate at the 10 to 15 ppm level. Precision,
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usually within ±10% for a given extract, was ±30% for six different
samples of the same foam.
Lepri et al. (1976) investigated the behavior of several
coloring agents (including 2,4—DT) used in oxidation—type hair dyes
on thin layers of various ion exchangers. Some of the systems
provided unique separations. Examples of such systems are AG l—X4
(CH 3 CO 2 —) developed with 0.1 mol acetate buffer in a 4:1
water—methanol mixture; BD—cellulose developed with 0.5 mol acetate
buffer solution; Dowex 50—X4 (H+) with 0.1 mol acetate buffer in a
1:1 mixture of water—methanol; Rexyn 102 (H+) developed with
various mixtures of dimethylformamide—water; and AG 3—X4A developed
with 95% ethyl alcohol. Rf values were tabulated for some 25
compounds.
Two direct gas chromatography (CC) procedures for separation and
analysis of isomeric diaminotoluenes were reported in 1968.
Willeboordse et al. (1968) separated mixtures of 2,3—, 3,4—, 2,4—,
2,5—, and 2,6—DT using a mixed partitioning agent of Carbowax 20M
and Saponate DS—10 on base—loaded Chromosorb C, followed by Saponate
DS—10 on the same solid support. Boufford (1968) separated a
mixture of 3,4—, 2,3—, 2,4—, 2,5—, and 2,6—DT on a column of 5%
Bentone 34 plus 155 Hyprose SP—80 [ octakis(2—hydroxypropyl)—sucrosej
on potassium—hydroxide—treated Chromosorb W at 170°C. Helium
carrier gas and a hydrogen flame ionization detector (FID) were used.
The analysis of 2,4—DT was specifically mentioned in several of
the procedures described for primary aromatic amines. Additional
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discussions in the literature (details unavailable) involve in situ
TL.C determinations of toluenediamirie and methylenedianiline isomers
in the products of hydrolytic degradation of polyurethanes (Lesiak
and Orlikowska, 1978) and an ultrasonic method to assay amine
solutions, including toluenediamine (Bogdanova et al., 1976).
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HEALTH EFFECTS
Me ta bol is m
2,4—DT is rapidly absorbed after intraperitoneal injection into
rats and mice, and peak levels in blood or serum are attained within
1 hour after injection (Grantham etal., 1979). Less than 1.5% of
the dose is excreted unchanged. Metabolites are rapidly excreted,
predominantly in urine (Grantham et a].., 1979; waring and Pheasant
1976). Unger et al. (1980) confirmed these observations in male
B6C3F mice administered a single intraperitoneal dose of
2,4—1 14 C)tlT. They noted rapid absorbtion from the peritoneal
cavity with the dominant route of excretion via the kidneys; one
hour after dosing almost 50% of the radioactivity was recovered in
the urine. They also noted that the three tissues which have shown
a carcinogenic response to 2,4—DT (hepatocarcinoma,
rhabdomyosarcoma, and fibrosarcoma), either acquired an initial high
concentration of 2,4—DT (liver) or demonstrated an extended
elimination half—life (muscle and skin)
Major metabolic reactions include acetylation of one or both
amino groups oxidation of the methyl group to the benzylic alcohol
and benzoic acid functions and ring hydroxylation, primarily at the
3 and 5 positions. Glinsukon et a].. (1975) examined the ability of
liver cytosol from various species to N—acetylate 2,4—DT. The liver
cytosol from hamsters had the most enzymatic activity followed by
guinea—pig, rabbit, mouse and rat. Human liver cytosol formed only
trace amounts of acetyl derivatives while dog liver cytosol had no
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activity. Although the liver cytosol showed the greatest
acetylating activity, the cytosol from the kidney, intestinal
mucosa, and lung also were able to produce significant amounts of
various acetylaminotoluenes. In this study
4—acetylamino—2—aminotoluene was the major metabolite and
2,4-diacetylaminotoluene the minor metabolite. Glucuronide and
sulfate conjugation of these primary metabolites also occurs,
varying with the species examined. No information is available on
the mechanism of’rnetabolic activation of 2,4-DT.
Acute Toxicity
National Institute of Occupational Safety and Health (1979)
reported that the oral LDLO (lowest published lethal dose) of 2,4—DT
in rats was 500 mg/kg, and that the LDLO’S by subcutaneous injection
to rats, dogs, and rabbits were 50, 200, and 400 mg/kg,
respectively. More recent data on the acute toxicity of 2,4—DT
after intraperitoneal injection in to male Fischer rats and female
NIH Swiss strain mice indicate LD 50 values of 325 mg/kg and 480
mg/kg, respectively (Grantham et al., 1979).
Gosselin et al. (1976) reported that no poisonings of humans by
2,4—DT were known. Nonetheless, they rated the compound as very
toxic, reporting a probable oral LDLO in humans of 50—500 mg/kg. On
the other hand, National Institute for Occupational Safety and
Health (1979) 1ist the oral LDLO of 2,4—DT in humans as 50 mg/kg.
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Teratogenicity
No data are available on the embryotoxicity or teratogenicity of
2 , 4— DT.
Carcinogenic ity
Subcutaneous Administration . Twenty rats (mixed strains and
sex) were given 0.5 ml of 0.4% solution of 2,4—DT in propylene
glycol subcutaneously at weekly intervals for approximately 8 months
(Uineda, 1955) . The survival rate at 8 months was only 45%. All
rats surviving 8 months developed subcutaneous sarcomas. The total
dosage of 2,4—DT ranged from 60—90 mg per rat. No contemporary
controls were used in this experiment.
Oral Administration . Ito et al. (1969) first reported the
carcinogenicity of 2,4—DT after oral administration. They observed
carcinoma of the liver in male Wistar rats fed diets containing 600
or 1,000 ppm 2,4—OT for 33 to 34 weeks. Tumor incidences at 36
weeks were 9/9 (100%) after a dosage of 1,000 ppm 2,4—DT and 7/li
(64%) after a dosage of 600 ppm. Histologically, all the liver
tumors were hepatocellular carcinomas. Many of the rats also had
multiple metastatic tumors in lymph nodes, omentum, lungs, and
epididynds. The livers of six control rats fed the basal diet
without 2,4—DT were essentially normal. Weisburger et al. (1978)
confirmed the hepatocarcinogenicity of 2,4—DT (as the
dihydroch]otide) after oral administration in CD—i (Sprague—Dawley)
rats and in HaM/1CR mice.
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More recently, the National Cancer Institute (1979) selected
2,4—DT for additional bioassay in the Carcinogenesis Testing
Program. Groups of 50 F344 rats of each sex received 2,4—DT in feed
at two dose levels. Time—weighted average doses were 79 ppm for 103
weeks (low dose) and 171 to 176 ppm for 79 to 84 weeks (high dose).
The incidences of hepatocellular carcinomas or neoplastic nodules
were dose related (males: controls 0/20, low—dose 5/49, high—dose
10/50; females: controls 0/20, low—dose 0/50, high—dose 6/49). In
addition, carcinomas or adenomas of the mammary gland occurred in
female rats (controls 1/20, low—dose 38/50, high—dose 41/50). The
most common type of mammary tumor by far was the fibroadenoma.
Other types of tumors appeared less frequently than did liver or
mammary tumors, but were assumed to be related to exposure to
2,4—DT. These included lung tumor5, squamous cell carcinoma of the
skin and preputial gland, pancreatic acinar cell adenoinas,
subcutaneous fibromas and fibrosarcomas, and mesotheliomas.
In parallel studies, groups of 50 B6C3F1 mice of each sex
received 2,4—DT in feed at two dose levels, either 100 ppm or 200
ppm, for 101 weeks. Hepatocellular carcinomas occurred in female
mice (control 0/19, low dose 13/47, high—dose 18/46). In addition,
lymphomas occurred at a significant incidence in the low—dose female
mice (controls 2/10, low—dose 29/47, high—dose 11/46). No tumors
occurred at significantly increased incidences in the dosed male
mice.
Skin Application . Two studies (Burnett et al., 1975; Giles et
al., 1976) involving the skin painting of mice with hair dye
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formulations that included 2,4—DT have been performed. In one study
(Giles et al,, 1976) , mice were given weekly topical applications of 0.05
ml of either a 6% solution of 2,4—DT alone or hair dye formulations
containing 2,4—DT. The results of this study were judged inadequate
(International Agency for Reserarch on Cancer, 1978) for an evaluation of
the carcinogenicity of 2,4—DT because a large number of animals were
unaccounted for in the final analysis of tumor incidence.
The second study (Burnett et al., 1975) involved the painting of 0.05
ml of a hair dye formulation containing 0.2% of 2,4—DT on the skin of
Swiss—Webster mice weekly or fortnightly for 18 months. Carcinogenicity
of 2,4—DT could not be evaluated on the basis of this study because of
the complexity of the applied mixture and because of the reported high
incidence of tumors observed in control mice (International Agency for
Research on Cancer, 1978)
Mutagenicity
The data from mutagenicity and other genotoxic tests of 2,4—DT are
summarized in Table 8—4. The substance was mutagenic in both the
Salmonella and Escherichia coli systems (Ames et al., 1975; Aune et al.,
1979; Pienta et al., 1977; Venitt, 1978) and in Drosophila melanogaster
(Blijleven, 1977; Fahmy and Fahmy, 1977). Negative results were obtained
in Neurospora crassa (Ong, 1978) and in the micronucleus test in the rat
(Hossack and Richardson, 1977). Potentially genotoxic effects (chromatid
and chromosomal gaps and breaks in peripheral lymphocytes) from hair dyes
which may have contained 2,4—DT, among other ingrediants, were observed
in professional hair colorists and others with a history of hair dye use
(Kirkland et al., 1978).
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Salmonella , TA 1538, TA 98
E. coli , 343/113 (arg reversion)
N. crassa , ad—3A
D. melanogaster , Oregon K, Berlin K
Chromosome Damage
Micronucleus test in rat
Chromosome damage humans
Mutagenic in the presence of S-9 (metabolic activation).
Tested without metabolic activation.
X—Chromosome recessive (lethals and visibles).
Chromatid and chromosome gaps and breaks.
neg Hossack and Richardson, 1977
pOs /neg Kirkland et al., 1978
TABLE 8-4
Mutagenicity Tests of 2,4—DT
Species/Strain Results
Observations
Gene Mutation
Bacterial
Yeast
Insect
Reference
pos .
pos!
neg
posS
Ames et al. 1975
Venitt, 1978
0mg, 1976
Fabmy and Fahmy, 1977
Blijieven, 1977
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Bacterial Tests . The studies of Ames et al. (1975), Aune et al.
(1979), and Pienta et al. (1977) clearly demonstrate that 2,4—DT is
a potent mutagen in the Salmonella system. Like many other aromatic
amines, 2,4—DT requires metabolic activation (S—9 or microsomal
fractions) for mutagenesis. Principally, it causes a frameshift
type of change in strains TA 1538 and TA 98. Pienta et al. (1977)
reported a correlation between the 2,4—DT—induced morphologic
transformation in an in vitro carcinogenesis system (using secondary
culture target cells prepared from cryopreserved, primary Syrian
hamster embryo cells) and the inutagenicity of 2,4—DT in the
Salmonella system. Venitt (1978) reported mutagenicity of 2,4—DT in
the E. coli system.
Yeast . Ong (197 ) used the adenine—3 (ad—3) forward—mutation
system of N. crassia to test the mutagenicity of environmental
agents and chemical carcinogens, including 2,4—DT. 2,4—DT was
nonmutagenic in the N. crassa system; however, since no mammalian
metabolic activation system was included in the test, these data
must be regarded as inconclusive.
Insect . Fahmy and Fahmy (1977) examined the comparative
mutagenic effect of 2,4—Dr as well as that of phenylenediamine and
benzidine in D. melanogaster . The compounds were injected at
equimolar dose range (5—20 mM) into the abdomens of adult males, and
mutagenicy was measured separately on the various stages of
spermatogenesis. Genes-ic activity was assayed with respect to the
overall induction of the X—chromosome recessive (lethals
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and visibles) mutations relative to the specific effects on
ribosomal DNA (bobbed locus). All the compounds exerted
mutagenicity, both on the X—chromosome and on the RNA genes,
but activity on the different genic sites varied between
compounds and as a function of cell stage. The mutagenic
potency toward the bobbed locus was benzidine 2,4-DT>
4—N—o—phenylenediamine, which correlates with the carcinogenic
potency of these compounds.
Blijieven (1977) observed similar mutagenicity by feeding
2,4—DT to male D. melanogaster and measuring sex—linked
recessive lethal mutations. Metabolically active germ cells
(spermatids and spermatocytes) had the highest mutagenic
activity.
Chromosomal Damage.
Micronucleus Test . Hossack and Richardson (1977) obtained
negative results in micronucleus tests of 2,4—DT and 11 other
hair dye constituents. Groups of five male and five female
rats were gastricly intubated with the test compounds at 0.5%
(w/v) in gum tragacanth containing 0.05% sodium sulpfite. The
total dosages were close to the lethal doses and were
administered in two equal parts, separated by an interval of 24
hours. The animals were killed 6 hours after the second dose
and bone—marrow smears were prepared. The incidence of
micronucleated cells per 2,000 polychromatic erythrocytes was
compared with the values from the control group. The mean
values and ranges of micronucleated cells were not
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significantly different for the control and 2,4—D —treated groups.
Similar results were obtained for the other compounds.
Chromosomal Damage in Humans . No data are available on the
possible genotoxic effects of 2,4—DT se in humans. However, in
a recent epidemiologic study of the potentially genotoxic effects
of hair dyes (Kirkland et al., 1978), chromosomal damage was
investigated in peripheral—blood lymphocytes of professional hair
colorists. The authors found no significant differences in
chromosomal damage in cultured peripheral—blood lymphocytes from 60
professional hair colorists as compared with those of 36 control
subjects c1ose1y matched for age and sex. There was a
statistically significant excess of chromosomal damage (mainly
chromatid breaks) in women with dyed hair when age—matched women
were regrouped according to whether their hair was dyed or not.
Men (mean age 22.9 years) with dyed hair had significantly less
chromosomal damage than did men (mean age 31.5 years) whose hair
was not dyed.
Possible confounding factors in these findings are that most
tinters wear gloves when applying hair dyes and, even without
gloves, percutaneous absorption of hair dye constituents may be
effectively impeded by the horny surface of the hands and by the
lack of sebaceous glands in the palms; hair dye constituents are
readily absorbed through the scalp, which contains numerous
sebaceous glands; and the lower frequency of chromosomal
aberrations in young men with dyed hair (compared with the
frequency in slightly older men without dyed hair) is
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probably due to an age effect. This preliminary evidence on
the genotoxic effects of hair dyes in humans warrants further
study in view of the known inutagenicity and carcinogenicity in
animals of several hair dye constituents.
CONCLiJS IONS
2,4—DT is carcinogenic in rats and mice after oral
administration; the produces amine produces liver and mammary
gland tumors. There is some evidence that it also induces
sarcomas at the site of subcutaneous injection in rats.
Published data on the carcinogenicity of 2,4—DT after
application to mouse skin are not adequate for evaluation.
2,4—DT is a potent mutagen in microbial test systems and
causes germ cell mutation in D. melanogaster . The substance
may cause chromosomel damage to (chromatid and chromosome gaps
and breaks) in humans. It also induces cell transformation in
an in vitro mammalian carcinogenesis test system. However,
there are no data, such as case reports or epidemiologic
studies, on the carcinogenicity of 2,4—DT in humans.
RECOMMENDAP IONS
There is absolutely no information available on the
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mechanism by which 2,4—DT is activated in susceptible species,
including rats and mice. However, given the demonstration of
carcinogenicity in two species of animals and the data on the
genotoxic effects in in vitro systems, it is prudent to assume
that humans are under some increased risk from exposure to
2,4—DT. Recomn endatjons for future research with 2,4—DT
include studies of the mechanism by which the chemical is
activated in rats, mice and humans, testing for carcinogenicity
in additional species to obtain more data on the relationship
between metabolism and carcinogenicity, and examination of the
in vitro metabolism of 2,4—DT in human tissues to supplement
the preliminary observations of Glinsukon et al. (1975). This
kind of additional data would make it easier to estimate the
extent of risk to humans exposed to 2,4—DT.
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REFERENCES
Production, Uses, Exposure
Chemical Economics Handbook. 1980. Stanford Research Institute
International, Menlo Park, Calif.
Code of Federal Regulations. 1980. Title 21, Part 177. Indirect
food and additives: Polymers. Office of the Federal Register,
National Archives and Records Service, General Services
Administration, Washington, D.C.
Colour Index. 1971. Third edition, volume 4. The Society of
Dyers and Colourists, Bradford, Yorkshire.
International Agency for Research on Cancer. 1978.
2,4—Diaminotoluene. Pp. 83—95 in IARC Monographs on the
Evaluation of the Carcinogenic Risk of Chemicals to Man. Volume
16. International Agency for Research on Cancer, Lyon.
Milligan, B., and K.E. Gilbert. 1978. M ines,
aromatic——Diaminotoluenes. Pp. 321—329 in Kirk—Othmer
Encyclopedia of Chemical Technology, Third edition, volume 2.
John Wiley & Sons, New York.
SRI International. 1979. 1979 Directory of Chemical Producers:
United States of America. Stanford Research Institute
International, Menlo Park, Calif. 1122 p.
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U.S. International Trade Commission. 1977. Synthetic Organic
Chemicals. United States Production and Sales, 1976. USITC
Publication 833. U.S. Government Printing Office, Washington,
D.C. 357 pp.
U.S. International Trade Commission. 1978. Synthetic Organic
Chemicals. United States Production and Sales, 1977. USI
Publication 920. U.S. Government Printing Office, Washington,
D.C. 417 pp.
U.S. International Trade Commission. 1979. Synthetic Organic
Chemicals. United States Production and Sales, 1978. USITC
Publication 1001. U.S. Government Printing Office, Washington,
D.C. 369 pp.
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Analytic Methods
Bogdanova, T.M., P.V. Mulyanov, N.M. Moncharzh, and M.L. Pirozhkova.
1976. Ultrasonic method for analysis of amine solutions. Zavod.
Lab. 42:1486—1487. [ Chem. Abs. 86:199421s, 1977.]
Boufford, C.E. 1968. Determination of isomeric diaminotoluenes by
direct gas—liquid chromatography. J. Gas. Chromatogr. 6:438—440.
Guthrie, J.L., and R.W. McKinney. 1977. Determination of 2,4— and
2,6—diaminotoluene in flexible urethane foams. Anal. Chem.
49:1676—1680.
Jones, A.D., D. Hoar, and S.G. Sellings. 1978. Separation of non—dye
components of Brown FK by high—performance liquid chromatography.
J. Chromatogr. 166:619—622.
Lepri, L., P.G. Desideri, and V. Coas. 1976. Separation and
identification of colouring agents in the oxidation—type hair dyes
by ion exchange thin—layer chromatography. Ann. Chim. (Rome)
66:451—460.
Lesiak, T., and H. Orlikowska. 1978. “In situ” determination of
toluenediamine and methylenedianiline isomers in the products of
hydrolyltic degradation of polyurethanes after separation by
thin—layer chromatography. Chem. Anal. (Warsaw) 23:469—475. (Chem.
Abs. 89:216094u, 1978.]
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tJnger, P.D., and M.A. Friedman. 1979. High—performance liquid
chromatography of 2,6— and 2,4—diaminotoluene and its
application to the determination of 2,4—diaminotoluene in
urine and plasma. J. Chromatogr. 174:379—384.
Willeboordse, F. , Q. Quick, and E.T. Bishop. 1968. Direct gas
chromatographic analysis of isomeric diaminotoluenes. Anal.
Chem. 40:1455—1458.
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Health Effects
Ames, B.N., H.O. Kammen, and E. Yamasakj. 1975. Hair dyes are
mutagenic: Identification of a variety of niutagenic
ingredients. Proc. Natl. Acad. Sci. U.S.A. 72:2423—2427.
Aune, T., S.D. Nelson, and E. Dybing. 1979. Mutagenicity and
irreversible binding of the hepatocarcinogen
2 , 4 —diamjnotoluene. Chem. Biol. Interact. 25:23—33.
Blijieven, W.G.H. 1977. Mutagenicjty of four hair dyes in
Drosophila melanoqaster . Mutat. Res. 48:181—186.
Burnett, C., B. Lanman, R. Giovacchinj, G. Wolcott, R. Scala,
and M. Keplinger. 1975. Long—term toxicity studies on
oxidation of hair dyes. Food Cosmet. Toxicol. 13:353—357.
Fahmy, M.J., and O.G. Fahmy. 1977. Mutagenicity of hair dye
components relative to the carcinogen benzidine in Drosophila
melanogaster . Mutat. Res. 56: 31—38.
Giles, A.L., Jr., C.W. Chung, and C. Konunineni. 1976. Dermal
carcinogenicity study by mouse—skin painting with
2 , 4 —toluenedjamjne alone or in representative hair dye
formulations. J. Toxicol. Environ. Health 1:433—440.
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Glinsukon, T., P. Benjamin, P. Grantham, E. Weisburger, and P.
Roller. 1975. Enzymatic N—acetylatiOn of
2,4—toluenediamine by liver cyto8ols from various species.
Xenobiotica 5(8): 475—483.
Gosselin, R.E., H.C. Hodge, R.P. Smith, and M.N. Gleason.
1976. Section II, p. 141 in Clinical Toxicology of
Commercial Products: Acute Poisoning. Fourth ed. Williams
and Wilkins, Baltimore.
Grantham, P.R., L. Mohan, P. Benjamin, pp. Roller, J.R.
Miller, and E.K. Weisburger. 1979. Comparison of the
metabolism of 2,4—toluenediamine in rats and mice. J.
Environ. Pathol. !Ibxicol. 3:149—166.
Rossack, D.J.N., and J.C. Richardson. 1977. Examination of
the potential mutagenicity of hair dye constituents using the
micronucleus test. Experientia 33:377—378.
International Agency for Research on Cancer. 1978.
2,4—Diaminotoluene. Pp. 83—95 in IARC Monographs on the
Evaluation of the Carcinogenic Risk of Chemicals to Man.
Volume 16. International Agency for Research on Cancer, Lyon.
Ito, N., Y. Hiasa, Y. Konishi, and M. Marugami. 1969. The
development of carcinoma in liver of rats’ treated with
rn—toluylenediamine and the synergistic and antagonistic
effects with other chemicals. Cancer Res. 29:1137—1145.
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Kirkland, D.J., S .D. Lawler, and S. Venitt. 1978. Chromosomal
damage and hair dyes. Lancet 2:124—128.
National Cancer Institute. 1979. Bioassay of
2,4—Diaminotoluene for Possible Carcinogenicity. CAS No.
95-80—7. ITS Carcinogenesis Technical Report Series No.
162. DHEW Publication No. (NIH) 79—1718. U.S. Dept. of
Health, Education, and Welfare, Public Health Service,
National Institutes of Health, Bethesda, Md. 122 pp.
National Institute for Occupational Safety and Health. 1978.
Pp. 1233 in Registry of Toxic Effects of Chemical
Substances, 1978, Lewis, R.J., Sr., ed. DHEW (NIOSH)
Publication No. 79—100. U.S. Department of Health,
Education, and Welfare, Public Health Service, Center for
Disease Control, National Institute for Occupational Safety
and Health, Cincinnati, Ohio.
Ong, P. 1978. Use of the spot, plate and suspension test
systems for the detection of the mutagenicity of
environmental agents and chemical carcinogens in Neurospora
crassa . Mutat. Res. 53:297—308.
Pienta, R.J., M.J. Shah, W.B. Lebherz III, and A.W. Andrews.
1977. Correlation of bacterial mutagenicity and hamster cell
transformation with tumorigenicity induced by
2,4—toluenediamine. Cancer Lett. 3:45—52.
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Umeda, M. 1955. Production of rat sarcoma by injections of
propylene glycol solution of m—toluylenediamine. Gann.
46:597—604.
Unger, P.D., A.J. Salerno, W.C. Ness, and M.A. Friedman.
1980. Tissue distribution and excretion of 2,4—( 14 C)
Toluenediamine in the mouse. J. Pox. Environ. Health.
6:107—114.
Venitt, S. 1978. Mutagenicity of hair dyes: Some more
evidence and the problems of its interpretation. Mutat. Res.
53:278—279. (Abstract No. 214).
Waring, R.}l., and A.E. Pheasant. 1976. Some phenolic
metabolites of 2,4—diaminotoluene in the rabbit, rat and
guinea—pig. Xenobiotica 6:257—262.
weisburger, E.K., A.B. Russfield, F. Homburger, J.H.
Weisburger, E. Boger, C.G. Van Dongen, and K.C. Chu. 1978.
Testing of twenty—one environmental aromatic amines or
derivatives for long—term toxicity or carcinogenicity. J.
Environ. Pathol. Toxicol. 2:325—356.
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Chapter 9
TRIFLURALIN AND ORYZALIN
NO NO 2
F 3 C_1c-_N CH 3 NH 2 02 S —(f >—--N —CH 2 —CH 3
==< ‘ CH 2 —CU 2 —CH 3 CH 2 —CH 2 —CH 3
NO 2 NO 2
Trifluraljn (c ,c ,c —trif1uoro—2 ,6—dinitro—N,N—dipropyl—p—
toluidine; also known as Treflan) and oryzalin (3,5—dinitro—N 4 ,
N 4 —dipropylsulfanilamjde) are both members of a class of compounds
known as dinitroanjljnes. Trifluralin is an orange crystalline solid
that melts at 49°C. It has a low solubility in water (0.3 ppm) and a
low vapor pressure (2 x l0 urni Hg at 30°C). Oryzalin is a
yellow—orange crystalline solid that melts at 141°C. It has a much
lower vapor pressure (less than l0 mm Hg at 30°C) than
trifluralin, but is slightly more soluble in water (2.5 ppm at 25°C).
Trjf luralin is produced via a series of reactions beginning with
the reaction of hydrogen fluoride with 2—chiorotoluene to produce
4 —trifluoromethylchlorobenzene. The latter compound is then nitrated
followed by reaction with di—N—propylamine, which replaces the
chlorine to form trifluralin. The production of oryzalin begins with
the nitration of 2—chlorobenzenesulfonjc acid, followed by the
addition of chlorine to form 2,6—dinitro—2—chlorobenzenesulfony l
chloride, followed by the addition of ammonia and di—N—propylamine to
yield oryzalin.
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PRODUCTI ON
Eli Lilly and Co. (Elanco Products Division) is the sole U.S.
producer of trifluralin (Stanford Research Institute International,
1979; U.S. International Trade Commission, 1978). The plant at
Lafayette, md., has an estimated 23,000—metric ton capacity for
production of trifluralin.
Lilly owns the patent rights for oryzalin, but has contracted
production to various other firms since receiving approval of
oryzalin use in 1975. GM’ Corporation made oryzalin for Lilly at
Rensselaer, N.Y., from January 1975 to June 1976 (Chemical Week,
1979). Proctor Chemical at Salisbury, N.C., produced oryzalin from
January 1975 to January 1976. The U.S. International Trade
Commission listed the Sodyeco Division of Martin Marietta
Corporation, Sodyeco, N.C., as a producer of oryzalin in 1977 and
1978. Other processors and formulators of oryzalin for Lilly have
included Bold Chemical of Tif ton, Ga., Central Chemical of
Hagerstown, Ark., and Helena Chemical of Helena, Ark. (Chemical Week,
1979). Although no estimates of production capacity are available
for these plants, the production of trifluralin in 1975 and 1978 is
estimated at 12,000 and 16,000 metric tons, active ingredient,
respectively. Oryzalin production in 1975 is estimated at 45 metric
tons, active ingredient; 1978 production is estimated at 450 metric
tons of active ingredient.
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USES
Trifluralin and oryzalin are both used almost exclusively as
herbicides. Consumption estimates for each in 1978 are shown in
Table 9—1.
The Environmental Protection Agency (EPA) has approved both
trifluralin and oryzalin for use as herbicides; however, both
compounds have been challenged on several occasions. Trifluralin was
challenged on the grounds that it contained nitrosamine contaminants
and that its use therefore posed an unacceptable carcinogenic risk.
Risk—benefit studies initiated by EPA concluded that benefits of
trifluralin outweigh any risks if nitrosamine contamination is kept
below 1 ppm (Chemical Marketing Reporter, 1979). Subsequently, Lilly
has produced trifluralin with a nitrosamine content of less than 1
ppm, and has continued to receive approval for its use as an
herbicide.
Oryzalin has been suspected of causing heart—related birth
defects among children fathered by workers involved in its
manufacture and of causing skin rashes in workers. However, EPA
investigations did not disclose any adverse effects, and no
regulatory action is planned (Chemical Marketing Reporter, 1980).
Nonetheless, Lilly is required to maintain ongoing teratological
studies to support the continued registration of oryzalin (Pesticide
and Toxic Chemical News, 1980).
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Table 9—1.
Consumption of Trifluralin and Oryzalin
Percent of 1O 3 Metric Tons of
Product Containing Compound Total Active Ingredient
Tr if lu ra 1 in
Cotton 21% 2.7
Deciduous fruits/nuts 1. 0.05
Peanuts 1 0.1
SOybeans 74 9.7
Sugar beets 1 0.05
Vegetables 2 0.3
Other field crops 1 0.14
Industrial/commercial 1
Total 100 13.1
Oryzalin
Cotton 10 0.05
Deciduous fruits/nuts 10 0.05
Soybeans 60 0.27
Industrial/commercial 20 0.09
Total. 100 0.45
Stanford Research Institute, 1979
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EXPOSURE
The principal routes of exposure to trifluralin and oryzalin are
the following; workers are exposed during manufacturing process;
agricultural workers are exposed while applying the substances to
crops; and the general public is exposed through releases of the
compounds into air and water during manufacturing processes, through
drift, volatilization, and runoff as a result of application, and via
contaminated food crops. Because more trifluralin is used than
oryzalin, and because much more data about trifluralin exist, this
chapter focuses on exposure to trifluralin.
Trifluralin is produced at a single site in Lafayette, md.
(population, 45,000); thus, exposure of the public to trifluralin
during its manufacture is of only local importance. Although no data
are available on trifluralin plant discharges, small quantities are
known to be discharged into the Wabash River as waste from the
manufacturing processes. The treatment procedure involves activated
sludge, followed by settling, and then filtration through activated
carbon (Spacie and Hamelink, 1979).
Parts per trillion (ppt) concentrations of trifluralin have been
measured in the water downstream from the plant. However, the
tissues of fish captured in these waters contain concentrations of
trifluralin several thousand times greater than those in the water
(Spacie and Hamelink, 1979).
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The most likely source from which the general public is exposed
to trifluralin is the herbicide’s application to soils to control
grass and broadleaf weeds. This is practiced primarily in the North
Central and South Central States, especially in Illinois, Iowa, and
Mississippi (Nelling, 1976).
Trifluralin is not an especially persistent chemical. There have
been some studies of the persistence of trifluralin in soil under
actual field conditions (Golab et al., 1979; Golab and Amundsen,
1975; Probst et a]., 1967) These studies show that trifluralin
concentrations decreased from 10% to 15% of their original value I
year after application. Trifluralin is lost through volatilization
as well as thçough degradation. At this rate of loss, trifluralin
does not tend to accumulate in soils receiving repeated applications.
Although the rate of volatilization of trifluralin depends to a
great extent on the method of its application, such dissipation
provides a significant potential exposure route for persons living
downwind of treated fields. If trifluralin is applied to the soil
surface, up to 90% of the compound may be volatilized within 2—3 days
of application. If it is incorporated into the soil, volatilization
losses can be as little as 3% or 4% in 90 days (Taylor, 1978). In an
experiment in which trifluralin was sprayed onto soil and then tilled
into the soil, White et a]. (1977) measured a maximum concentration of
16.5 g/m 3 20 cm above the soil during application. The maximum
concentration was 3.4 pg/rn’ 3 20 cm above the soil on the second day
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after the compound was tilled into the soil, decreasing to 100
ng/m 3 after the 35th day. They estimated total volatilization
losses to be at 22.4% during the growing season (120 days) and vapor
losses during spraying to be at 35%.
Exposure of persons living downwind of sprayed fields is somewhat
reduced because trifluralin is subject to fairly rapid photochemical
decomposition. Measurements of trifluralin degradation in air
indicate a half—life of 20 minutes under midday summer sunlight
conditions. Woodrow et a].. (1978) observed that the half—life
increased to 193 minutes in the fall. Triflura].in is stable in the
dark.
Although relatively immobile in soil, trifluralin may be
transported long distances from its initial source of application via
runoff into streams and rivers. Indeed, trifluralin has been detected
at levels of up to 0.2 pg/l in the Cape Fear River, N.C.
Trifluralin concentrations tended to correlate with periods of
greatest runoff and soil erosion on agricultural lands adjacent to
tributaries of that waterway (Horden, 1977). Although there is
probably minimal direct exposure of humans via this route, the
tendency of triflura].jn to accumulate in fish must not be overlooked
(Spacie and Hamelink, 1979).
Humans may also be exposed to trifluraljn through food crops
treated with the herbicide. However, analyses of residues in a wide
variety of tolerant crops indicate that trifluralin is not
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incorporated into the leaves, seeds, or fruits (measurement
sensitivity, 5—10 ppb). In treated root crops, such as onions and
garlic, trifluralin residues are found only in the outer shell, which
is usually discarded before consumption. The exception is carrots:
31% of the total residue of 0.65 ppm was found in the interior of the
carrot proper—--69% was concentrated in the peel (Probst 1967).
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ANALYTIC METHODS
Methods to analyze trifluralin in a variety of vegetables, plant
tissues, soil, water, oily crops, wheat grain and straw, and mint and
mint hay are described in the Pesticide Analytical Manual of the Food
and Drug administration (1973). Trifluralin is extracted from crops
by blending it with methanol and subsequent extraction from the
solvent into dichloromethane. After evaporation of the
dichioromethane, the extract is dissolved in hexane, cleaned up on a
column of Florisil, and analyzed by electron—capture gas
chromatography (EC—GC). GC columns (1.83 m) packed with 3% XE—60 or
5% SE—30 on Chromosorb W are used at approximately 180°C with the
carrier gas flowing at 90 ml/minute. The retention time (tR) for
trifluralin is 3 to 4 minutes. The analytic procedure is modified as
required for aqueous or oily samples. Recoveries of approximately
80% or more can be expected, and the sensitivity ranges from 5 to 10
ppb. Samples containing residues of benzene hexachloride, ethion,
and/or zineb require additional cleaning. The interfering compounds
are separated by thin—layer chromatography (TLC). The trifluralin is
then scraped from the plate, eluted, and assayed by EC—GC.
The procedure used at North Carolina State University (T.J.
Sheets, North Carolina State University, personal communication,
1980) to determine trifluralin residues in soils requires a 4—hour
Soxhiet extraction of the oven—dried sample with benzene—isopropyl
alcohol (1:2). The extract is evaporated just to dryness, dissolved
in hexane, cleaned on a Florisil column and subsequently analyzed
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by EC—GC. Glass columns (1.83 m long 0.635 cm outside diameter)
packed with 4% SE—30 plus 6% QF—1 or with 5% Carbowax 20M on Gas
Chrom Q are operated at 155°C with a gas flow of 80 to 100
mi/minutes. Under these conditions, the tR for trifluralin is
approximately 2.0 minutes.
Recently reported analytic methods have focused mainly on CC
techniques, coupled with various types of detectors including mass
spectrometers. Payne et al. (1974) developed a procedure to analyze
trifluralin, diphenamid, and paraguat in admixture in soil,
sediment, and water. The procedure permits simultaneous CC assays
of trifluralin and diphenamid, without cleanup, by employing a
Coulson electrolytic conductivity detector. Paraquat is determined
colorimetrically. The 1.83 m long glass GC column (0.635 cm
outside diameter) was packed with 10% DC-200 on Gas Chrom Q.
Recoveries of trifluralin spiked into water at concentrations of
0.05 to 10 ppb were 82% to 91%; those from soil spiked at 0.05 and
1.0 ppm were 86% and 94%.
Smith (1974) used acetonitrile—water (9:1) and ultrasonication to
extract residues of four herbicides, including trifluralin, from
three types of soil. The addition of excessive amounts of water and
saturated sodium sulfate solution to the extract facilitated
subsequent partitioning of the herbicide residues into hexane. The
hexane extract was then concentrated and assayed by EC—GC. The GC
system consisted of a 1.5 in long glass column (6 mm outside
diameter), packed with 10% OV—1 on thromosorb G—HP operated at
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mi/minute, tRS for dichiorobenji, trifluralin, dinitramine, and
triallate were approximately 1.0, 2.9, 4.5, and 5.0 minutes,
respectively. Recoveries of all four compounds spiked into the
three types of soil at 0.05 and 0.5 ppm levels were 92% or more.
Lawrence (1976) examined the separation characteristics of
several GC liquid phases (i.e., 3% OV—l, 4% SE—30—6% SP—2401, 10%
DC—200, and 5% DEGS on Chromosorb W HP) for 12 pesticides, including
trifluralin, by using the Coulson detector. Sensitivities (50%
scale) for trifluralin at a tR of approximately 3 minutes on OV—1
at 175°C were reported as 6 ng and 3 ng for the nitrogen and
chlorine modes, respectively. Later, Lawrence et al. (1977)
reported a confirmatory procedure for pesticides that contain
nitrogen dioxide (NO 2 ). The pesticide residue in 1 ml of benzene
was shaken with 0.5 ml of aqueous chromous chloride to convert the
NO 2 groups to NH 2 . The solution was made basic and the product
extracted with benzene for analysis by GC with a Coulson detector
(nitrogen mode). The products were found to be approximately as
sensitive as the parents and had tR values of 0.4 to 0.9 minutes
relative to the parent compound on the SE—30/SP—2401 column. The
procedure was used to confirm trifiuralin residues in extracts of
potato spiked at levels of 0.5 to 1.0 ppm.
Woodrow et al. (1978) used EC—GC to study the behavior of
trifluralin vapors released into the atmosphere as emulsifiable
concentrate sprays. The experiment was conducted with a 1.8 m long
glass column (3 mm, internal diameter), packed with 3% OV— 17
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ng/m 3 . In a similar study, Soderguist et al. (1975) studied
photoproducts of trifluralin in air by using EC—CC, TLC, and GC—mass
spectrometry (MS). The photoproducts were subjected to TIC on 0.5 nun
plates of silica gel G containing 1% zinc orthosilicate phosphor and
developed with hexane—acetone (3:1). The resulting bands were
scraped of f, eluted with acetone, and quantitated CC—MS by using a
1.5 m long glass column (3 mm, internal diameter) containing 25 OV—l
on Chromosorb C. The column temperature was programmed to rise from
150°C to 270°C at 10°/minute with a helium carrier flow of 16
mi/minute. As each compound eluted, its mass spectrum was recorded
and identified by comparison with an authentic specimen. Samples of
analysis by C—GC were analyzed under conditions similar to those
described by Woodrow (1978).
Downer et al. (1976) compared analytical results for residues of
benef in and trifluralin in soil by using a CC—MS procedure and an
established EC—GC assay. Comparable sensitivities (<10 ppb) and
results were obtained for both the mass fragmentógraphy (multi—ion
detector, MID) and the EC—GC procedures. The MID procedure was said
to have a shorter analysis time and to be less susceptible to
contamination than were EC—CC assays.
Heck et al. (1977) reported a high—pressure liquid chromatography
(HPLC)—MS procedure developed to obtain pharmacokinetic data from
rats dosed with trifluralin. Isotope dilution analysis, with
nonfragn nting mass spectrometry offered several advantages over
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other techniques for determining trace quantities of organic
compounds in biologic materials. Residues of the compound were
separated from rat tissue and excreta by sequential
high—pressure gel permeation and reverse—phase liquid
chromatography. The cleaned samples were then quantitated by
field ionization MS.
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HEALTH EFFECTS
The mutagenicity and chronic toxicity of both trifluralin
and a contaminant, N—nitrosodipropylamifle (NDPA), have been
reviewed extensively by EPA’s Special Pesticide Review
Division, Office of Pesticide Programs (OPP), and is reported
in “Trifluralin (Treflan) Position Document” (Environmental
Protection Agency, 1979). A discussion of specific needs of
EPA with several representatives from the OPP led to the
decision that this committee should focus only on a review of
the possible genetic toxicity of trifluralin. Consequently,
the committee limited its study to a review of the EPA’s
documents, which is summarized below with minor editorial
changes and updated information from several studies.
Trifluralin Mutagenicity Data
Technical trifluralin, containing known and undetermined
levels of NDPA, has been tested in a number of system without
producing mutagenic effects. The results of these studies are
presented in Tables 9—2 and 9—3.
24].
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TABLE 9-2
Mutagenicity Tests of Trifluralin (Part I) and NDPA (Part II)
( Part I )
Technical Trifluralin
Test System Species/Strain Result References
Gene Mutations
Bacterial Salmonella typhimurium Neg . Anderson et al., 1972
(8 Strains)
Salmonella typhimurium ,2 .
TA 100 Neg Simmon et al., 1977
TA 1535 Neg Simmon eta].., 1977
TA1537 Neg Simmon et a].., 1977
TA1538 Neg Simmon et al., 1977
Escherichja coli ’
WP2 Neg Simmon et al., 1977
Insect Drosophila melanogaster Neg Murnik, 1978
Other Escherichia coli with Neg ’ Anderson et al., 1972
T 4 bacteriophage
Chromosomal Mutations
Insect Drosophila melanogaster Neg Murnik, 1978
Primary DNA Damage
DNA repair, bacteria Escherichia coli
W3110/p 3478 Neg ’ Simmon et al., 1977
Bacillus subtilis
H17(Rec )/rna 5 U ) Neg ’ . Simmon etal., 1977
Yeast mitotic recombina— Saccharomyces cerevisiae
tion and/or gene conver— D3 Neg ’E Simmon et al., 1977
sion
Mammalian cell Human Fibroblasts
Unscheduled DNA synthesis WI—38 Cells Neg ’ Simmon et al., 1977
-------
TABLE 9-2
Mutagenicity Tests of Trifluralin (Part I) and NDPA (Part II)
( Part II )
NDPA
Test System Species/Strain Result References
Gene Mutations
Bacterial Salmonella typhimurium McCann et al., 1975
Unspecified Post Yahagi et al., 1977
TA 98 Negt Yahagi et al., 1977
TA 100 Post Yahagi et al., 1977
TA 1530 Post Bartsch et al., 1976
Camus et al., 1976
TA 1535 Post Olajos and Cornish,
1976
Escherichia coli
Sd—B(TC) Post Nakajima etal., 1974
Mammalian somatic Chinese hamster
cells in culture V79 lung cells Post Kuroki et al., 1977
Ch romosoma 1 Mutations
Mammalian cells in Chinese hamster
culture CHL cells Pos . Matsuoka etal., 1979
Primary DNA Damage
Yeast mitotic recombina— Saccharoniyces cerevisiae
tion and/or gene D3
conversion post Brusick and Mayer,
1973
! No metabolic activation used.
Strains tested with and without metabolic activation.
2 Test material contained 87 ppm NDPA as a contaminant.
Test for rh mutation
! Preliminary data — sample of test material with NDPA
removed in the laboratory.
In vitro chemical hydroxylation system used.
-------
TABLE 9-3
Mutagenicity and Related Tests with Formulated Treflan (Part I) and
Unspecified Forms of Trifluralin (Part II)
Part I: Treflan Formulations
Test Systems
Species/Strain
Result References
Gene Mutations
Bacterial
Salmonella typhimurium
(8 strains)
Neg!
Anderson et al., 1972
Insect
Drosophila melanogaster
Neg ’ Murnik, 1978
Chromosomal Mutations
Insect
Other studies
Drosophila melano9aster
Exposed Humans
Neurospora
Sordaria
Mouse bone marrow P06
Yoder et al., 1973
Griffiths, 1979
Bond and McMillan, 1979
Nehez et al., 1979
Part II: Unspecified Trifluralin
Chromosomal Mutations
Plants
Salmonella typhimurium !
TA 1535
TA 1536
TA 1537
TA 1538
Eacherichia coli !
B/r WP2 her+
WP2 her
Haemanthus katheriniae
Tradescantia paludosa
f
Pod
Pod
Shirasu et al.,
Shirasu et al.
Shirasu et al.,
Shirasu et al.,
Shirasu et al.,
1976
1976
1976
1976
1976
Jackson and Stetler, 1973
Sawamura and Jackson, 1968
Triturus helveticus
Pleurodeles walti
Sentein, 1977
Sentein, 1977
Primary DNA Damage
DNA Repair, Bacteria
Bacillus subtilis
H17 (RecF)/M45 (Rec)
Neg!
Shirasu et al., 1976
Pos ’ Murnik, 1978
P0s .
Pos
Gene Mutations
Bacter ial
Neg
Neg
Neg
Neg
Neg
Neg
Neg
e
f
Shirasu et al., 1976
Shirasu et al., 1976
Salamanders
Vicia faba
Triticuis aestivuni
Zea maya
Sawamura and Jackson, 1968
Bartels and Hilton, 1973
Bartels and Hilton, 1973
I
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! Metabolic activation not used.
Inconclusive results.
2 Product sample used contained 177 ppm NDPA.
Chromatid lesions in lymphocytes of workers exposed to
herbicides.
. Decreased number of microtubules, accumulation of large
vesicles at the cell plate region.
Disruption of mitotic process temporarily impeded
chromosome movement.
. Inhibition of mitosis as a consequence of spindle disruption.
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Bacterial Tests . Simmon et al. (1977) tested 20 pesticides,
including technical trifluralin (90%) containing 87 ppm NDPA as a
contaminant in reversion—type mutagenic assays. Four Salmonella
typhimurium strains and the WP—2 strain of E. coli were used, both
with and without mammalian metabolic activation systems. Activation
was obtained by using liver preparations from rats pretreated with
polychlorinated biphenyl (PCB) Arochlor 1254. Trifluralin produced
negative results in this study.
Simmon et al. (1977) also tested the same trifluralin sample for
unscheduled DNA synthesis in human fibroblasts (WI—38 cells),
mitotic recombination in the yeast Saccharomyces cerevisiae strain
D3, and preferential toxicity in repair—deficient strains of E. coli
and Bacillus subtilis , as compared to strains that could repair DNA
damage. Each of these assays was performed both with and without
mammalian metabolic activation over a wide range of trifluralin
concentrations. Findings for trifluralin were negative in all
assays. The experimental and data—reporting procedures used in this
study appear to have been adequate.
Anderson et al. (1972) evaluated 109 herbicides, including both
technical and formulated (44.5%) trifluralin. Specifically, the
investigaters looked for induction of point mutations in a battery
of standard bacterial and viral plate assays involving base air
substitution and frameshift reversions as well as forward mutation.
They compared their results to positive results with known mutagens
specific for each of four assays. Single doses of all test
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chemicals, including formulated trifluralin (20 or 25
pg/plate), did not induce changes significantly different from the
spontaneous rates of mutation in eight histidine—requiring mutants
of Salmonella typhimurium , two ru mutants of bacteriophage T 4 , or
E. coli strain KB. This study did not conclusively demonstrate lack
of mutagenicity because the investigators provided no exogenous
metabolic activation to mimic possible conversion of the chemical to
potentially active intermediates and they administered only one dose.
Shirasu et al. (1976) studied the mutagenicity of an unspecified
form of trifluralin in four histidine—requiring strains of
Salmonella typhimurium in a standard Ames assay as well as in
differential toxicity assays with Bacillus subtilis strains R17
(Rec+) and M45 (Rec) and in reversion assays with two strains
of E. coli that require tryptophan. In each of these assays, the
investigators treated bacterial cultures with a single saturated
paper disc containing a 0.02 ml solution of a standard sample made
up at a concentration of 1 mg trifluralin per milliliter of
dimethylsulfoxide. Trifluralin showed negative results for
mutagenicity in this study. However,the investigators did not
provide mammalian metabolic activation, and they tested only one
concentration. Thus, these results are inconclusive.
Insect Studies . Preliminary results from a study of both
genetic and chromosomal effects in Drosophila melanogaster (Murnik,
1978 and I. Mauer, EPA, personal communication, 1978) showed
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“no evidence ... that trifluralin induces point mutations in
Drosophila, ” but some of the results of these two separate studies
are contradictory. In one portion of the first study, larvae were
fed a diet containing 0.01% formulated trifluralin (44.5% Al)
throughout their stages of development, and the number of
sex—linked deaths was recorded in the F 2 (second) generation. In
replicate tests, no significant differences were found in mortality
rates between the treated (0.10% for the first test) and combined
control (untreated spontaneous, 0.12%) groups. No positive
controls were reported, and the formulated trifluralin tested
contained approximately 177 ppm NDPA. In the second portion, adult
males fed 0.02% of the formulated trifluralin for 2 days also
incurred no increase in sex—linked deaths.
Although preliminary results from the two studies showed no
evidence that trifluralin induces point mutations in Drosophila ,
Murnik (1978) reported an increase in chromosoma]. nondisjunction.
Her first study involved the chronic feeding of 0.01% formulated
trifluralin throughout the larval stages. Feeding at this stage
resulted in a significant increase (0.12%) of XXY males compared to
those of a control population (0.04%). However, feeding 0.02%
formulated trifluralin to adult male Drosophila for 2 days resulted
in no increase in nondisjunction. XXY nondisjunction was the only
chromosomal aberration reported in this test. There were no
increases over controls in chromosoma]. loss (XO progeny) or
breakage. When the chromosomal portion of the first study was
repeated, with technical trifluralin having no detectable NDPA,
nondisjunction was not observed in test animals at a level
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significantly different from that of the control population. Thus,
the cytogenetic results from these two studies are inconclusive as
well as contradictory (Chaisson, 1978).
Studies with Fungi . Trifluralin has also been tested for
nondisjunction in Neurospora crassa (Criffiths, 1979) as well as in
Sordaria brevicollis (Bond and McMillan, 1979). Griffiths
concluded that trifluralin produced aneuploidy in Neurospora .
Nondose—related increases were found over a dosage range of 1—75 mg
trifluralin/liter of culture. No information was given on the
purity or source of the trifluralin nor of its ability to induce
other genetic effects in Neurospora . The authors considered the
results obtained in Sordaria to be inconclusive.
Rodent Tests . Male mice were injected with a trifluralin
formulation containing 26% trifluralin (OLITREF ) in the following
dosage regimen: A single dose of 200 mg/kg, two 100 mg/kg doses
four 50 lug/kg doses, and a single 0.6 mg/kg dose. The fractional
doses were administered at 3—day intervals (Nehez et al., 1979).
Significant increases in chromosomal aberrations in the bone marrow
were observed at all but the lowest dosage regimen. The LD 50 of
the trifluralin formulation was 600 mg/kg. The authors concluded
that this preparation induced chromosomal aberrations in bone
marrow at total doses of as little as one—third the LD 50 .
Plant Studies . A nuither of plant studies have been conducted
to determine whether trifluralin can disrupt the cellular spindle
apparatus. The studies were not performed specifically to assess
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the issue of mutagenicity. In an in vitro and ultrastructural
study of celiwall free endosperm cells of the African blood lily
( Haemanthus katherinae ) , Jackson and Stetler (1973) reported that
concentrations of trifluralin, ranging from 0.1—100.0 ppb,
inhibited the rate at which cells progressed through all stages of
mitosis from prophase to cell plate appearance. They observed
these effects by time—lapse phase microscopy during a 2—hour
period. Since 0.1 ppb had a near—maximum inhibitory effect, the
data presented from all concentrations were pooled. Electron
microscopic studies showed a decreased number of microtobules and
an accumulation of large vesicles in the cell plate region.
The ultrastructural and mitotic index studies appear to have
been conducted according to established protocols, but the bioassay
used to assess these effects is not well documented. Furthermore,
Jackson and Stetler did not establish a dose—response relationship,
included no positive control in the study, and provided no
information on the amount of NDPA that contaminated the study
material. Nonetheless, this study does indicate that trifluralin
interferes with the formation and function of plant cell
microtubules, and the substance, may therefore, disrupt the mitotic
spindle, thereby inducing numerical chromosomal aberrations.
Sawamura and Jackson (1968) treated staminal hair cells of the
tetraploid Tradescantia paludosa and leaf cells of Vicia faba with
0.2 to 1.6 ppb of trifluralin. The degree of NDPA contamination
for this material was unknown. At the highest dose (1.6 ppb) , the
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authors reported the appearance of “dicentric bridges” in late
stages of mitosis (anaphase and telophase) in both cell types, and
cell elongation in staminal hair cells only. This study
demonstrates that trifluralin can disrupt various stages of plant
cell mitosis; however, the report is of limited value because the
system tested is questionable, the data are not quantitative, and
the study was not designed to assess mutagenicity.
Bartels and Hilton (1973) treated wheat ( Triticum aestivum L.,
C.I. 5303) and corn (Za s L., Yellow Dent U.S. 13) with
technical—grade trifluralin at 10 4 M. Cell division in the roots
of the germinating seedlings appeared to be arrested at metaphase.
The arrested cells showed no microtubule formation. Trifluralin
did not inhibit microtubule assembly nor did it bind to the
microtubule protein as does colchicine. The investigators
concluded that trifluralin acts on the microtubular organizing
centers, rather than on the microtubules se.
Salamander Study . Sentein (1977) reported that trifluralin
inhibited mitosis by interfering with the spindle apparatus in two
urodele salarnanders, Triturus helveticus and Pleurodeles walti .
Eggs of these species were incubated in various concentrations (1/8
through full saturation) of an unspecified form of trifluralin for
1 to 10 mitotic cycles prior to the beginning of cleavage, or at
the 2—, 4—, 8—, and l6—blastomere stages. Cytologic observations
were made during treatment and after various periods of incubation
following transfer of the eggs to a trifluralin—free culture
medium. At similar concentrations, the effects were more severe in
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Pleurodeles than in Triturus eggs, but multinucleate blastomeres
and disorganized mitotic figures occurred in both species. Sentein
also reported disturbances in chromosomal condensation, especially
at chromosomal sites associated with the mitotic spindle
attachments, and gaps (discontinuities) at prophase. According to
the author, the cytologic effects of trifluralin resembled those
induced by classic antimitotic agents, but trifluralin was much
less potent. The author concluded that these effects demonstrate
that trifluralin interferes with the formation or function of
cellular microtubu].ar elements.
This study is difficult to interpret because of the lack of
details pertaining to its protocol. For example, the source and
composition of the trifluraljn were not stated, nor were control
data included (except that the solvent was reported to be
polyethylene glycol). However, the study does confirm, in an
animal test system, the potential antimitotic action of trifluralin
previously found in plant cytologic studies.
The cytologic studies also support the genetic studies in
Neurospora and Drosophila that indicate possible nondisjunctional
activity of trifluralin (Mauer, 1978)
Human Survey . Yoder et al. (1973) observed chromosomal
alterations in lymphocyte cultures prepared from samples obtained
from people who apply pesticides. Blood was drawn once during the
midwinter lull in spraying operations and again during the peak
summer spraying period. Forty—two white male workers, who had from
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1 to 25 years (mean exposure, 8.5 years) of prior occupational
exposure to a variety of pesticides, were matched as closely as
possible in age and physical characteristics to a control group of
16 businessmen, students, and teachers with no history of
involvement with pesticides. The exposed group was divided into
two subgroups. One consisted of 16 people who had been exposed to
a variety of 17 insecticides; the other consisted of 26 employees
of weed control agencies who had been exposed to 14 herbicides
(most frequently to 2,4—D, amitrole, and atrazine, but also to
formulated trifluralin).
The incidence of chromatid lesions per person in the worker
groups increased significantly over that in the control group, but
only in blood samples taken in the summer. Although Yoder et
observed no heteroploidy (which may be indicative of
nondisjunction) in any of the exposed or control cells, the authors
did note a small number of chromatid exchange figures among the
exposed groups. This study must be regarded as inconclusive in
implicating trifluralin as a chromosome breaker, because it was
only one of many pesticides used by these workers.
NDPA Mutagenicity Data
The principal contaminant of technical trifluralin preparations
is NDPA, which is a demonstrated oncogen in rodents (Montesano and
Bartsch, 1976). NDPA has been studied in in vitro mutagenicity
assays with bacteria and yeasts, as well as in mammalian cell
culture, coupled with appropriate mammalian metabolic activation
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systems (see Table 9—2) . In bacterial assays, NDPA has caused
reverse mutations by base—pair substitution at concentrations of up
to 1.0 mmol, but only in the Presence of complete liver enzyme
preparations from rodents. When the cofactors for the microsomal
mixed—function oxidase were omitted, the mutagenic effect was
absent. Positive results for gene mutation as well as for
chromosoma]. aberrations were also obtained in Chinese hamster lung
cell cultures treated with 20 rnmol NDPA and a rat liver enzyme
preparation (Kuroki et al., 1977; Matsuoka et al., 1979).
Krueger (1973) also found direct evidence that NDPA alters
genetic material in vivo . He reported the presence of alkylated
guanine residues in DNA of rats given NDPA.
Trifluralin Derivatives
The mutagenic potential of degradation and/or metabolic
products of trifluralin has also been assessed. Evidence indicates
that trifluralin may degrade into a series of products, including
substituted benzimidazoles in a mammalian—derived in vitro
microsome system (Nelson et al., 1977). Such conversion has been
reported to occur under ultraviolet photodecornposition conditions,
especially in the vapor phase above treated soil, as well as in the
soil. This finding is of concern because some benzimidazoles have
been shown to be mutagenic (Seiler, 1972).
A report by Nelson et al. (1977) presents some interim results
from bacterial mutagenicity assays performed with nine trifluralin
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metabolites, including some benzimidazoles. In these analysis, the
investigators used plate incorporation at concentrations of up to
200 jig/plate for a standard battery of five test strains of S.
typhimurium , both with and without metabolic activation.
Summarizing the results, Nelson reported that he had found “no
potent mutagens among these trifluralin derivatives tested thus
far,” as compared to the expected response of positive controls
appropriate to each of the test strains.
Mutagenic Risk Assessment
Neither technical nor formulated trifluralin (containing NDPA
at levels of up to 177 ppm) has shown any mutagenic activity in the
studies cited here. The principal contaminant, NDPA at
concentrations greater than 20 times those contained in current
formulations of trifluralin (< 1.0 ppm), has induced mutations in
various test systems (Mauer, 1978). NDPA is therefore considered
to be a mutagen.
At least two situations of potential mutagenic risk exist: The
direct effects on DNA and genes related to the NDPA contaminant,
and the potential effects trifluralin induces on the spindle
apparatus.
DNA and Gene Effects . When tests were performed with metabolic
activation, technical trifluralin (containing approximately 87 ppm
NDPA) did not produce significant gene mutations or primary DNA
damage. Formulated trifluralin (as Treflan or unspecified) also
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produced negative results in some of the same tests; however, the
results of these latter tests are inconclusive because they were
performed without exogenous metabolic activation. Other
preliminary studies indicate that Treflan, containing 177 ppm NDPA,
as well as trif].uralin with no detectable NDPA, produce negative
results in the Drosophila sex—linked recessive lethal test. On the
other hand, NDPA by itself has been shown to be mutagenic in
several in vitro microbial test systems by causing base—pair
substitution and primary DNA damage (Chaisson and Burkhalter,
1978). NDPA concentrations in the trifluralin preparations tested
may have been too low to produce gene mutations or direct DNA
interaction, especially in the presence of trifluralin (Chaisson
and Burkhalter, 1978).
Triflura].in—NDPA mutagenicity data are not adequate to
determine, much less to quantify, any risk for gene or DNA
interactions posed by trifluralin. Any potential DNA and gene
effects are associated with the NDPA contaminant of trifluralin
formulations. To pose a potential, heritable genetic risk to
humans, a chemical must be mutagenic and must be capable of
reaching mammalian germ cells in a metabolically active form.
There is no evidence showing whether mutagenically active forms of
trifluralin or NDPA do or do not reach mammalian germinal tissue or
whether these compounds are metabolized in situ to active forms if
they do reach these tissues. The NDPA data in Table 9—2 indicate
the need for metabolic activation of this compound before it can
induce mutagenic responses in test organisms. Although NDPA has
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mutagenic activity in some in vitro test systems, including
mammalian cells in culture, no in vivo tests have been performed.
Data on the structurally related aliphatic nitrosamines,
dimethylnitrosamine (DMN), and diethylnitrosamine (DEN) can be used
to bridge the information deficits concerning the in vivo mutagenic
activity of NDPA. DMN and DEN are mutagenic in both the Ames S.
typhimurium and Drosophila sex—linked lethal tests. Three mouse
dominant lethal studies on these chemicals have also been
performed. A single intraperitoneal dose of DEN (13.5 mg/kg body
weight) did not significantly increase the number of mutations in
the offspring of treated males (Propping et al., 1972). DMN also
produced negative results when male mice were administered DEN at 8
or 9 mg/kg body weight by the same route (Epstein et al., 1972).
DMN was reported to produce a weak dominant lethal effect in a
second study with male mice (Propping et al., 1972). The second
study has positive results with a DMN dosage lower than that
yielding a negative response in the first study; however, the mouse
strain and route of administration differed. Propping et al. used
only a single treatment group. The lack of varying treatment
levels precluded any within—experiment replication of the results
or knowledge of dose—response relationships. The authors did state
that the 4.4 mg/kg dose was the highest dosage of DMN compatible
with survival.
Because of the great variation in the responses of animals in
the dominant lethal test and the positive finding at a level of
significance just meeting the accepted critical level, the study
seems inconclusive. At face value, it suggests that DMN can reach
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the mammalian gonad. A negative interpretation, however, is consistent
with the finding that neither DMN nor DEN stimulated unscheduled DNA
synthesis in the mouse testis following intraperitoneal administration
of the test compounds and tritiated thymidine (Gary Sega, personal
communication, 1979). Also, DEN produced negative results in a
specific locus test with mice (Russell, 1977). NDPA itself has not
been tested for germinal or in vivo mammalian mutation. The available
evidence does not support that Treflan—containing NDPA causes a
significant risk to DNA and genes for the following reasons:
o NDPA appears to have point mutagenic activity in some in vitro
systems, but information is lacking from in vivo tests. Some
other short—chain alkylnitrosamjnes have been reported to
produced positive results in the Drosophila sex—linked
recessive lethal test.
o There is no direct evidence that NDPA does or does not reach
the mammalian gonad in a genetically active form. As for
other nitrosamines, it has been reported that neither DMN nor
DEN stimulates unscheduled DNA synthesis in the mouse testis.
Only one of three dominant lethal studies with these chemicals
in mice suggests a positive effect, and that study reported a
very weak positive finding with DMN. However, DEN produced
negative results in a specific locus test with mice.
o Testing of trifluralin products containing 87 ppm NDPA has
shown negative results for both mutagenic and DNA—damaging
activity.
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o A preliminary study with Treflan containing 177 ppm NDPA
produced negative results in the Drosophila sex-linked
recessive lethal test.
o Exposure of humans to NDPA through trifluralin use is expected
to be very low.
At this time, it is not possible to quantify the mutageniC hazard
that might be associated with the use of trifluralin contaminated with
NDPA because information on the presence of the active compound in the
mammalian gonad and the results of germinal testing are lacking.
Occupational exposures to NDPA ( 5.05 pg/yr) and exposures of the
general population through consumption of treated food (approximately
1.92 x lO mg/kg body weight/day assuming the presence of a residue
and a 5 ppm level of NDPA contamination in Treflan) are very low.
Further, even plants and runoff water from fields treated with
trifluralin (containing NDPA) did not contain any NDPA. The
manufacturer has already lowered the contamination to 1 ppm or less.
Thus, any risk is reduced further by a factor of approximately five.
Because risks of adverse effects are intimately related to exposure
and because the expected exposure of humans to NDPA is low, it is also
expected that any risk from point mutagenic effects would be mininal.
To obtain a better evaluation of point mutagenic risks, other tests
should be conducted on NDPA, including studies assessing its ability to
reach the mammalian gonad in a metabolically active form.
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Spindle Effects . The limited studies that have been conducted
appear to show that high concentrations of trifluralin (with or
without stated levels of NDPA) can disrupt formation or function of
the spindle apparatus in dividing cells, and thus have the potential
to cause abnormal segregation of chromosomes (nondisjunction).
Tests with formulated trifluralin (containing approximately 177 ppm
NDPA) in Drosophila showed nondisjunction. Replication of these
tests with technical trifluralin having no detectable NDPA produced
negative results. However, positive results, showing effects on the
spindle, were reported when formulated trifluralin (NDPA content
unknown) was tested on Neurospora .
The positive chromosonial effects reported in plants and
salamariders indicate that trifluralin (or trifluralin plus NDPA) may
affect spindle fibers by interfering with microtubule formation or
function. However no comparable studies in mammalian test systems,
either in vitro or in vivo , have been reported. Because the
mechanism of cell division does not differ significantly between
plants and animals, similar spindle effects might be expected to
occur in mammals exposed to trifluralin.
With this in mind, the committee surveyed mammalian and fish
studies for evidence of tnitotic disturbances, abnormalities in
treated embryos, or any other chroniosoma]., spindle, or cellular
effect of triflura].in on developmental processes. Overt
manifestations of such effects include depressed cell formation and
maturation, decreased viability of embryos, high resorption rates,
260
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or delayed tissue maturation (such as slow rates of ossification in
neonates) . Reports of vertebral hypertrophy in treated fish (Couch
et al., 1978) and variations in skeletal development in mice (Beck,
1977) are not evidence of mitotic spindle effects and do not support
the theory of a mutagenic effect of trifluralin in mammalian
systems. Evaluation of hematologic values from chronic toxicity
studies also did not elicit any such evidence (Mauer, 1978).
Thus, several lines of evidence from both the plant and animal
kingdoms suggest that trifluralin products, containing known or
unknown levels of NDPA, can interfere with the cell division
spindle. Mammalian somatic or germinal cells have not been studied,
but mammalian cells would probably respond similarly to cells of
other organisms.
The existing data regarding effects on the cell division spindle
at estimated trifluralin or NDPA exposure levels are inadequate for
an assessment of risk. Furthermore, it is not clear whether
trifluralin itself, one of its metabolites, or a contaminant is the
active cause of the noted effects. Additional studies are needed to
clarify these uncertainties.
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CONCLUSIONS
Extensive mutagenicity testing has been performed with
trifluralin, with positive results in some instances. These results
are tainted because they were obtained using undefined trifluralin
or trifluralin contaminated with the known mutagen—carcinogen NDPA
at levels as high as 177 ppm. Therefore, it is quite possible that
the mutagenicity (and CarCiflOgenicity) attributed to trifluralin
could be related to the contaminant. NDPA—free trifluralin should
not be considered mutagenic (or carcinogenic) until a carcinogenesis
bioassay on NDPA—free trifluralin is performed for comparison with
the existing NCI study.
The majority of the mutagenicity studies with trifluralin
produced negative results. Those that were positive for chromosomal
damage and aneuploidy may be due to the presence of NDPA. Parallel
studies with NDPA have not been reported. NDPA—free trifluralin and
pure NDPA need to be tested, in tandem, for their abilities to
induce chromosomal damage and aneuploidy.
Examination of the mutagenicity data on trifluralin preparations
(including the formulated product) containing NDPA reveals an
inadequate data base on which to evaluate potential hazards to DNA
and spindles in laboratory animals or humans. NDPA may induce
mutagenic effects, but the expected low exposures to this chemical
suggest that the degree of hazard, even if NDPA are a germinal
mutagen, are low.
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Golab, T., and M. E. Amundson. 1975. Degradation of trifluralin,
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Golab, T., W.A. Aithaus, and H.L. Wooten. 1979. Fate of
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Horden, w. 1977. A Report Ofl the Use of the Macroreticular Resin
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Probst, G.W., T. Golab, R.J. Herberg, F.H. Holzer, S.J. Parka,
C.C. Van der Schans, and J.B. Tepe. 1967. Fate of trifluralin in
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Spacie, A., and J.L. Hamelink. 1979. Dynamics of trifluralin
accumulation in river fishes. Environ. Sci. Technol. 13:817—822.
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United States of America. Stanford Research Institute
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Taylor, A.W. 1978. Post—application volatilization of pesticides
under field conditions. 3. Air Pollut. Control. Assoc. 28:922—927.
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Chemicals. United States Production and Sales, 1977. ISITC
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White, A.W., Jr., L.A. Harper, R.A. Leonard, and J.W. Turnbull.
1977. Trifluralin volatilization losses from a soybean field. J.
Environ. Qual. 6:105—110.
Woodrow, .3.E., D.G. Crosby, T. Mast, K.W. Moilanen, and J.N.
Seiber. 1978. Rates of transformation of trifluralin and
parathion vapors in air. J. Agric. Food Chem. 26:1312—1316.
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Analytic Methods
Downer, G.B., t4. Hall, and D.N.B. Mallen. 1976. Determination of
benefin and trif].uralifl residues by quantitative gas—liquid
chromatography/mass spectrometry. 3. Agric. Food Chem.
24:1223—1225.
Food and Drug Administration. l973• Pesticide Analytical Manual.
Heck, H.d’A., R.L. Dyer, A.C. Scott, and M. Anbar. 1977.
Determination and disposition of trifluraliri in the rat:
Separation by sequential high—pressure liquid chromatography and
quantitation by field ionization mass spectrometry. J. Agric. Food
Chem. 25:901—908.
Lawrence, J.F. 1976. Gas chromatographic separation of herbicides
of major interest in Canada, with electrolytic conductivity
detection in the nitrogen and chlorine modes. 3. Chromatogr. 121:
85—87.
Lawrence, J.F,, D. Lewis, and H.A. McLeod. 1977. Confirmation of
some N0 2 _ containing pesticides by chemical reduction and gas
chromatography with electrolytic conductivity detection. J. Agric.
Food Chem. 25:1359—1361.
Payne, W.R., Jr., J.D. Pope, Jr., and J.E. Benner. 1974. An
integrated method for trifluralin, diphenamid, and paraquat in soil
and runoff from agricultural land. J. Agric. Food Chem. 22:79—82.
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Smith, A.E. 1974. A multi—residue extraction procedure for the gas
chromatographic determination of the herbicides dichiobenil,
dinitramine, triallate and trifluralin in soils. J. ChromatOgr.
97:103—106.
Soderquist, C.J., D.G. Crosby, K.W. Moilanen, J.N. Seiber, and,
J.E . Woodrow. 1975. Occurrence of trifluralin and its
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Health Effects
Andersen, K.J., E.G. Leightly, and M.T. Takahashj. 1972.
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Bartels, P.G., and J.L. Hilton. 1973. Comparison of trifluralin,
oryzalin, pronamide, propham and coichicine treatments on
microtubu].es. Pestic. Biochem. Physiol. 3:462—472.
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H. Bartsch, and L. Tomatis, eds. Screening Tests in Chemical
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herbicides in mice, using normally occurring variations in
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Bond, D.J., and L. McMillan. 1979. Meiotic aneuploidy: Its
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brevicollis . Environ. Health Perspect. 31:67—74.
Brusick, D.J., and V.W. Mayer. 1973. New developments in
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Chapter 10
p-CRESIDINE
NH 2
(j (OCh1 3
—Cresidine (2—methoxy—5—inethylanaline) is a white crystalline
solid that melts at 51°C. It is sparingly soluble in water, but
volatilizes in the presence of steam. 2 —Cresidine is also known as
2—methoxy—5—methylbenzeneamine, 5—methyl—o—anisidifle and, rn—amino—
—cresol methyl ether.
E—Cresidine is obtained from the methylation and reduction by
hydrogen of p—cresol, which is derived from the action of nitrous
acid and excess nitric acid on 2 —toluidine.
PRODUCTION
Currently, the sole U.S. producer of p—cresidine is the
Sherwin—Williams Company in St. Bernard, Ohio (Stanford Research
Institute International, 1979). There is no record of plant
capacity.
The production of p—cresidine was reported by the U.S.
International Trade Commission in 1976—1977, implying that
commercial production was greater than 2,300 kg/year (U.S.
International Trade Commission, 1978). At least 450 kg/year
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was produced commercially in the United States in 1978—1979
(Stanford Research Institute International, 1979). ImportS (in
thousands of kilograms) through principal U.S. customs districts for
1976 to 1979 are as follows: 1976, 262.3; 1977, 40.2; and 1978,
125.7 (U.S. International Trade Commission, 1977, 1978, 1979).
USES
—Cresidine appears to be used solely as a chemical intermediate
in the production of dyes. The most commercially important of these
dyes is the food, drug, and cosmetic dye (FD&C) Red No. 40, (of
which 864 metric tons were produced in the United States during 1978
by the following companies: Buffalo Color Corp., Buffalo, N. Y.;
Crompton & Knowles Corp., Gibraltar, Pa.; H. Kohnstamm & Co., Inc.,
Brooklyn, N. Y.; Hilton Davis Chemical Co., Division of Sterling
Drug, Inc., Cincinnati, Ohio; and Warner—Jenkinson Co., St. Louis,
Mo. (U.S. International Trade Commission, 1979).
2—Cresidine can also be used to manufacture six other dyes
produced commercially in the United States, although no separate
production figures were reported: C. I. Direct Blue 67, produced by
Crompton & Knowles Corp., Fairlawn, N. 3.; Direct Blue 126, produced
by Harshaw Chemical Co., subsidiary of Gulf Oil Corp., Louisville
Ky.; Direct Green 26, produced by Torns River Chemical Corp., Toms
River, N. 3.; Direct Orange 34, produced by Crompton & Knowles
Corp., Fairlawn, N. J. and E. I. du Pont de Nemours & Co., Inc.,
Deepwater, N. 3.; and Direct Red 79 and Direct Violet 9, produced by
275
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Crompton & Knowles Corp , Fairlawn, N. J., and Toms River Chemical
Corp., Toms River, N. J. (Colour Index, 1971; U.S. International
Trade Commission, 1979)
EXPOSURE
There are few data to support an estimation of human exposure to
E—cresidine. Small quantities of the chemical appear to be produced
and imported and most exposures probably occur during the synthesis
of dyes that use 2—cresidine as an intermediate.
Some 2—cresidine may appear as an impurity in FD&C Red No. 40.
There are no Food and Drug Administration (FDA) regulations for
2—cresidine; however, the FDA regulations for Red No. 40 allow for
the presence of up to 1% each of sulfonated subsidiary dyes used in
its production (21 CFR 74). The subsidiary dyes may result from the
presence of impurities, such as 2—cresidine, in the FD&C Red No. 40
intermediates, one of which is diazotized cresidine—E—sulfonic acid
(Bell, 1976)
276
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ANALYTIC METHODS
Although £—cresidine should respond to many of the procedures
for primary aromatic amines, no information was available
concerning analytic chemical methods for the compound. Sherwin
Williams Company kindly provided the following information in the
ensuing paragraphs (private communication, 1980).
Assays for Purity
Analysis is accomplished by using a gas chromatograph equipped
with 1.5 m long column (0.32 cm inner diameter) packed with 20%
SE—30 on Chromosorb W AW DMCS (80—100 mesh) and a flame ionization
detector. The column oven is operated at 180°C and the injector
and detector at 300°C; the nitrogen carrier flows at 20 mi/minute.
Under these conditions, a methanol solution (0.4 jil) containing
400 jig of the compound is injected into the instrument for
analysis; quantification is based on the area under the 2—cresidine
peak. Although the retention time (tR) cannot be determined from
the available information, it appears to be approximately 6 minutes
or less. The peak is symmetrical. The sensitivity of the assay
can easily be enhanced by a factor of approximately 1,000 by
injecting a larger volume and using less attenuation; however, this
technique offers no particular advantage to the purity analysis.
277
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Analysis of Atmosphere in the Workroom .
Atmosphere in a Sherwin—Williams workroom is sampled by using an
apparatus consisting of a midget bubbler charged with 10 ml of
acetic arihydride followed by an activated carbon impinger and a
membrane filter to protect the vacuum pump. Air samples are
collected at a rate of approximately 1 1/minute for 1 to 2 hours.
Any 2—cresidifle in the air reacts with the acetic anhydride to form
the corresponding acetamide, which is analyzed by high—pressure
liquid chromatography (HPLC).
The acetic anhydride from the bubbler is diluted to 25 ml with
the same solvent, and 10 )Jl is injected into an HPLC equipped with a
column of Partisil 1025 ODS (0.46 X 25 cm) and a LJV absorption
detector set at 280 nm. The mobile phase consists of 55%
methanol—45% water containing 0.5% acetic acid; the flow rate is 1.0
mi/minute. Under these conditions the tR of 2—cresidine acetamide
is approximately 7.0 minutes. Quantitation is accomplished by
relating the areas under the peaks from samples of unknown
p—cresidine content to those of standards of
acetylarnino-p—cresidine. The minimum detectable level of the
compound in the diluted solution from the bubbler is 0.5 ]Jg/ml.
278
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HEALTH EFFECTS
Data on the toxicity of E—cresidine in animals and humans are
virtually nonexistent. The National Institute for Occupational
Safety and Health (1976) has reported an oral LD 50 in rats of
1,450 mg/kg.
In dose range—finding studies conducted by the National Cancer
Institute (NCI) Bioassay Program (National Cancer Institute, 1979)
groups of five male and female F—344 rats and five male and female
B6C3F1 mice were placed on diets containing 0, 1 and 3% E—cresidine
for 8 weeks. Deaths (number unspecified) occurred in male and
female mice and in female rats receiving the 3% diet. No deaths
occurred in the male rats receiving 3% 2—cresidine in any of the
groups receiving the 1% diet or in the control group.
Chronic Toxicity
Carcinogenicity .
In the NCI Bioassay Program, 2—cresidine was administered to
male and female F—344 rats at concentrations of 0.5% and 1.0% in the
diet for 104 weeks. The chemical was also administered for 104
weeks at time—weighted concentrations of 0.22% and 0.44% to female
B6C3F1 mice for 104 weeks at a time—weighted concentration of 0.22%
to male mice of the same strain and for 92 weeks at a time—weighted
concentration of 0.46% to a separate group of male B6C3F1 mice.
279
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Bladder carcinomas and olfactory neuroblastomas were observed in
dosed rats of both sexes. A statistically significant incidence of
neoplastic nodules of the liver, hepatocellular carcinomas, or mixed
hepatocholangiocarcinomas also occurred in the low—dose male rats.
A statistically significant number of bladder carcinomas were
also observed in both high— and low—dose male and female mice. In
addition, both high— and low—dose female mice had a significant
incidence rate of hepatocellular carcinoma.
Comparisons of 2—cresidine to other single—ring aromatic amines
and to 2-FAA (N—2—fluorenylacetamide) for potency in producing
urinary bladder tumors in rats and mice are shown in Figures 10—1
and 10—2.
280
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FIGURE 10-1
URINARY BLADDER TUMORS, RATS
100 F 344 OF
RATS
4 -chioro- NH 2 /
o-pheriylene- ( )-NH2,
diamine
clv
NH2
j0CH3 /
o-anisldlne A NH 2
/ j j0CH3
p /IH 3 C
p-cresld lne
p
/
NH2
JCH3
o-to lu ldlne
-n
-S
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-J
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C)
0
-no)
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- 0
-S -4
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—
o
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- . _5
—
-S o
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• 0
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-I-
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I- .
U i
C-)
z
Ui
0
U
0
.10
MOL/KG DIET X WEEKS OF FEEDING
-------
!IGU 10-2
URI NARY
AM
B6C3F1 • F
MICE
NCTR: • F
MICE
N
I
I
I
I
i J J ..NHAc
I
2-FAA
I
I
I
I
I
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p
05
. .
.50
.
1.0
V • •
5.0
11
-I.
-S
(D
- C,
0
I 0
T D i
-S
cn
—. 0
0
_5 -t
0
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‘ 0
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- -t
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—
o to
1 -
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—4
-a.
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BLADDER TUMORS, MICE
0
z
Lu
0
C .,
z
100-
80-
60
40
20 ”
0
MOL/KG DIET x WEEKS OF FEEDING
-------
Mutagenicity
The only data on the mutagenicity of E—cresidine were obtained
from the FDA Bureau of Foods. The results were from tests conducted
by Stanford Research Institute International and Inveresk Research
International (IRI) in the NCI collaborative study evaluating the
reproducibility of results obtained in the Salmonella microsoine and
Escherichia coli , WP2 uvrA assays. The data from four Salmonella
strains and one E. coli strain WP2 uvrA show that there are
differences among the responses obtained by the two laboratories.
For example, at IRI, E—cresidlne induced mutations in TA 1537, TA
1538, TA 98, and TA 100 strains without metabolic activation.
Similar differences for other chemicals were also obtained in this
collaborative study. Overall, however, a positive response was
reported by both laboratories for E—cresidine, with mutagenic
dose—responses in TA 1538, TA 98, and TA 100.
Teratogenicity
No data were available to evaluate the teratogenicity or
reproductive toxicity of 2—cresidine.
CONCLUSIONS AND RECOMMENDATIONS
Other than the cancer bioassay conducted by Nd, there is
virtually no other biologic data on 2—cresidine. The lack of data
makes it difficult to assess the potential health effects of this
compound. Because of the positive carcinogenicity demonstrated
283
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in rats and mice, 2 —cresidine must also be considered potentially
carcinogenic in humans. The preliminary mutagenicity data appear to
show a positive response for 2—cresidine, but confirmation is
needed. Additional data are also needed on metabolism, metabolic
activation, mutagenicity and genetic toxicity in both animal and
human in vitro test systems.
284
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REFERENCES
Production, Uses, Exposure
Bell, S.J. 1976. Preparation and spectral compilation of FD&C Red
No. 40 intermediates and subsidiary dyes. J. Assoc. Off. Anal.
Chem. 59:1294—1311.
Code of Federal Regulations. 1980. Title 21, Part 24. Listing of
Color additives subject to certification. Office of the Federal
Register, National Archives and Records Service, General Services
Administration, Washington, D.C.
SRI International. 1979. 1979 Directory of Chemical Producers:
United States of America. Stanford Research Institute
International, Menlo Park, Calif. 1122 pp.
The Society of Dyers and Colourists. 1971. Colour Index, Volume 4,
Lund Humphries Printers, London.
U.S. International Trade Commission. 1977. Synthetic Organic
Chemicals. United States Production and Sales, 1976. USITC
Publication 833. U. S. Government Printing Office, Washington,
D.C. 357 pp.
U.S. International Trade Commission. 1978. Synthetic Organic
Chemicals. United States Production and Sales, 1977. USITC
Publication 920. U.S. Government Printing Office, Washington,
D.C. 417 pp.
285
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U.S. International Trade Commission. 1979. Synthetic Organic
Chemicals. United States Production and Sales, 1978. USITC
Publication 1001. u.S. Government Printing Office, Washington,
D.C. 369 pp.
286
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Health Effects
National Cancer Institute. 1979. Bioassay of 2 —cresidine for
possible carcinogenicity. Tech. Report Series 142,
NCI—CG—TR—142. U.S. Department of Health, Education, and Welfare,
Bethesda, Md.
National Institute for Occupational Safety and Health. 1976.
Registry of Toxic Effects of Chemical Substances. H.E.
Christensen, ed. U.S. Department of Health, Education, and
Welfare, Bethesda, Md.
287
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Chapter 11
FURAZ OLI DONE
02
Furazolidone occurs as an odorless, yellow crystals that melt at
275°C. The crystals will darken under strong light and are
decomposed by alkali. Furazolidone’s solubility in water (pH 6) is
approximately 40 mg/i. It is also known by the following synonyms
and trade names: 3—( [ (5—nitro—2—furanyl)methylene]amino)—
2—oxazolidinone, 3—(5—nitrofurfurylideneamino)—2—oxazolidinone,
N—(5—nitro—2—furfurylidene)—3—amino—2—oxazolidone, NF 180, Furovag,
Furoxane, Puroxone, Giarlam, Giardil, Medaron, Neftin, Nicolen,
Nifulidone, Ortazol, Roptazol, Tikofuran, and Topazone. Furazolidone
is produced synthetically from furfural, hydroxyethylhydrazine, and
diethyl carbonate.
PRODUCTION
The sole u.s. producer of furazolidone is the Norwich
Pharmaceutical Company in Norwich, N.Y. (Stanford Research Institute
International 1979). The compound is prepared according to
procedures described in U.S. patents 2,759,931 and 2,927,110 to
Norwich Pharmaceutical Co. It was first produced in 1953 as a
veterinary medicinal and feed additive, and in 1957 as a human
288
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systemic medicinel for U.S. and worldwide use (Federal Register,
1976a,b; Bryan, 1978).
The U.S. International Trade Commission (1976—1978) reports
furazolidone data obtained on group of 20 pharmaceutical chemicals
whose combined production totals approximately 3.2 million kg/year.
The fact that production is reported to and by the commission
indicates that annual production is 450 kg or more.
USES
Furazolidone is one of five 5—nitrofurans currently approved for
use as systemic veterinary medicines in the United States (Federal
Register, 1976a,b; Bryan, 1978). Its use was approved in 1953 to
treat turkeys and chickens for fowl typhoid, paratyphoid, and
pullorum; blackhead (histomoniasis); nonspecific enteritis (blue
comb, mud fever), ulcerative enteritis (quail disease), and synovitis
(arthritis due to filterable virus); and paracolon infection
( Paracolobactrwn ) . Furazolidone use is permitted in chickens for
infectious hepatitis and coccidiosis, in turkeys for hexamitiasis,
and in swine for bacterial enteritis (necrotic enteritis, black
scours) or vibrionic (bloody) dysentery (Federal Register, 1976a).
Furazolidone is one of two 5—nitrofurans that have been
approved in the United States as veterinary feed additives (Federal
Register, 1976a,b), and it accounts for approximately 97% of the
5—nitrofurans administered to food—producing animals (Federal
Register, 1976b). As a feed additive, the compound has been approved
289
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for use in chickens and turkeys to prevent fowl typhoid, paratyphoid,
pullorum, air—sac infection and paracolon infection, and to enhance
growth and feed efficiency. In swine, it is approved for the
prevention of bacterial enteritis and vibrionic dysentery, and for
growth promotion and enhanced teed efficiency (Federal Register,
1976a) . In 1976, the Food and Drug Administration (FDA) published
proposals to withdraw approval of furazolidone for the veterinary
purposes for which it is now used (Federal Register, 1976a,b) . To
date, no final action on these proposals has been taken.
Furazolidone has been used in humans to treat bacillary
dysentery, typhoid and paratyphoid fevers, giardiasis, brucellosis,
and intestinal infections of undetermined etiology (Bryan, 1978;
Mjura and Reckendorf, 1967; Paul and Paul, 1964, 1966).
EXPOSURE
Furazo].jdone has been the subject of controversy in recent
years. The chemical has been determined to cause cancer when
ingested by rats and mice, although the producer has challenged this
finding. In 1976, the FDA proposed to withdraw approval for use of
the drug in food—producing animals (Federal Register, 1976a). At
that time, the agency ruled that data were not adequate to determine
the total drug—related residues that can occur, that the analytic
techniques for measuring the drug were not reliable for the lower
concentrations found in food, that the drug was present in edible
tissue following medication when no withdrawal period was observed,
290
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and that a reliable withdrawal period could not be established from
the information available.
The results of testing for furazolidone in controlled experiments
are shown in Table 11—1. As indicated, each study concluded that
residues were undetectable (usually with a detection limit of 1 to 5
ing/g) after a 2— to 19—day withdrawal period.
The 1976 proposal to ban the use of furazolidone in
food—producing animals was withdrawn because of problems in obtaining
supportive data, but another proposal is being issued in 1980 (Moy,
Food Animal Additive Branch, FDA, personal communication, 1980).
Although FDA requires that food products contain no residue,
compliance is handled by the U.S. Department of Agriculture. Because
analytic techniques for sampling at low concentrations are
unreliable, the existing regulations have not been enforced.
It is not possible to estimate the degree to which humans are
exposed to this drug. The largest potential for exposure occurs from
its presence in food. However, the residue disappears or, at the
very least, becomes undetectable, within 20 days after the withdrawal
of medication. Thus, the concentrations expected in
furazolidone—treated food products cannot be estimated. Moreover,
there Is no information concerning the percentage of the total
poultry and swine treated with the drug. Minute quantities may be
released to the air, water, and solid waste in the vicinity of the
plant.
291
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Table 11—i
Results of Testing for Furazolidone in Food
Food Type Dose 1 esidue Reference
Chicken “Excessive” None after 2 days Krieg and Loeliger,
1973
Eggs 100 mg/l00 ml water 8 mg/g on 3rd day; Krieg, 1972
for 3 days, and none after 10
5 mg/bC my for 2 days
days
Eggs 40 mg/lOG g water 23 micrograms/g Krieg, 1972
for 15 days on 6th day none
after 19th day
Veal Unknown None found Nouws, 1973
292
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ANALYTIC METHODS
Most of the analytic methods for furazolidone are based on
thin—layer chromatography (TLC), high—pressure liquid
chromatography (HPLC), UV absorption spectrophotometry, or
combinations of these techniques.
A general procedure using TLC to identify 18 drugs, including
furazolidone, in animal feed was reported by Williams (1979). The
sample was extracted with acetonitrile—chioroform (4:1), and an
aliquot of the extract was cleaned on a column of aluminum oxide.
The concentrated eluate was then subjected to mc on silica gel G
by using chloroform-methanol (9:1) to develop the plate. Spots
were made visible on the plates by spraying with 1,2—diaminoethane
or with Dragendorff’s reagent.
Cieri (1978) also reported a method for determining
furazolidone in animal feed. The sample was extracted with
acetone and cleaned on TLC plates of silica gel H by using
chloroform—methanol (9:1) to develop the plates. The appropriate
zone was scraped from the plate, eluted with ethyl alcohol, and
guantitated at its absorption maximum near 360 nm. The method was
reported not to be applicable at levels below 0.005%.
Moretain et al. (1979) described a procedure for determining
furazolidone and furaltadone in admixture at the levels expected
in animal feed (i.e., 50 to 200 ppm). The sample was extracted
293
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with dimethylformamide (DMF) and subjected to TLC on silica gel
plates developed with chloroform—acetone (7:3) . The appropriate
zones were then scraped from the plates, extracted with DMF, and
determined spectrophotometrically at 370 nm.
Methods reported recently have all been based on HPLC.
Lefebvre (1979) demonstrated that furazolidone could be assayed by
HPLC, using a glassy carbon or carbon paste electrode, coupled
with a voltametric—amperometric detector. Detection limits were
reported to be in the low nanogram range; however, no actual
residue assays were performed. Using HPLC Jones et al. (1978)
determined furazolidone levels in swine and poultry feed at levels
as low as 5ppm, with a silica column and a mobile phase of
water—saturated dichioromethone. The UV—visible detector was set
at 360 nm. The sample preparation consisted only of extracting
the feed with methanol and 2 N hydrochloric acid (1:1),
partitioning the residue into dichloromethane, and concentrating
the solvent for a 20 p1 injection into the HPLC system. Again
using HPLC, Cieri (1979) determined residues of furazolidone and
nitrofurazone in feeds at levels as low as 0.5 ppm. The sample
was extracted with DMF—acetone (1:1), cleaned on a column of
silica gel, and analyzed on a reverse—phase column with 30%
acetonjtrjle as the mobile phase. The detector was set at 365 nm.
Hoener et al. (1979) recently reported an HPLC procedure for
determining residues of furazoljdone in turkey tissue at levels as
low as 2 ppb. The tissue was ground with methanol and centrifuged.
294
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The extract then was either injected directly or concentrated
before being injected into the HPLC system. A Bondapak C 18
column was used with a mobile phase consisting of methanol and
0.01 mol sodium acetate (1:4). The UV absorption detector was set
at 365 nm. Recoveries from muscle and liver spiked at the 2 ppb
level were 103 + 19% and 112 + 12%, respectively.
Bagon (1979) used a Spherisorb S5—ODS column for HPLC
separations of several antibiotics and nitrofurans in
pharmaceutical preparations. Furazolidone was separated from
nitrofurazone by using a mobile phase of water—methanol (11:9)
with the absorption detector set at 375 nm. Although no samples
other than pharmaceutical preparations were analyzed, the
procedure is said to be generally capable of distinguishing the
parent compounds from decomposition products and likely congeners.
Problems associated with the analysis of furazolidone in
edible tissues of at levels of 0.5 to 4.0 ppb with methods
available prior to 1976 are well documented (Federal Register,
1976). Various spectrophotometric and PLC procedures failed to
yield satisfactory and reproducible recoveries at these levels.
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HEALTH EFFECTS
Metabolism
Furazolidone is biotransformed in vivo in mammals to a variable
but major extent (Swaminathan and Lower, 1978). Two to five
metabolites have been suggested from in vitro or in vivo analyses
(Federal Register, 1976a; Tatsumi et al., 1978). One
biotransformation product, 3 —(4—cyano—2—oxobutylideneamino)—2—
oxazolidone, was characterized by mass—, ultraviolet—, and
nuclear—magnetic—resonance—spectroscopic methods following
furazolidone incubation in vitro with milk xanthine oxidase or
administration in vivo to rabbits, when it was identified in urine.
In contrast to furazolidone, this metabolite was not an active
mutagen in Salmonella typhimurium TA 100 (Tatsumi et al., 1978).
Acute Toxicity
Humans . Reported symptons of acute toxicity of furazolidone in
humans include nausea, emesis, occasional diarrhea, abdominal pain,
and bleeding (Cohen, 1978). Infrequently, there have been reports of
an Antabuse—like reaction to alcohol (Cohen, 1978) or, rarely,
idiosyncratic or hypersensitivity reactions such as pneumonitis
(Cohen, 1978; Collins and Thomas, 1973; Cortez and Pankey, 1972;
Jirasek and Kalensky, 1975). Hemolytic anemia due to
glucose—6—phosphate— dehydrogenase deficiency has been reported with
furazolidone (Cohen, 1978). Furazolidone demonstrated
nonoamine—oxidase inhibition (Pettinger and Oates, 1968) and
296
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required precautions when coadministered with other monoamine—oxidase
inhibitors, sympathomimetic amines, or tyrainine—containing foods. No
data have been reported concerning chronic toxic effects of
furazolidone in humans.
Animals . Furazolidone induced emesis and neurologic changes in
dogs (Miura and Rekkendorf, 1967; Rogers et al., 1956). No published
oral LD 50 data exist for mammals. The drug also induced
cardiomyopathy in turkeys (Czarnecki et al., 1978; Staley et al.,
1978).
Chronic Toxicity
Carcinogenicity
Humans . No furazolidone—associated carcinogenicity in humans has
been reported.
Animals . Furazolidone was evaluated for carcinogenic
activity in six studies in rats and one study in mice (Federal
Register, l976a,b; Cohen, 1978), but no review of these studies has
yet appeared. However, the FDA has summarized the statistically
significant effects resulting from its analyses of the data submitted
(Federal Register, l976a,b) and has used these evaluations as the
basis for proposed regulatory action (Federal Register, 1976a,b).
Statistical comparisons of significant effects in rats and mice of
both sexes exposed to varying dose levels of furazolidone administered
orally for 18 or more months are presented in Tables 11—2 through 11—5.
297
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TABLE 11—2
Statistical Comparison of Significant Effects Produced in Groups of 50
Male and Female Sprague—Dawley Rats Fed Furazolidone!
Dose of Furazoljdone, ppm
Males Females
Effect 0 250 500 1,000 0 250 500 1,000
Malignant mammary tumors <0.052
Benign mammary tumors <0.2 <0.01
Mammary tumors <0.05 <0.05 <0.05
Multiple mammary tumors <0.05 <0.05
Nonmamniary tumors . <0.05
Mortality, 18 months <0.05 <0.05 <0.05
Mortality, 26 months <0.05 <0.01 <0.02
! Abstracted from Federal Register, 1976a.
Fed diet for approximately 18 months (553 days); surviving rats
maintained on furazo].jdone—free diet until mortality in each group
reached 90%, at which time the remaining 10% were killed approximately
13 months after furazolidone was withdrawn from the diet.
£ Value of P compared to rats fed furazolidone-free diet.
Included squamous cell carcinoma, derinal fibroma, pituitary neoplasms,
leukemia, and lymphosarcoma.
-------
TABLE 11-3
Statistical Comparison of Significant Effects Produced in Groups of 50
Male and Female Fischer 344 Rats Fed Furazolidone
Dose of Furazolidone, ppm
Males Females
Effect 0 250 500 1,000 0 250 500 1,000
Malignant mammary tumors <0.05
Mammary tumors <0.01 <0.01 <0.01
Multiple mammary tumors <0.01 <0.01 <0.01
Thyroid aclenomas <0.05 <0.05 <0.05 <0.05
Sebaceous adenomas <0.05 <0.05 <0.05
Testicular mesothe].jomas <0.05
Basal cell epitheliomas <0.05
Mortality <0.05 <0.05
Abstracted from Federal Register, 1976a.
Fed diet for 20 months; surviving rats maintained on furazolidione—free
diet until mortality in each group reached 90%, at which time the remaining
10% were killed approximately 11 months after furazolidone was withdrawn
from the diet.
£ Value of p compared to rats fed furazolidone—free diet.
-------
TABLE 11—4
Statistical Comparison of Significant Effects Produced in Groups of 40
Male and Female Sprague—Dowley Rats Fed Low—Levels of Furazolidone.-
mg/kg/day x 265 days
( ppm beyond 266 days)
Males Females
0 1 15 15 0 1 5 15
Effect (0) (25) (125) (375) (0) (25) (125) (375)
No. of rats with tumors
Multiple mammary tumors <0.05 <0.05
Onset of palpable mammary
tumors, 16 months <0.05
Mortality, 20 months <0.05
g Mortality, 24 months <0.05 <0.05
. Abstracted from Federal Register, 1976a.
Fed diet for 265 days on basis of mg/kg of body weight/day; thereafter
until termination of study at 2 years (731 days) on basis of ppm in diet.
£ Value of P compared to rats fed furazolidone—free diet.
!. Majority of tumors were in mammary gland; in addition there were pituitary
adenomas and lymphoreticular neoplasms, including leukemia, lymphosarcoma,
and reticulum cell sarcoma.
-------
TABLE 11-5
Statistical Comparison of Significant Effects Produced in Groups of 50
Male and Female Swiss MBR/ICR Mice Fed Furazolidone .
Dose of Furazolidone, ppm
Males Females
Effect 0 75 150 300 0 75 150 300
Bronchial adenocarcinoma <0.05 <0.05 <0.05
Malignant tumors <0.05 <0.05 <0.05
Benign plus malignant tumors <0.05 <0.05 <0.05
Multiple tissue tumors <0.05
Mortality <0.05 <0.05 <0.05
. Abstracted from Federal Register, 1976a.
Fed diet for 18 months; surviving mice maintained on furazolidone—free
diet for an additional 10 months at which time study was terminated.
-------
Furazolidone was fed to 35 female Sprague—Dawley (Holtzman
strain) rats for 45 weeks followed by a drug—free diet for 8
additional weeks before the rats were sacrificed. A control group
of 35 rats was fed a drug—free diet for 53 weeks. Rats fed
furazolidone had a significantly higher incidence of mammary tumors
than did control rats (Federal Register, 1976a).
In second study, 20 male and 20 female rats (CFE strain) were
fed furazolidone for 45 weeks. The animals then received a
drug—free diet for 7 more weeks. Control groups of both sexes were
fed a drug—free diet for the 52 weeks. The female rats fed
furazolidone had a higher incidence of mammary tumors than did the
female controls. No significant effects on tumor development were
noted in male rats (Federal Register, 1976a)
A third 2—year evaluation of furazolidone toxicity was also
reported (Federal Register, 1976a) for 60 rats divided into three
groups, each consisting of 10 males and 10 females. One group of
each sex was fed a furazolidone—free control diet. The diets for
the other groups contained two levels of furazolidone. There were
three times as many tumors in rats fed furazolidone at a level of
0.01% in the diet as compared to the number of tumors in the control
group (Federal Regigte , 1976a).
A fourth study involved 2—month-old male and female
Sprague—Dawley (Charles River strain) rats fed furazolidone (Table
11—2). The number of female rats with malignant mammary tumors
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increased as the dosage level increased, and the dose—response
relationship was reported as linear and significant (P <0.01) . At
the highest dose level (1,000 ppm), the proportion of female rats
with malignant tumors was significantly higher (P <0.05) than that
of the control rats fed the furazolidone—free diet. Furazolidone
also concurrently induced benign mammary tumors in female rats.
Furazolidone did not induce mammary tumors in male rats; however, it
did randomly induce tumors in other body tissues. The proportion of
male rats that developed tumors of other tissues at the 1,000 ppm
dose level was significantly higher (P <0.05) than that of
controls. Finally, furazoljdone—treated male and female rats
exhibited a drug—related early mortality (Federal Register, l976a).
A fifth study in which furazoljdone was fed to 2—month—old male
and female Fischer 344 rats is summarized in Table 11—3. The
proportion of female rats with malignant mammary tumors at the
1,000—ppm level was significantly higher (P <0.05) than that of
control rats. Malignant mammary tumors were diagnosed only in
female rats fed the highest dosage of furazolidone. At all dosage
levels, the proportion of female rats with mammary tumors was
significantly higher (P <0.01) than that of control rats. A
significant relationship between the dosage of furazolidone and the
proportion of female rats with mammary tumors was reported (Federal
Register, 1976a). The proportion of female rats with multiple
mammary tumors was significantly higher (P <0.01) for all treatment
groups than in controls. Other tumors that occurred more
significantly (P <0.05) in male and female rats fed furazolidone
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include thyroid and sebaceous adenomas (both sexes) , testicular
rnasotheliomas, and basal cell epitheliomas in male rats (Table
11—3). A dose—response (early mortality) relationship was
significant for both male and female rats (Federal Register, 1976a).
A sixth study, in which furazolidone was fed to 2—month—old male
and female Sprague—Dawley (Charles River strain) rats, is summarized
in Table 11—4. There was a significant (P <0.05) linear
dose—response relationship for the number of female rats with
tumors, and a significant increase (P <0.05) in the proportion of
female rats at the 375—ppm dose level with tumors, as compared to
females fed a furazolidone—free diet. The types of tumors observed
are shown in Table 11—4. A significant increase (P <0.05) of the
incidence of multiple mammary tumors for both the 125— and 375—ppm
dose groups (as compared to controls) was noted, with a significant
(P <0.05) linear dose—response relationship. Furazolidone resulted
in significant and early onset and development of mammary tumors in
female rats at the 375—ppm dose level as compared with that of
controls. There was a significant linear dose response for mammary
tumor development in female rats during the first 16 months of the
study. Early significant mortality (P <0.05), which was dose
related, was reported for female rats. No significant tumor or
mortality rate differences were found for male rats (Federal
Register, 1976a).
A study of furazolidone fed to 2—month—old male and female Swiss
MBR/ICR mice is summarized in Table 11—5. Bronchial adenocarcinoinaS
were significantly higher (P <0.05) in both male and female mice
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than in controls fed a furazolidone—free diet. Incidence of this
growth in both sexes showed a significant (P <0.01) and linear dose
response (Federal Register, 1976a). The dose—response relationships
for malignancies of all tissue types as well as for the incidence of
all tumors were significant (P <0.05) and linear for both sexes.
There was a significant increase (P <0.05) both in the development
of malignancies and in the development of benign and malignant
tumors combined in male mice at the 150 and 300 ppm levels, and in
female mice at the 300 ppm level, as compared with those of control
mice. Male mice that received the 300 ppm dose had significantly
more (P <0.05) multiple tumors than did control mice. There was
also a significant linear dose—response relationship for tumor
multiplicities for female mice fed furazolidone. Male and female
mice fed furazolidone both exhibited significant (P <0.05) early
mortality which was linear in dose response (Federal Register,
l976a)
Mutagen icity
The data from mutagenicity tests of furazolidone are summarized
in Table 11—6. Furazolidone was found to be mutagenic in
Escherjchja coli (Lu et al., 1979; McCalla and Voutsinos, 1974) and
Drosophila melanogaster (Blijleveh et al., 1977) caused chromosomal
damage in human lymphocytes (Cohen and Sagi, 1979), and
cross—linking of DNA in Vibrio cholerae (Chatterjee et al., 1977).
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TABLE 11—6
Mutagenicity Tests of Furazolidone
Test System Species—Strain Results Reference
Gene Mutation
Bacterial E. coli — WP2 uvr A post McCalla and Voutsinos,
1974
E. coli — WP2,WP2 uvr
A, CM561 (lex A), CM 571
(rec A) , CM 611 (uvr A,
rec A) pos . Lu et al., 1979
insect D. melanogaster (Berlin—K,
Oregon—K) pos Blijieven et al., 1977
0 ’
Chromosomal Damage
Manialian cells
in culture Human lymphocytes neg Tonomura and Sasaki 1973
Human lymphocytes pos Cohen and Sagi, 1979
Bacterial Vibrio cholerae pos Chatterjee et al., 1977
. Tester strains without metabolic activation.
-------
Bacterial Tests . Furazo].jdone was among 22 nitrofurans that McCalla
and Voutsinos (1974) found to be mutagenic in E. coli WP2 and its
uvrA—derjvatjve by reversion from trp to trp+. ExrA—or recA tester
strains were not induced to mutate by the nitrofurans, indicating that
mutants arise when the lesions induced by these compounds are repaired by
the “error—prone” repair system, components of which are coded for by rec
and exr genes. Results obtained with the nfr—straing, which lack
nitrofuran reductase I, suggest that the nitro group is a key structural
component, and that only when the nitro group is reduced and the drug
converted into a more reactive compound are mutants induced. In this
respect, mutagenesis of nitrofurans is similar to the formation of
alkali—labile DNA lesions by these compounds (McCalla et al., 1971).
The mutagenic activity of the nitrofurans so far tested covers a wide
range (Lu et al., 1979, McCann et al., 1975). Lu et al. (1979) reported
an approximately 10,000—fold range in mutagenicity of eight nitrofurans,
including furazolidone. The most active compound was
2 —( 2 —furyl)— 3 —(5—nitro—2—furyl)acry lamide, (AF—2) which was approximately
6 times as mutagenic as furazoljdone. Lu et al. (1979) ranked these
compounds from most to least toxic to !• coli as follows:
AF—2 > N—( (5—nitro—2—furyl) thiazolyl]formamide (FANFT) ).
2 —amino—4—(5—nitro—2—furyl)thjazole (ANPT) furazolidone
(furagin) > nitrofurazone> inethylnitrofuroete > nitrofuroate. In general,
mutagenic activity was found to parallel toxicity.
Insect Tests . The genetic effects of furazolidone were determined in
D. melanogaster by the induction of sex—linked recessive ].ethals,
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which is the most sensitive mutation test in this organism
(Blijleveh et al. 1977). Furazo].jdone was fed to adult males, which
were then mated with fresh virgins for three consecutive periods of
3, 2, and 2 days. A consistent increase in the frequency of
sex—linked recessive lethals was observed for furazolidone under
these COfldj j 0 5 , indicating that the spermatids are sensitive to
this compound, as they were to AF—2 (Blijleven et al., 1977).
Although the mutation frequency for furazolidone is low in this
system (as in AF—2), and a demonstration of dose—response effect is
lacking, the present data indicate that furazolidone and AF—2 are
mutagenic in Drosophila.
Chromosomal Damage
Mammalian Cells in Culture . The capacity of furazolidone to
induce chromosomal damage (chromosomal breaks, sister—chromatid
exchange, DNA repair synthesis, and inhibition of mitosis) in
cultured human peripheral lymphocytes was examined by Tonomura and
Sasaki (1973) and Cohen and Sagi (1979). Tomomura and Sasaki (1973)
did not report either a significant amount of chromosomal abberation
or unscheduled DNA synthesis for furazolidone over an 0.5 to 100 x
io6 M dose range. In contrast, Cohen and Sagi (1979) found that
furazolidone produced dose—dependent mitotic suppression,
chromosoma]. breakage, and sister—chromatjd exchanges (SCE’s). The
different results are surprising and difficult to explain, in that
the experimental design of the two studies (e.g., range of drug
concentration used, exposure times, solvents, methods of cytologic
scoring) were similar. However, the fact that furazolidone induced
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dose—dependent SCE in the Cohen and Sagi study (1979) and that this
type of chromosomal damage was not reported by Tomomura and Sasaki
(1973) suggests that this compound is capable of inducing
chromosomal damage in human cells. Further studies are needed to
clarify this point.
Bacterial DNA . Furazolidone inhibits DNA synthesis in V.
cholerae cells while stimulating RNA and protein synthesis and
causing filamentation of these cells (Chatterjee and Maiti, 1973).
Chatterlee et al. (1977) reported that interstrand cross—linking in
DNA takes place within the furazolidone—treated V. cholerae cells,
which therefore might explain the actual mechanism of inhibition of
DNA biosynthesis by this drug. The in vivo action of furazolidone
has considerable similarity to that of mitomycin C (Iyer and
Szybalski, 1964). Both agents induce interstrand cross—linking in
DNA, inhibit DNA synthesis, and cause filamentation of the cells at
the appropriate dose level by inhibiting cell division.
Teratogenicity
No data were available to evaluate the teratogenicity or
embryotoxicity of furazolidone.
CONCLUS IONS
Furazolidone has exhibited carcinogenic effects in male and
female rats and mice in a variety of tissues. Susceptible tissues
are different for the two species. Furazolidone, in common with
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most 5—nitrofurans studied, is a Sigflifjca carcinogen in rodents
(Cohen, 1978)
Furazolidone is highly mutaqenic in both microbial (E. coli ) and
insect (D. melanogaster ) test systems, produces chromosoinal damage
(breakage, SCE, mitotic suppression) in human lymphocytes, and forms
interstrand cross—linking in bacterial (V. cholere ) DNA.
It is for the above reasons that the use of furazolidone is now
being reviewed by the FDA. Resolution of this matter awaits the
development of a sufficiently sensitive and reliable analytic
method. If risk to human health does exist, it most certainly would
be associated with the use of furazolidone for veterinary purposes.
A °solution” may involve the substitution of an efficacious product
that could be demonstrated not to have the mutagenic and
carcinogenic potential of furazolidone.
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