EPA-560/11-80-014
June 1980
SUPPORT DOCUMENT
HEALTH EFFECTS TEST RULE:
CHLORINATED BENZENES
ASSESSMENT DIVISION
OFFICE OF TOXIC SUBSTANCES
Washington, D.C. 20460
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
WASHINGTON, D.C. 20460
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PREFACE
Under the authority of the Toxic Substances Control Act
(TSCA), the Environmental Protection Agency has proposed the
requirement of health effects testing of a representative group
of chlorinated benzenes (Federal Register, June 28, 1980). This
action follows on the recommendations of the Interagency Testing
Committee. The Agency has reviewed the available information,
including TSCA Section 8(d) submissions, on chlorinated benzenes
and discussed the significant scientific and economic issues both
in Agency Workgroup meetings and in public meetings. The results
of this effort are reflected in this document which supports the
proposed health effects test rule with specifics from the
literature and rationales for decisions.
The EPA encourages all interested parties to review the
scientific and economic reasoning expressed in the Support
Document and provide comment to the Agency. Such contributions
can significantly benefit the development of the Final Health
Effects Test Rule. All comments will be carefully reviewed by
EPA, and all major points will be addressed in the final Support
Document.
Written comments should bear the document number EPA 560/11-
80-014 and should be submitted to the Document Control Officer
(TS-793), Office of Pesticides and Toxic Substances, U.S.
Environmental Protection Agency, 401 M St., SW, Washington, DC
20460.
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CONTENTS
Introduction 1
Summary 3
I. Chemical Identity
A. Category Definition 7
B. Category Characteristics 7
C. Physical Properties 11
D. Chemical Properties 11
II. Exposure Aspects 13
A. Monochlorobenzene 13
1. Nature of the Substance 13
2. Manufacture 13
3 . Production Volume and Trends 14
4. Uses 14
5. Occupational Exposure 16
6. General Population Exposure 18
7. Environmental Release 18
8. Environmental Transformation 19
9. Biological Uptake 20
B. Dichlorobenzenes 22
1. Nature of Substances 22
2. Manufacture 23
3. Production Volume and Trends 23
4. Uses 25
5 . Occupational Exposure 26
6. General Population Exposure 28
7. Environmental Release 29
8. Environmental Transformation 32
9. Biological Uptake 32
C. Trichlorobenzenes 33
1. Nature of the Substances 33
2. Manufacture 33
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3 . Production Volume and Trends 34
4. Uses 35
5. Occupational Exposure 35
6. General Population Exposure 36
7. Environmental Release 36
8. Environmental Transformation 37
9. Biological Uptake 37
D. Tetrachlorobenzenes 38
1. Nature of the Substances 38
2. Manufacture 38
3. Production Volume and Trends 38
4. Uses 39
5. Occupational Exposure 40
6. General Population Exposure 40
7. Environmental Release 40
8. Environmental Transformation 41
9. Biological Uptake 41
E. Pentachlorobenzene 42
III. Health Effects 44
A. Acute Effects 44
1. Evaluation of Pertinent Studies 44
a. Human Case Reports 44
b. Animal Studies 46
2. Decision 50
B . Subchronic Effects 51
1. Evaluation of Pertinent Studies 51
a . Human Case Reports 51
b. Animal Studies 53
(1) Monochlorobenzene 53
(2) Dichlorobenzenes 57
( 3) Trichlorobenzenes 59
(4) Tetrachlorobenzenes 60
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( 5) Pentachlorobenzene 61
c. Other Indicators of Systemic Toxicity..71
(1) Metabolism 71
( 2) Porphyria 85
(3) Structural Relationships 86
2. Decision 87
3. Proposed Testing:
Subchronic & Chronic Effects Testing 89
4. Testing Under Consideration:
Metabolism Studies 90
C. Neurotoxic Effects 90
1. Evaluation of Pertinent Studies 90
2. Decision 94
3. Testing Under Consideration 95
D. Reproductive Effects 96
1. Evaluation of Pertinent Studies 96
2. Decision 98
3. Proposed Testing 99
E. Teratogenic Effects 99
1. Review of Pertinent Studies 99
a. Morphological Teratogenicity 99
b. Behavioral Teratogenicity 103
2. Decision 104
3. Proposed Testing: Morphological
Teratogenicity 104
4. Testing Under Consideration: Behavioral
Teratogenicity 104
F. Mutagenic Effects 105
1. Evaluation of Pertinent Studies 105
a. Gene Mutation Studies 105
b. Chromosomal Aberration Studies 108
c. DNA Repair Studies.. 109
2. Decision 110
3. Testing to be Sponsored by EPA Ill
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G . Oncogenic Effects 112
1. Evaluation of Pertinent Studies 112
a. Human Case Reports 112
b. Animal Studies 113
c. Other Indicators of Oncogenic
Potential 113
2. Decision 118
3. Proposed Testing 120
H. Epidemiology 120
IV. Materials to be Tested and Justification
for Sampling 122
V. Route of Administration 125
Appendix A: Use of information From the
TSCA Inventory 127
Appendix B: Proposed Mutagenicity Testing
Sequence 129
Appendix C: Occupational Exposure Limits for
Chlorinated Benzenes 141
References 142
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INTRODUCTION
In October 1977, the Interagency Testing Committee
recommended in its first report to the EPA that monochlorobenzene
and the three isomers of dichlorobenzene be given priority con-
sideration for the development of testing requirements under
Section 4 of TSCA (CEQ 1977). In its third report to EPA in
October 1978, the Committee made similar testing recommendations
for the trichlorobenzenes, tetrachlorobenzenes, and pentachloro-
benzene (TSCA ITC 1978). This document presents detailed support
for EPA's decision: (1) to propose test rules to assess the
potential of chlorinated benzenes to cause chronic, reproductive,
morphological teratological (birth defect), and oncogenic
effects; (2) to propose test rules at a later date for neurotoxic
effects, behavioral teratological effects, and metabolism
studies, following resolution of methodology issues raised in the
Preamble and in this document; (3) not to propose test rules for
acute toxicity and epidemiological studies; (4) to have the EPA
sponsor certain lower level mutagenicity tests.
It should be noted that while hexachlorobenzene is a
chlorinated benzene, it was not included with the other chlori-
nated benzenes in the Testing Committee's recommendations. This
substance has been evaluated by a separate Agency process, and
referred to EPA's Office of Solid Waste for control of the major
source of hexachlorobenzene release to the environment under
regulations promulgated on May 19, 1980 (44 FR 33063). This
document does not, therefore, consider the need for health
effects testing of hexachlorobenzene, and the term "chlorinated
benzenes" when used in this document does not include hexachloro-
benzene as a member of the test rule category. However,
information on hexachlorobenzene is included wherever it is
relevant to the technical analysis.
In this document the exposure aspects section (Section II)
contains separate discussions on monochlorobenzene, the dichloro-
benzenes, the trichlorobenzenes, the tetrachlorobenzenes, and
pentachlorobenzene. In Section III on health effects, all
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chlorobenzenes for which data are available are discussed
together for each effect.
The EPA is aware that guidelines for the level of exposure
to some chlorinated benzenes have been prescribed by the American
Conference of Governmental Industrial Hygienists (ACGIH), the
Food and Drug Administration, and the EPA [Clean Water Act, Safe
Drinking Water Act, and Federal Insecticide, Fungicide and
Rodenticide Act (FIFRA)] and that the Department of Transporta-
tion has regulations for handling chlorobenzenes and for report-
ing accidental spills. These rules are based on existing data;
the results of the testing proposed by EPA may necessitate
additional regulations or changes in existing guidelines,
standards, and regulations. A list of the existing guidelines
for occupational exposure is attached as Appendix C.
Non-Confidential information from the TSCA §8(b) Chemical
Inventory is cited in this document. For a discussion of the use
and limitations of this information, the reader should refer to
Appendix A.
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SUMMARY
Exposure
The total annual production of chlorobenzenes is on the
order of 450 million pounds or about half of the available
capacity. Most of the production is of mono- and dichloro-
benzenes; nevertheless, annual production of the commercially
important tri- and tetrachlorobenzenes and pentachlorobenzene is
in the millions of pounds. Total chlorinated benzene imports
range up to several million pounds each year.
Chlorinated benzenes are produced by the controlled cataly-
tic chlorination of benzene; the reaction conditions selected
determine which chlorobenzenes are obtained. There are always
some unwanted chlorobenzenes produced in addition to the desired
product(s). Some of these by-products are recycled in order to
produce higher chlorinated benzenes, while others have commercial
uses. Most commercial chlorinated benzenes can thus be expected
to contain some congeneric and/or isomeric chlorobenzenes as
impurities. Workers involved in chlorinated benzene production
and in their uses are potentially exposed to various members of
the category, and various members may enter the environment as
emissions and production wastes.
The potential release of chlorinated benzenes to the
environment is very large because of the combination of high
annual production and a wide variety of industrial and consumer
uses. Many industrial uses and disposal practices may result in
the ultimate discharge of chlorinated benzenes into the environ-
ment rather than in their recovery and reuse. Much of this
release occurs to the atmosphere, but nearly all the chlorinated
benzenes have been detected in the aquatic environment. Adsorp-
tion from water onto solids occurs to some extent. Atmospheric
decomposition of some chlorinated benzenes occurs fairly readily,
and there is some evidence of degradation by microorganisms under
certain conditions; in most cases environmental transformation
products have not been identified.
Since chlorinated benzenes are used as chemical intermedi-
ates and for other industrial purposes as well as in consumer
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products, there is very broad potential exposure. Thus, there is
known or potential exposure of workers involved in chlorobenzene
production, processing, and use, and of the general population,
both directly from consumer products and indirectly through the
environment.
Acute Effects
The Agency believes that the acute effects of the chlori- v
nated benzenes, chiefly tissue irritation and nervous system
depression, are adequately characterized as a result of human
case studies and tests in several animal species. Further acute
testing is unnecessary.
Subchronic/Chronic Effects
A number of subchronic studies were reported which indicate
that the chlorinated benzenes produce damage to the liver and the
hematopoietic system in humans and several animal species and to
the kidney in dogs and rats. No reports of chronic studies were
found. The data from these subchronic studies are inadequate to
define the hazards of chronic exposure to chlorinated benzenes.
Therefore, EPA is proposing that 90-day subchronic studies be
done using rats, the most sensitive of species thus far tested.
No chronic studies are being proposed because EPA believes that
for chlorinated benzenes the vast majority of the chronic effects
will appear in a 90-day study. Subchronic testing is not being
proposed for pentachlorobenzene, which has been adequately
tested.
Neurotoxic Effects
There are studies indicating adverse effects on the central
nervous system of humans and animals after exposure to some
chlorobenzenes. Since the data are not adequate to provide a
complete characterization and assessment of the hazard, testing
is desirable. But, because the Agency has not yet developed test
standards for such testing, EPA is not proposing specific
neurotoxicity or behavioral effects testing at this time.
4
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Metabolism Studies
The metabolism studies available deal primarily with the
products of chlorinated benzene metabolism and provide little
information on the pharmacokinetic aspects. The studies lead EPA
to the conclusion that the chlorinated benzenes are metabolized
at least in part to epoxide (arene oxide) intermediates.
EPA is soliciting comments on the appropriateness of and
approach to studies (a) to determine the distribution of
chlorinated benzenes to tissues and organs, (b) to learn the
rates of their clearance from these tissues, and (c) to
ascertain whether or not chlorinated benzenes form covalent
compounds with macromolecules, particularly in the brain and
gonads and in organs from which excretion is slow.
Reproductive Effects
Studies in rats of hexach}.orobenzene, which is structurally
related to the chlorinated benzenes, have revealed that the
substance passes the placenta, appears in the milk supply, and
affects fertility. Further, ovarian effects have been noted in
monkeys treated with hexachlorobenzene and in rats treated with
monochlorobenzene. On the basis of available data and of struc-
tural relationships, EPA suspects that exposure to the chlori-
nated benzenes may cause reproductive effects. Because the
existing data are insufficient to determine and predict effects
on fertility, EPA proposes reproductive studies to develop
additional data for chlorinated benzenes except 1,2,4-trichloro-
benzene on which testing has recently been performed.
Teratogenic Effects; Morphological and Behavioral
Hexachlorobenzene causes teratogenic effects in mice.
Pentachlorobenzene in rats causes rib abnormalities which are
dose-related. Certain phenolic metabolites of the chlorinated
benzenes are also known to cause embryo- and fetotoxic responses
in rats. This evidence causes EPA to propose testing to evaluate
the morphological teratogenic potential of chlorinated benzenes
except pentachlorobenzene, which has been adequately tested.
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Chlorinated benzenes are also known to be non-specific
central nervous system (CNS) depressants in adults, and they or
their toxic metabolites are likely to cross the placenta.
Because the CNS is especially susceptible to toxic insult during
its development, EPA is considering the requiring of behavioral
teratogenicity testing of chlorinated benzenes. The Agency
desires comments on the behavioral tests suggested in this
document.
Mutagenic Effects
Evidence indicates that the chlorinated benzenes are
mutagenic in certain bacterial and eukaryotic systems that detect
gene mutations, produce differential cell killing in DNA repair-
deficient strains of bacteria, and induce C-mitosis and chromoso-
mal breaks in plant systems. For EPA to determine whether the
chlorinated benzenes pose a genetic hazard to humans, the Agency
will sponsor some initial, relatively inexpensive tests to
determine whether these substances cause gene or chromosomal
mutations in higher organisms. The Agency will arrange for this
testing because it has not yet proposed test standards for some
mutagenicity tests or decision criteria for a mutagenicity
testing sequence. After evaluating the test results, EPA will
decide whether to propose further testing to provide information
for mutagenicity hazard assessments on chlorinated benzenes.
Oncogenic Effects
The weight of suggestive evidence leads EPA to the
conclusion that chlorinated benzenes may be oncogenic. This
evidence includes: (a) case reports of human leukemia, (b)
positive mutagenicity test results which may have a correlation
with oncogenicity, (c) structural and metabolic similarities
with known oncogens, and (d) reports of tumor enhancement
potential. Therefore, EPA is proposing that the chlorinated
benzenes be tested for oncogenicity in a two-year study using
rodents. Monochlorobenzene and _o_- and j^-dichlorobenzene are
excluded from the proposed testing because of bioassays now
planned or in progress at the National Cancer Institute.
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I. Chemical Identity
A. Category Definition
In EPA's proposed test rule for the "chlorobenzenes" and its
Support Document, "chlorinated benzenes" and "chlorobenzenes"
mean the group of substituted benzene compounds in which one to
five hydrogen atoms of benzene are replaced by chlorine atoms,
with no substituents present other than chlorine and hydrogen.
As explained in the Introduction, hexachlorobenzene is not
included in the chlorinated benzenes category in this document.
The chemical structures of the eleven chlorinated benzenes are
shown in Figure 1.
B. Category Characteristics
In this document the Agency considers the chlorinated
benzenes as a group. However, because most of the studies deal
with the compounds individually, for the sake of clarity some of
the material presented here is organized separately for monochlo-
robenzene, dichlorobenzenes, trichlorobenzenes, and so on.
The chlorobenzenes comprise a category of closely related
chemical compounds that have been shown to cause or would be
expected to cause similar biological consequences upon exposure.
The chlorobenzene group is formally constructed by substituting
one hydrogen of benzene after another with chlorine, in all
possible structural arrangements, resulting in corresponding
gradual changes in properties across the series. Proceeding from
less chlorinated to more highly chlorinated benzenes, we observe
regular changes in characteristics or numerical values over a
broad range of categories: chemical and physical properties,
method of manufacture, use patterns, nature of impurities, and
biological and environmental behavior. Some irregularities do
occur within the group that result from different steric and
electronic effects among isomers of the same degree of substitu-
tion, but these are not significant enough to negate the overall
consistency of the group's behavior. For example, p-dichloro-
benzene is unlike the other two dichlorobenzenes in that it is a
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Figure 1. Chemical Structures of the Chlorinated Benzenes
Cl
Monochlorobenzene
Cl
Cl
1,2-Dichlorobenzene
o-Dichlorobenzene
Cl
Cl
Cl
1,2,3-Trichlorobenzene
Cl
Cl
1,3-Dichlorobenzene
m-Dichlorobenzene
Cl
Cl
.Cl
Cl
Cl
1,4-Dichlorobenzene
£-Dichlorobenzene
Cl
Cl
Cl
1,2,4-Tr ichlorobenzene 1,3,5-Tnchlorobenzene
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
1,2,3,4-Tetrachlorobenzene
1,2,3,5-Tetrachlorobenzene 1,2,4,5-Tetrachlorobenzene
Cl
Cl
Pentachlorobenzene
8
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solid at room temperature; it is also somewhat less readily
chlorinated than the ortho isomer. Yet, like the other dichloro-
benzenes, it is more resistant than monochlorobenzene and less
resistant than trichlorobenzene to chlorination. Such departures
from strict regularity indicate that caution should be exercised
in extrapolating biological characteristics from one chloroben-
zene to another on the basis of structure, physicochemical
properties, and limited test data. However, because of the
consistency of the overall trends in physicochemical properties
and the similarities in metabolism and health effects discussed
in Section III, the EPA believes that once a representative
number of compounds has been characterized toxicologically,
extrapolation will be possible.
Further, knowledge of the commercial methods for producing
and handling the chemicals and the possibility that the chemicals
may interconvert to some extent encourage EPA to regard chloro-
benzenes as a group. All industrial chlorobenzenes are produced
by the chlorination of "benzene or of other chlorobenzenes. This
practice ensures that most commercial chlorobenzenes will contain
other chlorobenzenes as impurities and that chlorobenzene produc-
tion wastes will also contain various chlorobenzenes. The
estimation of relative environmental levels of the various
chlorobenzenes is complicated! by the possibility that some
interconversion of isomers might occur in the environment. This
could be the result either of conversion to more highly chlori-
nated compounds during water treatment by chlorination or of
reductive dechlorination by photo-degradative mechanisms or by
microorganisms to form the less-chlorinated derivatives. There
is little information on this point, although interconversions by
dechlorination apparently do occur to some extent during the
mammalian metabolism of some chlorinated benzenes (Section
III.B.I.e.(1). The potential for human exposure to various
chlorobenzenes in unknown proportions thus increases the
desirability of focusing on the risks associated with
chlorobenzenes as a group.
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The concern of EPA for those chlorinated benzenes that have
lesser or little commercial importance is based on several
factors. One is that a seemingly small contaminant in a large
quantity of material can represent a sizable contribution to
environmental contamination. Thus, the jn-dichlorobenzene content
of commercial c>-dichlorobenzene ranges from 0.5 percent to at
least 2 percent (see Section II.B.l.); if the average content is
only one percent, this still represents approximately one-half
million pounds annually that may ultimately enter the environ-
ment. Furthermore, about the same amount of _m-dichlorobenzene
was imported in 1977 (Section II.B.3), increasing the potential
for exposure to the chemical. Both 1,3,5-trichlorobenzene and
1,2,3,5-tetrachlorobenzene are produced as by-products of more
commercially important isomers (Section II.C.3 and Section
II.D.2,3). The disposition of the 1,3,5-trichlorobenzene was not
identified but the 1,2,3,5-tetrachlorobenzene is disposed as
waste. Ten thousand pounds of 1,3,5-trichlorobenzene was
imported in 1977 (Section II.C.3). The Agency needs more
information on the fate of these materials before it can evaluate
their potential risk to human health or the environment. Another
factor in the concern for the less commercially important chlori-
nated benzenes is that they as well as the more important
category members have been detected in air and water. m-Dichlo-
robenzene has been found in drinking water at a concentration
similar to those of the other two dichlorobenzenes (Section
II.B.7.). Furthermore, concentrations of airborne _m-dichloroben-
zene near a disposal site exceeded those of the ortho and para
isomers. Both 1,3,5-trichlorobenzene and 1,2,3,5-tetrachloroben-
zene have been found in fish tissue (Section II.C.7 and Section
II.D.7). Both isomers have also been detected in ambient water
systems. Hence, even chlorinated benzenes of lesser commercial
value have the potential for human exposure.
10
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C. Physical properties
The physical properties of the chlorobenzenes vary in an
approximately regular way with increasing substitution. Some of
these properties appear in Table I. In general, the chlorinated
benzenes have low water solubility, low flammability, moderate to
high octanol/water partition coefficients, and low to moderate
dielectric constants.
D. Chemical reactivity
Because of the electron-withdrawing character of the
chlorine atom relative to carbon, monochlorobenzene is less
reactive toward electrophilic attack (e.g., chlorination) than is
benzene. Each additional chlorine substituent further lowers the
reactivity of the compound. Thus, the more highly substituted
chlorobenzenes are expected to be the most stable members of the
category toward this type of reaction. When electrophilic
substitution does occur on monochlorobenzene, it takes place
primarily in the ortho and para positions. Nucleophilic substi-
tution on chlorobenzenes (see equation) is possible under forcing
conditions, but in most cases should occur only very slowly, if
at all, at environmental temperatures and pH values.
o
NaQH-H20
340"
pH ~15
o
In fact, the reaction shown probably does not proceed by a true
substitution mechanism (Roberts and Caserio 1965).
11
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II. Exposure Aspects
Note; This section contains information on pesticide uses for
several chlorinated benzenes. This information is included in
order to present a more complete picture of use patterns rather
than to support the case for a test rule under TSCA.
A. Monochlorobenzene
1. Nature of the Substance
At room temperature, monochlorobenzene is a colorless,
flammable liquid that is heavier than water (Hawley 1977).
According to the Merck Index, it is soluble in alcohol, benzene,
chloroform, and ether. Impurities in a reportedly typical
analysis were dichlorobenzenes at less than 0.1 percent and
benzene at less than 0.05 percent (Kao and Poffenberger 1979),
implying a purity of 99.8 percent or higher for the sample. A
Dow product data sheet claims 99.9 percent purity for monochlo-
robenzene (Dow 1977), while Allied states a purity of 99.0
percent for its product (Allied 1973).
2. Manufacture
The manufacture of chlorobenzenes is well described by
Hardie (1964) in the Kirk-Othmer Encyclopedia of Chemical
Technology. In brief, monochlorobenzene is manufactured by
chlorination of benzene at temperatures between 30° and 50°C in
the presence of a catalyst. During this process, c^-dichloroben-
zene, j>-dichlorobenzene, and hydrogen chloride are also
produced. However, production of the dichlorobenzenes may be
minimized by selecting the proper catalyst or an additive such as
fuller's earth, and keeping the reaction temperature close to
30°C. The hydrogen chloride is removed by washing with alkali to
yield a product with as much as 95 percent monochlorobenzene;
subsequent distillation provides a purer product (see II.A.I,
above).
13
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3. Production Volume and Trends
According to the 1978 Directory of Chemical Producers (SRI
1978), six companies produce monochlorobenzene in the United
States, with a combined annual production capacity of 615 million
pounds. Domestic production of monochlorobenzene averaged about
440 million pounds per year during the period 1966 through 1977
(USITC 1966-1977). During this period, production was highest in
1969 at 601 million pounds, and lowest in 1975 at 306 million
pounds. In 1976 and 1977, monochlorobenzene production was 329
and 325 million pounds, respectively. In a 1980 report prepared
by Hull and Company for the Synthetic Organic Chemicals Manufac-
turers Association (SOCMA), 1978 production of monochlorobenzene
was stated to be 303 million pounds, of which 302 million pounds
was reported to be consumed within the United States (Hull 1980).
Imports of monochlorobenzene are small compared to domestic
production. Imports were about 1.5 million pounds in 1974 and
8.4 million pounds in 1975 (Allport et al. 1977). According to
nonconfidential TSCA Inventory information, five importers
reported no imports of monochlorobenzene in 1977 (OPTS 1979a).
4. Uses
Monochlorobenzene consumption has been in a state of flux
over the past several years. According to Hardie (1964), the
bulk of monochlorobenzene consumption in the early 1960's went to
the production of phenol (60 percent) and the insecticide DDT (25
percent), with the remainder used to produce a variety of dye
intermediates. However, phenol production from monochlorobenzene
was gradually phased out in favor of the cumene process before
1975 (Lowenheim and Moran 1975), while DDT production in the
United States dropped from 123 million pounds in 1969 to 54.3
million pounds in 1970 (USITC 1966-1977) as a result of regula-
tions, effective in 1972, severely restricting its sale and
use. No production data have been available for DDT since 1970.
The current consumption pattern of monochlorobenzene is not
known in detail, but some general information on consumption is
available for recent years from three sources. SRI International
(Allport et al. 1977) reports that the monochlorobenzene consump-
tion pattern in 1974 was:
14
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Solvent (for pesticides, degreasing) 49%
Chloronitrobenzenes intermediate 30%
Diphenyl oxide intermediate 8%
DDT intermediate 7%
Other uses 6%
The Chemical Marketing Reporter (1977) presented the following
consumption pattern:
Solvent 30%
Chloronitrobenzenes intermediate 35%
Diphenyl oxide intermediate 10%
Rubber intermediate* 10%
Synthesis of DDT, silicones, isocyanates, others 15%
Lastly, Dow Chemical (1978b) provided these consumption
data:
Chemical intermediate 50-70%
(chloronitrobenzenes, herbicides,
diphenyl oxide, DDT, silicones,
others)
Solvent for herbicide formulations 30-50%
Other (chemical process solvent; <5%
degreasing solvent; other uses)
It appears that the two major categories of monochloroben-
zene consumption are as an intermediate and as a solvent. Among
its uses as an intermediate, the largest is for the production of
2_-chloronitrobenzene and jv-chloronitrobenzene. Both of these
monochlorobenzene derivatives are used as dye intermediates; _p_-
chloronitrobenzene is also used to produce p-nitrophenol.
A Dow product bulletin refers to monochlorobenzene1s use as a
solvent for synthetic rubbers (Dow 1975).
15
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Another derivative, diphenyl oxide (diphenyl ether) is used in
perfumery, especially in soaps, and as a heat transfer medium
(Hawley 1977, Merck 1976). The silicon derivatives made from
monochlorobenzene are phenyltrichlorosilane and diphenyldichloro-
silane (Meals 1964), which are used in the production of silicone
lubricants (Hawley 1977). No specific information could be found
on the other intermediate uses of monochlorobenzene.
There is very little specific information on the use of
monochlorobenzene as a solvent. Allied Chemical (1973) indicated
in their product data sheet for monochlorobenzene that it has
industrial process solvent applications in the manufacture of
adhesives, paints, polishes, waxes, diisocyanates, pharmaceu-
ticals, and natural rubber. This source also notes the use of
monochlorobenzene as a dye carrier in textile dyeing. The extent
of actual use of monochlorobenzene in these areas could not be
determined. Dow Chemical (Dow 1978b) stated that the bulk of the
solvent use was for unspecified herbicide formulations, with only
a small amount of monochlorobenzene going to other solvent uses
such as chemical processing or degreasing. Hull and Company
(Hull 1980) indicate that monochlorobenzene is used as a "formu-
lation solvent for a consumer auto radiator flush." An EPA
(USEPA 1980) survey found monochlorobenzene in industrial water
samples from the following industrial classifications (listed in
decreasing order of frequency of observation): organics and
plastics, petroleum refining, inorganic chemicals, textiles,
pesticides, Pharmaceuticals, leather, paint and ink, auto and
other laundries, printing and publishing, landfill, coal mining,
mechanical products, and photographic. This list indicates the
variety of possible applications of this substance.
5. Occupational Exposure
Dow Chemical Co. (Dow 1978b) states that the industrial
manufacturers and processors of monochlorobenzene employ
approximately 10,000 people. The National Occupational Hazard
Survey (NIOSH 1979), which includes in addition to manufacturers
and processors those potentially exposed on the job to products
16
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containing monochlorobenzene, indicates that slightly more than 1
million people may be exposed to monochlorobenzene in the
workplace. See Exposure Support Document for a general
discussion of the NOHS data.
Hull and Co. estimated that 3,146 people were exposed to
monochlorobenzene through its production, its captive use, and
receipt of direct shipments from producers (Hull 1980). Several
aspects of this report indicate that the worker exposure esti-
mates may be biased toward the low range of exposure (Versar
1980). In any event, the document contains no citations from
which the data can be verified (Versar 1980). The survey
questionnaire used by Hull and Co. requested best estimates of
numbers of people exposed and allowed rounding of these numbers
to the nearest 10. Certain key questions relating to workroom
exposure due to equipment wear and volatilization during
processing were omitted from the form. There was no indication
that the data were substantiated by any visits by the survey team
or by any means other than follow-up phone calls to the respon-
dents. The numbers of workers exposed do not agree with the
worker population identified with the industrial use component of
potential exposure to monochlorobenzene (Versar 1980). Finally,
the percentage of companies identified as being involved with the
manufacture, distribution, and use of monochlorobenzene that
responded to the questionnaire was not identified. Consequently,
the Hull and Co. figures may represent an underestimate of
occupational exposure to chlorinated benzenes.
OSHA (1979) reports two instances in which monochlorobenzene
was detected at levels below or equal to the threshold limit
value (TLV) in air samples collected from companies using
monochlorobenzene. The companies employed 50 and 67 people,
respectively. No other information was given. The TLV for
monochlorobenzene in workroom air is 75 ppm (350 mg/rn^) (ACGIH
1971).
-------
Pure mono- and dichlorobenzenes tend to be corrosive to
metals (Versar 1980). This increases the potential for worker
exposure to fugitive emissions from the pump seals, compressor
seals, valves and pressure relief devices of the manufacturing
machinery (Versar 1980).
No other information was found on occupational exposure to
monochlorobenzene in its manufacture, processing or subsequent
industrial applications.
6. General Population Exposure
Exposure of the general population to monochlorobenzene
appears most likely to occur through release of the compound to
the environment through activities such as manufacturing,
industrial use, processing and disposal.
7. Environmental Release
Dow Chemical (Dow 1978b) provided an estimate that 30 to 50
percent of the monochlorobenzene produced annually, or 98 million
to 162 million pounds, is ultimately released into the air, and
less than 0.1 percent (300,000 pounds) into water. The estimated
range of losses to the air includes Dow's estimate of annual
consumption of monochlorobenzene as a herbicide solvent, a use
that would presumably result in virtually complete release to the
air. Monsanto (1978c) presented data that showed that direct
application of monochlorobenzene to both soil and to plants
resulted in a minimum of 80 percent of the monochlorobenzene
evaporating within 6 hours after application.
EPA studies have detected monochlorobenzene in air samples
collected from the Kin-Buc Land Disposal Site, Edison, N.J. and
the air of Birmingham, Alabama (Pellizzari 1978) and in an
industrially produced cloud in Henderson, Nevada (Wojinski et al.
1979). Monochlorobenzene levels ranging from 0-12 ug/m3 (0-2.6
ppb) were detected in the samples from Kin-Buc Land Disposal Site
and 38-1000 ng/m3 (0.008-0.2 ppb) in Birmingham. The Henderson
cloud contained 11-40 ug/m3 (2-8 ppb) of monochlorobenzene,
compared to a level of 0.45 ug/m3 (0.1 ppb) measured in nearby
Las Vegas.
18
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In a report by the EPA (Gruber 1975), it was estimated that
during the batch manufacture of monochlorobenzene, 0.00088 kg of
monochlorobenzene enters the water stream, and 0.004 kg of mono-
chlorobenzene goes to land disposal per kg of monochlorobenzene
produced. In 1977, therefore, 130,000 kg (86,000 pounds) would
have been released into water and 591,000 kg (1,300,000 pounds)
to land; these are maximum figures based on an assumption of 100
percent production by batch processes. Monochlorobenzene was
reported to be present in a textile mill effluent (Erisman and
Gordon 1975), in an industrial discharge in South Carolina, and
in a municipal discharge in Georgia (Ware et al. 1977). Organic
priority pollutant surveys conducted by EPA of industrial water
and wastewater samples found monochlorobenzene in 147 of 3194
samples with levels ranging from 11 to 6400 ppm (USEPA 1980).
The EPA (1975) reported that monochlorobenzene was detected in
finished drinking water in 9 out of 10 cities sampled in the
National Organics Reconnaissance Survey, with concentrations
ranging from 0.1 ug/1 to 5.6 ug/1 (0.1-5.6 ppb).
Lombardo (1979) reported detection of unspecified
concentrations of monochlorobenzene in fresh water fish under the
PDA chemical contaminants program.
8. Environmental Transformation
Data from simulated atmospheric photodecomposition experi-
ments indicate that monochlorobenzene may degrade rapidly in the
atmosphere. In an experiment performed by Dilling et al. (1976),
a half-life of 8.7 hours was found for monochlorobenzene in
air. However, the intensity of the ultraviolet light in the test
chamber was about 2.6 times that of normal sunlight (as measured
at noon on a summer day in Freeport, Texas). No information was
presented on the products of monochlorobenzene photodecomposi-
tion.
No experimental data were found in the literature on the
hydrolysis of monochlorobenzene under environmental conditions.
However, this chemical probably will not hydrolyze under environ-
mental pH and temperature conditions (see Section I.D).
19
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In a Monsanto study (1978a), -^c-monochlorobenzene was added
to samples of Mississippi River water, and evolved ^-^CQ^ was
measured over a 63-day period. Twenty-seven percent of the
theoretical 14C had been recovered as 14C02 after 63 days.
Addition of acclimated activated sludge to a sample of
Mississippi River water plus l4C-monochlorobenzene resulted in
much slower 14C02 evolution; no explanation was offered for this
observation. Gibson et al. (1968) found that the bacterium
Pseudomonas putida, growing on toluene, oxidized monochloro-
benzene to 3-chlorocatechol. No oxidation or bacterial growth
occurred on monochlorobenzene in the absence of toluene,
suggesting that biodegradation of the former may proceed
relatively slowly.
9. Biological Uptake
The octanol/water partition coefficient of monochlorobenzene
is approximately 700 (Leo et al. 1971), suggesting moderate
bioconcentration potential. Dow Chemical (Dow 1978b) reported
that Japanese experiments indicate a fish bioconcentration factor
of less than 300 for monochlorobenzene.
Lu and Metcalf (1975) studied the fate and biomagnification
of monochlorobenzene in a model ecosystem using Daphnia, mosquito
larvae, fish, snails, and green algae. The results are
summarized in Table 1.
20
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Table 1
24-Hour Ecological Magnification3 (EM) of Monochlorobenzene in
Various Aquatic Species
•L4C6H5C1 accumulated x 100
Species EMb Total 14C accumulated
Alga, Oedogonium cardiacum 4185 70
Water flea, Daphnia magna 2789 49
Snail, Physa sp. 1313 53
Mosquito larva, Culex quinquifasciatus 1292 43
Mosquito fish, Gambusia affinis 645 46
aDefined as (concentration of compound in organism)/(concentration in water)
Initial concentration = 0.01-0.1 ppn. It appears that the
magnifications observed for algae and Daphnia are not maintained
at the upper end of the food chain in this test system. It is
worth noting that the extent of metabolism of monochlorobenzene,
as reflected in the last column of the table, was much less than
that found for some other compounds tested under the same
conditions. For example, in mosquito larvae 14 percent of
anisole and 0 percent of aniline were retained in the form of the
unconjugated parent compound, whereas for monochlorobenzene,
43 percent was retained in the unmetabolized form. This order of
reactivity conforms with what is expected on chemical grounds and
demonstrates the deactivating influence of an aromatic chlorine
substituent in a biochemical reaction.
Conclusion; Monochlorobenzene is manufactured on the order
of hundreds of millions of pounds annually, more than half of
which goes to TSCA uses, primarily as a chemical intermediate.
Opportunities for human exposure are both direct, including
occupational contact and contact with releases from plants and
disposal sites, and indirect, including ingestion via drinking
water and, to some extent, the food chain.
21
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B.
Dichlorobenzenes
1.
Nature of the Substances
At room temperature, c>-dichlorobenzene and jn-dichlorobenzene
are combustible, neutral, mobile, colorless liquids while p-
dichlorobenzene is a combustible white crystalline solid that
sublimes readily. The dichlorobenzenes are all very soluble in
nonpolar solvents (Hawley 1977, Merck 1976) but are poorly water-
soluble (see Table 1, Section I.). The lack of industry-wide
standards of purity for chlorobenzenes (Kao and Poffenberger
1979) is illustrated by the compositions reported for ^-dichlo-
robenzene by different sources as compiled in Table 2.
Table 2
Reported Percentage Compositions of Commercial
o-Dichlorobenzenes
Information Source
and
Percentage Composition
Constituent
C6H5C1
0-C6H4C12
jn-C6H4Cl2
Dow
(1977)
80
2
Allied
(1973)
Standard
Grade
0.07
82.7
0.5
1
C
Mechan:
Grade
75-85
0.5
17
(all Isomers)
1,2,4-C6H3C13
15.4
1.6
MCA Kao and Poffenberger
(1974) (1979)
High
Tech. Purified
15-25
Grade
99.0
>"balance"
Grade Grade
<0.05
80
r!9
<0.05
98
<1
<0.02
22
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The commercially available technical grade of p-dichlorobenzene
contains 0.08 percent by weight of the meta and ortho isomers of
dichlorobenzene, but may also contain monochlorobenzene and
trichlorobenzenes (IARC 1974, Brown et al. 1975, Kao and
Poffenberger 1979). A Dow product data sheet (1977) states a
purity of 99.95% for that company's p-dichlorobenzene. The para
isomer is commercially available in either the liquid or the
solid form (Anon. 1979a). Product information from Montrose
Chemical (1972) describes a mixture of 35 percent _p_- and 65
percent p-dichlorobenzene. Contaminants of jn-dichlorobenzene are
the ortho and para isomers in unspecified proportions (Hardie
1964).
2. Manufacture
o-Dichlorobenzene and p-dichlorobenzene are produced by
chlorinating benzene or monochlorobenzene at 150°-190°C over a
ferric chloride (FeCl3) catalyst. An orienting catalyst such as
benzenesulfonic acid or p-methylbenzenesulfonic acid will
increase yields of p-dichlorobenzene. The isomers can be
separated by fractional distillation, or by crystallizing the p-
dichlorobenzene. Another method of obtaining the para isomer is
by chlorination of crude dichlorobenzene over Fed3, whereby the
more reactive ortho isomer is converted to 1,2,4-trichloro-
benzene. p-Dichlorobenzene can then be separated by distilla-
tion. Instead of chlorination, selective sulfonation of the
ortho isomer with chlorosulfonic acid allows a similar separa-
tion. The purified grade of ^-dichlorobenzene is obtained by
efficient redistillation of the technical product (Kao and
Poffenberger 1979). m-Dichlorobenzene can be prepared by
isomerization of ^-dichlorobenzene and p-dichlorobenzene with
heat under pressure in the presence of a ratalyst (Hardie 1964).
3. Production Volume and Trends
TSCA Inventory data indicate that in the United States in
1977 there were eight manufacturers of o_- and p-dichlorobenzene
and five manufacturers of m-dichlorobenzene (OPTS 1979a). The
23
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total combined production capacity for j>-dichlorobenzene and
£-dichlorobenzene was 279 million pounds per year in 1978 (SRI
1978) and 307 million pounds per year in 1979 (Anon. 1979a,b).
The Dow Chemical Company reports that jn-dichlorobenzene is
produced as a by-product in the manufacture of other chlorinated
benzenes. It is separated from the ortho and para isomers, and
further chlorinated to tri- and tetrachlorobenzenes. Only very
small quantities are produced for research purposes (Dow
1978b). The EPA estimates on the basis of TSCA Inventory
information that 666,000 pounds of the meta isomer was produced
in 1977; some of this production was site-limited (OPTS 1979a).
An average of 60 million pounds of ^-dichlorobenzene per
year was produced from 1966 through 1973. In 1976 the amounts of
^-dichlorobenzene and j>-dichlorobenzene produced annually were
48.6 million pounds and 36.7 million pounds, respectively. The
average sales for ^-dichlorobenzene from 1966 through 1975 was 52
million pounds per year. The industry expects only very slow
growth in the market for £>-dichlorobenzene through 1983 because
almost all end-use sectors are mature (Anon. 1979a). In 1978,
demand equalled 55 million pounds, and in 1979, it was projected
at 56 million pounds (Anon. 1979b). Hull (1980) reports that 59
million pounds of ^-dichlorobenzene was produced and that
domestic sales and captive use accounted for 52.5 million pounds
in 1978. ja-Dichlorobenzene sales averaged 67 million pounds per
year from 1966 through 1973. During 1975, £-dichlorobenzene
sales dropped to a 10-year low of 34 million pounds (USITC
1976). By 1978, demand was back up to 68 million pounds and a
projected 67 million pounds in 1979. Hull (1980) estimated 1978
production of jD-dichlorobenzene to be 72.5 million pounds and
domestic consumption to be 53.2 million pounds. A market decline
of two to three percent per year is expected through 1983 because
space odorant and mothproofing markets, which comprise 80 percent
of para-dichlorobenzene uses, are essentially mature. In
addition, rising production costs, the appearance of cheaper sub-
stitutes, and the popularity of synthetic fabrics which require
no mothproofing, will combine to decrease production and sales
24
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volumes until 1983. However, a new use, as an intermediate in
the production of the resin polyphenylene sulfide, may help
maintain demand. EPA TSCA Inventory-based estimates of 1977
imports of _o_-, _m_-, and jD-dichlorobenzene are 2 thousand, 551
thousand, and 56 thousand pounds, respectively (OPTS 1979a).
Exports of dichlorobenzenes for the months of June and July, 1979
totaled 4.39 million pounds (Anon. 1979c).
4. Uses
Sixty-five percent of the ^-dichlorobenzene produced goes to
organic synthesis, primarily as a pesticide intermediate.
Fifteen percent is used as a solvent in the production of toluene
diisocyanate (Anon. 1979b). Miscellaneous solvent uses, such as
for oxides of nonferrous metals (Hawley 1977), for soft carbon
deposits, for tars and wool oils in the textile industry (Dow
1975) and for degreasing leather and automobile and aircraft
engine parts (Allied 1973), account for most of the rest of the
annual production of cv-dichlorobenzene. It is also a solvent in
formulated toilet bowl cleaners and drain cleaners (Allied
1973). Other uses in metal polishes, in industrial odor control,
as a heat transfer fluid, and in rustproofing mixtures account
for 4 percent of annual js-dichlorobenzene production (Lowenheim
and Moran 1975). c^-Dichlorobenzene is registered as a fumigant
and insecticide against termites, beetles, bacteria, slime, and
fungi (Hawley 1977). Dow Chemical indicated that c>-dichloro-
benzene is used in the following ways (Dow 1978b).
Chemical Intermediate 70-75%
(herbicides, other)
Process Solvent 15-20%
(toluene diisocyanate)
Miscellaneous 5-10%
(solvent, heat transfer fluid, other)
25
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Eighty percent of the annual production of p-dichlorobenzene
goes to home and industrial use as a moth control agent (30
percent) and as a space odorant (50 percent), especially in
toilets and rest rooms.
Miscellaneous uses as a dye intermediate, insecticide,
extreme pressure lubricant, forming agent for grinding wheels,
disintegrating paste for molding concrete and stoneware, and as
an intermediate in the manufacture of 2,5-dichloroaniline and
polyphenylenesulfide resins account for 10 to 20 percent of the
consumption of jD-dichlorobenzene (Allied 1973, Dow 1975, Anon.
1979a) . No uses were identified for ju-dichlorobenzene apart from
its conversion to higher chlorobenzenes (Section II.B.3).
In an EPA industrial wastewater survey (USEPA 1980) the
industrial categories with dichlorobenzenes present (listed in
decreasing order of frequency of occurance) were: organics and
plastics, steam and electric, leather, textiles, auto and other
laundries, mechanical products, printing and publishing, pesti-
cides, nonferrous metals, electrical, pharmaceutical, adhesives
and sealants.
5. Occupational Exposure
Occupational exposure to j^-dichlorobenzene can occur through
the inhalation of vapors during its production, processing, and
use as an industrial solvent and in commercial products. The
potentially occupationally exposed population figure given by the
National Occupational Hazard Survey (NOHS) for all occupational
activities involving jD-dichlorobenzene is approximately 2 million
(NIOSH 1979); Dow estimates that 10,000 workers are potentially
occupationally exposed as a result of production, processing and
industrial solvent use (Dow 1978b). For p-dichlorobenzene, the
NIOHS estimate for potential worker exposure during its produc-
tion, processing into space odorants, and use as an intermediate
is approximately 1 million (NIOSH 1979). Hull (1980) estimated
that 1,311 people were exposed to ^3-dichlorobenzene and 821
people were exposed to p-dichlorobenzene during production,
26
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captive use, and shipment from producers. Several aspects of
this report indicate that the worker exposure estimates may be
biased toward the low range of exposure (Versar 1980). In any
event, the document contains no citations from which the data can
be verified (Versar 1980). The survey questionnaire used by Hull
and Co. requested best estimates of numbers to the nearest 10.
Certain key questions relating to workroom exposure due to
equipment wear and volatilization during processing were omitted
from the form. There was no indication that the data were
substantiated by any visits by the survey team or by any means
other than follow-up phone calls to the respondents. The numbers
of workers exposed do not agree with the worker population
identified with the industrial use component of potential
exposure to dichlorobenzenes (Versar 1980). Finally, the
percentage of companies identified as being involved with the
manufacture, distribution, and use of dichlorobenzenes that
responded to the questionnaire was not identified. Consequently,
the Hull and Co. figures may represent an underestimate of
occupational exposure to chlorinated benzenes.
No information was found on the number of workers exposed
to jn-dichlorobenzene. Between 1973 and March of 1979, OSHA
inspectors collected, from a total of 15 companies throughout the
United States, air samples that contained dichlorobenzenes at
levels less than or equal to the TLV standards in effect at the
time of sampling. A total of 8000 people were involved. The air
samples from nine companies (6750 people) contained cv-dichloro-
benzene; samples from four companies contained p_-dichlorobenzene
(400 people), and samples from two companies (1800 people)
contained both isomers (USOSHA 1979). The TLV's for o_- and p_-di-
chlorobenzene are 50 ppm and 75 ppm, respectively (ACGIH 1978).
No TLV has been established for nv-dichlorobenzene.
A 1940 air-monitoring study in a Dow p_-dichlorobenzene plant
found concentrations in working areas ranging from 12 ppm to 550
ppm; more than half the values were in the range 50-175 ppm.
Insufficient details were available to determine whether the
27
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measurements were selective for the para isomer or whether other
chlorinated benzenes might have been included in the figures
reported. Some of the exposed workers had developed a local
dermatitis (Dow 1978c).
Another study (Pagnotto and Walkley 1965) reported levels of
dichlorobenzenes in a chlorobenzenes manufacturing plant, in a
factory where jD-dichlorobenzene was processed into moth cakes,
and in a plant where p-dichlorobenzene was used in an abrasive
wheel-forming process. Average levels of £-dichlorobenzene for
various working areas ranged from 24-34 ppm in the chlorobenzenes
plant (high value 49 ppm), from 9-25 ppm in the moth cake opera-
tion (high value 34 ppm), and from 8-11.5 ppm in the abrasive
wheel facility (high value 14.5 ppm). The authors also referred
to workers in the moth cake plant being struck occasionally by a
fine dust burst from a p-dichlorobenzene pulverizing machine. In
the chlorobenzenes plant, ^-dichlorobenzene was detected at
levels up to 25 percent of those for the para isomer. A metabo-
lite of jD-dichlorobenzene, 2,5-dichlorophenol, was found in the
urine of exposed workers; although metabolite excretion dropped
off rapidly when exposure was terminated, it was still in
evidence after several days, suggesting that some retention of
j^-dichlorobenzene or its metabolites occurs after exposure.
Hull and Co. (Hull 1980) indicate that the major use of cjr
dichlorobenzene in formulated products is as an industrial
cleaner. Over 4 million pounds of o-dichlorobenzene went into
degreasing and decarbonizing formulations for use in the
automotive, trucking, and aircraft industries. During the use of
these formulations, workers are exposed to fumes throughout the
entire working day. Hull (1980) indicates that 200 workers may
be exposed in transmission shops alone.
6. General Population Exposure
General population exposure to ^-dichlorobenzene and p-di-
chlorobenzene occurs because the chemicals are present in many
household products. Sources of consumer exposure to ^-dichloro-
benzene are auto engine parts cleaners, toilet bowl cleaners, and
28
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drain cleaners. Household exposure to jD-dichlorobenzene occurs
because of its use in deodorizers (Hull 1980). Morita and Ohi
(1975) reported indoor concentrations of p_-dichlorobenzene from
its use as a space odorizer or moth repellant of 1700 ug/nP (283
ppb) (inside wardrobe); 315 ug/m3 (52 ppb) (closet); and 105
ug/m3 (18 ppb) (bedroom). Further exposure to dichlorobenzenes
may occur by way of the environment (see Environmental Release,
below).
7. Environmental Release
Because jo-dichlorobenzene is relatively volatile and has
widespread use in industry as a solvent and as an intermediate in
the manufacture of other chemicals, release to the atmosphere may
occur readily. Because jD-dichlorobenzene readily sublimes and is
used extensively as a domestic and industrial space deodorant,
extensive quantities are released into the atmosphere.
Dow Chemical's estimates for the release of the dichloro-
benzenes into the environment as a percentage of their annual
production are 5 to 10 percent in air (2.4 to 4.8 million pounds
based on 1976 production),and less than 0.1 percent (0.49 million
pounds) in water for jD-dichlorobenzene, and 70 to 90 percent (26
to 33 million pounds) in air and less than 5 percent (1.8 million
pounds) in water for ja-dichlorobenzene (Dow 1978b) . Other
estimates of the environmental release rates are 43 percent (21
million pounds) for o-dichlorobenzene and 91 percent (34 million
pounds) for p-dichlorobenzene (calculated from data in Allport et
al. 1977). No release rate was found for ^n-dichlorobenzene.
c»-Dichlorobenzene was reported to be lost to the environment
during manufacture at the rate of 0.9 million pounds per year in
1972. These losses arose through venting and scrubber washings
(Brown et al. 1975). When used as a solvent in the manufacture
of toluene diisocyanate (TDI), ^-dichlorobenzene is lost to the
environment through venting, scrubbers, and leaking equipment;
the sludge and hydrogen chloride remaining after TDI manufacture
contain less than a few hundred parts per million of c^-dichloro-
benzene (Brown et al. 1975). Environmental losses during manu-
29
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facture for jo-dichlorobenzene were estimated to be 1.2 million
pounds in 1972 (Brown et al. 1975). Dow Chemical estimates that
between 73 and 99 percent of the js-dichlorobenzene used in deodo-
rant blocks evaporates into the air (Dow 1978b).
Dichlorobenzenes also are lost to the environment as a
result of monochlorobenzene manufacture. According to an EPA
report (USEPA 1975), the loss of dichlorobenzenes to waste waters
from this source is 0.37 percent of monochlorobenzene production,
or 1.2 million pounds per year on a 1977 basis; land disposal
amounts to 0.01 percent, or 32,500 pounds in 1977.
Dichlorobenzenes have been detected in air samples from the
Kin-Buc Disposal Site Edison, N.J., Birmingham, Ala. the Chambers
Works site of the E.I. DuPont de Nemours Co, at Deepwater, N.J.
(Pellizzari 1978) and in an industrial cloud which periodically
develops over Henderson, Nevada (Wojinski et al. 1979). All
three isomers of dichlorobenzene were detected within a 5-mile
radius of the Edison, N.J. disposal site. ^-Dichlorobenzene
concentrations ranged from 0-10 ug/m3 (0-2 ppb); jjv-
dichlorobenzene from 0-0.5 ug/m3 (0-0.08 ppb); and _m-
dichlorobenzene from 0.2-33 ug/m3 (0.03-5.5 ppb). o_-
Dichlorobenzene was also detected at the Chambers Works site
(1.319 ug/m3) and in Birmingham (0.348 ug/m3). jn-Dichlorobenzene
was detected in Birmingham (0.258-0.557 ug/m3) (Pellizzari
1978). The Henderson cloud contained a total of dichlorobenzene
isomers of 1.6-33 ug/m3 (0.3-5.5 ppb) compared to a range of 0-
6.5 ug/m3 (0-1.1 ppb) found in nearby Las Vegas (Wojinski et al.
1979).
^-Dichlorobenzene used in toilet blocks is flushed into
sewer systems. A Dow study (1978d) found that jD-dichlorobenzene
urinal blocks, when subjected to intermittent water washes to
simulate flushing conditions, dissolved to the extent of 0.26-
0.67 mg/L on each washing. Continuous dropwise washing dissolved
up to 18 mg/L. The solvent effect of urine was not investi-
gated. ^-Dichlorobenzene also has been detected in textile
finishing plant effluents (Erisman and Gordon 1975 ). EPA
monitoring data have indicated the presence of ^-dichlorobenzene
and pj-dichlorobenzene in industrial discharges.
30
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Young and Heesen (1977) examined the effluent from the Los
Angeles County, Los Angeles City, and Orange County sewage
treatment plants along the Southern California Bight. Both
ja-dichlorobenzene and ^o_-dichlorobenzene were detected in the
water samples. Values were generally in the range of 2-12 ug/L,
although levels as high as 230 ug/L (para isomer) and 435 ug/L
(ortho isomer) were reported for samples from the Los Angeles
City treatment plant effluent. None of the three treatment
plants treated their sewage by chlorination. Sediments collected
from the vicinity of the Los Angeles County sewage plants
contained detectable levels of both dichlorobenzene isomers as
did the muscle and liver tissue of fish collected from the same
area. Up to 0.010 mg/L of an unspecified isomer of dichloro-
benzene was detected in cooling water and in seepage lagoons at a
detergent manufacturing plant in Muskegon, Michigan (Christensen
and Long 1976). Organic priority pollutant surveys conducted by
EPA of industrial water and wastewater samples found all three
dichlorobenzene isomers in 178 out of 3,268 samples at levels
greater than 10 ppb (USEPA 1980). As the isomers were difficult
to determine by the procedures used, the three isomers were
combined for frequency of occurance analysis. Samples collected
from the wastewater treatment plants of Dalton, Calhoun, and
Rome, Georgia and from the Coosa River contained from 0.004 mg/L
to 0.268 mg/L of unspecified dichlorobenzene. Concentrations of
the dichlorobenzenes tended to increase in the downstream direc-
tion and in some cases were higher in the finished water than in
the treatment plant water. The Coosa River is the center of the
tufted carpet and rug industy (Gaffney 1976). A survey of
environmental monitoring data on the occurrence of volatile
organics in drinking water indicated that j>-dichlorobenzene was
present in 12.5% of the finished drinking water and in 12.9% of
the finished ground water samples from stations reporting usable
data. In a New Jersey study, 7 out of 717 samples contained
detectable levels of unspecified dichlorobenzenes (Coniglio et
al. 1980). The highest concentrations of ^-dichlorobenzene,
jn-dichlorobenzene, and ^-dichlorobenzene found in drinking water
31
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were 0.0010 mg/L, 0.0005 mg/L, and 0.0005 mg/L, respectively (EPA
1975). The fact that maximum levels are similar for the three
isomers is of interest in view of the low production volume of
the meta isomer relative to the other two dichlorobenzenes. Fish
specimens collected from the Great Lakes drainage system have had
detectable levels of dichlorobenzenes in their tissue (Veith et
al. 1979), as did those from other fresh water sources (Lombardo
1979).
The dichlorobenzenes can volatilize from water. Some
laboratory data indicate that these compounds volatilize nearly
completely from nonaerated distilled water in less than three
days using initial concentrations of 100 mg/L for jD-dichloro-
benzene and 300 mg/L for ^-dichlorobenzene (Garrison and Hill
1972).
8. Environmental Transformation
No experimental data have been found on the environmental
hydrolysis of dichlorobenzenes. However, the dichlorobenzenes
are probably not readily susceptible to hydrolysis under environ-
mental pH and temperature conditions (see Section I.B.2.). In
the atmosphere, o- and p-dichlorobenzene react with hydroxyl
radicals and have a half-life of 3 days (Brown et al. 1975). No
data were found on the rates of photolytic decomposition in the
atmosphere.
A study done for Monsanto (1978b) showed that both _o_- and
jp_-dichlorobenzene are biodegradable by acclimated microorganisms.
The total biodegradation time (determined from 27-day C02 evolu-
tion rates) was calculated to be 55 days for ^-dichlorobenzene
and 58 days for j>-dichlorobenzene. A Dow study (1978d) showed
that acclimated activated sludge can remove 95 percent of _p_-di-
chlorobenzene from the water within 7 days.
9. Biological Uptalee
The octanol/water partition coefficient for both _o_-dichlo-
robenzene and ja-dichlorobenzene is 2400 (calculated from Table 1,
Section I), suggesting some bioconcentration potential. Dow
32
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found bioconcentration factors for j3-dichlorobenzene of 231 for
trout and 15 for bluegill. After 10 weeks' exposure to j>-di-
chlorobenzene, the bioconcentrated compound was not detected in
bluegills within one week after they were transferred to clean
water (Dow 1978a); analogous data were not reported for the
trout.
Conclusion; About 100 million pounds of dichlorobenzenes is
produced each year. They have chemical intermediate, space
odorant, and solvent uses. Considerable loss to the environment
occurs during their manufacture, processing, and use and as a
result of their formation during monochlorobenzene manufacture.
Potential for exposure to dichlorobenzenes results from occupa-
tional contact during manufacture and industrial use, from
consumer product use, and from consumption of drinking water and
fish, and also occurs as a result of disposal.
C. Trichlorobenzenes
1. Nature of the Substances
At room temperature, 1,2,4-trichlorobenzene is a colorless
liquid while 1,2,3- and 1,3,5-trichlorobenzene are crystalline
solids (Hardie 1964). All are soluble in ethanol (Merck 1976)
and have low water solubility (see Table 1, Section I.). Com-
mercial 97 percent 1,2,4-trichlorobenzene may contain mono-, di-
and tetrachlorobenzenes (Kao and Poffenberger 1979). One company
is reported to produce both pure and technical grades of 1,2,4-
trichlorobenzene, with no further details given (Anon. 1979d). A
Dow Chemical information sheet states a purity of 100 percent for
that company's 1,2,4-trichlorobenzene (Dow 1977). An entry on
the TSCA Inventory, Pyranol 1478, is identified only as trichlo-
robenzene (USEPA 1979a).
2. Manufacture
1,2,4-Trichlorobenzene is produced along with 1,2,3-trichlo-
robenzene by chlorination of ^-dichlorobenzene at 25°-30°C in the
presence of ferric chloride, then separated from the 1,2,3-tri-
chlorobenzene by distillation (Hardie 1964). 1,3,5-Trichloro-
33
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benzene can be produced readily only by special methods such as
by the diazotization of 2,4,6-trichloroaniline followed by
treatment with alcohol (Merck 1976). Additional methods of
synthesis for trichlorobenzenes are reviewed in the report by
Ware and West (1977).
3. Production Volume and Trends
Production of 1,2,4- and 1,2,3-trichlorobenzene, and
mixtures of the two, increased from 9.3 million pounds in 1970 to
28.3 million pounds in 1973 (USITC 1966-1977). International
Trade Commission production figures for the trichlorobenzenes
have not been available since 1973. In that year, 1,2,4-tri-
chlorobenzene was produced by four manufacturers in the United
States. Since 1974, according to the International Trade
Commission there have been only two manufacturers of 1,2,4-tri-
chlorobenzene (USITC 1966-1977); the TSCA Inventory lists four
for 1977 (OPTS 1979a). Dow (1979) estimated the annual domestic
production at less than 20 million pounds. There is one manufac-
turer of a mixture of 1,2,3- and 1,2,4-trichlorobenzene (Dow
1979). Imports for 1977 of the 1,2,4-isomer are estimated at
around one million pounds by EPA using Inventory data (OPTS
1979b).
Most trichlorobenzene produced in the United States for
commercial purposes consists of the 1,2,4-isomer. In the produc-
tion of the 1,2,4-isomer, 1,2,3-trichlorobenzene is also
produced, but can be separated from the 1,2,4-isomer by distil-
lation. A very small quantity of 1,3,5-trichlorobenzene is also
produced as a by-product in this process, but according to Dow
Chemical, this quantity amounts to less than 6,000 pounds per
year (Dow 1979). Nevertheless, TSCA Inventory data indicate that
at least 20 thousand pounds of 1,3,5-trichlorobenzene was
produced in 1977 by *->ur manufacturers and that a fifth either
produced or imported at least 10 thousand pounds. Production and
imports of the 1,2,3-isomer were both in the one to ten million
pound range for 1977 (OPTS 1979b). Dow's estimate for 1,2,3-tri-
chlorobenzene production is four million pounds per year maximum
(Dow 1979).
34
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4. Uses
The major uses of 1,2,4-trichlorobenzene are as a dye
carrier (20-30 percent), a synthesis intermediate (50-60 per-
cent), as a dielectric fluid (5-10 percent), and as a solvent
(5-10 percent) (Dow 1979). As a dye carrier, 1,2,4-trichloro-
benzene is used to facilitate the dyeing of textiles (Jones
1973). A carrier swells the fibers of a material and allows the
dye to diffuse through the fiber more quickly. Upon completion
of the dyeing process, the dye carrier is removed from the fabric
and disposed (Mark 1966). As an herbicide intermediate, 1,2,4-
trichlorobenzene is used to manufacture 2,5-dichlorobenzoic acid
(Dow 1975). Other uses of 1,2,4-trichlorobenzene are as a
solvent for coal tar products such as asphalt, for oil-soluble
dyes, and for wood preservatives (Dow 1975); its use in lubri-
cants has also been cited (Hawley 1977). The quantities of
1,2,4-trichlorobenzene utilized in each case are not known. Its
high thermal conductivity and heat capacity along with its
anticorrosion characteristics make 1,2,4-trichlorobenzene useful
as a heat transfer fluid. It is also used as an insulating
(dielectric) fluid in transformers and capacitors (Dow 1975,
1979) and is thus a potential substitute for PCB's; at least one
U.S. company plans to import such a product, Iralec®, from a
French manufacturer (Prodolec 1979). For all except chemical
intermediate uses, the uses of 1,2,3- and 1,2,4-trichlorobenzene
are the same, and mixtures of the two can be employed (Dow
1979). For example, a sample of transformer fluid involved in a
spill proved to contain 28% 1,2,4-trichlorobenzene and 4% 1,2,3-
trichlorobenzene by weight (Lewis 1979). No data were found on
uses for 1,3,5-trichlorobenzene other than its possible use as a
starting material for tetrachlorobenzenes.
5. Occupational Exposure
NIOSH has estimated that 86,340 people are potentially
exposed to 1,2,4-trichlorobenzene in the workplace each year and
35
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that about 1,867 are exposed to 1,3,5-trichlorobenzene* (NIOSH
1979). Dow estimates that fewer than 12,500 persons are exposed
to trichlorobenzenes used as dye carriers, less than 2,500 as a
result of dielectric applications, and less than 300 in connec-
tion with synthetic chemical production (Dow 1979). No estimate
was provided for exposure resulting from miscellaneous solvent
uses. The TLV for 1,2,4-trichlorobenzene is 5 ppm (ACGIH
1971). No TLVs for the other two trichlorobenzene isomers have
been established.
6. General Population Exposure
The uses of trichlorobenzenes are such that any general
population exposure is most likely to occur as a result of their
release to the environment - e.g. by disposal or leakage of
dielectric fluid.
7. Environmental Release
In a data review by Coniglio et al. (1980), 11.5% of the
finished ground water stations surveyed for volatile organic
substances had 1,2,4-trichlorobenzene present in the water. A
New Jersey ground water survey covered in the same review found
trichlorobenzene present in 13 of 396 samples. 1,3,5-
Trichlorobenzene has been detected in wastewater discharges in
Southern California (Young and Heeson 1978). 1, 2, 4-Trichlo-
robenzene has been found in textile plant effluents (Erisman and
Gordon 1975). Organic priority pollutant surveys conducted by EPA
of industrial water and wastewater samples found 1,2,4- trichlo-
robenzene at levels greater than 10 ppb in 30 out of 3266 samples
(EPA 1980). The water levels ranged from 12 to 607 ppb from the
following industrial classes (listed in decreasing order of
occurrence): textiles, organics and plastics, foundries, nonfer-
rous metals, pesticides, electrical, and paint and ink. Trichlo-
robenzenes have also been detected in both waste water treatment
EPA is attempting to confirm that the 1,3,5- isomer and not the
higher-production 1,2,3- isomer is intended.
36
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plant and Coosa River water samples in Georgia (Gaffney 1976).
Garrison and Hill (1972) indicated that 1,2,4-trichlorobenzene at
a concentration of 100 mg/L volatilized from aerated water in
less than 4 hours and from unaerated water in less than 48
hours. Thus, initial entry of this isomer into water may in some
situations lead to fairly rapid transfer of most of the substance
to the atmosphere (but see part 8 below).
Tissues of freshwater fish from the Great Lakes and the
Arkansas River (Veith et al. 1979) and from other locations
(Lombardo 1979) have been found to contain unspecified amounts of
trichlorobenzenes Muscle and liver tissue of Dover sole collec-
ted from the Southern California Bight contained detectable
levels of 1,2,4- and 1,3,5-trichlorobenzene (Young and Heeson
1977).
r
t
8. Environmental -Transformation
Few data have been published on the physical and chemical
properties of the trichlorobenzenes that govern their transport
and transformation in the environment.
1,2,4-Trichlorobenzene is slowly biodegradable. Experiments
by Simmons et al. (1977) showed that a residence time in excess
of five days in a wastewater treatment plant may be required to
significantly reduce its concentration. The relatively small
amount that was not degraded in an activated sludge system was
mostly bound to the waste sludge (Simmons et al. 1977). In an
aerated mixed aerobic microbial culture, 1,2,4-trichlorobenzene
was still detectable after 9 days (Garrison and Hill 1972). From
this information and that in part 7 above, it appears that 1,2,4-
trichlorobenzene in water is subject to both evaporation and
microbial degradation, but that both processes may be retarded by
the presence of undissolved matter.
9. Biological Uptake
Although no experimental data are available on the
bioconcentration potential of trichlorobenzenes, the reported
octanol/water partition coefficient (log P = 4.1 - see Table
37
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1, Section I) implies a potential for bioaccuraulation.
Conclusion; Trichlorobenzene production and importation
total around 20 million pounds yearly. They are used principally
as synthesis intermediates, dye carriers, solvents, and dielec-
tric fluid components. People may be exposed to trichloroben-
zenes occupationally during their manufacture, processing, and
use, and indirectly by way of drinking water and fish consump-
tion. The extent of occurrence of trichlorobenzenes in consumer
products is poorly characterized but there may be some direct
exposure from this source. Because trichlorobenzenes occur in
some commercial dichlorobenzenes, there is additional exposure
potential as a result of activities involving the latter. The
physicochemical properties of trichlorobenzenes imply some
potential for bioaccumulation.
D. Tetrachlorobenzenes
1. Nature of the Substances
All three of the tetrachlorobenzenes occur as white needles
that are soluble in most organic solvents and slightly soluble in
alcohol (Hardie 1964). A commercial 1,2,4,5-tetrachlorobenzene
was analyzed as 97.0 percent pure; impurities were not identified
(Kao and Poffenberger 1979, Dow 1977).
2. Manufacture
1,2,3,4-Tetrachlorobenzene can be produced by chlorinating
1,2,3-trichlorobenzene in the presence of a catalyst. 1,2,4,5-
Tetrachlorobenzene is manufactured by chlorinating 1,2,4-trichlo-
robenzene over aluminum amalgam. To produce 1,2,3,5-tetrachloro-
benzene, 1,3,5-trichlorobenzene can be chlorinated over aluminum
amalgam (Hardie 1964). In practice, 1,2,3,4- and 1,2,3,5-tetra-
chlorobenzene are produced only as by-products in the manufacture
of 1,2,4,5-tetrachlorobenzene (Dow 1979).
3. Production Volume and Trends
According to the U.S. International Trade Commission, there
is only one producer of 1,2,4,5-tetrachlorobenzene in the United
38
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States. In 1973, consumption of the material reportedly amounted
to approximately 18 million pounds (USITC 1969-1977). However,
the TSCA Inventory data (OPTS 1979b) indicate that two manufac-
turers produced 1,2,4,5-tetrachlorobenzene in 1977, with one
reporting a production volume range of 10 to 50 million pounds;
the other reported site-limited production. The Inventory
includes three companies importing the chemical in 1977, with one
reporting 1977 imports of 1 to 10 million pounds. Dow (1979)
estimated U.S. production of 1,2,4,5-tetrachlorobenzene as less
than 12 million pounds/year.
1,2,3,4- and 1,2,3,5-Tetrachlorobenzene are formed as by-
products during the production of 1,2,4,5-tetrachlorobenzene.
The TSCA Inventory data (OPTS 1979b) indicate that there was one
active manufacturer of 1,2,3,4-tetrachlorobenzene in 1977. Dow
(1979) estimates that the annual production volume of the
1,2,3,4-isomer is about 66 percent of 1,2,4,5-tetrachlorobenzene
production; this would be less than 12 million pounds on the
basis of both 1973 consumption and a Dow estimate of current
production (Dow 1979). Dow also states that the 1,2,3,5-isomer
formed during the production of 1,2,4,5-tetrachlorobenzene is
separated by distillation and disposed of as waste (Dow 1979).
The method of disposal was not given.
Tetrachlorobenzenes are found in some commercial trichlo-
robenzenes. A sample of transformer fluid was found to contain
16% by weight of two tetrachlorobenzene isomers (Lewis 1979) that
were not further identified.
4. Uses
The most widely used isomer of tetrachlorobenzene is
1,2,4,5-tetrachlorobenzene, which is used primarily as an
intermediate in chemical synthesis (Dow 1979). Of the
approximately 18 million pounds consumed in 1973, six million
pounds went to produce the fungicide and bactericide 2,4,5-tri-
chlorophenol, 10 million pounds went to the production of the
herbicide 2,4,5-T (2,4,5-trichlorophenoxyacetic acid), and the
remaining 2 million pounds went to miscellaneous uses. 1,2,4,5-
39
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Tetrachlorobenzene may also be used as an impregnant for moisture
resistance, as electrical insulation and as temporary packing
protection (Hawley 1977). According to a recent review, 1,2,4,5-
tetrachlorobenzene is now used exclusively to make 2,4,5-T and
its esters (Kao and Poffenberger 1979). However, 1,2, 4,5-tetra-
chlorobenzene is a component of the transformer fluid, Iralec®,
referred to in Section II.e.4. Thus, the use pattern for this
material appears to be in flux. EPA has no information on uses
of 1,2,3,4- and 1,2,3,5- tetrachlorobenzenes except that the
former, as a mixture with the 1,2,4,5-isomer, is an intermediate
in the synthesis of the fungicide pentachloronitrobenzene (Dow
1979).
5. Occupational Exposure
According to Dow Chemical (1979), manufacture and use of
tetrachlorobenzenes are conducted in closed systems, and fewer
than 200 people are occupationally exposed to them. There are no
TLVs established for the tetrachlorobenzenes. Occupational
exposure to trichlorobenzenes (Section II.C.5) may also involve
exposure to tetrachlorobenzenes present as impurities.
6. General Population Exposure
General population exposure to tetrachlorobenzenes appears
most likely to occur as a result of their release to the environ-
ment, through activities such as manufacturing, industrial use,
processing and disposal.
7. Environmental Release
The extent to which tetrachlorobenzenes enter the environ-
ment is not known. Small amounts of 1,2,3,4-tetrachlorobenzene
and 1,2,3,5-tetrachlorobenzene have been found in fish taken from
the vicinity of a kraft pulp and paper mill discharge in Nipigon
Bay, Lake Superior (Kaiser 1977). This may be the result of
discharges from the chlorine bleaching process used to lighten
the color of the pulp. Tetrachlorobenzenes have also been found
in fish from the Saginaw (Michigan) and Ashtabula (Ohio) Rivers
40
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(Veith et al. 1979) and other fresh water locations (Lombardo
1979). The exact amounts of the tetrachlorobenzenes found were
not determined in these studies. Levels of tetrachlorobenzenes
ranging up to 42 ppb have been found in herring gull eggs from
several sampling areas in the Great Lakes region (International
Joint Commission 1979).
8. Environmental Transformation
No information was found on the environmental transformation
of tetrachlorobenzenes. On the basis of their known and expected
physicochemical properties, they could be expected to exceed
trichlorobenzenes in persistence in the environment. Thus,
tetrachlorobenzenes were still readily detectable in roadside
soil samples seven months after a spill of used transformer fluid
has occurred. Although some loss of tetrachlorobenzenes had
occurred (presumably by evaporation), particularly from the top
inch of affected soil, 43% of the original tetrachlorobenzenes
was present in the next lower inch (Lewis 1979).
9. Biological Uptake
There are no experimental data on the bioconcentration
potential of tetrachlorobenzenes. They can be expected to have
some bioaccumulation potential, and on the basis of their known
and expected physicochemical properties probably exceed the
trichlorobenzenes in this respect.
Conclusion; About 20 million pounds per year of tetrachlo-
robenzenes is produced as a chemical synthesis intermediate, as a
dielectric fluid component, and for miscellaneous minor uses.
They are also found as contaminants of trichlorobenzenes and,
therefore, there is potential exposure to tetrachlorobenzenes
where exposure to the former occurs. Thus, occupational exposure
during manufacturing, processing, and use is likely. Human
exposure via the food chain is also a possibility. Exposure
potential is increased because the physicochemical properties of
tetrachlorobenzenes indicate a potential for persistence in soil
and water systems and for bioaccumulation.
41
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E. Pentachlorobenzene
Pentachlorobenzene, also known as quintochlorobenzene, is a
crystalline solid. It is moderately soluble in benzene, carbon
tetrachloride, chloroform, ether, and carbon disulfide but
insoluble in cold alcohol. Its water solubility is expected to
be lower than those of tri- and tetrachlorobenzenes (compare
trend of data in Table 1, Section I). Pentachlorobenzene can be
produced by the catalyzed chlorination of any of the tetra-
chlorobenzenes. It may also be formed in small amounts when
trichloroethylene is heated to 700°C (Hardie 1964).
The TSCA Inventory lists one manufacturer of pentachloroben-
zene for 1977, with production volume reported in the one to ten
million pounds per year range (OPTS 1979b). Pentachlorobenzene
is nitrated to produce the soil fungicide and seed disinfectant
pentachloronitrobenzene (PCNB), also called Quintozene^ (Thomson
1975); Dow (1979) states that all pentachlorobenzene produced
intentionally (i.e., not as a by-product) is converted to PCNB at
the site of manufacture. The final product contains about 0.2
percent pentachlorobenzene (Beck and Hansen 1974). According to
Dow, the pentachlorobenzene that is formed during the manufacture
of other chlorinated benzenes amounts to less than one million
pounds per year and is all disposed of as waste (Dow 1979); the
method of disposal was not discussed. However, pentachloroben-
zene was shown to comprise 2.5% by weight of a transformer fluid
(Lewis 1979), a use of chlorinated benzenes that may be increas-
ing because of controls that are being placed on PCB's.
Pentachlorobenzene has been identified in the tissues of
fish from the Saginaw River (Mich.), Ashtabula River (Ohio),
Wabash River (Ind.), Arkansas River (Ark.), Mississippi River
(La., Tenn.), White Lake and Tittabawassee River (Mich.), Niagara
River (N.Y.) and Lake Ontario (Veith et al. 1979, Yurawecz
1980). It has been detected in Great Lakes herring gull eggs at
concentrations up to 30 ppb (International Joint Commission 1979)
and in Great Lakes coho salmon at unspecified levels (Interna-
tional Joint Commission 1978).
42
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Pentachlorobenzene has been found at levels of 0.001 to
0.006 ppm in oils, fats and shortening, and has been detected in
sugar at a concentration of about 0.002 ppm (Johnson and Manske
1977). Recently a level of 0.11 ppm in peanut oil has been
reported (Lombardo 1979). The occurrence of pentachlorobenzene
in agricultural products may be a result of pesticide uses.
Pentachlorobenzene is persistent and may remain in soil for
up to two or three years after application (Beck and Hansen
1974).
Conclusion; Pentachlorobenzene is manufactured in the one
to ten million pounds/year range for use as a chemical inter-
mediate. It also is a contaminant of lower chlorobenzenes and a
by-product of their manufacture and, therefore, may have similar
exposure patterns; its occurrence in a transformer fluid
containing tri- and tetrachlorobenzenes is an example. The known
soil persistence of pentachlorobenzene, its occurrence in natural
waters and in fish and bird tissues, and its physicochemical
properties all point to a potential for persistence and
bioaccumulation that magnifies the above-noted exposure concern.
43
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III. Health Effects
A. Acute Effects
1. Evaluation of Pertinent Studies
a. Human Case Reports
Most reported incidents of human toxicity due to chlorinated
benzenes resulted from inhalation exposure. Accidental inhala-
tion can occur either in the home or at the workplace. There are
also incidents in which accidental or deliberate ingestion of
chlorinated benzenes has occurred.
Monochlorobenzene is a central nervous system depressant and
will cause symptoms typical of,its anesthetic effect. Exposure
to monochlorobenzene causes headaches, irritation of the eyes and
upper respiratory tract, numbness, and eventual loss of
consciousness (Irish 1963). Ehrlicher (1968) described a case in
which a worker inhaled massive amounts of monochlorobenzene while
repairing a pump which was spewing a thick spray of the
chemical. After a few hours he experienced massive hemoptysis
(coughing up of blood); examination with X-rays did not identify
an alternative cause for the reaction.
Sensitization to ^-dichlorobenzene was reported by Downing
(1939) for a man who regularly handled window sashes dipped in a
mixture containing the compound. Subsequently when _o_-
dichlorobenzene was applied to the skin of this individual there
was a positive reaction within 2 minutes. Intense erythema and
edema developed at the site of application and for one-half inch
surrounding it. Later a large bullous lesion formed in the
center of this area.
An industrial hygiene survey (Dow 1978c) noted that, in the
production of p_-dichlorobenzene, "at least some of the operators
had a dermatitis on the inside of the forearms, apparently from
their exposure during the pulling of the cakes." They reported
further that jD-dichlorobenzene vapor at concentrations of 1-3
mg/liter (17-500 ppm) produced "severe eye irritation, even in
those men who are accustomed to the vapor." Although these
observations do not prove that this compound caused dermal
sensitization, they appear to rule out any lessening of
sensitivity through habituation.
44
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Odor-threshold data have been reported on monochlorobenzene
and _o_- and ja-dichlorobenzenes (Varshavskaya 1967, Tarkhova
1965). Although odor is not usually considered to be a health
hazard, it may be useful to consider olfactory data. Odor
signals the fact of exposure and of increasing ambient
concentrations and thus may presage the development of adverse
health effects as the concentration in air increases. It may be
useful to know the margin between the olfactory threshold and the
concentration(s) at which adverse health effects occur. Odor
triggers more complaints from the general public than do some of
the more significant adverse health effects. Vague health
complaints, such as disturbances of sleep or digestion, are
frequently associated with odor nuisances.
No-effect levels for eye and respiratory irritation in
humans have also been reported for j>-dichlorobenzene and JD-
dichlorobenzene. Hollingsworth et al. (1958) reported that
neither eye nor respiratory irritation was evident in workers
exposed to £>-dichlorobenzene at concentrations of 1-44 ppm (6-265
mg/m3) with an average concentration of 15 ppm (90 mg/m3).
Exposure at 100 ppm (600 mg/m3) had produced such irritation
(Elkins 1950).
Workers exposed to jo-dichlorobenzene at concentrations of
15-88 ppm (90-510 mg/m3), with an average of 45 ppm (270 mg/m3),
did not consider these levels to be objectionable, although they
had complained of eye and nasal irritation at 50-170 ppm (300-
1,020 mg/m3) with an average exposure of 105 ppm (630 mg/m3)
(Hollingsworth et al. 1956).
Mussell et al. (1958) asserted that 1,2,4,5-tetrachloroben-
zene might be slightly irritating to the eyes or skin if direct
contact with the eyes or prolonged or repeated contact with the
skin occurred. Supporting data and quantitative levels were not
presented.
There may be two explanations, not mutually exclusive, for
the different responses seen with similar exposure conditions as,
for example, in the concentration ranges of p-dichlorobenzene
45
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reported by Hollingsworth et a.I. (1956) to be irritating or non-
irritating. One explanation is that individual differences in
sensitivity or susceptibility are most evident at threshold
concentrations. The other explanation may reside in differences
in the duration of exposure to a given concentration, as
suggested by Mussell et al. (1958), with longer exposure
producing greater irritation.
Dupont (1938) described the experiences of French sewage
workers exposed to the effluents of a dry cleaning establishment
using j>-dichlorobenzene. Vapors emanating from the sewer floor
caused lacrimation and vomiting, and the workers experienced a
distaste for cigarette smoking.
There is more experience with human exposure to jo-dichlo-
robenzene than to other chlorobenzenes, due to its widespread use
as a moth repellent and deodorizer. Exposed individuals have
exhibited various responses including weakness, headache,
rhinitis, twitching of facial muscles, and acute hemolytic anemia
with methemoglobinuria (Cotter 1953, Hallowell 1959).
One case of acute p-dichlorobenzene poisoning was reported
by Cotter (1953). It involved a 35-year old housewife who,
following use of a commercial moth killer, experienced
periorbital swelling, intense headache, and profuse rhinitis; all
symptoms subsided in 24 hours.
The case of a 3-year-old boy who presumably ingested
"Demothing Crystals" containing _p_-dichlorobenzene was reported by
Hallowell (1959). The child developed acute hemolytic anemia
with methemoglobinuria. Analysis of his urine, which was
markedly reduced in volume during the first 2 days after
poisoning, revealed traces of 2,5-dichloroquinol, but no 2,5-
dichlorophenol. Examination approximately one month after the
incident indicated an apparently complete recovery.
b. Animal Studies
Animal toxicity determinations have been conducted for
monochlorobenzene, ^-dichlorobenzene , jp_-dichlorobenzene , and
1,2,4-trichlorobenzene in various animal species including mice,
46
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rats, rabbits, and guinea pigs. The LD5Q values determined in
these studies are presented in Table 1.
Additional studies include reports of mortality following
short term exposure to chlorobenzenes. Irish (1963) cited the
results of an acute study of monochlorobenzene vapor in cats.
The cats were exposed presemably continuously, to mono-
chlorobenzene vapor at concentrations of 220 to 8,000 ppm.
Concentrations of 220-660 ppm were tolerated well for an hour;
definite narcotic signs were noted at 1,200 ppm; unsteadiness,
tremors, and twitching occurred at concentrations of 2,400 to
2,900 ppm. Death occurred after 7 hours in animals exposed to
3,700 ppm. At the highest concentration, 8,000 ppm, severe
narcosis occurred after one-half hour, and the cats died two
hours after removal from exposure.
The Eastman Kodak Company (1978) reported that acute
inhalation of 22,000 ppm of monochlorobenzene killed two-thirds
of the rats tested in 2.5 hours while inhalation of 9,000 ppm of
monochlorobenzene killed two-thirds of the rats in 3 hours.
Hollingsworth et al. (1958) exposed male rats via inhalation
to one of four different concentrations of £>-dichlorobenzene for
1 to 10 hours. The results are presented in Table 2. The
average vapor concentration ranged from approximately 500 to
1,000 ppm and the numbers of animals ranged from 5 to 20 per
group. During the exposure period, the rats exhibited drowsi-
ness, unsteadiness, eye irritation, difficulty in breathing, and
anesthesia. The intensity of visible responses was dependent on
the concentration and the duration of exposure. Most of the
deaths occurred within three days after removal of the animals
47
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from the chamber. The same investigators also exposed matched
groups of 20 male rats to 977 ppm of ^-dichlorobenzene for 1 hour
and 0.5 hour and to 539 ppm for 6.5 hours and 3 hours. Central
lobular necrosis in the liver and cloudy swelling of the kidney
tubular epithelium were observed in all groups except those
exposed to 997 ppm for 0.5 hour. The degree of damage was
greater in the case of the longer exposures.
TABLE 2
Mortality of Male Rats Exposed to Various Concentrations of _o_-
Dichlorobenzene Vapor for Single Periods (Ho11ingsworth et al.
1958)
Average Vapor Concentration
By Analysis (ppm)
977
941
821
539
Period of
Exposure (hr)
10
7
2
1
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4
2
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Exposed
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Hollingsworth et al. (1958) also fed ^-dichlorobenzene to
guinea pigs by intubation as a 50 percent solution in olive
oil. All of 10 animals receiving a single dose of 0.8 g/kg
survived, while a dose of 2.0 g/kg produced 100 percent mortality
in 10 guinea pigs.
Cameron et al. (1939) studied the acute toxicity of o-
dichlorobenzene in rabbits and mice. Intravenous injection of
0.25 to 0.5 ml/kg was fatal to rabbits within 24 hours. In mice
the minimal lethal dose following injection was found to be about
0.4 ml/kg. Mice, rats, and guinea pigs were exposed to air
containing 0.005 to 0.080 percent ^x-dichlorobenzene from 30
minutes to 50 hours. Under these conditions, the animals
49
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exhibited reduced activity, drowsiness, and irritation of eyes,
followed by death. Autopsy revealed liver necrosis in nearly all
animials.
Varshavskaya (1967) studied acute exposure to monochloro-
benzene, j>-dichlorobenzene, and p-dichlorobenzene in mice, rats,
rabbits, and guinea pigs. The adverse effects included asthenia,
inactivity, loss of weight and appetite, and distinct narcotic
effects followed by death within three days due to paralysis of
the respiratory center.
Results of an acute toxicity study in rats and mice on
pentachlorobenzene carried out at EPA's Health Effects Research
Laboratory have been submitted for publication and will be
included in this document when the prepublication review is
complete. The LD^Q'S for this compound are around 1 g/kg,
consistent with other chlorobenzenes tested.
2. Decision
The two major acute effects of the chlorinated benzenes are
tissue irritation and central nervous system depression. Eye,
skin, and respiratory irritation, rhinitis, and periorbital
swelling have been associated with exposure to the lower
chlorinated benzenes (monochlorobenzene, _o_- and p-
dichlorobenzene). Skin sensitization may also occur. Central
nervous system depression leading to loss of consciousness,
headache, and vomiting have been reported following acute human
exposures, which generally occur by accidental inhalation.
Although high concentrations of chlorinated benzenes may be
lethal to laboratory animals, human exposures of comparable
magnitude are unlikely. Threshold limit values in the air of the
work place adopted for several chlorinated benzenes by the U.S.
Occupational Safety and Health Administration are given in
Appendix C of this support document.
In general, the acute toxicity of the chlorinated benzenes
has been adequately characterized. The available LD^Q data are on
the order of the 1000 mg/kg or higher, and there is no reason to
expeat significant deviations from this range for the untested
50
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category members. Two reports (Downing 1939, Dow 1978c)
indicated that prolonged, heavy exposure to £_- or ja_-
dichlorobenzene may induce dermal sensitization; however, the
Agency is not aware of any indication that there is a serious or
widespread problem posed by dermal exposure to the chlorinated
benzenes. The Agency is, therefore, not requiring testing for
dermal sensitization at this time. Although a more rigorous and
systematic determination of the thresholds for acute skin, eye,
and mucous membrane irritation and for acute central nervous
system depression could be undertaken for all of the chlorinated
benzenes, such testing is not needed at this time for regulatory
purposes and is not being proposed.
B. Subchronic and Chronic Effects
1. Evaluation of Pertinent Studies
Note: This part excludes effects on the central nervous system,
which are discussed under Neurotoxic Effects, part C of this
Section.
a. Human Case Reports
The case histories presented here provide only suggestive
evidence because the absence of controlled conditions makes it
difficult to determine cause and effect.
A number of incidents of systemic toxicity in humans exposed
to chlorinated benzenes have been reported. Toxic effects on the
liver have been reported by several authors (Berliner 1939,
Sumers et al. 1952, Cotter 1953). The report by Cotter presents
an example of the type of liver toxicity generally noted. He
described several cases of liver damage associated with exposure
to jD-dichlorobenzene. The first case involved a woman who
demonstrated products containing p-dichlorobenzene and who
complained of tiredness, nausea, headache, and vomiting. Her
conjunctivae were muddy with a yellow tinge, and she had elevated
serum bilirubin. An x-ray of the esophagus showed varices. The
final diagnosis of her case was subacute yellow atrophy and
cirrhosis of the liver. The second case involved the death of a
51
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husband and wife whose house had been saturated with mothball
vapor for a period of at least three to four months. Autopsy
confirmed the cause of death as acute yellow atrophy of the liver
for both patients and Laennec's cirrhosis and splenomegaly for
the wife. Both had elevated serum alkaline phosphatase (SAP) and
serum bilirubin. Cotter also reported the case of a 52-year-old
trapper who had used j>-dichlorobenzene for two years in the
curing of raw furs. The subject showed a consistent picture of
subacute yellow atrophy of the liver, accompanied by jaundice,
elevated serum bilirubin and SAP, and a palpable spleen. All of
the above cases displayed a picture of yellow atrophy with a
tendency toward connective tissue formation rather than liver
cell regeneration. The husband and wife showed anemia and
borderline anemia, respectively.
Toxic effects on the hematopoietic system and the formed
blood elements due to exposure to chlorinated benzenes have also
been observed by several authors (Perrin 1941, Petit and Champeix
1948, Wallgren 1953, Gadrat et al. 1962, Campbell and Davidson
1970, Girard et al. 1969). The following three reports indicate
the variety of effects noted .
Wallgren (1953) reported the case of eight men who worked
from one to seven months in a factory manufacturing mothproofing
agents produced from ^pj-dichlorobenzene that contained
approximately one percent of an unidentified nitrogen-containing
impurity and a small amount of ^>-dichlorobenzene. Blood
examinations showed methemoglobinemia and varying degrees of
lymphocytosis in all cases, thrombocytopenia in four cases, and
leukopenia and peripheral granulocytopenia in one case.
Campbell and Davidson (1970) reported the unusual case
history of a pregnant woman who developed a pica for
j3-dichlorobenzene. Each week throughout her pregnancy, the 21-
year-old woman consumed one to two toilet air-freshener blocks
composed primarily of p-dichlorobenzene. She was diagnosed as
having hypochromic anemia and toxic hemolytic anemia with Heinz
body formation. When she stopped eating the chemical, the woman
made an apparently complete recovery. In blood tests six weeks
52
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after delivery, hemoglobin levels, erythrocyte morphology, and
reticulocyte numbers were normal. Neonatal examination of the
child revealed no apparent abnormalities.
Girard et al. (1969) reported severe aplastic anemia in a
68-year-old woman exposed to trichlorobenzene. Although the
exact composition of the fluid to which she was exposed was
unknown, it was believed by the authors to be primarily
trichlorobenzene. Following the woman's hospitalization, an
examination revealed severe aplastic anemia. When the patient
died one month later, she was hemorrhaging. The autopsy results
indicated a retroperitoneal hematoma.
b. Animal Studies
Summaries of the available animal studies are given in
Tables 3-5 at the end of this Section b. Only those reports most
significant for hazard assessment are presented here.
(1) Monochlorobenzene
(a) Oral Studies (capsule or gavage)
A 13-week oral toxicity study was reported by Monsanto Co.
(1967a). Thirty-two beagles, 8 animals per group (4 males, 4
females), were administered 0, 0.025, 0.050, and 0.250 ml/kg/day
(27.3 mg/kg/day, 54.6 mg/kg/day, and 272.5 mg/kg/day) of
monochlorobenzene orally by capsule 5 days per week for 13
weeks. At the highest dose, two of the 8 dogs died, and two
other dogs were sacrificed in moribund condition between the
third and fifth weeks of the study. The surviving animals were
sacrificed after 13 weeks of administration. All of the dogs in
the high dose group showed weight loss: 3.1 kg for each of the
two dogs that died; 4.2 kg for each of the two dogs sacrificed;
and 0.7-2.0 kg for the four dogs surviving to termination of the
study. Clinical studies conducted on three of the four dogs that
died or were sacrificed early revealed the following: increased
count of juvenile and band cells (marked in two dogs); increased
count of monocytes (marked in one dog); sharply decreased lympho-
cyte count; low blood sugar levels; elevated serum glutamic-
53
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pyruvic transaminase (SGPT), serum alkaline phosphatase (SAP),
and total bilirubin (bilirubin in the urine of two of the dogs
was significantly elevated also); and elevated total cholesterol
in two of the dogs. The results of hematological studies and
urine analyses for the four animals surviving to termination were
at one and three months comparable to corresponding initial
values. Biochemical studies on these latter animals showed a
slight increase in SGPT values for the two male dogs and a slight
increase in SAP values for one male and one female. Histologic
changes were observed in the liver (centrolobular fibrosis),
kidney, gastrointestinal mucosa, and hematopoietic tissue in all
eight dogs in the high dose group. Slight and somewhat inconsis-
tent histologic changes were observed in the livers of the dogs
at the intermediate dose. No reported toxic effects were
observed in the dogs at the low dose.
Monsanto (1967b) reported the results of a 90-day oral study
on monochlorobenzene using albino rats. Repeated doses of 12.5
mg/kg/day for 90 days produced an increase in salivation for 5 to
10 minutes in 1/36 rats (18 males, 18 females tested) and
alopecia in 1 rat. No clinical signs or histopathological
alterations were noted. Doses of 50.0 mg/kg/day for 90 days
produced an increase in salivation in 18/36 rats, and 1/36
animals died just prior to sacrifice. No compound-related
clinical changes or histopathological alterations were
observed. Doses of 100 mg/kg/day produced increased salivation
in the majority of animals and alopecia in 1/36 rats. One animal
died on day 88. No clinical signs of histopathological
alterations were observed. Doses of 250/mg/kg/day produced
increased salivation in all of the animals, alopecia in 4/36
animals, and a significant decrease in growth rate for males.
One animal died on day 90. No clinical signs or
histopathological lesions were observed. In this study no
vehicle controls were used (only negative controls) and
histopathology was performed only on 5 animals of each sex per
dose group. Extensive microscopic studies were done on the
highest dose group and on controls and only the thyroid, heart,
54
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kidney, liver, and adrenals were examined in the low and
intermediate dose groups.
Varshavskaya (1967) administered 0, 0.1, 0.01, and 0.001
mg/kg of monochlorobenzene in sunflower oil by stomach tube daily
for nine months to male albino rats (7 animals/dose level). At
the highest dose the compound caused a statistically significant
inhibition of erythropoiesis, thrombocytosis, and an inhibition
of mitotic activity in the marrow. Eosinophilia was also noted
as well as an increase in the phagocytic activity of leukocytes,
SAP, serum transaminase activity, and the gamma-globulin plasma
protein fractions. The intermediate dose produced all the
effects noted above except the phagocytic activity of
leukocytes. These effects occurred to a lesser degree, but it
was not reported whether they were statistically significant. No
effects were noted at the lowest dose level. No indication was
given whether the term "daily" meant 7 days/week or 5 days/week.
(b) Inhalation Studies
The Monsanto Co. (1978g) reported the results of a 90-day
inhalation toxicity study on monochlorobenzene using beagle dogs
and Charles River albino rats. Prolonged inhalation of 0.75 mg/1
air, 6 hours per day, 5 days per week for a total of 62 exposures
(12.4 weeks) did not cause any adverse effects in dogs (4 males,
4 females). However, only urinalysis, clinical chemistry, and
blood studies were done at this dose and no histopathological
examinations were performed. Prolonged inhalation of 1.50 mg/1
air (325 ppm) for the same time period caused a decrease in
weight, conjunctivitis in 2/8 dogs, and hypoactivity in 2/8
dogs. The dogs were sacrificed in moribund condition before the
thirty-first day. Gross examination revealed evidence of icterus
in 1 of these 2 dogs. No histopathological examination was
performed in any of the dogs at this dose level and no clinical
chemistry, urinalysis or blood studies were done on the 2 dogs
sacrificed prior to termination of the experiment. Prolonged
inhalation of 2.00 mg/1 air (434 ppm) for the same time period
caused a significant decrease in weight, hypoactivity, and
55
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conjunctivitis in 8/8 dogs (4 males, 4 females). Mean values for
total leukocyte counts after 45 and 90 days of treatment were
lower than corresponding controls and mean values for SAP, serum
glutamate oxaloacetate transaminase (SCOT), and SGPT were
elevated after 38 days of treatment. Histopathologic examination
revealed vacuolation of hepatocytes and aplastic bone marrow in 5
dogs, hypoplastic bone marrow in 1 dog, cytoplasmic vacuolation
of the epithelium of the renal collecting tubules in 4 dogs, and
bilateral atrophy of the seminiferous epithelium in the testes of
2 dogs. Five dogs were sacrificed in moribund condition between
the twenty-fifth and thirty-eighth day of the experiment.
Prolonged inhalation of 0.75, 1.50, or 2.00 mg/1 of
monochlorobenzene by rats for the same time period as for the
dogs revealed erythemia and alopecia in 2/30 animals in the
lowest dose group and variable organ weights in all dose groups
(15 males, 15 fentales per group). Clinical chemistry,
urinalysis, and blood chemistry were done on only 5 males and 5
females from the highest dose group and from the controls ; the
values determined for these parameters fell within the normal
range. Histopathology was performed on controls and on the
highest dose group (all animals in these groups); no tissue
changes attributable to the effects of monochlorobenzene were
observed.
Dilley (1977) exposed 3 groups of 32 male rats and rabbits
to 0, 75 and 250 ppm of monochlorobenzene for 7 hours/day, 5
days/week, for up to 120 exposure days (24 weeks). Clinical
changes included hematological changes in both treated rat groups
at 11 and 24 weeks (changes at 11 weeks were significant), a
decrease in SCOT activity in high dose rats, and minor
hematological changes in both groups of rabbits at 11 and 24
weeks. No significant histopathologic changes were observed in
the rabbits or rats at 24 weeks.
Khanin (1977) exposed rats to 0.1 and 1.0 ug/1 (0.022 ppm
and 0.22 ppm) of monochlorobenzene 24 hours/day for 70-82 days.
Three to five rats were used in each group and corresponding
controls were tested. Toxic encephalopathy, granular albumin,
56
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dystrophy of the heart and liver, non-acute nephrosis of the
kidneys with limited glomerulonephritis, hyperplasia of the
follicles of the spleen, and proliferation of reticular cells of
the arterial sheaths were noted. Inflammation of the heart and
liver were also observed. The effects were greater with the
higher dose.
Other monochlorobenzene animal studies are summarized in
Table 3 at the end of this Section b.
(2) Dichlorobenzenes
(a) Oral Studies
Groups of 10 young adult female white rats were given doses
of 18.8, 188, or 376 mg/kg of j>-dichlorobenzene (10 controls) in
olive oil emulsified with about 2 ml of 5 to 10 percent acacia
(gum arabic) (Hoilingsworth et al. 1956), or ^-dichlorobenzene in
olive oil (20 controls) (Hollingsworth et al. 1958) by stomach
tube 5 days per week for a total of 138 doses in 192 days.
Animals were sacrificed one day after the final dose. At the
highest dose level both compounds were associated with a moderate
increase in liver weight; p_-dichlorobenzene was associated with
slight cirrhosis and focal necrosis of the liver; and ^-dichloro-
benzene was associated with slight to moderate swelling of the
liver. For both compounds only slight increases in liver and
kidney weights were observed with the intermediate dose level;
the lowest level produced no changes.
A group of five rabbits of both sexes were administered a
25.0 percent solution of j>-dichlorobenzene in olive oil (intuba-
tion) 5 days a week for a total of 92 doses in 219 days
(Hollingsworth et al. 1956). The dosage level was 1000
mg/kg/day. Another group of seven rabbits received the compound
5 days a week for a total of 263 doses in 367 days at a level of
500 mg/kg/day. Slight changes in the liver characterized by
cloudy swelling and a few areas of focal, caseous necrosis were
found at both dose levels.
Varshavskaya (1967) reported the same type of studies for _o_-
dichlorobenzene that were done for monochlorobenzene. Increases
57
-------
in urinary 17-ketosteroids, in the phagocytic activity of
leukocytes (at 0.1 mg/kg/day), and in the gamma-globulin plasma
protein fractions (at 0.01 and 0.1 mg/kg/day) were found, as well
as a statistically significant inhibition of erythropoiesis,
thrombocytosis, and inhabition of mitotic activity in the bone
marrow (at 0.1 mg/kg/day), when the chemical was administered
orally to rats for a period of 9 months. The same effects were
produced with 0.01 mg/kg/day but to a lesser degree. No effects
were reported at 0.001 mg/kg/day.
(b) Inhalation Studies
Hollingsworth et al. (1956) exposed various sized groups of
rats, rabbits, guinea pigs, and other species to jv-dichloroben-
zene vapors at each of five concentrations for 7 hours/day (8
hours/day for the highest dose group), 5 days/week. In the rat,
19 males and 15 females were exposed to 4.8 mg/1 (798 ppm) for up
to 69 exposures. Cloudy swelling and central necrosis of the
liver were observed in both sexes, and slight cloudy swelling of
the tubular epithelium of the kidneys was observed in the
females. Increased liver and kidney weights were observed at
concentrations of 2.05 mg/1 (341 ppm: 20 male rats tested for 6
months) and 1.04 mg/1 (173 ppm: 5 male, 5 female rats tested for
16 days). At 0.95 mg/1 (158 ppm), 20 rats (10 males, 10 females)
were given 139 exposures over 199 days. Increased liver weights
and centrolobular hepatocellular cloudy swelling or granular
degeneration of questionable significance in the parenchymal
cells in the central zones were noted. No adverse effects were
observed in 20 rats (equal number of males and females) exposed
to 0.58 mg/1 (96 ppm) for six to seven months.
Liver toxicity was observed in guinea pigs exposed to
similar concentrations as the rats. Cloudy swelling and central
necrosis of the liver were seen at 798 ppm (16 males, 7 females
tested for up to 23 exposures); vacuolization, fatty degenera-
tion, focal necrosis, and slight cirrhosis were seen in some
males at 341 ppm for 6 months (8 males, 8 females tested).
Decreased spleen weights in males were reported at 173 ppm
58
-------
(exposed for 16 days), increased liver weights in females were
observed at 158 ppm (exposure period was 157-219 days), and no
adverse effects were noted at 96 ppm.
The picture was similar for rabbits. Cloudy swelling and
central necrosis of the liver were reported at 798 ppm and some
lung effects at 173 ppm. No other adverse effects were noted at
any of the other dose levels. Mo effects were reported for mice
and a monkey exposed to either 158 ppm from 157 to 219 days or 96
ppm for 6 to 7 months.
There appeared to be no rationale for the choice of dose
levels, number of animals, and species. However, the results of
the study indicated systemic effects in several species.
Hollingsworth et al. (1958) also exposed animals by inhala-
tion to ^-dichlorobenzene. Groups of 20 rats, R guinea pigs, and
2 rabbits of each sex and 2 female monkeys were exposed to 0.56
mg/1 (93 ppm) 7 hrs./day, 5 days/week for periods ranging from 6
to 7 months. The only toxic effect noted was a statistically
significant decrease in the average spleen weight of male guinea
pigs. When groups of 20 rats and 8 guinea pigs of each sex and
10 female mice received 0.29 mg/1 (49 ppm) of c>-dichlorobenzene 7
hrs./day, 5 days/week for six and one half months, no gross or
microscopic abnormalities were observed.
Imperial Chemical Industries in Great Britain is reported to
be carrying out a long-term inhalation study in rats on _p_-
dichlorobenzene (Dow 1978b). EPA is trying to learn more about
this study, and the Agency will include any available data in its
ongoing evaluation of the chlorinated benzenes.
Animal studies involving dichlorobenzenes are summarized in
Table 4 at the end of this Section b.
(3) Trichlorobenzenes
Coate et al. (1977) exposed 4 groups of 30 young male
Sprague-Dawley rats, 4 groups of 16 male albino rabbits, and 4
groups of 9 male cynomolgus monkeys to 0, 0.183, 0.37, or 0.73
mg/1 (0, 25, 50, or 100 ppm) of 1,2,4-trichlorobenzene (99.07
percent pure) for 26 weeks, 7 hours per day, 5 days per week.
59
-------
The microscopic change observed was hepatocytomegaly in rats
after 4 weeks of exposure. The changes were greater in animals
exposed to 492 and 95.8 ppm than in those exposed to 25 ppm.
Hyaline degeneration was present in the inner zone of the kidney
cortex in all test groups after 4 weeks and 13 weeks of expo-
sure. These changes were transient, however, and no exposure-
related abnormalities were seen after 26 weeks. A no-effect dose
was not established. No toxic effects were observed in the other
species at any dose level.
(4) Tetrachlorobenzenes
Fomenko (1965) reported toxic effects on the liver and
hematopoietic system in rabbits and rats following administration
of 1,2,4,5-tetrachlorobenzene. Rats were given 75 mg/kg by
gavage daily for two months. Decreased liver function was
reported. The prothrombin index dropped by almost one-third in
comparison to the control group. There was a statistically
significant increase (p= 0.01) in the activity of the blood
cholinesterase (author did not state whether pseudo- or
acetylcholinsterase). The number of reticulocytes in the
peripheral blood first decreased (p= 0.02) and then increased; at
the end of the experiment, the toxic signs included erythemia (p=
0.01), an increased number of large-diameter erythrocytes, and
some decrease in the serum potassium. Microscopic examination
revealed no tissue abnormalities. The same tetrachlorobenzene
isomer was administered in vegetable oil by gastric intubation to
rabbits at 0, 0.05, 0.005, and 0.001 mg/kg daily for eight
months. Increased retention of an intravenous load of galactose
in the blood began at 6 months in the group receiving 0.05 mg/kg,
suggesting to the author interference with the glycogen-forming
function of the liver. A significant (p= 0.01) reticulocytosis
was noted by the end of the eighth month. In rabbits given 0.005
mg/kg, a brief disorder of glycogen formation was reported in the
eighth month of the study. No effects were observed at the
lowest dose level. The number of animals tested was not given in
the report.
60
-------
Trichloro- and tetrachlorobenzene animal studies are
summarized in Table 5 at the end of this Section b.
(5) Pentachlorobenzene
A study of subchronic toxicity of pentachlorobenzene in rats
performed at EPA's Health Effects Research Laboratory, Research
Triangle Park, NC., has recently been submitted for publication
(Linder et. al., 1980). Initial evaluation of the study by EPA
indicates that the toxicity of pentachlorobenzene is similar to
that of other chlorinated benzenes: the liver, kidney and
nervous system are the primary targets. Provided that editorial
review of the study raises no major questions, EPA considers this
study to characterize adequately the subchronic toxicity of
pentachlorobenzene for risk assessment purposes.
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c. Other Indicators of Systemic Toxicity
(1) Metabolism
The metabolic pathways of benzene, monochlorobenzene,
dichlorobenzenes, trichlorobenzenes, tetrachlorobenzenes, penta-
chlorobenzene, and hexachlorobenzene are treated sequentially in
the following text and in Figures 1-7. These metabolic reactions
represent classic conversions of chlorinated aromatic compounds
in the liver.
(a) Benzene
Benzene is absorbed readily from the gastrointestinal
tract. Rusch et al. (1977) concluded from the available
experimental findings that the metabolism and elimination of
benzene in humans and in all animal species studied follow very
similar pathways (Figure 1). About 40 percent is eliminated
unchanged in the expired air. The remainder is oxidized to
benzene oxide by the aryl hydrocarbon hydroxylase (AHH) enzyme
system. The benzene oxide then breaks down via one of three
pathways:
(i) It can rearrange spontaneously to yield phenol by the
"NIH shift". The majority of the phenol formed subsequently
conjugates with glucuronic and sulfuric acids. However, some of
the phenol undergoes further oxidation to hydroquinone.
(ii) Benzene oxide can be acted upon by epoxide hydrase to
give benzene glycol, which then yields predominantly catechol via
enzymatic dehydrogenation, and some trans-trans-muconic acid
following subsequent oxidation. The majority of the catechol is
conjugated and eliminated with a small amount being oxidized to
hydroxyhydroquinone.
(iii) The benzene oxide can react with glutathione in the
presence of an epoxidetransferase, followed by dehydration to
produce phenylmercapturic acid.
71
-------
(b) Monochlorobenzene
Monochlorobenzene resembles benzene with regard to
absorption, excretion and metabolism. It is absorbed easily from
the gastrointestinal tract and excreted in the urine (Smith et
al. 1950, Spencer and Williams 1950, Azouz et al. 1953).
Unchanged monochlorobenzene is eliminated with expired air (25-30
percent), though not as extensively as benzene (40 percent). As
illustrated in Figure 2, monochlorobenzene undergoes three
primary enzymic attacks in the production of the major urinary
metabolites, 4-chlorocatechol and 4-chlorophenylmercapturic
acid. 3-Chlorocatechol and the three isomeric chlorophenols are
produced as minor metabolites.
(i) Formation of 3-chlorobenzene oxide which either
isomerizes to 2-chlorophenol or goes to 2,3-dihydro-2,3-dihy-
droxychlorobenzene via epoxide hydrase and finally forms 3-
chlorocatechol (Spencer and Williams 1950, Selander et al. 1975
a,b) .
(ii) Formation of 3-chlorophenol via a direct oxidative
pathway (Selander et al. 1975a).
(iii) Formation of 4-chlorobenzene oxide which either
conjugates with glutathione to produce 4-chlorophenylmercapturic
acid (Smith et al. 1950, Azouz et al. 1953, Parke and Williams
1955, Williams 1959, 1975), rearranges to 4-chlorophenol (Spencer
and Williams 1950, Smith et al. 1950, Selander 1975a), or is con-
verted to 3,4-dihydro-3,4-dihydroxychlorobenzene by the action of
epoxide hydrase and finally to 4-chlorocatechol via enzymic
dehydrogenation (Azouz et al. 1953, Smith et al. 1950, Williams
1959, Williams et al. 1975).
(c) Dichlorobenzene
Dichlorobenzenes are absorbed less readily than are
benzene and monochlorobenzene. Metabolic attack upon at least
the 1,2- and 1,3-isomers of this compound appears to involve the
72
-------
formation of arene oxides by epoxidase (Figure 3). Unlike
monochlorobenzene, however, mercapturic acids and catechols are
not major metabolites of dichlorobenzenes. They are formed
merely as minor metabolites of 1,2- and 1,3-dichlorobenzenes and
are not formed at all by the 1,4-isomer (Azouz et al. 1953, 1954,
Parke and Williams 1955). The major metabolites of 1,2- and 1,3-
dichlorobenzene are 3,4- and 2,4-dichlorophenol respectively,
which are excreted as conjugates of glucuronic and sulfuric
acids. 1,4-Dichlorobenzene is mainly oxidized to 2,5-
dichlorophenol and 2,5-dichloroquinol (Azouz et al. 1954, 1955).
(d) Tr ichlorobenzenes
Trichlorobenzenes are absorbed and excreted slowly. As
indicated in Figure 4, these compounds are metabolized initially
to arene oxides and are excreted in urine mainly as trichloro-
phenols. The major metabolite of the 1,2,3-isomer is 2,3,4-
trichlorophenol, but small amounts of 3,4,5-trichlorocatechol and
3,4,5- and 2,3,6-trichlorophenols are also formed (Jondorf et al.
1954, 1955b, Kohli et al. 1976).
1,2,4-Trichlorobenzene is mainly metabolized to 2,4,5- and
2,3,5-trichlorophenols. They are excreted together with small
amounts of 3, 4,'6-trichlorocatechol, and 2,3,5- and 2,4,5-
trichlorophenylmercapturic acids.
2,3,5-Trichlorophenol and 2,4,6-trichlorophenol were
identified as the major metabolites of 1,3,5-trichlorobenzene,
which was also shown to declilorinate and produce 4-chlorophenol
and 4-chlorocatechol as minor urinary metabolites (Kohli et al.
1976, Parke and Williams 1960, Jondorf et al. 1954, 1955a).
(e) Tetrachlorobenzenes
Tetrachlorobenzenes are absorbed with still greater
difficulty. They are excreted slowly and yield no mercapturic
acids or catechols. 1,2,3,4-Tetrachlorobenzene gives two
phenolic metabolites, 2,3,4,6- and 2,3,4,5-tetrachlorophenol
which can be generated from 2,3,4,5-tetrachlorobenzene, oxide
(Jondorf et al. 1958, Kohli et al. 1976).
73
-------
1,3,4,5- and 2,3,4,6-Tetrachlorobenzene oxides are the
proposed intermediates in the conversion of 1,2,3,5-
tetrachlorobenzene into 2,3,4,5-, 2,3,4,6-, and 2,3,5,6-
tetrachlorophenols (Jondorf et al 1958, Kohli et al. 1976). All
phenols are derived via the "NIH shift".
1,2,4,5-Tetrachlorobenzene gives only one metabolite,
2,3,5,6-tetrachlorophenol. There is evidence that this isomer is
also partly dechlorinated in the gut to less chlorinated benzenes
(Jondorf et al. 1958, Kohli et al. 1976).
(f) Pentachlorobenzenes
Pentachlorobenzene is absorbed poorly from the gastroin-
testinal tract and is removed slowly from the tissues. Its major
metabolites are pentachlorophenol and 2,3,4,5-tetrachlorophenol
(Kohli et al. 1976, Koss and Koransky 1978), As indicated in
Figure 6, pentachlorophenol can be formed by direct oxidation,
but formation of 2,3,4,5-tetrachlorophenol may involve reductive
dechlorination and the production of an arene oxide intermedi-
ate. Other metabolites identified include tetrachloroquinone,
2,3,5,6-tetrachlorophenol and a hydroxylated chlorothiocompound
(Koss and Koransky 1978). The mechanism of formation of these
metabolites, however, has not been established.
(g) Hexachlorobenzene
Hexachlorobenzene is very poorly absorbed from the gastro-
intestinal tract, is excreted predominantly in feces, and is
removed very slowly from tissues. The mechanism of formation of
the known metabolites of hexachlorobenzene is unclear. Like
pentachlorobenzene, it undergoes reductive dechlorination since
metabolites containing only three, four, or five chlorine atoms
have been identified (Figure 7). The urinary metabolites present
at the highest levels were identified as pentachlorophenol,
tetrachlorohydroquinone, and pentachlorothiophenol (Cooper 1978,
Koss et al. 1976, Rozman et al. 1977, Yang et al. 1975, Lui and
Sweeney 1975). Small amounts of 2,3,4,6- and 2,3,5,6-tetrachlo-
rophenols and traces of 2,3,4,-, 2,4,5- and 2,4,6-trichloro-
74
-------
phenols are also present (Mehendale et al. 1975, Engst et al.
1976, Renner and Schuster 1977, Cooper 1978) .
Discussion of Metabolism Data. Several points should be
kept in mind in evaluating metabolism data on aromatic
compounds. The formation of phenolic metabolites can occur by
more than one mechanism and, therefore, does not by itself show
that arene oxide intermediates are involved (Jerina and Daly
1976). Such intermediates can be suspected if the product phenol
appears to be the result of a ring substituent shift to an
adjacent position (the "NIH shift") or if certain other
metabolites--!,2-dihydrodiols, catechols, or arylmercapturic
acids—are detected. In some cases, the intermediacy of an arene
oxide can be demonstrated only by more sophisticated experiments
involving isotopically-labeled starting material or chemically
synthesized arene oxides, and such experiments may be crucial in
determining how much of a given phenol is formed by arene oxide
and how much by nonarene oxide pathways (Jerina and Daly 1976).
Authors of metabolism papers sometimes suggest uncritically an
arene oxide pathway where the evidence for this is ambiguous or
nonexistent. In other cases the intermediacy of arene oxides is
a possibility but not demonstrable from the available data; that
is, the evidence is neither for nor against this pathway for the
compound in question. In Figures 1-7 arene oxide intermediates
proposed in the literature without sufficient evidence are
indicated by a question mark below the structure.
Another problem encountered with much published metabolism
data is the failure to account for all of the administered
compound. Among published reports on chlorinated benzenes, the
quantity accounted for ranges from 85% (Parke and Williams 1960)
to as little as 2% (e.g., Kohli et al. 1976). For the higher
chlorinated benzenes in particular, much of the missing chemical
may have been simply excreted unchanged, but the lack of a
materials balance leaves open the question of whether a
significant metabolic pathway has escaped detection. These
shortcomings merely reflect the incompleteness of the available
75
-------
data and do not preclude some generalization on the basis of the
data in hand.
Thus, it is clear from the metabolism data and from Figures
1-7 that the patterns of absorption, excretion, and metabolism of
the chlorinated benzenes shift with the degree of chlorination,
with monochlorobenzene more closely resembling benzene and
pentachlorobenzene more closely resembling hexachlorobenzene. It
is noteworthy that in general mono-, di-, tri-, and tetrachloro-
benzenes can be metabolized initially to arene oxides, compounds
that can bind covalently to vital cellular macromolecules, which
may initiate oncogenesis, teratogenesis, or mutagenesis. Another
potentially toxic effect of the metabolism of chlorinated
benzenes is the formation of tumor promoters. 2-Chlorophenol and
3-chlorophenol, metabolites of monochlorobenzene, and 2,4,5-
trichlorophenol, a metabolite of 1,2,4-trichlorobenzene, are
known to promote oncogenesis [see Section III.G.1.c.(4) for more
discussion].
Although there appears to be only a single study of the
metabolism of a chlorinated benzene in humans, the data are
valuable because other mammalian species were investigated for
comparison. Monochlorobenzene was metabolized to the same major
metabolites (i.e., 4-chlorophenol, 4-chlorocatechol, and 4-
chlorophenylmercapturic acid) in man and in 12 other mammalian
species (Williams et al» 1975). This suggests that, at least for
this chlorinated benzene, metabolism studies performed in lower
species are relevant to man.
76
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FIGURE 1
PATHWAYS OF BENZENE METABOLISM AND ELIMINATION
expired unchanged (40%)
benzene (100%)
AHH
trans-trans-
mucomc acid (1.3%)
benzene glycol (0.3%)
CIS-CIS-
muconic acid
srcaptunc
0.5%)
-H O
^C*N ** 2. ^
X NHAc
1 1
SCH,-CH
2 1
COOH
LJd li.G1 1C
(in) oxide
glutathione x£X^
\^^ jfc
S-epoxide
transferase
i
>
(0
, H (10
* H epoxide
hydrase
' spontaneous
^JX.
1
^
COOH
H r- -ix
.__B^»-
OH
fi^COOH
j
_^CO°H_
//
— »«-\ -HCO,-h
1 M u ^ COOH
dehydrogenase
hydroquinone (5%) phenol (23-50%;
OH ^v^ OH
conjugations-
catechol (3-25%)
.OH
OH
UDPG HO
hi
glucuromdes
hydroxyhydroqumone (0.3%)
|T
alkaline salts'
potassium phenylsulfate
(50-100%)
0 CH-(CHOH)3 CHC02 K
phenyl gtucuronide
(0-50%)
eliminated in urine
Reference: Rusch at al. (1977)
AHH = aryl hydrocarbon hydroxylase
UDPG = undine dipnosonate glucuronvi
transferase
PAPS = 3'-pnospho-adenosm-
5 '-phosphosuifate
77
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FIGURE 2
METABOLISM OF MONOCHLOROBENZENE
chloro-
benzene
dehy-
OH drogenase
2,3—dihydro—
2,3—dihydroxychlorobenzene
H
4—chloro
benzene
oxide
OH 3,4—dihydro —
3,4—dihydroxy-
chlorobenzene*
3—cnlorocatechol*
I
conjugates"
4—chloroohenyl mercaptunc acid*
4—chlorophenol
^conjugates*
4—chlorocatechol * *
I
conjugates**
NHAc
-CH, -CH
(• \
COOH
Urinary metabolite
"Major urinary metaoolite
References; Spencer and Williams (1950); Smith et al. (1950);
Azouz et al. (1953); Parks and Williams (1955); Williams (1959);
Lindsay-Smith et al. (1972); Selander et al. (1975a, 19756); Williams
etal. (1975).
78
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FIGURE 3
METABOLISM OF DICHLOROBENZENES
dehydrogenase
HJ |1 ,
epoxide ^ OH ft \ HO
hydrase jS H OH
1.2—dichloro
benzene
ci
CI
OH
epoxide CI
hydrase ^ ^Cl dehydrogenase
HOH
CI
OH
4,5—dichloro-
catecnol*
3,4—dichlorophenol1
3.4—dichlorophenyl-
mercaptunc acid
3.4— dichloro-
catechol*
2.3,—dichloropnenoi'
CI
1,3—dichloro-
benzene
CI
O]
CI
CI
O
C|
dehydrc- , OH
genase
CI
CI
HO
H'OH OH
glutathione
CI
CI
CI
,OH
:OT—-o
CI
H JL .OH
c:
3.5-and2.4—
dichlorophenoi
3,5—dichloro-
catechol*
2.4—dichlorophenyl-
mercaptunc acid*
CI
SR
HO
OH
2.5—dichloro-
hydroqumone*
C!
•?
1,4—dichloro-
benzene
CI
2,5—dichloro-
phenoi **
CI
CI
NHAc
I
R = -CH,-CH
< I
COOH
* Urinary metabolite
''Major urinary metabolite
References: Azouz et ai. (1953, 1954, 1955);
Parke and Williams (1955).
79
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FIGURE 4
METABOLISM OF TRICHLOROBENZENES
ci
RS
t
CI 3,4,5—trichlorophenyl-
mercapturic acid*
CI
glutathione
CI
CI
epoxidase
epoxidase
3,4,5—trichlorophenol *
2,3,4—trichlorophenol *'
3,4,5—tnchlorocatechol'
2.3.6—trichloropnenol *
OH
CI
"NIH
epoxidase
CI
1,3,5—trichlorobenzene
dechlorination
CI
CI shift
H
CI
.» CI
CI
CI
C!
CI
CI
CI
epoxidase
chlorobenzene
HO
epoxide
hydrase
CI / CI
dehydrogenase
H
OH
HO H
OH
OH
80
2,3,5—tnchlorophenol'
2,4,6—trichlorophenol'
4—chlorophenol*
4—chlorocatechol*
-------
FIGURE 4
METABOLISM OF TRICHLOROBENZENES
(CONTINUED)
CI
CI
CI
2,3,5—tnchlorophenyl-
mercapturic acid*
gluta-
thione
CI
C!
1,2.4—tncnloro-
benzene
CI
RS
CI
CI
HO
HO
HO dehydrogenase
CI
CI
CI
2,4,5—trichlorophenyl-
mercapturic acid*
2.3.5—tncntorophenol'
2.4.5—tncrttorophenol'
3.4,6—tncrilcrocatechol'
'Urinary metabolite
"Major urinary metabolite
References: Jondorf et al. (1954, 1955a, 1955b); Parke
and Williams (1960); Kohli et al. (1976V
NHAc
a = -CH, -CH
COOH
81
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FIGURE 5
METABOLISM OF TETRACHLOROBENZENES
ci
epoxidase
CI T CI
CI
1,2,3,4—tetrachloro-
benzene
CI
CI
CI
2,3,4,5—tetrachloro-
benzene oxide
OH
CI
CI
CI
2.3,4,6,—tetrachlorophenol'
2,3,4,5—tetrachlorophenol"
C!
CI
CI
1,2.3,5—tetrachloro-
benzene
1,3,4.5—tetracnloro-
benzene oxide
CI
2,3,4,5—tetrachloro-
benzene oxide
CI
CI
CI
OH
CI
CI
OH
c,
2.3,4,5—tetracnloroohenol'
2,3,4.6—tetrachlorophenoi'
2,3,5,6—tetrachlorophenol'
Ci
Ci
1.2.4.5 — tetrachloro
benzene
CI
CI
2.3.5.6 —tetrachloro-
benzene oxide
Urinary metabolite
Major urinary metaoolite
References' Jondorf et al. (1958); Kohh et al. (1976).
82
OH
2.3.5.6—tetrachlorophenol *
-------
FIGURE 6
METABOLISM OF PENTACHLOROBENZENE
pentachlorobenzene
dechlonnation
pentachlorophenol"
Cl epoxidase
conjugate*
'Urinary metabolite
'Major urinary metabolite.
Reference: Kohli et al. (1976); Leber et al.
(1977): Koss-and Koransky (1978).
2.3,4,5—tetachlorophenol"
83
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FIGURE 7
METABOLISM OF HEXACHLOROBENZENE
C!
hexachlorobenzene
Ci
C!-v^N^C1
*_ ICVl pentachlorophenol'
pentachlorobenzene *
dechlorination
OH
pentachlorothiophenol **
2,3,4,6—tetrachlorophenol *
2,2,5,6—tetrachlorophenol *
tetraonlorohydroquinone**
2,4,5—tricrtlorophenol *
^*"
trichlorobenzenes sr—*- 2.3,4—trichlorophenol*
2,4,6—trichlorophenol*
tetrachlorobenzenes"
dechiorination
'Urinary metabolite
•Major urinary metabolite
References: Cccper (1978); Engst et al. (1976); Kohli et al. (1976);
Kcss and Koransky (1978); Koss et at. (1975, 197Sa, I978b);
Menencaie et a!. (1975); Rczman et al. (1377, 1978; Renner
and Scr-usier (1977); Bedford (1979); Yang gt al. (1975);
LJI anc Sweeney (1975).
84
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(2) Porphyria
Porphyria is any one of a group of diseases that result from
a disturbance of porphyrin metabolism, usually in the liver,
which leads to an increase in the tissue and circulating blood
levels of porphyrins and related compounds.
Hexachlorobenzene consumption produces hepatic porphyria in
humans, with cutanea tarda lesions and porphyrinuria (Schmid
1960). This effect can also be produced in rats, rabbits, guinea
pigs and mice (Ockner and Schmid 1961, DeMatteis et al. 1961, San
Martin de Viale et al. 1970, Taljaard et al. 1972, Rajamanickam
et al. 1972, Stonard 1974). Some of the lower chlorinated ben-
zenes will also affect porphyrin metabolism, but not to the same
extent. Rimington and Ziegler (1963) found that high doses (455-
1140 mg/kg) of monochloro-, ortho- and para-dichloro-, 1,2,3- and
1,2,4-trichloro-, and 1,2,3,4-tetrachlorobenzene given to rats
for brief periods resulted in porphyria which varied in intensity
and expression from compound to compound. 1,2,4,5-
Tetrachlorobenzene (905 mg/kg) did not affect liver or urinary
porphyrins. The increases in urinary porphyrin excretion, caused
by other chlorinated benzenes, were fairly small considering the
large doses administered. Even the substantial increase in
uroporphyrin excretion seen after para-dichlorobenzene
administration is modest (10 ug/day) when compared with the huge
increases following hexachlorobenzene poisoning (100-400 ug/day)
(Ockner and Schmid 1961).
Carlson (1977) compared para-dichlorobenzene, 1,2,4- tri-
chlorobenzene and hexachlorobenzene in their ability to induce
porphyria in rats. Doses of 50 mg/kg/day of hexachlorobenzene
(p.o.) for 30 days in female rats (chosen because of their
greater susceptibility to the porphyrinogenic effects of hexa-
chlorobenzene) caused a statistically significant increase in
both liver and urine porphyrins. However, doses of up to 200
mg/kg/day for 120 days of para-dichlorobenzene and 1,2,4-tri-
chlorobenzene caused no porphyrinuria and only minor changes in
the porphyrin content of the liver. It is possible that the
porphyrinuria seen acutely with large doses (Rimington and
85
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Ziegler 1963) represents massive liver damage, rather than
porphyria. In addition, although Rimington and Ziegler noted skin
lesions with 1,2,3,4-tetrachlorobenzene, they were not the
cutanea tarda lesions seen in cases of hexachlorobenzene-induced
porphyria, as they were not a photosensitive response, but were
apparently similar to chloracne. The large doses used by
Rimington and Ziegler, in some cases, approach the LDcQ for the
compounds, while very small amounts of hexachlorobenzene, which
has a high LDgo, suffice to initiate porphyria, a further
indication that liver damage, rather than a specific effect on
porphyrin metabolism, may be the cause of the effects seen by
Rimington and Ziegler (1963). The spectrum of porphyrins
excreted (coproporphyrin and porphobilinogen) is also different
from that seen in hexachlorobenzene poisoning (7- and 8-carboxy-
porphyrin). While it is possible that the porphyrias induced by
hexachlorobenzene and the lower chlorinated benzenes are similar
and have a common mechanism, it seems more likely that they
result from different causes.
(3) Structural Relationships
The chlorinated benzenes are structurally related to benzene
and hexachlorobenzene, both of which cause chronic effects
(Schmid 1960, Aksoy et al. 1971, 1972, 1976, Tareeff et al.
1963), as discussed below. The oncogenicity of benzene and
hexachlorobenzene are discussed in Section III.G.I.e.(2) .
(a) Benzene
Benzene has been shown to produce damage to the hematopoie-
tic system (Aksoy et al. 1971). Leukopenia, thrombocytopenia,
and pancytopenia are among the health effects reported among
workers who were chronically exposed to the solvent. Similar
effects have been reported in dogs (Svirbely et al. 1944, Hough
et al. 1944), rats, rabbits, and guinea pigs (Wolf et al. 1956,
Deichmann et al. 1963). These effects appear similar to those
such as thrombocytopenia, leukopenia, and aplastic anemia among
other blood effects attributed to chlorinated benzenes and
discussed earlier in this subchronic effects section.
86
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Since it is unknown what aspect of benzene metabolism is
responsible for the observed effects, or even whether there is a
single cause of all of benzene's effects, the significance of the
structural similarity with chlorinated benzenes is uncertain.
Until more information is available, this structural relationship
remains only suggestive of the potential of chlorinated benzenes
to cause similar toxicity.
(b) Hexachlorobenzene
Hexachlorobenzene has been shown to produce histopathologi-
cal changes in the liver and spleen in rats (Kuiper-Goodman
1977). Neutrophilia and some hepatotoxicity were found in beagle
dogs after exposure to hexachlorobenzene (Gralla and Fleischman
1976). All of the chlorinated benzenes tested induced similar
liver damage.
2. Decision
An assessment of the literature to date reveals that most of
the chlorinated benzenes that have been tested thus far produce
similar health effects. Mono- through tetrachlorobenzenes have
been noted for their effects on the liver and the hematopoietic
system in humans, and animals (Varshavskaya 1967, Monsanto 1967a,
Hollingsworth et al. 1956, 1958, Coate et al. 1977, Fomenko 1965,
Cotter 1953, Campbell and Davidson 1970). Mono-, di- and
trichlorobenzenes have been shown to produce variable changes in
the kidney (Khanin 1977, Monsanto 1978g, Hollingsworth et al.
1956, 1958, Coate et al. 1977). The results of an unpublished
subchronic study indicate that pentachlorobenzene also induces
effects in the liver and kidney (Linder et al. 1980). Although
the experimental designs of some of these tests have weaknesses,
the test results lead to the common conclusion that the
chlorinated benzenes produce similar chronic health effects and
may present an unreasonable risk to human health.
Determination of the most sensitive species, route of
administration, and health effect cannot be done because of
conflicting results from the studies reported in the
87
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literature. For example, for monochlorobenzene the Monsanto
results (1978g) indicate that the dog is more sensitive via the
inhalation route than the rat, but Khanin (1977) observed effects
in rats at doses considerably lower than those used in the
Monsanto study. Another Monsanto study (1967a and 1967b)
indicates that the dog is more sensitive via the oral route than
the rat, but Varshavskaya (1967) observed effects at doses
considerably lower than those used in this Monsanto study as
well. A similar discrepancy occurs with ^-dichlorobenzene
(Varshavskaya 1967, Hollingsworth et al. 1958).
The chlorinated benzenes are structurally related to two
compounds with recognized chronic effects: benzene and hexachlo-
robenzene. Benzene has been noted for its effects on the
hematopoietic system in both humans and animals (Aksoy et al.
1971, Svirbely et al. 1944, Hough et al. 1944, Wolf et al.
1956). Chlorinated benzenes may produce similar effects (Perrin
1941, Petit and Champeix 1948, Wallgren 1953, Monsanto 1978g).
Hexachlorobenzene has been shown to produce changes in the liver
and spleen (Schmid 1960, Kuiper-Goodman 1977). Chlorinated
benzenes also damage these organs (Monsanto 1967a, Hollingsworth
et al. 1956, 1958, Coate et al. 1977, Fomenko 1965).
Both monochlorobenzene and its analog monobromobenzene are
metabolized to arene oxides. Bromobenzene has been found to bind
covalently to liver proteins and to produce liver necrosis,
particularly after depleting 90 percent of hepatic glutathione
via formation of mercapturic acid metabolites (Reid and Krishna
1973, Jollow et al. 1974). Mono-, di- and trichlorobenzenes
(except j>-dichlorobenzene, for which the evidence is ambiguous)
are metabolized to arene oxides and excreted partly as
mercapturic acids (see Figures 2, 3, and 4, Metabolism Section),
indicating their potential to react with proteins and possibly
cause necrosis. This may explain the liver damage associated
with all of the chlorinated benzenes tested and the fact that
monochlorobenzene has been reported to cause renal necrosis (Reid
1973).
88
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In summary, the subchronic studies reported in the
literature show that the chlorinated benzenes produce similar
chronic health effects when given by several routes of adminis-
tration. These compounds are structurally related to two
compounds of known chronic toxicity: benzene and hexachloroben-
zene. They are metabolized to reactive compounds that may bind
to macromolecules in the cell and thus produce a variety of toxic
effects. Available information does not permit the Agency to
complete an assessment of the nature and extent of chronic
toxicity risks associated with exposure to the chlorinated
benzenes because the reports in the literature give insufficient
detail or apparently inconsistent results.
3. Proposed Testing; Subchronic and Chronic
Effects Testing
EPA proposes that the chlorinated benzenes, except penta-
chlorobenzene, be tested for chronic health effects. Pentachlo-
robenzene has already undergone adequate subchronic testing.
A number of reviewers have suggested that short-term (90
day) animal studies can in some cases gives reasonable
indications of the long-term (lifetime) effects of chemicals and
that long-term studies therefore need not always be performed
(Weil and McCollister 1963, Peck 1968, McNamara 1976). On the
basis of the test data available for chlorinated benzenes,
EPA believes that the nature and degree of chronic effects
induced by chlorinated benzenes can be determined in 90-day
subchronic toxicity studies following the test standards proposed
in the Federal Register on July 26, 1979 (USEPA 1979c) .
EPA has proposed standards recommending the use of a rodent
and a nonrodent species, with the dog as the preferred nonro-
dent. Because the studies by Khanin (1977) and Varshavskaya
(1967) indicate that the rat may be the more sensitive species
(see above under "Decision"). EPA is proposing that the 90-day
subchronic testing be done only in rats. The results of this
study will assist the Agency in determining whether a safe level
of human exposure can be established for these effects.
89
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The Agency is aware that a 90-day inhalation study, of Which
only an abstract has been published (Watanabe et al. 1978), has
been performed on 1,2,4-trichlorobenzene in rats that may suffice
to characterize the subchronic toxicity of the compound. EPA
will try to obtain more details of this study.
4. Testing Under Consideration; Metabolism Studies
EPA believes that valuable information would be obtained
from studies designed (a) to determine the distribution of
chlorinated benzenes to tissues and organs of one species, (b) to
learn the rates of their clearance from these tissues, and (c) to
ascertain whether or not chlorinated benzenes or their
metabolites form covalent compounds with macromolecules,
particularly in the brain and gonads and in organs from which
excretion is slow. If any of these compounds do form covalent
compounds with macromolecules, experiments should determine
further whether binding is to DNA, protein, or both. EPA
believes that this information could strengthen the basis for
using the results from tested chlorobenzenes to characterize the
category (see Section IV of this document). EPA is not proposing
specific metabolism studies at this time since the Agency now has
standards only for studies on absorption and excretion. EPA is
interested in comments from all sectors on what other kinds of
metabolism studies are needed.
C. Neurotoxic Effects
1. Evaluation of Pertinent Studies
a. Human Case Reports
Reich (1934) reported an instance of a two-year-old male who
swallowed an estimated 5 to 10 ml of monochlorobenzene. Within
two hours, the child was pale, his lips cyanotic, and he had no
detectable reflexes to strong stimuli. When hospitalized, he was
unconscious and cyanotic, with twitching in the head and neck
90
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regions. The child began to regain consciousness after three
hours, and all signs were normal within eight hours. There was
no long-term follow-up of this patient.
Monochlorobenzene and ja-dichlorobenzene have been associated
with CNS depression in humans. Rozenbaum et al. (1947) examined
54 people, 28 of whom worked together in a factory where they had
been exposed to monochlorobenzene vapors for one to two years.
Headache, dizziness, somnolence, and dyspeptic disorders were
reported by many of these workers. Examination revealed acro-
paresthesia (tingling, numbness, stiffness in extremities) in
eight individuals, spastic contraction of finger muscles in nine
individuals, hyperesthesia (excessive sensitiveness) of hands in
four individuals, and spastic contraction of the gastrocnemius
muscle in two individuals. The other 26 people, who had either
short-term exposure to monochlorobenzene or were exposed to
benzene and monochlorobenzene fumes, displayed no characteristic
symptoms.
Wallgren (1953) reported on the examination of eight men who
worked for one to seven months in a factory manufacturing moth-
proofing agents. These agents were produced from j>-dichloroben-
zene, which contained about 1% of an unidentified nitrogen-
containing* impurity and small amounts of o-dichlorobenzene. The
workers developed neural disorders including intensified muscular
reflexes, mild clonus of the ankle, and tremors of the fingers.
The workers also experienced loss of appetite and hematopoietic
changes.
Tarkhova (1965) studied the electroencephalographic patterns
in 4 human subjects exposed by inhalation to 0.1, 0.2, or 0.3
mg/m^ (0.02, 0.04 or 0.06 ppm) of monochlorobenzene. Changes
were noted within minutes in the patterns of response to 10 nsec
light flashes of 8-10 Hz and varying intensities by subjects
exposed to the two higher concentrations. The lowest
concentration appeared to produce no effect.
91
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b. Animal Studies
S
In animal studies, nervous system effects have been shown
following exposure to monochlorobenzene, j>-dichlorobenzene, _p_-
dichlorobenzene, and 1,2,4,5-tetrachlorobenzene.
Tarkhova (1965) exposed two groups of adult male white rats
(15 per group) to monochlorobenzene by inhalation, at concentra-
tions of either 0.1 or 1.0 rng/m^ (0.02 or 0.2 ppm) continuously
for 60 days. The higher-dose group showed increased cholines-
terase activity in, whole blood at 36 days and a reversal of the
normal chronaxy ratio of antagonistic muscles at 39 days.
Normally, the chronaxy (the relationship between a stimulus
intensity and latency of response of the excitable tissue) of
flexors is shorter than that of extensors, but exposure to
certain toxic substances reverses that relationship. The group
receiving 0.1 mg/m^ of monochlorobenzene did not manifest these
changes.
In studies by Varshavskaya (1967), groups of seven male
albino rats were treated daily for nine months with monochloro-
benzene or _o_-dichlorobenzene, in doses of 0.1, 0.01, or 0.001
ing/kg by stomach tube. With both monochlorobenzene and ^-dichlo-
robenzene, the dose of 0.1 mg/kg produced deficits in the
acquisition and performance of a conditioned response. Effects
at the 0.01 mg/kg level were minimal. _
In an investigation carried out on rats, Pislaru (1960)
found that inhalation exposure to monochlorobenzene in
concentrations of 0.1, 1.25, and 1.5 mg/1 (22-326 ppm) produced
chronaximetric modifications. As a result of exposure for 37
weeks at a concentration of 0.1 mg/1, chronaximetric inhibition
was observed between weeks 7 to 14, after which the inhibition
subsided. The author stated that chronaximetric changes preceded
enzymatic changes (not specified) by 3 to 4 weeks.
Gabor and Raucher (1960) reported the effects on white rats
of exposure to monochlorobenzene vapors at a concentration of 0.1
mg/1 for three hours every other day for 37 weeks. Nervous
system disturbances, indicated by inhibiting the chronaxy of the
extensor tibialis, were observed from week 7 to 14; animals
returned to normal by week 20.
92
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Rabbits, rats, and guinea pigs exposed by inhalation to
approximately 100 mg of £-dichlorobenzene for 20-30 minutes/day
for 1-5 weeks exhibited tremors and twitches of the extremities,
a "mark time" reflex, a loss of the righting reflex, a definite
nystagmus, and rapid but labored respiration (Zupko and Edwards
1949).
Pike (1944) subjected several rabbits to as many as 62
exposures of eight-hour duration to j>-dichlorobenzene vapors at a
concentration of 4.6-4.8 mg/1 (770-800 ppm) over a period of 83
days. Marked tremors and weakness were observed in all of the
animals; only three survived 62 exposures. Lateral nystagmus and
transitory edema of the cornea and optic nerve head were also
observed. Repeated oral doses of £-dichlorobenzene at 1.0 g/kg
by stomach tube five days/week produced marked intoxication,
weakness, tremors, loss of weight, and death in some animals, but
no eye effects were reported. Oral doses of 0.5 g/kg on the same
schedule produced definite tremors and other signs of intoxica-
tion, but no deaths throughout one year of exposure.
Hollingsworth et al. (1956) found symptoms of central
nervous system effects including tremors and weakness in rats,
guinea pigs, and rabbits exposed to 4.8 mg/1 (798 ppm) of j>-di-
chlorobenzene vapors, 8 hours/day, 5 days/week, for up to 69
exposures.-- No neurological effects were reported at 341 ppm or
less in 6 months.
Fomenko (1965) reported central nervous system effects in
rats treated with 1,2,4,5-tetrachlorobenzene. The rats were
treated after a conditioned response to a stimulus had been
established. A dose-dependent alteration of conditioned
responses was observed in rats intubated with 0.005 mg/kg/day and
0.05 mg/kg/day for eight months. The conditioned responses
became slower and decreased in magnitude. More responses during
extinction and a longer time to restoration of the response were
also noted. At 0.05 mg/kg, the response was not restored, i.e.,
was completely inhibited.
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2. Decision
Evidence cited in this document indicates that the chlori-
nated benzene congeners are hazardous to the integrity of
neurobehavioral functioning in humans and animals. Signs and
symptoms of adverse effects on the nervous system in various
species, including humans, rats, rabbits, and guinea pigs have
been associated with exposure to four of the congeners (mono-
chlorobenzene, j>-dichlorobenzene, ja-dichlorobenzene/ and 1,2,4,5-
tetrachlorobenzene).
The substances are non-specific central nervous system (CNS)
depressants. While the chlorinated benzenes vary in potency, the
neural effects produced in humans and animals by the various
compounds are similar. Effects observed in humans include
somnolence and loss of consciousness, dizziness, headache, loss
of appetite, clonus, disturbance of innate reflexes, tremors, and
spastic contractions; the last three effects occurred in animals
as well. Also found in animals were disturbance of conditioned
reflexes, changes in the chronaxy ratio of antagonistic muscles,
nystagmus, and weakness.
For the chlorobenzene compounds that have been tested for
neurotoxic and behavioral effects, the dose-response characteri-
zation is incomplete. Moreover, available observational data are
poorly quantified, subjective, and therefore, relatively insen-
sitive. Subchronic studies of electrophysiological functions are
inadequately detailed. Better data are needed for a more
complete characterization and assessment of the hazard from
exposure to the chlorinated benzenes.
Russian reports suggest that a no-effect level for
neurobehavioral effects of the chlorobenzene congeners may be
very low. Adverse' effects have been reported in continuous
inhalation experiments on rats using as little as 0.2 ppm of
monochlorobenzene in air. These studies suggest that functional
effects may ensue from exposure to chlorinated benzenes at
concentrations lower than those that produce overt pathology.
The Agency believes that testing of the chlorinated benzenes
for their neurotoxicity will provide data relevant to determining
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whether exposure to these substances presents an unreasonable
risk to human health from neurotoxic and behavioral effects and
is therefore considering what testing might be undertaken with
respect to these effects. EPA is not proposing specific neuro-
toxicity or behavioral effects testing at this time because the
Agency has not yet developed test standards for such testing.
3. Testing Under Consideration
The following discussion sets forth the Agency1s current
views on testing chlorinated benzenes for neurotoxicity and
behavioral effects. EPA is proposing that such tests include
both acute and subchronic (repeated exposure for 90 days or
longer) tests on rodents using locomotor activity, a functional
observational battery, a neurophysiologic test of chronaxy, and
an appropriate electrodiagnostic test. Histopathology of the
nervous system of subchronic test animals is also recommended.
This examination should include: longitudinal and cross sections
of the spinal cord, i.e., thoracic and lumbar regions; cross
sections of the forebrain, midbrain, and brainstem; and
representative sections of the sciatic nerve. Tissue should be
fixed in situ^ with formaldehyde or glutaraldehyde and
paraformaldehyde.
Tests of locomotor activity have been widely used in
screening drugs and have been proposed as screening tests for
environmental chemicals. A recent survey by Reiter and MacPhail
(1979) of locomotor activity measures discusses some of the
problems involved in generating comparable data from different
types of devices as well as the influence of other important
variables. They conclude that in general, when combined with
observational measurements of other central nervous system
functions, automated activity devices provide more reliable and
better quantified measures of locomotor activity.
Observational assessment by means of screening tests that
measure objective physiologic signs, unconditioned reflexes,
elicited responses, and operants are essential for detecting the
spectrum of an agent's effects and providing a basis for
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determining the agent's functional anatomical targets. Tilson
and Cabe (1978) and Tilson, Mitchell, and Cabe (1979) present
useful examples of a screening battery and discuss some factors
important to development of screening batteries.
The neurobehavioral functions assessed in the literature on
chlorinated benzenes by means other than observation are acquisi-
tion of conditioned responses, chronaxy measurements of nerves or
muscles, and electroencephalography. EPA is proposing that
subchronic studies of the effects of chlorobenzenes include
measurement of chronaxy and some other neural function. Among
such functional tests, conduction velocity of a mixed large and
small diameter fiber population (see, for example, Glatt et al.
1979) is a well-known parameter for evaluating nerve damage.
However, other tests such as frequent impulse series transmission
(e.g. Tackmann et al. 1975) or other electrodiagnostic procedures
should be considered. The Agency is interested in comments on
the suggested neurotoxicity tests, particularly on the adequacy
of rodents as the proposed test species and on the appropriate
duration of exposure for obtaining data for risk assessment.
D• Reproductive Effects
1. Evaluation of Pertinent Studies
a. Human Case Reports
No reports of reproductive effects in humans exposed to
chlorinated benzenes are known to EPA.
b. Animal Studies
The Monsanto Co. (1978g) reported gonadal effects in a study
in which dogs were exposed to monochlorobenzene vapor at 0, 0.76,
1.47, and 2.00 mg/L for 6 hours/day, 5 days/week for a total of
62 exposures. In the high dose group, two of four male dogs
developed bilateral atrophy of epithelial tissue in the
seminiferous tubules. These effects are consistent with effects
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found in an earlier subchronic study in which groups of four male
and four female dogs were dosed with 0.025, 0.050, and 0.250
nig/kg/day orally for 13 weeks (Monsanto 1967a) . In this earlier
study, decreased spermatogenesis was seen in three of the four
dogs in the high dose group. Tubular atrophy and epithelial
degeneration were also seen in this group.
In the Monsanto (1978g) study, rats exposed to the same
concentrations and test conditions as the dogs showed less
definite gonadal responses; female rats exposed to 2.0 mg/1 of
monochlorobenzene exhibited a significantly higher gonad to body
weight ratio than untreated females (p < 0.01).
c. Other Indicators for Reproductive Effects
Evidence for the potential of chlorinated benzenes to affect
reproduction can be inferred from the effects of hexachloroben-
zene on reproduction. This compound is related to chlorobenzenes
structurally, and in producing some effects such as hepatotoxic-
ity it is similar to the other chemicals of the group. Hexachlo-
robenzene was given orally to five female monkeys for 60 days
(latropoulos et al. 1976). Three monkeys received 8, 32, and 64
mg/kg/day, respectively, and two others received 128 mg/kg of
hexachlorobenzene. One control monkey received vehicle only.
Fourteen monkeys of similar weight and age were also used as
controls but did not receive the vehicle. At the lowest dose
given, changes occurred in the germinal epithelium of the •
ovaries. Multiple follicular cysts were observed in all
hexachlorobenzene-treated animals except in the animal treated
with 32 mg/kg. The ovarian cortices of monkeys from all treated
groups showed varying degrees of degeneration. The number of
primary follicles was markedly reduced by 17, 17, 71, and 80% for
the monkeys given 8, 32, 64, or 128 mg/kg/ respectively. The
changes involved all ovarian elements including primary
follicles, germinal epithelium, and stroma; they were
morphologically similar to postmenopausal changes. Apparently,
the corpora lutea were not receptive to gonadotropin stimulation
or were deficient in steroidogenesis, or the uterus failed to
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respond to luteal transition, since the endometrium of all dosed
animals remained in the follicular phase. Minor liver changes as
well as the changes in the ovarian pathology and function
occurred in the low dose animal. Increased ovarian and liver
pathology occurred at the higher doses.
A four generation reproduction study in the rat in which
groups of 20 female and 10 male rats per group were given,
respectively, 0, 10, 20, 40, 80, 160, 320, and 640 ppm of
hexachlorobenzene in the feed demonstrated several reproductive
effects (Grant et al. 1977). The two highest dietary concen-
trations caused 10% and 50% death respectively, in the parental
generation. Viability indices of the F^ generation were severely
decreased in groups fed 160 ppm or more in the diet. The
lactation index was reduced in the Fj generation of the 80 ppm
group, the highest dose group for which there was an P.,
generation. Fertility was affected in the two highest dose
groups. The relative liver weights and aniline hydroxylase
activities were increased in weanlings from dams fed 40 ppm of
hexachlorobenzene, the only dose for which the enzyme activity
was reported.
The levels of hexachlorobenzene in the plasma, brain,
kidney, liver, and body fat of 21 day old pups from dams fed 0,
10, 20, 40, 80, and 160 ppm of hexachlorobenzene were determined
(Grant et al 1977). The highest concentration of hexachloro-
benzene was found in the body fat. The hexachlorobenzene content
of the pups increased with the dietary content of the compound
fed to the dams. The high concentrations observed in the pups in
relation to the dietary intake of the dam suggest a high rate of
excretion of HCB via the milk. The residue data also indicate
that the pups can tolerate relatively high body burdens without
showing any adverse effects on the various parameters measured in
this study.
2. Decision
Studies in monkeys indicate that hexachlorobenzene causes
dose-related ovarian effects after 60 days' exposure to a dose as
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low as 8 mg/kg/day. A multigeneration study in rats dosed with
hexachlorobenzene reported fertility problems in animals given
320 or 640 ppm in feed, and decreased fetal viability in F3
generation animals exposed to as little as 80 ppm. Decreased
spermatogenesis and atrophy of seminiferous tubule tissue has
been noted in dogs receiving monochlorobenzene. The totality of
these results raises concern that other chlorinated benzenes may
elicit similar responses and that consequently they should be
tested for reproductive effects.
3. Proposed Testing
EPA is proposing testing of the chlorinated benzenes except
1,2,4-trichlorobenzene for reproductive effects. Since a repro-
ductive effects study on 1,2,4-trichlorobenzene, requested by the
EPA Office of Drinking Water, is nearing completion at EPA's
Health Effects Research Laboratory in Research Triangle Park,
North Carolina, further testing of this compound appears
unnecessary unless evaluation of the final report reveals
deficiencies in the EPA study. Standards for development of test
data on reproductive effects have been proposed (USEPA 1979d).
Adherence to these standards should produce data necessary to
define the hazard to reproduction from exposure to chlorinated
benzenes.
E. Teratogenic Effects
1. Review of Pertinent Studies
a. Morphological Teratogenicity
i. Human Case Reports
No epidemiology studies or human case reports were found
which indicate that exposure to chlorinated benzenes is
associated with teratogenic effects.
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ii. Animal Studies
Investigators have demonstrated fetal anomalies and
malformations resulting from the exposure of pregnant rats and
mice to pentachlorobenzene and hexachlorobenzene.
Khera and Villeneuve (1975) reported the testing of penta-
chlorobenzene for teratogenic potential. Groups of pregnant
Wistar rats were given pentachlorobenzene dissolved in corn oil
at doses of 0, 50, 100, or 200 mg/kg/day by gavage on days 6-15
of gestation. No signs of toxicity were reported in dams
throughout the test. There were no significant differences in
the average number of live fetuses/litter and in the ratio of
fetal deaths to total implants. There was a statistically
significant increase in the incidence of unilateral and bilateral
extra rib formation in fetuses in all test groups. Unilateral
extra ribs occurred in 1.6% of the controls while pentachloroben-
zene-treated animals showed 14%, 8%, and 17% incidence, at 50,
100, and 200 mg/kg, respectively. The incidence of bilateral
extra ribs was 1.6% for controls, 8% at 50 mg/kg, 9% at 100
mg/kg, and 46% at 200 mg/kg. In addition, the number of litters
in which one or more fetuses had rib anomalies (14th and 15th
combined) were 3 of 19 for controls, 14 of 19 at 50 mg/kg, 11 of
19 at 100 mg/kg, and 15 of 19 at 200 mg/kg. The increased
incidences were significant (p<0.001) at all dose levels. Other
workers treated pregnant CD-I mice with 50 or 100 mg/kg of
purified pentachlorobenzene by gastric intubation in corn oil on
days 6 to 15 of gestation (Courtney et al. 1977). The maternal
liver to body weight ratio was significantly increased in both
dosage groups. Although the fetal weight was reduced in both
dose groups and one cleft palate occurred (10 litters with 11^ 4
live/litter) in the 50 mg/kg dose group, no dose-related
malformations occurred.
A screening-type teratology study on 1,2,4-trichlorobenzene,
requested by the EPA Office of Drinking Water, is nearing comple-
tion at EPA's Health Effects Research Laboratory in Research
Triangle Park, North Carolina. When a report on the results of
this study becomes available, and if the screen becomes validated
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as an indicator of teratogenic potential, EPA will take the test
results into consideration in determining the need to include
1,2,4-trichlorobenzene in a final teratology test rule for the
chlorinated benzenes.
The chlorobenzene producers are reportedly (Dow 1978b)
planning jointly sponsored teratology studies on monochloroben-
zene, jD-dichlorobenzene and js-dichlorobenzene. The National
Toxicology Program nominated p_-dichlorobenzene for teratogenicity
testing, but it has not yet made a final decision to test the
chemical (USDHEW 1979).
i i i. Other Indicators of Teratogenic Potential
(1) Structural Relationship with
Hexachlorobenzene
Hexachlorobenzene was studied for teratogenic potential in
mice. Ten pregnant CD-I mice were given hexachlorobezene (100
mg/kg by oral intubation) on days 7 through 16 of gestation.
Small kidneys, renal agenesis, enlarged renal pelvis, club foot,
and cleft palate were observed (p<0.05) (Courtney et al. 1976).
Although only one dose group and ten animals were used, the study
indicates that hexachlorobenzene is teratogenic in mice.
In another study, hexachlorobenzene was administered orally
to rats at a dose of 0, 10, 20, 40, 60, 80, or 120 mg/kg on each
of days 6-9, 10-13, 6-16, and 6-21 of gestation (Khera 1974). A
significant increase in the incidence of 14t^1 rib (uni- and
bilateral) in hexachlorobenzene-treated groups compared with that
in the relevant control group was observed in the groups treated
on days 10-13, 6-16, and 6-21 of gestation. The incidence
increased as a function of dose and length of treatment. Sternal
defects and retarded ossification were significantly more
frequent in the group treated on days 6-21 and were dose-
related. The report indicates the rib anomalies were not
confirmed in subsequent trials at doses up to 80 mg/kg. No test
results or further discussion were presented.
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(2) Potential for Placental Transfer
It is likely that the chlorinated benzenes can pass from the
maternal circulation into the fetal unit. Non-ionized chemicals
with high lipid solubility readily cross the placental membranes
and gain access to the developing embryo (Fingl and Woodbury
1970, Nishimura and Tanimura 1976). Monochlorobenzene has an
octanol/water partition coefficient of log Poct = 2>84 and those
of higher chlorinated benzenes range from 3.38 to an upper limit
expected to fall between the 4.1 observed for a trichlorobenzene
and the 5.8 found for hexachlorobenzene (Table 1, Section I).
The relatively low molecular weights and the lipid solubility of
the chlorinated benzenes indicate a potential for rapid diffusion
across the placenta. Furthermore, because chlorobenzenes are non-
specific CNS depressants in adults and therefore must cross the
blood-brain barrier, the chlorinated benzenes or their toxic
metabolites can probably cross the placenta as well. Khera
(1974) pointed out in the introduction of his paper that the
appearance of hexachlorobenzene in the body fat and milk of
exposed humans has been reported by several other
investigators. Thus the exposure of pregnant females to
chlorinated benzenes may result in exposure of the embryo, fetus,
and neonate.
(3) Teratogenicity of Chlorinated Benzene
Metabolites
The fact that chlorinated benzenes are metabolized to
chlorinated phenols (Parke and Williams 1955, Kohli et al. 1976;
see Section III.B.1.c.(1), Figures 2-6) is of concern because one
chlorinated phenol has shown embryotoxicity and another caused
minor developmental effects in animal tests.
Pentachlorophenol was given to rats at dose levels of 5, 15,
30, and 50 mg/kg on days 6-15 inclusive of gestation; the 30
m9/T
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as delayed skull ossification, lumbar spurs, vertebral and
sternal anomalies, and subcutaneous edema, each statistically
significant.
2,3,4,6-Tetrachlorophenol has been tested for teratogenic
potential at doses of 10 or 30 mg/kg on days 6-15 of pregnancy
(Schwetz et al. 1974b). A statistically significant increase in
subcutaneous edema was observed in the 10 mg/kg dose group but it
did not appear to be dose-related. An increase in delayed skull
ossification observed at the 30 mg/kg dose was not statistically
significant. Some of the minor anomalies observed in this study
also occurred with hexachlorobenzene, pentachlorobenzene and
pentachlorophenol.
b. Behavioral Teratogenicity
Additional concern for the teratogenic potential of chloro-
benzenes is based on their documented neurotoxicity. Acute and
repeated exposure to all of the tested chlorinated benzenes (in
animals: monochlorobenzene, ortho- and para-dichloro- and
1,2,4,5-tetrachlorobenzene; in humans: monochlorobenzene and
para-dichlorobenzene) have produced adverse effects on the
central nervous system (CNS) (see Section III.C.). The central
nervous system appears to be especially susceptible to toxic
insult during its development (Buelke-Sam and Kimmel 1979). The
period during which the CNS develops is an extended one and
vulnerability to toxic insult continues into the post-natal
period. The possibility of fetal exposure to neurotoxicants such
as the chlorinated benzenes warrants their evaluation as terato-
gens. Evidence has been presented that suggests that both
structural and behavioral deficits in adult and developing
systems are associated with exposure to other non-specific CNS
depressant chemicals (van Stee 1976). Few purely behavioral
teratogens are known at this time, but psychotropic drugs which
have little or no structural teratogenic potential have been
identified as behavioral teratogens (Vorhees et al. 1979a).
Chlorinated benzenes therefore may have the potential for causing
neurobehavioral problems in newborns, even if no classical terata
are produced.
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2. Decision
Several factors indicate that the chlorinated benzenes may
have teratogenic potential. They are related structurally to
hexachlorobenzene, which is teratogenic in mice. Pentachloroben-
zene causes rib abnormalities in rats similar to those caused by
hexachlorobenzene. Although pentachlorobenzene does not cause
overt malformation in the rat, the rib abnormalities are dose-
related. EPA considers the appearance of extra ribs in the rat
to be suggestive but not conclusive evidence for teratogenic
potential, and is interested in receiving comment on this
point. Certain phenolic metabolites of the chlorinated benzenes
are also known to cause embryo- and fetotoxic responses in the
rat. The chlorinated benzenes have been associated with adverse
central nervous system effects in humans and animalsr these
substances and some of their toxic metabolites are likely to
cross the placenta and thus may pose a hazard to the developing
embryo or fetus for both morphological and behavioral terato-
genicity. The EPA is proposing teratogenicity testing on the
chlorinated benzenes except pentachlorobenzene, which has been
adequately tested and needs no further studies to evaluate its
teratogenic potential.
3. Proposed Testing; Morphological Teratogenicity
Standards for development of data for teratogenic health
effects have been proposed (USEPA 1979c). Adherence to these
standards should produce data necessary to define the structural
teratological hazard from exposure to chlorinated benzenes.
4. Testing Under Consideration; Behavioral
Teratogenicity
EPA is considering requiring behavioral teratogenicity
testing of chlorinated benzenes. The Agency believes that
behavioral teratogenicity testing should include evaluation of
behavioral and neurological development in the offspring of
pregnant animals exposed to environmental contaminants and
industrial agents (Vorhees et al. 1979b). In addition to routine
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parameters of physical development that may reflect toxicity,
such as body weight, the proposed testing should include specific
tests to assess in offspring the known effects of chlorinated
benzenes in adults. As non-speqific CNS depressants, the
chlorinated benzenes cause narcosis, reflex changes, and other
neurological motor signs as well as changes in food intake and
body weight (see Section III.C, Neurotoxic Effects). Screening
batteries specifically designed for examining these behaviors in
developing organisms should include measures shown to be related
to intoxication by chlorinated benzenes. Neuropathology should
also be included. The Agency is interested in comments on the
suggested behavioral teratogenicity tests.
F. Mutagenic Effects
1. Evaluation of Pertinent Studies
Chlorinated benzenes have been tested for mutagenic activity
in several systems including bacteria, yeast, mammalian cells in
culture, and plants. The results of these studies are summarized
below.
a. Gene Mutation Studies
Keskinova (1968) reported that the frequency of induced
mutations in Streptomyces antibioticus (in Russian nomenclature,
Aotinomyces antibioticus) 400 after exposure to gaseous
monochlorobenzene was 1400 times the mutation frequency seen in
negative control cultures. Liver activation was not employed.
Monochlorobenzene induced point mutations in this system.
Although it appears that monochlorobenzene is a direct-acting
mutagen (i.e., does not require metabolic activation), it is
possible that the organism used in this assay possesses the
enzymes necessary to metabolize the compound to a mutagenic form.
Monochlorobenzene and jo_-, nv-, and p-dichlorobenzene were
tested at a concentration of 200 ug/ml to determine their ability
to induce reverse mutation in methionine and pyridoxine auxo-
trophs of Aspergillus nidulans (Prasad 1970). Liver enzyme
activation was not employed. Monochlorobenzene was nonmutagenic
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in this assay. The dichlorobenzenes appeared to have mutagenic
activity, however. This activity increased in the order ^-di-
chlorobenzene, ji^-dichlorobenzene, j>-dichlorobenzene, with
p-dichlorobenzene being approximately twice as active as
^-dichlorobenzene. As stated above for monochlorobenzene, the
dichlorobenzenes may be direct-acting mutagens or may be
activated by enzymes present in Aspergillus. Although the
dichlorobenzenes appear to be active in this study, there are
several points that should be taken into consideration. The
spontaneous mutation rate was much lower than normal for
Aspergillus? the dichlorobenzenes were tested at only one
concentration, 200 ug/ml; and the number of plates per experiment
was not given although it was stated that duplicate experiments
were done. To be considered fully valid, a test should be
performed with 15 plates per point for at least three dose
levels. Nevertheless, the EPA considers this study to be an
indication of the activity of the dichlorobenzenes in Aspergillus
and feels that this is an appropriate system for initial testing
of the chlorinated benzenes for gene mutational activity (see
Appendix B).
Chlorinated benzenes have been reported to be inactive in
bacterial tests for mutagenicity using histidine auxotrophs of
Salmonella typhimurium as indicators of mutagenic potential (E.
I. duPont 1977, Merck 1978, Monsanto 1976a, 1976b, Monsanto
1977a, 1977b, 1977c, Monsanto 1978d, 1978e, 1978f, Simmon et al.
1979).
Monochlorobenzene was tested in plate incorpation assays
with Salmonella strains TA 1535, TA 1537, TA 1538, TA 92, TA 98,
and TA 100 both with and without liver enzyme activation over a
range of 0.01 ul - 5.0 ul per plate (Monsanto 1976a, 1976b,
Simmon et al. 1979) and 100 ml per plate (Merck 1978), and at
concentrations of 150-3000 ug/plate (E. I. duPont 1977); the
compound was negative at all levels.
jD-Dichlorobenzene was tested in plate incorporation assays
with Salmonella strains TA 1535, TA 1537, TA 1538, TA 98, and TA
100, both with and without liver enzyme activation, at concentra-
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tions of 0.6-1300 ug/plate (Monsanto 1977a, 1978e) and 0.05-1.0
ul/plate (Simmon et al. 1979). It was found to be nonmutagenic
in these assays.
jiv-Dichlorobenzene was tested both with and without liver
enzyme activation in plate incorporation assays with Salmonella
strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100 (Monsanto
1977b, 1978d, Simmon et al. 1979) at concentrations of 0.6-1300
ug per plate. It was found to be nonmutagenic at all
concentrations tested.
j>-Dichlorobenzene at concentrations of 0.5-1000 ug/plate was
found to be nonmutagenic for Salmonella strains TA 1535, TA 1537,
TA 1538, TA 98, and TA 100 when tested both with and without rat
liver enzyme activation (Monsanto 1978f, Simmon et al. 1979) in a
plate incorporation assay.
Schoeny et al. (1979) reported that 1,2,4-trichlorobenzene
at concentrations of 102-2914 ug/plate was nonmutagenic for
Salmonella strains TA 1535, TA 1537, TA 98, and TA 100 when
tested both with and without liver enzyme activation.
The National Institute of Environmental Health Sciences has
scheduled all of the chlorinated benzenes for mutagenicity
testing under the National Toxicology Program. The compounds
will be tested in the Salmonella/microsomal assay. Tests on the
six di- and trichlorobenzenes are scheduled for completion in
fiscal year 1980; testing of the remaining five compounds will
begin before the end of fiscal year 1980 (USDHEW 1979).
Monochlorobenzene and _o_-, m-, and ja-dichlorobenzene tested
at the same concentrations as those reported above for Salmonella
were found to be nonmutagenic for E. coli WP2 (Simmon et al.
1979) when tested both with and without liver enzyme activation.
Monochlorobenzene was tested for its ability to induce
specific locus forward mutations in the mouse lymphoma L5178Y
TK+ /~ assay system (Monsanto 1976c). The test was performed
both with and without mouse liver enzyme activation. Monochloro-
benzene was tested at concentrations of 0.001 ul/ml to 0.1 ul/ml
without activation and at concentrations of 0.0001 ul to 0.01
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ul/ml with activation. Monochlorobenzene was found to be
nonmutagenic for L5178Y cells in these studies. _o_-, _m- and
j>-Dichlorobenzene have not been tested in this system.
b. Chromosomal Aberration Studies
(1) Mitotic Gene Conversion and Recombination in
Yeast
Monochlorobenzene and o-t m-, and j>-dichlorobenzene were
tested for their ability to induce reciprocal recombination in
Saccharomyces cerevisiae strain D3 (Simmon et al. 1979). Mono-
chlorobenzene at concentrations of 0.05% and 6% in the presence
of an S-9 mix produced a reproducible increase in the number of
recombinant cells per 10^ survivors over that seen in negative
control cultures (Simmon et al. 1979). Little or no recombino-
genic activity was observed in the S. Cerevisiae D3 assays with
^-dichlorobenzene. Although enzyme activation enhanced the
recombinogenic activity of _m-dichlorobenzene, it was not
necessary. ^n-Dichlorobenzene at concentrations of 0.001% -
0.025% was inactive in this system both with and without
metabolic activation. Results with j>-dichlorobenzene were
inconsistent and not reproducible. In three experiments,
recombination was increased, but survival data were inconsis-
tent. In two subsequent experiments, survival data were more
consistent, but no recombinogenic activity was noted. Monochlo-
robenzene at concentrations of 0.01-5.0 ul/plate did not induce
mitotic conversion at the trp 5 locus of S. cerevisiae strain D4
when tested with and without liver enzyme activation (Monsanto
1976a,b).
(2) Tests for Chromosomal Effects in Plants
Chromosomal effects can be measured in microorganisms,
plants, mammalian cells in culture, and whole animals. Although
the events measured in plants are similar to those in mammalian
cells, their relevance to man is sometimes questioned. However,
an examination of the literature comparing chromosomal effects
demonstrated in plants with those in mammalian cells in culture
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shows an excellent degree of correlation between the two systems
(Flamm 1977).
Ostergren and Levan, in a preliminary report (1943), indi-
cated that monochlorobenzene, o- and jn-chlorobenzene, 1,2,3-tri-
chlorobenzene, and hexachlorobenzene display C-mitotic activity
toward plant cells of Aliiurn cepa. The authors proposed that the
C-mitotic properties of organic compounds are not due to their
chemical properties (e.g. reactivity) but to their physical
properties (e.g. water solubility).
jD-Dichlorobenzene has been reported to cause chromosome
breaks in a variety of plant systems (Sharma and Bhattacharyya
1956, Sharma and Sarkar 1957, Srivastava 1966, Gupta 1972).
Although these studies are difficult to evaluate because neither
control data nor quantitative dose-response data were presented,
it is apparent that ja-dichlorobenzene does have an effect on
chromosomal structure.
c. DNA Repair Studies
The relative toxicity of monochlorobenzene and _o-, m-, and
jD-dichlorobenzene was assessed in repair-proficient and repair-
deficient strains of E_. coli (pol A+/pol A") and Bacillus
subtilis (Rec-^/Rec") (Simmon et al. 1979). _o_- and jn-Dichlo-
robenzene produced differential cell kill of repair proficient
and deficient strains of E. coli.
Monochlorobenzene at concentrations of 10 and 20 ul/plate
was equally toxic to both the pol A+ and pol A~ strains of E_.
coli and to rec+ and rec~ strains of j^. subtilis.
_o_- and jn-Dichlorobenzene at 20 ul/plate were more toxic to
—' C°H strain p3478 (pol A~) then they were to _E_. coli strain
W3110 (pol A+). j^-Dichlorobenzene and jn-dichlorobenzene were
each equally toxic to both the rec~ and rec+ strains of _B_.
subtilis.
]>-Dichlorobenzene at concentrations of 1 and 5 ul/plate had
no effect upon either the J^. coli or the J3_. subtilis strains. It
may be that jD-dichlorobenzene is nontoxic at the concentrations
tested or that it failed to diffuse away from the filter paper
disc into the medium to come in contact with the bacteria.
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2. Decision
The chlorinated benzenes have been reported to produce gene
mutation in some bacteria and fungi, mitotic recombination in
yeast, DNA damage in bacteria, and C-mitosis and chromosome
breaks and abnormalities in plants.
Monochlorobenzene, &-, nv-, and j>-dichlorobenzene and 1,2,3-
trichlorobenzene have been tested and found positive in one or
more systems designed to assess these effects. The first four of
these compounds have also been tested in the E. cpli WP2 and
Salmonella/microsomal assays with negative results.
Positive results in a test system are clear indications of
mutagenicity. Negative results in such systems are a bit
ambiguous. A negative result in a back mutation test, for
example, may mean that the chemical was non-mutagenic under the
test conditions, i.e., that it is mutagenic but the activity
could not be expressed. The agent may not be able to interact
with DNA at the specified locus, or mutations may be occurring at
sites in the DNA which are not detected in the test system.
Inactivity in any test system may mean that the conditions of the
test including pH, cofactors, and time of exposure were not
optimal for the test agent or that the metabolic activation
system was inappropriate for the chemical under test. The
significance of negative results in either bacterial or mammalian
cell culture assays must be assessed in relation to results in
other assays and viewed as part of a continuum rather than as
absolute reflections of mutagenic potential.
The activity of monochlorobenzene in the 5. antibioticus 400
system and the activity of the dichlorobenzenes in A. nidulans
show that these chlorinated benzenes do cause point mutations.
The chlorobenzenes are also genetically active in yeast where
they induce mitotic recombination in S_. cerevisiae. Mitotic
recombination can result in the expression of a recessive
homozygous condition which would not be expressed in the
heterozygous state. There is evidence that mitotic recombination
occurs in all eukaryotes, including mammals (Plamm 1977).
Although the significance of mitotic recombination in the human
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population is unknown/ the ability of the chlorobenzenes to
induce recombination indicates that they are potentially
genetically active agents. This potential is further emphasized
by the ability of these agents to interact with bacterial and
plant DNA.
In summary, the chlorinated benzenes have mutagenic
activity. Given the weight of the evidence, EPA considers the
chlorinated benzenes to have the potential to mutate the human
genome, which may pose a genetic risk to the population. Further
testing of these agents is needed to define the degree of hazard
that these agents represent to humans. However, as explained
below, EPA is not proposing such testing at this time.
3. Testing to be Sponsored by EPA
EPA believes that mutagenic risk from exposure to chlori-
nated benzenes can most reasonably be determined by performing a
sequence of tests for both gene mutation and chromosomal
aberration; the testing sequence under development by EPA is
described in Appendix B. In such schemes, the performance of
certain tests is triggered by positive or negative results from
previous tests. At this time, EPA is not proposing test
requirements for the mutagenicity sequence because the Agency has
not yet defined the criteria for determining whether the results
of each test are positive or negative. Such criteria are
important if the Agency is to establish a sequential testing
*
process in one rulemaking under Section 4. In addition, EPA has
not yet developed test standards to be followed for the DNA
alkylation tests in the gene mutation sequence, which is the
uppermost test in the proposed testing sequence. Nevertheless,
testing of chlorobenzenes for their mutagenic effects should not
be delayed solely because EPA cannot yet put in place all
elements necessary for the testing sequence. Since the tests are
relatively inexpensive, EPA plans to sponsor all tests in the
sequences except the two DNA alkylation tests for gene mutation
and the heritable translocation assay for chromosomal
aberration. On the basis of its test results, EPA will decide
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whether to propose that the final tests of each sequence be
performed.
EPA wishes to avoid duplicative testing where possible.
Therefore the Agency will coordinate its planned mutagenicity
tests on chlorinated benzenes with any such testing planned or
contemplated by the National Toxicology Program, which, as
described above under "Gene Mutation Studies," has scheduled some
mutagenicity tests on chlorinated benzenes.
G. Oncogenic Effects
1. Evaluation of Pertinent Studies
No tests for the oncogenicity of the chlorobenzenes have
been identified in the published literature. Although toxicity
studies by Hollingsworth et al. (1956, 1958) on o- and ^-dichlo-
robenzene were termed carcinogenicity testing by Ware and West
(1977), these investigations were conducted for 5 to 7 months and
therefore are too short to be considered as oncogenicity tests.
There are, however, human case reports of leukemia associated
with exposure to the chlorobenzenes.
a. Human Case Reports
Four cases of human leukemia have been associated with
exposure to the chlorinated benzenes (Girard et al. 1969, Tolot
et al. 1969).
The first case (Girard et al. 1969) involved a man
hospitalized for chronic lymphoid leukemia, after having worked
with a solvent containing 80 percent j>-dichlorobenzene, 15
percent jD-dichlorobenzene and 2 percent jn-dichlorobenzene for 10
years. This same solvent was implicated in the development of
acute myeloblastic leukemia in a 55-year-old woman who used it
for an unspecified period to clean spots from her family's
clothes. In another case (Girard et al. 1969), a 15-year-old
girl was hospitalized with acute myeloblastic leukemia and died
10 months later of peripheral leukoblastosis. She had habitually
removed dirt and grease stains from the clothes she was wearing
with a product containing 37 percent j3~dichlorobenzene. No
further details of these incidents were given.
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Tolot et al. (1969) presented the case of a 40-year-old man
who had been exposed to ^-dichlorobenzene for 22 years in the
preparation of dye stuffs. The subject exhibited purpura, intense
anemia, marked hepatomegaly, and discrete splenic enlargement.
The man died four months after the case was diagnosed as
myelocytic (myeloblastic) leukemia.
The absence of controlled conditions at the time of exposure
in all these case reports makes it difficult to determine cause
and effect. Thus, the case reports discussed provide only
inconclusive evidence of the leukemogenic potential of the
chlorinated benzenes.
b. Animal Studies
The Imperial Chemical Industries long-term inhalation study
in rats on j>-dichlorobenzene referred to in Section III.B.1.b.(2)
may provide information on oncogenicity. Any such information
that becomes available will be included in EPA's evaluation of
the chlorinated benzenes.
Three of the chlorinated benzenes, monochlorobenzene, _o_-di-
chlorobenzene, and j>-dichlorobenzene are being tested in long-
term bioassays directed by the National Cancer Institute (NCI).
Testing of monochlorobenzene began in January of 1979 and is
scheduled to be completed in February 1981. jD-Dichlorobenzene
testing began in March 1979 and is scheduled for completion in
February 1981. Testing of p-dichlorobenzene will begin in June
of 1980 and is scheduled for completion in June of 1982.
c. Other Indicators of Oncogenic Potential
The following sections discuss information that is often
suggestive of oncogenic potential but which is usually inadequate
evidence by itself to address the oncogenic hazard of a
substance.
(1) Mutagenic: Activity
The concept that neoplasms arise from mutations in somatic
cells was originally postulated by Boveri in 1914 to account for
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the unlimited variety of tumor types and the fact that, on cell
division, the daughter cells maintain their neoplastic properties
(Boveri 1929, Chu et al. 1977, Trosko and Chang 1978). Oncogens
and mutagens have two properties in common: 1) the ability to
transmit newly induced properties to their daughter cells and
2) the ability to convert normal cells into irreversibly changed
cells (Suss et al. 1973). Although the mutation theory of
oncogenesis is still waiting for unequivocal experimental proof,
the theory has recently gained more attention because of three
important findings. First, in the 1960's, the Millers at the
University of Wisconsin discovered that a majority of oncogens
required metabolic activation (Miller and Miller 1974, Miller
1978, Miller 1979); second, in vitro metabolic activation systems
which could be incorporated into mutagenicity assay systems were
developed (Mailing and Chu 1974); and third, comparison of the
ultimate reactive metabolites of structurally diverse oncogens
and mutagens revealed that the common denominator of these
substances is their electrophilicity, (i.e., they are compounds
that are able to react with electron-rich sites, or nucleophiles,
in cellular nucleic acids and proteins) (Bartsch 1976, Miller
1979). These three findings have now been verified by a host of
experimental data which show that oncogens can induce different
types of mutations including gene mutations (both base-pair
substitution and frame-shift alterations), chromosomal
aberrations, and non-disjunctions. The oncogenic potential of a
chemical has been correlated with its ability to interact with
and modify DNA (Rosenkranz and Poirier 1979). These
considerations have resulted in the general acceptance of
mutagenicity tests as indicators of oncogenic potential (Miller
1978).
The chlorinated benzenes have been evaluated for mutagenic
activity in a number of test systems including bacteria, fungi,
yeast, mammalian cells in culture, and plants. The results of
these studies indicate that several of the chlorinated benzenes
possess mutagenic potential. In addition, the chlorinated
benzenes have been shown to produce DNA damage in bacterial
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systems and chromosomal damage in plants. These studies are
discussed in detail in the Mutagenicity Section of this document
(Section III. F) . Although the exact relationship between a
chemical's mutagenic activity in these systems and its
oncogencity is unknown, EPA must consider these positive
mutagenicity test results suggestive evidence of the oncogenic
potential of the chlorinated benzenes.
(2) Structural Relationships
Known chemical oncogens comprise a stsructurally diverse
group of substances. (Miller and Miller 1974, Miller 1979).
However, many chemical oncogens can be divided among a smaller
number of structural classes, and a structural relationship to a
known oncogen or class of oncogens is a reasonable basis for
suspecting an untested chemical of oncogenic activity (Arcos
1978).
The chlorinated benzenes are structurally similar to two
recognized oncogens: benzene, which has been shown to produce
tumors in Sprague-Dawley rats (Maltoni and Scarnato 1979) and
which has been implicated as a leukemogenic agent in humans
(Aksoy et al. 1972, 1976, Tareeff et al. 1963), and hexachloro-
benzene, which has been shown to produce tumors in Syrian golden
hamsters (Cabral et al. 1977) and in outbred Swiss mice (Cabral
et al. 1979).
(a) Benzene
There have been over 100 case reports, a number of epidemio-
logic studies, and an animal study implicating benzene as an
oncogen (Tareeff et al. 1963, Aksoy et al. 1972, 1976, USEPA
1978a, Maltoni and Scarnato 1979). It has been established that
benzene is toxic to the human hematopoietic system and may induce
blood and bone marrow changes that may in turn lead to the
appearance of leukemia in man (USEPA 1978a). Most individuals
and scientific groups have accepted a causative role for benzene
in the development of human acute myelogenous (myeloblastic)
leukemia (USEPA 1978a).
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The recent study by Maltoni and Scarnato (1979) reveals the
oncogenic potential of benzene in laboratory animals. In this
study, groups of 60 and 70 Sprague-Dawley rats (13 weeks old at
initial dosing) were given 50 mg/kg and 250 mg/kg of benzene,
respectively, via stomach tube, once daily, 4-5 days weekly, for
52 weeks. The animals were observed until spontaneous death 10-
62 weeks later. A significant incidence of Zymbal gland carcino-
mas was observed together with an increased incidence of mammary
carcinomas and leukemias. This study is significant because it
is the first demonstration of the carcinogenicity of benzene in
animals. In addition, the study reinforces the epidemiologic
evidence of the leukemogenicity of benzene in humans.
(b) Hexachlorobenzene
Hexachlorobenzene has been shown to be oncogenic in two
animal species. When fed diets consisting of 50, 100, and 200
ppm of hexachlorobenzene for life (from 50-70+ weeks), Syrian
golden hamsters developed significant numbers of tumors at all
dose levels in a variety of tissues including the liver, thyroid,
and spleen (Cabral et al. 1977). In a later study, Cabral et al.
(1979) demonstrated a much less pronounced oncogenicity (liver
cell tumors) in outbred Swiss mice given 100 or more ppm of
hexachlorobenzene in their diet for about 100 weeks.
The accidental poisoning of over 3000 people in Turkey
between 1955 and 1959 has identified a number of other hexachlo-
robenzene-induced toxic effects in humans; they include porphyria
with concomitant cutaneous, hepatic, and neurologic effects
(Cabral et al. 1979). Many of these undesirable symptoms have
persisted in these individuals throughout the past twenty
years. Enlarged thyroids observed in the exposed population are
presently being viewed with suspicion in light of Cabral's
earlier finding of thyroid tumors in hamsters. It has not been
determined, however, whether goiter problems are endemic to the
Turkish population as a whole. No significant incidence of any
oncogenic response has yet been reported in the population under
study.
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(3) Metabolism in Common with Known Oncogens
The metabolic pathways of benzene and its chlorinated
derivatives, illustrated in Figures 1-7 in Section III.B.1.c.(1),
have many similarities. One significant similarity is that these
compounds are metabolized to electrophilic arene oxides that
should be able to bind covalently to nucleophilic centers in
vital cellular macromolecules. Experimental evidence of this
capability is the covalent binding of monochlorobenzene, _p_-
dichlorobenzene, and several other halogenated benzenes to
cellular proteins (Reid and Krishna 1973).
Since benzene is metabolized to an arene oxide, and since
benzene has been shown to bind covalently to DNA (Lutz and
Schlatter 1977), its oncogenicity may be attributable to the
formation of covalent bonds with cellular nucleophiles (see also
discussion under part (1), Mutagenic Activity, of this
subsection), and it is a reasonable assumption that chlorinated
benzenes have the metabolic potential to initiate the same
process.
(4) Tumor Enhancement Potential
Preliminary evidence suggests that the chlorinated benzenes
and/or their metabolites may enhance the development of tumors in
animals exposed to carcinogens. Several phenolic metabolites of
the chlorinated benzenes have been shown to promote skin onco-
genicity in mice, while the structurally related hexachloroben-
zene has been implicated as an enhancing factor in the induction
of hepatic cancer in mice. These studies are discussed briefly
below.
Phenol, 2-chlorophenol, 3-chlorophenol, and 2,4,5-trichloro-
phenol were evaluated by Boutwell and Bosch (1959) as potential
promoters of oncogenicity in mice. The animals were painted for
12 to 24 weeks with the promoter following topical administration
of one dose of the initiator dimethylbenz(a)anthracene. They
were observed for periods of up to 40 weeks. The experiment
revealed promoter activity for all four compounds. This activity
is relevant to the chlorinated benzenes because skin has been
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shown to contain the enzyme system capable of hydroxylating
aromatic compounds to phenols (Pannatier et al. 1978). 2-Chloro-
phenol and 3-chlorophenol are known metabolites of monochloroben-
zene, and 2,4,5-trichlorophenol is a metabolite of 1,2,4-trichlo-
robenzene.
Hexachlorobenzene apparently enhances the hepatocarcino-
genicity of polychlorinated terphenyls in ICR mice (Shirai et al.
1978). While hexachlorobenzene is an oncogen in long-term tests
(Cabral et al. 1977, 1979), in a short-term study (24 weeks of
treatment followed by 16 weeks of observation) by Shirai et al.,
hexachlorobenzene at 50 ppm in the diet was not observed to give
an oncogenic response. The incidence of hepatocellular carcinoma
in mice fed polychlorinated terphenyls (250 ppm) plus hexachloro-
benzene (50 ppm) for 24 weeks was found to be significantly
higher (p <0.01) than when the same dose of polychlorinated
terphenyls was fed alone (30.8 percent and 14.3 percent,
respectively). The authors suggest that the enzyme-inducing
ability of hexachlorobenzene may be responsible for the
apparently enhanced carcinogenicity of the polychlorinated
terphenyls, or that if the hexachlorobenzene is weakly
carcinogenic toward these mice, then the two agents may act
synergistically to induce the liver tumors. However, the lack of
information as to the mechanism by which hexachlorobenzene causes
the observed effect makes it difficult to assess the significance
of this information for the chlorinated benzenes, and it can only
be considered suggestive.
2. Decision
There is considerable evidence that suggests that chlori-
nated benzenes may be oncogenic. This evidence includes:
a. Case reports of leukemia in humans exposed to
the chlorinated benzenes;
b. Mutagenic activity;
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c. Structural relationships;
d. Metabolism in common with "known oncogens; and
e. Reports of tumor-enhancement potential of
chlorinated benzene metabolites.
EPA believes the weight of this evidence demonstrates that
exposure to chlorinated benzenes may present an unreasonable risk
of oncogenic effects in humans.
The mutagenic activity of chlorinated benzenes has been
discussed in Section III.F. Evidence for oncogenicity or
leukemogenicity of chemicals structurally related to chlorinated
benzenes includes over 100 human case reports and results of an
animal study for benzene, and hamster and mouse studies for
hexachlorobenzene. Benzene appears to undergo metabolic
transformations similar to those found for many chlorinated
benzenes, such as conversion to phenolic and catecholic products
and to mercapturic acids(see Section III.B.1.c.(1).). It is
likely that such products are formed by way of intermediate
epoxides, which, in addition to producing the metabolites just
referred to, can react with nucleophilic macromolecules such as
DNA and protein. Tumor-promoting potential in mice has been
observed for the chlorinated benzene metabolites 2-chlorophenol,
3-chlorophenol, and 2,4,5-trichlorophenol.
Although the above information suggests oncogenic potential
for chlorinated benzenes, it is not sufficient for a determi-
nation of oncogenic hazard. Mutagenic activity as determined by
short-term _in vivo and in vitro testing cannot always be
correlated with oncogenic activity in mammalian species.
Similarly, although the likelihood that the chlorinated benzenes
as a group may be oncogenic is supported by structural relation-
ships, such evidence in the absence of test data on any
chlorinated benzenes is deemed insufficient to allow for the
adequate evaluation of oncogenic hazard for the chlorinated
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benzenes. EPA concludes on the basis of this evidence that
evidence is insufficient to determine whether exposure to
chlorinated benzenes presents a risk of oncogenicity.
3. Proposed Testing
EPA proposes that the chlorinated benzenes, with the
exceptions noted below, be tested for oncogenicity in a two-year
study in rodents according to the EPA guidelines proposed in the
Federal Register, May 9, 1979 (USEPA 1979b). The guidelines call
for the use of two species of rodent in the study, the rat and
the mouse. Because the Maltoni and Scarnato study has recently
found Sprague-Dawley rats to be sensitive to the oncogenic
effects of benzene (a structurally related compound), EPA is
proposing the use of this strain of rats in the proposed tests.
Monochlorobenzene, ^-dichlorobenzene, and p-dichlorobenzene
are not included in this proposed oncogenicity testing because
they are being tested under the direction of the National Cancer
Institute. While the NCI bioassay protocol differs from the
oncogenicity testing standards proposed by EPA, EPA is
tentatively accepting these differences in testing approaches.
When the results of the NCI tests become available, the Agency
will include them in its continuing evaluation of these
chemicals.
H. Epidemiology
At this time, the EPA is not in a position to develop a test
rule for an epidemiology study on chlorinated benzenes because
the Agency lacks specific information on a suitable cohort. The
EPA is proposing a rule under Section 8(a)(2)(F) of TSCA for
chlorobenzenes as well as other ITC chemicals. Section 8(a)
authorizes EPA to obtain readily accessible information from the
files of manufacturers, processors, and importers on use,
production, and worker exposure for specified chemicals. This
information will play a key role in the identification of a
cohort for epidemiologic studies. If a suitable cohort can be
found, the Agency will decide after reviewing any available
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results from Section 4 testing whether to propose that an
epidemiology study be undertaken.
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IV. Materials to be Tested and Justification for Sampling
EPA is proposing that a representative sample of chemicals
in the chlorinated benzenes group be tested initially. This
sample consists of the following chemicals:
1) Monochlorobenzene
2) 1,2-Dichlorobenzene (ortho-Dichlorobenzene)
3) 1,4-Dichlorobenzene (para-Dichlorobenzene)
4) 1,2,4-Trichlorobenzene
5) 1,2,4,5-Tetrachlorobenzene
6) Pentachlorobenzene
The chlorobenzenes used in health effects testing should be
substantially free of potentially interfering impurities in order
that the test results can be interpreted as unambiguously as
possible (for a general discussion of factors involved in the
selection of test materials, see the Preamble to the proposed
Chlorinated Benzenes test rule). The EPA believes that this
condition can be met by testing chlorobenzenes at 99.9 percent or
greater purity, with a benzene content of no greater than 0.05
percent and a hexachlorobenzene content of no greater than 0.05
percent; these limits are imposed because benzene and
hexachlorobenzene have toxic effects that could lead to ambiguous
test results. Commercial monochlorobenzene has been reported to
contain below .05 percent benzene (Section II.A.I), and EPA
believes that this level of benzene will not interfere with the
proposed toxicity testing. The Agency also feels that the
amounts of benzene in purified samples of the dichlorobenzenes
and higher chlorobenzenes are not likely to exceed 0.05
percent. EPA further believes that a hexachlorobenzene content
of 0.05 percent will not confound the test results, and that
these criteria can be satisfied without excessive difficulty by
the purification of commercially available materials. Thus,
monochlorobenzene is or has been commercially available at a
purity of 99.9 percent (see Section II.A.). Corresponding
figures for other members of the sample are 99.0 percent for _p_-
dichlorobenzene, 99.95 percent for ja-dichlorobenzene, 100 percent
for 1,2,4-trichlorobenzene, and 97.0 percent for 1,2,4,5-
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tetrachlorobenzene; no figure was found for pentachlorobenzene.
EPA welcomes comment on the ease of further purification of the
less pure materials to the 99.9 percent figure.
The Agency's decision to propose testing of a representative
sample rather than testing of all 11 category members rests in
part on the chemical nature of the category and in part on avail-
able data on the biological effects of chlorinated benzenes.
As discussed in Section I.A.2. of this Chlorinated Benzenes
Support Document, the structural relationships among the chlori-
nated benzenes lead to the expectation of regular progressive
changes in properties going through the series from mono- to
pentachlorinated benzene, with the discontinuities that arise
from different isomeric arrangements of chlorine and hydrogen
atoms being relatively minor in comparison with the overall
trends. This expectation is supported by trends in physicochemi-
cal data of which several appear in Table 1, Section I. Thus, in
proceeding from monochlorobenzene to pentachlorobenzene, densi-
ties, boiling points and partition coefficients show a gradual
increase, while water solubility decreases.
Since physicochemical properties determine, in a complex
fashion, the biological effects of a substance, the observed
regularity in these properties of the category provides a basis
for expecting that biological data on a well-chosen sample of
category members can be used to characterize the biological
behavior of the untested members. This is not to imply that all
*
category members will necessarily have identical effects or
similar potencies for a given effect. In choosing the category
test sample, an important factor is that, with increasing
chlorination, chlorobenzenes will be more resistant to metabolic
attack and more likely to be retained in body tissues (see
Section III.B.1.c.(1), Metabolism). Thus a sample including only
mono- and dichlorobenzenes would be unrepresentative because it
would include only the compounds most subject to metabolic attack
and least likely to be stored in tissues. EPA is, therefore,
proposing a test sample that includes all levels of
chlorination. Further, the Agency believes that relative
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production volume should be an important factor in the sample
selection. Applying these two criteria leads to the choice of
monochlorobenzene, jo_- or p_-dichlorobenzene, 1,2,4-
trichlorobenzene, 1,2,3,4- or 1,2,4,5-tetrachlorobenzene, and
pentachlorobenzene. EPA has decided to include both _o_- and ja-
dichlorobenzene in its test sample for two reasons. First, both
have widespread general population exposure. Second, it seems
prudent to include more than one isomer for at least one level of
chlorination in order to provide information on to what extent
the toxic effects of chlorobenzenes may be affected by the
distribution of chlorine atoms. The 1,2,4,5-isomer of tetrachlo-
robenzene was chosen because its production is somewhat higher
than that of the 1,2,3,4-isomer, and because there is not a more
compelling reason to distinguish between them.
The six sample chemicals thus represent all levels of
chlorination, the full range of physicochemical properties, and
compounds having the highest commercial production among the
chlorinated benzenes. Available data on chlorinated benzenes not
included in the testing sample will serve as additional data
points for evaluation of chlorobenzene toxicity when the test
results become available.
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V. Route of Administration
The selection of the route of administration of a test
substance emphasizes the following considerations: (a) the
physical and chemical properties of the test substance, such as
volatility under conditions of probable or actual human exposure,
(b) the predominant portal(s) of entry of the test substance in
man, (c) the practicability of experimentally approximating the
probable conditions of human exposure, given the physical and
chemical constants of the test substance and the relative
adaptability of the test species to the proposed route of
administration.
For subchronic, morphological teratogenicity, reproductive
effects, behavioral teratogenicity and neurotoxicity testing, EPA
is proposing that monochlorobenzene be tested with inhalation as
the route of administration. Monochlorobenzene is a volatile
liquid, used primarily as a solvent and as an intermediate for
the synthesis of chloronitrobenzenes. It appears that inhalation
would be the most likely exposure route for humans. It is
proposed that ortho- and para-dichlorobenzene likewise be tested
for these effects with inhalation as the route of
administration. Both of these compounds are used in a variety of
household products. para-Dichlorobenzene is a solid that
sublimes readily. Inhalation is the most likely exposure route
for humans for the two dichlorobenzenes in both the occupational
setting and in the home. It is proposed that 1,2,4-
trichlorobenzene, a liquid, be tested with oral gavage as the
route of administration for the teratogenicity and acute
neurotoxicity studies. It shall be administered in the diet for
the subchronic, oncogenicity, reproductive, and subchronic
neurotoxicity tests. This compound is partly used as a dye
carrier. Upon completion of the dyeing process, the carrier is
removed from the fabric and disposed. 1,2,4-Trichlorobenzene has
been identified in drinking water and it appears that the most
likely exposure route for humans would be orally through the
water supply. It is proposed that 1,2,4,5-tetrachlorobenzene and
pentachlorobenzene, both crystalline solids, be mixed in the diet
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for administration to the animals for the purposes of subchronic/
oncogenicity, reproductive, and subchronic neurotoxicity studies
and be administered by oral gavage for acute neurotoxicity
studies. In the case of 1,2,4,5-tetrachlorobenzene, the route of
administration for both morphologic and behavioral teratogenicity
studies should be oral gavage; for subchronic studies, it should
be mixed in the diet. For pentachlorobenzene, gavage is the
proposed route for behavioral teratogenicity (morphologic
teratogenicity and subchronic testing are not proposed for
pentachlorobenzene). In teratogenicity studies, the test
chemical should not be added to the feed or water since reduction
in food or water intake may seriously compromise the value of the
study. 1,2,4,5-Tetrachlorobenzene and pentachlorobenzene have
been found in fresh water fish and in herring gull eggs while
pentachlorobenzene has been found in many foods. Therefore, the
most likely exposure route for humans is orally through the food
supply.
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Appendix A
Use of Information From The TSCA Inventory
The TSCA Section 8(b) Chemical Inventory includes both
nonconfidential and confidential information. Because TSCA
Section 14 prohibits the disclosure of confidential data with
certain exceptions, only non-confidential data have been used in
this support document.
There are a number of qualifications associated with the use
of non-confidential Inventory information. Obviously, because a
manufacturer or importer could claim any or all of the
information submitted as confidential , the non-confidential data
exclude any confidential information. Another qualification is
that production volumes are reported for only one year, 1977.
Finally, production and imports are reported in ranges rather
than in specific figures, which not only increases uncertainty
but makes meaningful aggregation of data difficult. (A thorough
discussion of the meaning and the limitations of the chemical
data is available in the Introduction in Volume 1 of the
published TSCA Chemical Substance Inventory. A thorough
discussion of the meaning and the limitations of the
manufacturer's data is contained in the Inventory Reporting
Regulations (40 CFR 710).)
Nevertheless, the publicly available Inventory data are
often a useful addition to other information about a substance.
In the present document, they have been used particularly to
supplement fragmentary or out-of-date information from other
sources. Two conventions were adopted for presenting Inventory
information: 1) Where the number of importers or manufacturers
of a substance is given, those who specifically reported no
A manufacturer or importer could claim any of the following
items as confidential: company name; site; specific chemical
identity; whether the chemical substance is manufactured,
imported or processed; whether the activity in the chemical is
site-limited; and the quantity manufactured, processed, or
imported.
127
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activity for the chemical in 1977 are not included. In a few
cases, numbers of nonactive companies are also given but always
as an explicit separate item; 2) Production volumes are stated in
two ways. Where the highest range of production reported for a
substance is 100,000 to one million pounds or lower, either a
minimum figure is given or an aggregated figure is cited which is
essentially a crude order-of-magnitude estimate. In other cases,
the range is stated rather than an aggregate.
As an example, take the statement that, according to
Inventory data, six manufacturers produced substance A in 1977,
with two reporting production of 10 million to 100 million
pounds. This means that six manufacturers who did not claim
their identities confidential reported production in 1977. There
may have been others who reported manufacturing substance A since
1974 but who made none in 1977. There may also have been one or
more others who reported as both producer and importer but did
not specifically assigji the volume reported to either activity
(in the support document, such cases have sometimes been included
as a separate item). Finally, although at least 20 million
pounds of production of substance A has been reported, there may
have been additional 1977 production by one or more of the six
manufacturers that cannot be disclosed.
128
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Appendix B
Proposed Mutagenicity Testing Sequence
In recent years, mutagenicity experts have discussed and
provided guidance on hazard estimation procedures for determining
if a chemical is a potential human mutagen. The following
describes the basis for EPA's approach to mutagenicity testing
under Section 4 of TSCA.
Four major reports on the hazards of environmental mutagens
were issued between 1975 and 1979 (Drake 1975, Plamm 1977, NAS
1977, McElheny and Abrahamson 1979). In 1978 the Office of
Pesticide Programs proposed Guidelines for Registering Pesticides
in the U.S. (USEPA 1978b). Addendum III to these guidelines,
"Criteria for Evaluating the Mutagenicity of Chemicals," contains
a discussion of the scope and nature of the human genetic disease
burden. The Preamble to the guidelines contains EPA's rationale
for using mutagenicity data derived from non—human test systems.
These reports agree that to perform a mutagenicity hazard
estimation for humans, scientists must first demonstrate that a
substance and/or its metabolite(s) cause heritable gene or
chromosomal mutations (the two classes of mutagenic damage which
have been shown to be responsible for a portion of human genetic
disease) and that the mutagenically active form can reach the
genetically significant target molecules in mammalian germinal
tissue.
A discussion of the principles and practices of mutagenicity
testing in terms easily understood by persons unfamiliar with
mutagenicity is presented in the EPA's booklet "Short-Term Tests
for Carcinogens, Mutagens, and Other Genotoxic Agents" (Trontell
and Connery 1979).
The following points are essential to such a rationale and
are generally accepted by experts in the field of mutagenesis
(see e.g., Drake 1975, Flamm 1977, McElheny and Abrahamsom
1979). They are:
129
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(1) All organisms (except for a few viruses) have DNA as the
genetic material, which contains all of the information
necessary for survival and reproduction.
(2) The DNA code is the same in all organisms.
(3) The cellular machinery for decoding the information
stored in the DNA code is similar among all organisms.
(4) Eukaryotic cells contain nuclei within which DNA,
complexed with protein, forms complex bodies called
chromosomes. Prokaryotic organisms lack nuclei, and
their chromosome structure differs from that of
eukaryotic organisms.
(5) Unless a mutation occurs, the information in DNA is
faithfully replicated in each cell generation in both
unicellular organisms and somatic and germ cells of
multicellular organisms.
(6) DNA can be altered by chemicals. Tf this damage is
repaired properly there is no mutation. If it is
repaired with error or not repaired prior to replication
of DNA, mutation can result. A single lesion in DNA may
lead to a mutation.
(7) Point mutations usually involve changes in the bases of
the DNA chain. The replacement of one purine or
pyrimidine nucleotide by another is called base pair
substitution; insertion or deletion of a base pair into
the DNA chain is called a frameshift mutation.
(8) Breaks in DNA may lead to structural chromosomal
aberrations.
130
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(9) Disturbances in the distribution of individual
chromosomes or chromosome sets can occur during cell
division and result in numerical chromosomal aberrations.
(10) Mutations are generally considered to be deleterious to
an organism and to result in decreased survival and
reproduction. Although not all mutations are deleterious
(e.g., the Ames test measures a mutation which is
advantageous to the organism), it is impossible to
predict the effect of an unfamiliar alteration in the
genome.
Given the ubiquitous nature of DNA as the genetic material,
the universality of the genetic code, and the similarity in
response of genes and chromosomes of various lifeforms, a
rationale for using the results from different test systems
develops. Humans, as well as bacteria, fungi, and higher
organisms suffer DNA damage and gene mutations; man, as well as
other eukaryotes, shows structural and numerical chromosomal
aberrations. For these reasons, cells of any species may be used
to detect genetic changes and to predict genetic change or damage
in other species, and in vitro testing can thus provide an
initial indication of mutagenic potential.
1. Test for Gene Mutations
As just noted, a potential for causing gene mutations can be
detected by subjecting a substance to in vitro tests.
The ability of a substance to cause heritable mutations can
be detected by a sex-linked recessive lethal test in Drosophila
melanogaster. The frequency of induced mutations is also
determined in this test.
In order to show that a suggested mutagen can reach and
interact with mammalian germinal tissue, the mouse sperm
alkylation test can be carried out. This test gives a measure of
the relationship between mammalian body exposure to a mutagen and
the resulting mammalian gonadal dose. Methods for analysis of
131
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mouse sperm DNA alkylation using radiolabelled alkylating agents
have been published (Sega et al. 1974). To estimate genetic risk
from a dose measured as alkylations per nucleotide in the germ
cells (a large fraction of known mutagens either alkylate DNA or
are metabolized into alkylating agents), it is necessary to
determine a dose-response relation in an experimental system that
can be thoroughly analyzed for mutations induced in the different
germ cell stages. Methods for determination of dose of
alkylating agents measured as alkylations per nucleotide in the
germ cells of Drosophila melanogaster have been developed (Lee
1978, Aaron and Lee 1978). With the combination of the mouse and
Drosophila sperm alkylation systems, mammalian body exposure can
be related to mutagenic potential using molecular dosimetry as a
bridge between the two systems.
In summary, in vitro bacterial mutagenesis assays provide an
indication of basic mutagenic potential. In the case of a test
for DNA damage and repair, the test indicates whether the test
substance can interact with DNA without indicating whether the
interaction can lead to mutagenesis. The Drosophila sex-linked
recessive lethal test detects heritable gene mutations in an
insect system amenable also to molecular dosimetry. The mouse
sperm alkylation test measures the ability of a given chemical
and/or its metabolites to reach and to interact with the DNA of
mammalian germinal tissue. This information will be used by EPA
to estimate mutational risk to mammals from exposure to
chlorinated benzenes and ultimately to assess the genetic hazard
of human exposure.
It is possible that an agent capable of producing heritable
gene mutations in mammals would not be detected in Drosophila
because of species differences between insects and mammals. In
such an instance, a positive result in a second system such as
mammalian cell culture, together with a demonstration of the
ability of the chemical to reach and interact with mammalian
germinal tissue DNA, is sufficient evidence to classify a
chemical as a potential human mutagen. Data gained from
alkylation of the DNA in mammalian cell cultures may be used to
132
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estimate risk to man in a manner analogous to that described
above for Drosophila if data cannot be collected from the insect
system.
If it cannot be shown that a chemical or its reactive
metabolites can reach and interact with mammalian germinal tissue
DNA, there is no evidence the chemical is a potential human
mutagen, and hazard assessment is unwarranted.
A testing scheme for chlorinated benzenes based on the
foregoing discussion is shown in Figures 1-4. The first test in
the in vitro assay (Figures 2-4) utilizes Aspergillus nidulans,
since three of the chlorobenzenes have produced positive results
in this system and testing of chlorobenzenes in the
Salmonella/rnicrosomal system has been negative (a detailed
discussion of existing test data for chlorinated benzenes appears
in Section III.F. of this Support Document). If the Aspergillus
assay is negative, the higher chlorinated benzenes should be
tested for activity in both mammalian cell culture and DNA repair
assays before concluding that they do not exhibit mutagenic
potential. Not all compounds are proposed to be tested in the
entire test battery because some compounds have been adequately
characterized in some tests and can be started further along in
the sequence. Thus, testing of monochlorobenzene begins with the
sex-linked recessive lethal test in Drosophila because this agent
has already been adequately tested in assay systems for the
induction of point mutations in bacteria and fungi. On the other
hand, EPA does not believe that monochlorobenzene should be
tested in mammalian cells in culture because this agent has been
adequately tested in this system and found to be inactive.
EPA does not plan to require a mouse specific locus test for
the chlorinated benzenes to demonstrate the induction of
heritable gene mutations. The mouse sperm alkylation and
Drosophila sperm alkylation tests were both designed to detect
the ability of known alkylating agents to react with the DNA of
germinal tissue and are extremely sensitive (Sega et al. 1974,
Aaron and Lee 1978). For alkylating agents or for those agents
133
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MONOCHLOROBENZENE
Gene Mutation Testing Scheme
Known Positive in
Streptomyces
EPA
WILL
positive
Drosophila Sex-Linked
Recessive Lethal
positive
1
-negative-
•Stop
Mouse Sperm
Alkylation—
positive
Drosophila Sperm
Alkylation
-negative-
-•-Stop
FIGURE 1
134
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a- and E-DICHLOROBENZENE
Gene Mutation Testing Scheme
Known Positive
in Aspergillas
positive
EPA
r H
WILL
TEST
i
Sex- Linked
Recessive
Lethal
i
in Mamm
tation —
alian
Cell Culture
negative ^ Stop
positive
-I
positive
Stop «• negative Mouse Sperm
Alkylation
positive
Drosophila
Sperm Alkylation
Mouse Sperm-
Alky 1 at ion
positive
Mammalian Cell
Culture Alkylation
-negative m Stop
FIGURE 2
135
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TRICHLOROBENZENE AND HIGHER
Gene Mutation Testing Scheme I
(Positive Aspergillus Assay)
1
1
EPA
WIL
TES
Aspergillus
positive
Sex-Linked in Mammalian
Recessive Cell Culture
i
Mou
Alk
pos
^Lethal
positive positive
ylation Sperm
Alkylation
i :ive posi
tive
Drosophila Mammalian Cell
Sperm Culture
Alkylation Alkylation
FIGURE 3
136
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TRICHLOROBENZENE AND HIGHER
Gene Mutation Testing Scheme II
(Negative Aspergillus Assay)
EPA
WILL
TEST
Aspergillus
negative
Gene Mutation-
Mammalian
Cell Culture
negative
DNA Damage
and Repair-
positive
I
Drosophila-
Sex-Linked
Recessive
Lethal
positive
-positive
negative
-negative-
• Stop
Mouse Sperm-
Alkylation
positive
Drosophila Sperm
Alkylation
-negative-
•Stop-
•negative-
-Mouse Sperm
Alkylation
positive
Mammalian Cell
Culture
Alkylation
FIGURE 4
137
-------
such as chlorinated benzenes that are metabolized to alkylating
intermediates (see Section III.B.)/ DNA alkylation may possess
superior sensitivity to the mouse specific locus test. This test
is appropriate, therefore, for measuring the ability of the
chlorinated benzenes to interact with the DNA of germinal
tissue. For these reasons, EPA considers a mouse specific locus
test on the chlorinated benzenes not to be warranted at this
time.
As discussed in Section III.F.3, EPA will sponsor all gene
mutation tests on chlorinated benzenes except the two DNA
alkylation tests. EPA will follow its proposed test standards
(USEPA 1979c) for all tests that it sponsors except mammalian
cell culture alkylation. Protocols for rodent sperm alkylation,
Prosophila alkylation, and mammalian cell culture alkylation have
been published by Sega et al. (1974), Aaron and Lee (1978), and
Aaron et al. (1980).
2. Tests for Chromosomal Aberrations
Chromosomal aberrations may be detected in a variety of
animal and plant systems employing both in vitro cell culture and
whole animal techniques (Flamm 1977). Because EPA is unaware of
tests on chlorinated benzenes for chromosomal aberrations using
mammalian systems, the Agency intends to test these substances
for chromosomal aberrations in a sequence beginning with a test
using mammalian cells in culture. Because it is possible that
some agents that are not detected in in vitro systems may be
detected in whole animal systems, the sequence includes a test
for chromosomal aberrations in vivo following a negative in vitro
cytogenetics assay. No further testing for chromosomal
aberrations is indicated if both the in vitro and in vivo
cytogenetics tests are negative. A positive cytogenetics assay
is followed by a dominant lethal test to demonstrate the effect
of the chlorinated benzenes on germinal cell chromosomes. Brewen
et al. (1975) have shown that the incidence of chromosomal breaks
at first cleavage of the fertilized egg is proportional to the
number of dominant lethals that occur after treatment and mating.
138
-------
No further testing for chromosomal aberrations is done if the
dominant lethal test is negative. A heritable translocation test
shows the ability of a chemical to induce heritable chromosomal
aberrations. This test, therefore, can be used not only to
detect potential mutagens but also for purposes of assessing
mutagenic hazard. A positive dominant lethal assay is thus
followed by a heritable translocation test, the results of which
will be used for hazard assessment. No further testing for
chromosomal aberrations is indicated if the heritable
translocation test is negative.
The test sequence for chromosomal aberrations proposed by
EPA for the chlorinated benzenes is shown diagrammatically in
Figure 5. As discussed in Section III.F.3, EPA will sponsor all
chromosomal aberration studies on chlorinated benzenes except the
heritable translocation assay. The standards EPA intends to
follow for all these tests except the in vitro cytogenetics have
been proposed by EPA (Trontell and Connery 1979). The Agency is
seeking comment on the proposed sequence from all interested
parties.
139
-------
CHLORINATED BENZENES
Test Scheme for Chromosomal Aberrations
1
El
JO
T]
i
i
In Vitro
Cytoge
A P°Si
LLL (
"OT1 i
Lethal
posi
1
letics
;ive
lant
Assay
tive
— negative ^ In Vivo negative <» Stop
Cytog
pos
1
Dom
Letha
posi
1 i
snetics
itive
inant negative ^ Stop
1 Assay
tive
1
Heritable
Translocation
Assay
Heritable
Translocation
Assay
FIGURE 5
140
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Appendix C
Occupational Exposure Limits for Chlorinated Benzenes
The American Conference of Governmental Industrial Hygienists has
recommended the following threshold limit values (TLV), expressed
as a time-weighted average (TWA) or short-term exposure limit
(STEL) as indicated (ACGIH 1978). The TWA for the first three
compounds listed have been adopted as standards by the
Occupational Safety and Health Administration (USOSHA 1974).
monochlorobenzene
j>-dichlorobenzene
j£-dichlorobenzene
ja-dichlorobenzene
1,2,4-trichlorobenzene
75 ppm
50 ppm*
75 ppm
110 ppm
5 ppm*
(350 mg/m3)
(300 mg/m3)
(450 mg/m3)
(475 mg/m3)
(40 mg/m3)
TWA
TWA
TWA
STEL
TWA
* mixture notation—where mixtures are used, the limit
recommended for the most toxic compound must be taken into
consideration when evaluating the exposure.
141
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1 REPORT NO
iPA-560/11-80-014
3. RECIPIENT'S ACCESSION-NO.
4 TITLE AND SUBTITLE
Support Document Health Effects Test Rule
Chlorinated Benzenes
5. REPORT DATE
June 1980 (approved)
6. PERFORMING ORGANIZATION CODE
7 AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
9 PERFORMING ORGANIZATION NAME AND ADDRESS
10. PROGRAM ELEMENT NO.
Assessment Division/Office of Pesticides & Toxic Sub-
401 M Street, SW stances
Washington, DC 20460
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
13. TYPE OF REPORT AND PERIOD COVERED
U. S, Environmental Protection Agency
401 M Street, SW
Washington, DC 20460
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Since chlorobenzenes are used as chemical intermediates and for other
industrial purposes as well as in consumer products, there is very broad potential
exposure. Thus, there is known or potential exposure of workers involved in chloro-
benzene production, processing, and use, and of the general population, both directly
from consumer products and indirectly through the environment. For this reason and
on the basis of limited toxic effects studies, EPA has proposed that certain chloro-
benzenes be tested to assess their potential to cause chronic, reproductive, terato-
logical, and oncogenic effects. Following resolution of methodology issues the
Agency has raised, EPA will propose at a later date test rules for neurotoxic and
mutagenic effects. Further, the Agency has decided not to propose test rules for
acute toxicity and epidemiological studies.
Bibliography included.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OP-EN ENDED TERMS C. COSATI Field/Group
13. DISTRIBUTION STATEMENT
Release unlimited
19. SECURITY CLASS (ThisReport)
unclassified
21. NO. OF PAGES
20. SECURITY CLASS (This page)
unclassified
22. PRICE
EPA Form 2220-1 (9-73)
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