CHLOROMETHANE
DRAFT SUPPORT DOCUMENT FOR
TSCA SECTION 4 TEST .RULE
Test Rules Development Branch
Assessment Division
Office of Testing and Evaluation
Office of Toxic Substances
Environmental Protection Agency
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TABLE OF CONTENTS
Summary, Proposed Testing and Justification 1
A. Health Effects 1
B. Exposure 21
C. Material to Be Tested 21
I. Identity of Chloromethane 22
II. Exposure Aspects . 24
A. General 24
B. Direct Exposure. .26
C. Environmental Exposure 31
D. Existing and Proposed Guidelines and
Standards for Chloromethane 35
III. Health Effects 38
A. Systemic Effects 38
B. Neurotoxicity 49
C. Mutagenicity 58
D. Oncogenicity 66
E. Teratogenicity 74
F. Metabolism 76
IV. Current and Planned Testing 89
References 98
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Summary, Proposed Testing and Justification
In its first report to the EPA, October 1977, the Inter-
agency Testing Committee (ITC) recommended that chloromethane be
given priority consideration for the development of testing
requirements under Section 4 of the Toxic Substances Control Act
(ITC 1978). Specifically, the ITC recommended that chloromethane
be tested for its carcinogenicity, mutagenicity, teratogenicity,
and other chronic effects. With regard to chronic effects, the
ITC expressed particular concern for chloromethane's effects on
the central nervous system, liver, kidney, bone marrow, and the
cardiovascular system.
A. Health Effects
On the basis of information presented in the following
sections, the EPA is proposing that chloromethane be tested for
its potential chronic neurologic and behavioral effects, mutagen-
icity, oncogenicity, and teratogenicity, both morphologic and
behavioral. This document is in support of the EPA's proposed
test rules requiring such testing. As soon as test standards
have been developed and proposed covering the necessary neuro-
toxicity and behavioral teratogenesis effects, these will be
proposed in a subsequent rulemaking.
1. Chronic Neurotoxicity
a. Summary and Findings
Several investigators have detailed the permanent
neurobehavioral effects of long-term exposure to chloromethane.
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Klimkova-Deutschova (1957), Langauer-Lewowicka et al. (1974), and
Milkov et al. (1965) are representative of studies that deal with
groups of workers in the chloromethane industry (either direct or
indirect) who exhibited chronic neurologic or behavioral changes
from long-term exposure, with no known high-level acute
exposures. In a slightly different type of study, Repko et al.
(1977) found significant decrements in complex math tasks,
increases in resting tremor, and increases in the latency to
visual stimuli in a group of chloromethane-exposed workers, while
testing in the workplace. The EPA feels that while these studies
suggest that long-term exposure to chloromethane may pose an
unreasonable risk, they are inadequate to determine the extent of
that risk.
Animal studies have also been done, but many problems in
evaluating these studies occur. Smith and von Oettingen
(1947a,b) tested chloromethane in several species of animals and
concluded that 300 ppm had "no apparent effect in 64 weeks of
exposure" on any species tested, but that (a) the effects of 500
ppm in dogs and monkeys had much in common with symptoms
described for humans and, therefore, that (b) "then it is evident
that daily exposures to concentrations of 500 ppm are extremely
dangerous even for a period of two weeks or less".
More recent animal studies of chronic exposure have produced
suggestive evidence of functional and pathologic effects after a
shorter duration of exposure at lower concentrations. A Russian
study (Yevtushenko 1966), which the author cited as one basis for
the 1965 Soviet TLV of 2.5 ppm, reported effects in rats and
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rabbits at low levels in both acute and chronic exposures. This
study reported an increase in the time to acquire a conditioned
response in rats after 4 hours of exposure to as little as 114
ppm. Furthermore, after six months of exposure to 20 ppm rats
showed behavioral deficits. Pathologic changes in rabbits
exposed in the same experiment (Belova and Yevtushenko 1967)
occurred throughout the brain as well as in the eye at the low
dose.
b. Testing
One specific neurobehavioral effect of concern has been
identified for testing, namely, chronic effects on function and
morphology of the nervous system. While the Agency is not pre-
pared at this time to propose complete standards for the conduct
of such testing, set forth below are current views on the pro-
posed testing, and related issues relevant to the development of
these standards. Comments are solicited from all sectors on the
appropriateness and conduct of the suggested testing.
Based on the recent controlled laboratory studies of Putz et
al. (1979) and Stewart et al. (1977), and the worker study of
Repko et al. (1977), it appears that changes in complex cognitive
functions, and visual function as measured by behavioral tasks,
may be the most sensitive human indicators of exposure to
chloromethane. The report on exposed workers by Klimkova-
Deutschova (1957) as well as the 13 year follow-up study of
exposed fishermen by Gudmundsson (1977) suggest that chloro-
methane intoxication may induce damage that involve the cranial
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nerves or other structures controlling the eye, pyramidal and
extrapyramidal neurologic signs, a reduced tolerance to alcohol,
fatigue, and depression.
The EPA is considering proposing animal studies to determine
no-effect levels for neurobehavioral effects of chronic exposure.
Among the variables to be determined are choice of species,
length of test, days per week exposed, type of exposure and type
of testing. It has not yet been determined that in this case the
most sensitive human indicators of chloromethane neurotoxicity
would necessarily be those tested by choice in an animal model.
The EPA is asking for public comment on these issues in an ANPRM.
The choice of species for animal testing will involve
several considerations. First, Smith and von Oettingen (1947a)
have suggested that dogs and monkeys are more sensitive than the
other species they tested, and that effects in these species most
resemble human intoxication. The inappropriateness of rats as a
test species is suggested by the same authors' failure to observe
any overt effects in rats but not other mammalian species exposed
to 500 ppm. On the other hand, Yevtushenko (1966) reported
behavioral effects from both acute and chronic exposure to low
levels in rats; the apparent discrepancy may be due to their use
of quantified behavioral testing as compared to the presumably
less objective observational techniques of Smith and von
Oettingen (1947b). However, the reports of neither study are
adequate to determine if this is, in fact, the case. The ocular
conjunctivitis observed by Belova and Yevtushenko (1967) in
rabbits and more recently by CUT (1979a) in mice but not rats
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suggests that rats are less sensitive with respect to ocular
irritation as well.
The Agency is also considering the appropriateness of and
the best means of defining adequate post-exposure testing of
subjects from all exposed groups to assess the severity of
delayed effects, if any, and the persistence of any observed
effects. If exposure in chronic testing is noncontinuous, these
effects could be assessed in part during chronic exposure studies
prior to the beginning of daily or weekly exposure.
In addition, the Agency is considering whether testing for
abuse potential, interaction with ethanol and/or a mixed schedule
of exposures (long-term low-level plus acute high-level) would be
appropriate additions to the requirements. Details are presented
in an ANPRM.
2. Mutagenicity
a. Summary and Findings
There is evidence from bacteria and higher plants that
chloromethane is capable of causing both gene mutations and
chromosomal aberrations. In bacteria, chloromethane is a direct-
acting mutagen capable of inducing base pair substitutions in the
DNA of S. typhimurium strains TA 1535 and TA 100 (Andrews et al.
1976, DuPont 1978, Simmon 1978). In Tradescantia pollen grains,
chloromethane is more effective than ethylene oxide in inducing
chromatid breakage (Smith and Lotfy 1954). Although this infor-
mation indicates that exposure to chloromethane may present an
unreasonable risk of mutation to humans, it is insufficient by
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itself to assess chloromethane1s risk as a potential human
mutagen. Therefore, the EPA is requiring more extensive testing to
determine if chloromethane may be so classified and to generate the
information necessary to perform a mutagenicity hazard assessment
on chloromethane.
b. Testing
In recent years, mutagenicity experts have discussed, and
provided guidance on, hazard estimation procedures for determining
if a chemical is a potential human mutagen.
Four major reports on the hazards of environmental mutagens
were issued between 1975 and 1979 (Drake 1975, Flamm 1977a,b,
McElheny and Abrahamson 1979). In 1978 the Office of Pesticide
Programs proposed Guidelines for Registering Pesticides in the U.S.
(OPP 1978) and a report entitled "Mutagenicity Guidelines" is being
prepared by Dr. W.G. Flamm for the Office of Health Effects Assess-
ment describing mutagenicity risk assessment procedures for use by
the Agency (Flamm 1979).
The reports agree that to perform a mutagenicity hazard
estimation for humans, scientists must first demonstrate that a
substance and/or its metabolite(s) does or does not 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 whether or not the mutagenically active form
can reach the genetically significant target molecules in mammalian
germinal tissue.
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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 Genetoxic Agents" (EPA 1979a).
The rationale for utilizing mutagenicity data which are not
derived from humans (all present data) has been previously detailed
(OPP 1978) and is based on an extensive body of knowledge in the
field of genetics. 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 1977a,b, McElheny and
Abrahamsom 1979). They are:
(1) All organisms (except for a few viruses) have DNA as
the genetic material which is basic 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 organisms contain nuclei in their cells, and
their DNA is associated with protein to form complex
bodies called chromosomes. Prokaryotic organisms lack
nuclei, and their chromosome structure differs from
that of eukaryotic organisms;
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(5) Unless there is a mutational event, the information in
DNA is faithfully replicated in each cell generation in
unicellular organisms and in somatic and germ cells of
multicellular organisms;
(6) DNA can be altered by chemicals. If 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 DNA unit 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;
(9) Disturbances in the distribution of individual
chromosomes or chromosome sets can occur during cell
division and result in numerical chromosomal aber-
rations; and
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(10) Mutations are generally considered to be deleterious in
reference to the normal environment for 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 tell if any alteration in the genome would be
good, bad, or of no importance.
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 eukaryotes suffers
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.
There are two tests each of which measures one of the genetic
endpoints (gene mutation or chromosomal aberration) and the ability
of a mutagenically active form of a chemical to reach germinal
tissue. These tests are the mouse specific locus test and the
heritable translocation test. Both of these tests are performed in
mice. The mouse specific locus test detects gene mutations; the
heritable translocation test detects chromosomal aberrations as its
genetic endpoint.
The EPA is not requiring a mouse specific locus test for
chloromethane for the following reasons. Chloromethane is a known
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alkylating agent with the demonstrated capability to react with
cellular nucleophiles. The rodent alkylation and Drosophila sperm
alkylation tests discussed below were both designed to detect the
ability of alkylating agents to react with the DNA of germinal
tissue. These tests are extremely sensitive. Sega et al. (1974)
have shown DNA alkylation in mouse sperm at levels below those
detected in the mouse specific locus test with ethyl
methanesulfonate. Aaron and Lee (1978) reported a genetic
alkylation in Drosophila sperm at levels just over 4X the spon-
taneous mutation rate in this system. Detection of genetic events
at this level in the mouse has not been reported. For alkylating
agents, then, DNA alkylation may possess superior sensitivity to
the mouse specific locus test, and is appropriate, therefore, for
measuring the ability of chloromethane to interact with the DNA of
germinal tissue. For these reasons, the EPA believes a mouse
specific locus test on chloromethane is not warranted.
(1) Test Requirements for Determining
Gene Mutation
Tests will be required to demonstrate the potential of
chloromethane to induce heritable gene mutations in a higher
organism. In addition, the ability of chloromethane to interact
with mammalian germinal tissue will be determined. The tests to be
performed include:
(a) The sex-linked recessive lethal test (DSRL) in
Drosophila melanogaster;
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(b) DNA alkylation in rodent (RA) and Drosophila (DA) sperm
cells;
(c) Gene mutation in mammalian cell culture (MCC);
(d) DNA alkylation in mammalian cell culture (MCCA).
These tests will (1) generate information necessary to
classify chloromethane as a potential human mutagen and to perform
a mutagenicity hazard assessment; or (2) determine that
chloromethane does not appear to induce heritable gene mutations in
mammals and, therefore, cannot be classified as a potential human
mutagen. (See Chart on following page.)
The sex-linked recessive lethal test in Drosophila will be
the first test to be performed. If the DSRL is positive the next
test to be performed will be-rodent sperm alkylation. A positive
rodent sperm alkylation will be followed by a determination of
DNA alkylation in Drosophila sperm. If the results of the rodent
sperm alkylation test are negative, no further testing will be
required at this time.
A negative DSRL in Drosophila will be followed by a test for
gene mutations in cultured mammalian cells. A positive mammalian
cell assay will be followed by a determination of rodent sperm
alkylation. A positive rodent sperm alkylation will be followed
by a determination of the extent of DNA alkylation in mammalian
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CHLOROMETHANE
Gene Mutation Testing Scheme
brosophila
Confirmatory
testing
Risk
Evaluation
Mouse Germ
Cell Alkylation
A
I
Stop
Drosophila
Alkylation
Mammalian Cell
Mammalian Cell
Alkylation
Quantified Risk Assessment
Quantified Risk Assessment
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cell culture. No further testing will be required if chloro-
methane is inactive in either the mammalian cell culture or
rodent sperm alkylation assays.
The results of these tests will be used to perform a risk
assessment of the gene mutation hazard to the human genome.
The EPA believes that as a matter of policy, further testing
for gene mutations of a chemical that fails to induce heritable
sex-linked recessive lethal mutations in Drosophila or gene
mutations in mammalian cells in culture should not generally be
undertaken. Should chloromethane, therefore, be negative in
these assays, the EPA would require no further testing for gene
mutations at this time.
In the absence of evidence that a chemical and/or -its
reactive metabolites can reach mammalian germinal tissue and once
there interact with germinal tissue DNA (the rodent sperm alkyl-
ation assay), evidence—to classify the chemical as a potential
human mutagen is lacking and hazard assessment is unwarranted.
To determine the mutagenic risk to humans of a chemical such
as chloromethane, it is necessary to estimate the dose that human
germ cells will receive. This estimation can be made from
metabolic fate studies in mammals where the alkyl group is
labeled with a radionuclide and the alkylations per nucleotide of
DNA are determined, using methods developed by Sega et al.
(1974). The rodent sperm alkylation test gives a measure of mam-
malian body exposure to a mutagen and relates to mammalian
gonadal dose.
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TO estimate genetic risk from a dose measured as alkylations
per nucleotide (AN) in the germ cells, it is necessary to
determine a dose (AN)-response relation in an experimental system
that can be thoroughly analyzed genetically for mutations induced
in the different germ cell stages. The required experimental
data is obtained from the relationship of dose to mutagenic
response in D. melanogaster using several of the genetic tests
available in this species. To determine the mutational risk of a
compound present in low levels in our environment it is necessary
to determine the shape of the dose-response curve from the
Drosophila experiments and the exposure-dose curve from the
metabolic fate studies. Methods for determining the dose of
alkylating agents measured as alkylation per DNA nucleotide in
the germ cells of Drosophila melanogaster have been developed
(Lee 1978, Aaron and Lee 1978). Therefore a determination of the
mutation frequency induced by a given alkylation level can be
made.
This battery of tests has as its general justification the
assumptions regarding mutagenicity testing which were presented
above. Specifically, the Drosophila sex-linked recessive lethal
(DSRL) test detects heritable gene mutations in an insect. The
rodent 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. As described above, this
information can be used to estimate mutational risk to humans
from exposure to chloromethane..
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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
the case of chloromethane, a DSRL test is being required before a
test for mutations in mammalian cell culture (MCC). It is
believed that because of the volatile nature of chloromethane,
exposure of Drosophila will be technically easier to achieve than
tests in MCC. Both procedures are feasible, however, and a
negative DSLR will be followed by a test for mutations in MCC. A
positive result in 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
(Aaron et al. In press) may be used to estimate risk to man in a
manner analogous to that described above for Drosophila.
( 2 ) Test Requirements for
Chromosomal Aberrations
In addition, the EPA is requiring that chloromethane be
tested for its potential for causing chromosomal aberrations,
starting with a dominant lethal assay in rodents. If chloro-
methane is inactive in the dominant lethal assay, no further
testing will be required. A positive dominant lethal assay will
be followed by a heritable translocation assay. A quantified
chromosomal aberration hazard assessment will be performed if
chloromethane is positive in the heritable translocation assay.
This scheme is illustrated by the following diagram:
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Dominant lethal assay Heritable Quantified
Translocation Hazard
Assessment
(-) (-)
No further testing
The dominant lethal test is used to trigger a heritable
translocation test because all chemicals positive in the
heritable translocation test are positive in the dominant lethal
test. However, not all chemicals positive in the dominant lethal
are positive in the heritable translocation test (Flamm 1977b).
Therefore, a positive dominant lethal result triggers a heritable
translocation. Hazard assessment is performed on the basis of
results of the heritable translocation test.
(3) Other
In this series of required tests, the EPA is not specifying
test details such as mammalian cell line, or type of mutation in
the mammalian cell culture assay, or species to be used in the
dominant lethal assay. Protocols to be followed for all tests
except DNA alkylation have been published by the EPA (EPA
1979b). Protocols to be followed for sperm alkylation, mammalian
cell culture alkylation and Drosophila alkylation have been
published by Sega et al. (1974), Aaron et al. (1980) and Aaron
and Lee (1978) .
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3. Oncogenicity
a. Summary and Findings
After reviewing the evidence available on the oncogenic
potential of chloromethanes, the EPA finds that chloromethane may
be an oncogen. Although there is no direct evidence in humans or
in animals that chloromethane is an oncogen, various indirect
evidence indicates that it has oncogenic potential. The indirect
evidence is summarized as follows:
a. chloromethane is a mutagen: (1) inducing gene
mutations and (2) causing chromosomal aberrations.
b. chloromethane is a direct alkylating agent which is
known to alkylate human and animal tissues; and
c. chloromethane is a close structural analogue of two
potential human oncogens, carbon tetrachloride and
chloroform, and of a suspect human oncogen,
iodomethane. Another suspect analogue with weaker
evidence of potential oncogenicity is dichlorome-
thane. Chloromethane is metabolized to yet another
possible oncogen, formaldehyde.
b. Testing
The EPA will require that a 2-year oncogenicity study be
undertaken to determine the oncogenicity potential of
chloromethane in animals (EPA 1979c). The justification for such
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a requirement is two-fold: I) the available information
indicating that chloromethane possesses oncogenic potential is of
sufficient strength to make a finding that the chemical may
represent an unreasonable risk; and 2) the finding that the only
study available (see Section IV) to evaluate the oncogenic
potential of chloromethane in animals is inadequate for EPA
purposes, if the results are negative.
4. Teratogenicity
a. Summary and Findings
Because of the biological activity of chloromethane in
adults, its probable accessibility to the fetus, and the embryo-
fetal effects of closely related compounds, the EPA believes that
chloromethane may present an unreasonable risk of teratogenicity.
With regard to the teratogenic potential of chloromethane, the
EPA is concerned with the danger of both structural malformations
and behavioral alterations.
b. Testing
Standards for the development of data on morphologic
teratogenic effects have been proposed (EPA 1979b). These
standards relate to the development of data on anatomical
abnormalities. The EPA believes that these standards are
appropriate for the testing of chloromethane for the induction of
these effects.
The requirements for the testing of chloromethane for
teratogenic effects is based on the need to develop data for
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assessing this aspect of possible health effects under the Toxic
Substances Control Act (TSCA). This need has been developed with
the knowledge that certain toxic chemicals produce developmental
deviations (Wilson and Fraser 1977). These developmental
deviations may take the form of death, malformation, growth
retardation and/or functional disorder. It is an underlying
assumption that production of these effects in humans constitute
unreasonable risks.
The use of animal testing to evaluate teratogenic potential
in humans has been accepted world-wide (FDA 1970, MHW 1976, WHO
1967). In addition, the value of animal testing is supported by
the observation that all substances known to be teratogenic to
man can be shown to be teratogenic in laboratory animals (WHO
1967). Although the full theoretical relationship between animal
test data and human teratogenic potential has yet to be estab-
lished, it is generally accepted that substances shown to be
teratogenic in animals may be teratogenic to man under
appropriate conditions of dosage and timing. Positive data of
this type from animal studies will be considered by the EPA as
substantial evidence for teratogenic potential in man, surpassed
only by human epidemiologic studies and case histories.
In addition, the EPA is proposing to include an evaluation
of neurologic/behavioral abnormalities and of the acquisition of
developmental landmarks in its development of data on the pos-
sible teratogenicity of chloromethane (see, e.g., Vorhees et al.
1979b). Since no standards for the development of this type of
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data have been proposed, this topic will be included in the ANPRM
for chloromethane, and will be subject to public comment.
In addition to routine signs of physical development that
may reflect toxicity (e.g., body weight), the proposed testing
should include specific tests to assess in the offspring known
effects of chloromethane in adults. Acquisition of a conditioned
reflex was reported as a sensitive endpoint by Yevtushenko
(1966). Neurologic impairment of motor function in humans and
other mammals has been reported (see, e.g., Klimkova-Deutschova
1957, Smith and von Oettingen 1947b) and impairment of visual
functions in humans (see, e.g., Langauer-Lewowicka et al.
1974). These three types of endpoints should be considered as
well as thorough neuropathology.
5. Other Chronic Toxicity
Although the Interagency Testing Committee (ITC) recommended
testing to determine chronic effects on the liver, kidneys, bone
marrow, and cardiovascular system, the EPA is not proposing
requiring such studies. Results available from previous studies,
especially those of Smith and von Oettingen (1947a,b), Smith
(1947), Dunn and Smith (1947), and Yevtushenko (1966), and that
which will be available from the current CUT study (CUT 1979b)
are deemed by the Agency to provide sufficient information to
evaluate the chronic effects of chloromethane on the liver,
kidney, bone marrow, and cardiovascular system. In the earlier
studies the liver, kidneys, and bone marrow were affected, but at
exposure levels higher than those that induced CNS effects. This
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means that no-effect levels were in essence established for
liver, kidney, and bone marrow .toxicity. The no-effect levels
varied with the frequency of exposure. For example, in rats
exposed to 500 ppm, 6 hours per day, 6 days per week for nine
months, no signs of liver, kidney, or bone marrow toxicity were
detected (Dunn and Smith 1947), while exposure to less than 120
ppm was needed if the exposure frequency was 4 hours/day daily
for 6 months (Yevtushenko 1966). Because no-effect levels have
been determined for liver, kidney, and bone marrow toxicity under
a series of test conditions and because the most sensitive
indicator of toxicity appears to be the CNS, for which separate
testing is being recommended, the EPA finds that no further
chronic toxicity study to examine liver, kidney, and bone marrow
toxicity is needed.
Effects on the cardiovascular system are associated with
acute lethal concentrations of the chemical and not with non-
lethal chronic exposure. As discussed in Section III.A., human
and animal data are sufficient to evaluate the acute toxicity of
chloromethane. Because of these two factors the EPA is not
recommending further chronic studies to evaluate cardiovascular
toxicity.
6. Epidemiology
The EPA has determined that at this time a suitable cohort
for epidemiology studies cannot be identified and, therefore, is
not requiring such studies. However, if information becomes
available to the Agency through TSCA Section 8(a)(2)(F) leading
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to the development of a suitable cohort, the Agency may reexamine
this conclusion.
B. Exposure
Chloromethane was produced in the United States totally for
domestic consumption at approximately 497 million pounds in 1979.
Hydrochlorination of methanol is the process used for greater
than 98 percent of production. It is used almost exclusively as
an intermediate, primarily in the manufacture of silicones and
tetramethyllead. Although Chloromethane is present in the
atmosphere in parts per trillion levels from natural sources, and
in the parts per billion range from anthropogenic sources other
than manufacturing, processing and use, the greatest risk of
health effects is presented by exposure to Chloromethane in
local, high concentrations at the parts per million level found
in occupational settings.
On the basis of Chloromethane1s almost exclusive use as an
intermediate, reports prepared for NIOSH, and various reports of
exposure found in the literature, the EPA staff concludes that
the maximum potential for the possible risk associated with
direct exposure to Chloromethane exists during its manufacture,
processing and use.
C. Material To Be Tested
The EPA is proposing that a grade of Chloromethane of 99.97
percent or better be used as the test material in the required
tests. Chloromethane of this purity is available commercially,
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and is being used by CUT in their studies. The primary reason
for this proposal is that the EPA believes that the molecular
species CH-^Cl, may pose an unreasonable risk and that, therefore,
the most valuable information to evaluate the risks of exposure
to chloromethane would come from testing the purest form of the
compound available. The contaminants present in chloromethane
produced by both of the industrial processes in use today, are
either characterized or under deliberation for further testing
under Section 4(a) of TSCA. General considerations for selection
of the appropriate form of the substance for testing are
discussed in the Legal and Policy generic document (Appendix 1 of
Rule 1). Those manufacturers, processors or users of
chloromethane may apply for a blanket exemption as dealt with in
the Exemption Policy generic discussion (Appendix 3 of Rule 1).
I. Identity of Chloromethane
Chloromethane, CH^Cl, (also known as methyl chloride) is a
colorless, noncorrosive, liquefiable gas at room temperature and
normal atmospheric pressure. Other physical properties of this
chemical include: molecular weight, 50.49; boiling point,
-23.7°C; melting point, -97.6°C; specific gravity, 0.92 at 20°C;
solubility in water, 0.74 g/100 ml at 25°C (DeForest 1979); vapor
pressure, 5 atm at 20°C; and an estimated logarithm of the
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octanol/water partition coefficient (log Poct) of 1.08 (Leo et
al. 1971).1
Almost all of the chloromethane produced in this country
(greater than 98 percent) is made by the hydrochlorination of
methanol (Lowenheim and Moran 1975, CMR 1976). Ahlstrom and
Steele (1979) state that two grades of chloromethane are
produced, the technical and the refrigerant. The refrigerant
grade must be very pure, to prevent attack by impurities on the
refrigeration equipment, and generally contains less than 75 ppm
water. Continental Oil Company produces chloromethane of at
least 99.9 percent purity for the production of tetramethyllead,
while Dow Corning, which uses chloromethane both as a direct
contact refrigerant and in non-refrigerant uses, reports that the
chloromethane it produces is greater than 99 percent pure (SRI
1979a,b). In response to a questionnaire, industry reported
finding methanol, acetone, dimethyl ether, water, ethyl chloride,
and hydrogen chloride as impurities in chloromethane (NSF
1975). Ahlstrom and Steele (1979) state that the known
contaminants of a technical grade product are no more than 100
ppm H-O, vinyl chloride, ethyl chloride, and residue, 50 ppm
methanol and acetone, 20 ppm dimethyl ether and 10 ppm hydrogen
chloride. It has also been reported that chloroform
(trichloromethane) and carbon tetrachloride (tetrachloromethane)
are obtained as coproducts in the production of chloromethane by
With the exception of the solubility in water and the log of
the octanol/water partition coefficient, all physical properties
were obtained from recent editions of the Handbook of Chemistry
and Physics (Weast 1978) and the Merck Index (Windholz 1976).
23
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the hydrochlorination of methanol (SRI Undated, SRI 1979a), so
that possible contamination by these products may also occur.
A small amount of chloromethane may be produced by direct
chlorination of methane, in which case there is the potential for
contamination with, in decreasing order, dichloromethane, chloro-
form, and carbon tetrachloride (Lowenheim and Moran 1975).
II. Exposure Aspects
A. General
In 1976 chloromethane was produced in the United States by
11 manufacturers at 15 sites (OTS 1979, SRI 1979a). In 1979, the
Chemical Marketing Reporter (CMR) stated that U.S. production
capacity is approximately 625 million pounds as produced by nine
manufacturers at eleven sites (CMR 1979). The production volume
in the United States averaged about 450 million pounds per year
between 1970 and 1976, ranging from 544 million pounds in 1973 to
304 million pounds in 1975 (ITC 1970-1975). CMR reported that*
demand for chloromethane was 485 million pounds in 1978, 497
million pounds in 1979, and an estimated 550 million pounds in
1983, a growth rate of 2-3 percent per year through 1983, a
result mainly of the growth potential of silicones (CMR 1979).
The quantities of chloromethane that are either imported or
exported are insignificant (EPA 1977).
Chloromethane is used almost exclusively as an intermediate.
Approximately 50 percent of all chloromethane is consumed in the
manufacture of silicones which are used for a wide variety of
24
-------
products (CMR 1979). About 30 percent of chloromethane consump-
tion is for the production of tetramethyllead, an antiknock
compound used in gasoline formulations. This use is probably
declining in the United States as a result of recent restrictions
on the use of lead in gasoline, although tetramethyllead is being
exported (CMR 1979).
Minor uses of chloromethane as a methylating agent in the
production of methyl cellulose, as an intermediate in the
production of quaternary amines, and as an intermediate in the
production of certain pesticides account for about 4 percent each
of total consumption. A variety of other intermediate uses such
as in the production of Triptane©, an antiknock fuel additive,
and methyl mercaptan, used to produce jet fuel additives, account
for about 4 percent of total consumption.
The major nonintermediate use of chloromethane, which
accounts for about 4 percent of consumption, is as a catalyst-
solvent in the manufacture of butyl rubber. Minor noninter-
mediate uses of chloromethane are as a foam-blowing agent for
extruded polystyrene foams, e.g., StyrofoamR (EPA 1975, 1976, NAS
1978, SRI Undated) and as a direct contact refrigerant (SRI
1979b). At one time chloromethane was used widely as a
refrigerant in both domestic and industrial refrigerators.
Although there are some refrigeration devices using chloromethane
still in operation today, this use has been almost completely
replaced by other substances, notably the chlorofluorocarbons.
Chloromethane is also used as an aerosol propellant combined with
25
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dichloromethane, propane, and Freon 12 for various aerosol mixes
(Deforest 1979).
B. Direct Exposure
Virtually all of the information on direct exposure to
chloromethane involves occupational exposure. Because chloro-
methane is a gas at room temperature, the major route of human
exposure to chloromethane is almost certainly inhalation. The
1972-74 National Occupational Hazard Survey (NOHS) indicates that
an estimated 50,575 workers have the potential for exposure to
chloromethane (NIOSH 1972-74).
The National Institute for Occupational Safety and Health
(NIOSH) has sponsored studies in several plants that produce or
use chloromethane, to evaluate the extent of worker exposure in
various occupational settings. The exposure levels found in
these studies, described below, were generally at or below the
current threshold limit values (TLVs) for the time weighted
average (TWA) of 100 ppm (210 mg/m3) and the short-term exposure
limit (STEL) of 125 ppm (260 mg/m3) (ACGIH 1979). (The current
TLV, and the data used to arrive at it are discussed in Section
II.D.).
At the Dow Corning plant site in Midland, Michigan, chloro-
methane is used in the production of methyl chlorosilanes in
three buildings, and is used as a direct contact refrigerant in
three other buildings (SRI 1979b). In the first industrial
hygiene survey conducted by SRI International (SRI) at the Dow
Corning Corporation on September 27, 1977, (SRI 1979b) it was
26
-------
determined that airborne chloromethane levels in the working
environment ranged from less than 1 ppm to 51 ppm (as area
samples). In March 1979 SRI conducted a personal monitoring
survey of the operators at the Dow Corning plant site for full
shift TWA exposure concentrations, and determined levels of
chloromethane from below detection to 12.67 ppm on operators, and
short-term levels of 0.62 ppm to 5.81 ppm on maintenance
workers. Eight-hour TWA concentrations in four work areas were
determined to range from below detection to 31.66 ppm. The
highest levels were consistently found in chloromethane com-
pressor areas. No samples were taken from areas in which
chloromethane is used as a refrigerant. Although the chloro-
methane is stored, transferred, and reacted in relatively closed
systems, chloromethane is nevertheless present in the working
environment air. Furthermore, elevated short-term exposure
levels of chloromethane can occur through a leak or when
operators must collect quality-control samples. At least 38
employees work directly within areas of the plant that produce
and use chloromethane. Additional workers with the potential for
exposure include maintenance personnel, material handlers and
laboratory personnel.
Dow Chemical uses chloromethane as a foam-blowing agent in
its polystyrene (Styrofoam ) foam process (NIOSH 1978). The
StyrofoamR production occurs in a closed system until the mate-
rial comes through a die in the extruder and expands onto a
conveyer assembly. Employees are exposed to chloromethane in the
foam production area. Exposure is also known to occur when
27
-------
chloromethane is liberated from the foam product while it is
cooling and in storage, or when the residual chloromethane is
released by certain operational procedures such as cutting,
routing, drilling, and reaming of the finished product. Levels
of 105 parts per trillion (ppt) (0.0001 ppm) and 355 ppt (0.00036
ppm) were found in two air samples collected from the foam
storage warehouse (NIOSH 1978). The average eight-hour TWA
exposure to chloromethane found in an SRI study ranged from 15
ppm to 54 ppm at various sites in the Styrofoam plant, with the
highest eight-hour TWA level being 101 ppm (NIOSH 1978). In
another SRI study, average half-hour concentrations at sample
points in Dow's fabrication plants ranged from 2-1500 ppm (SRI
Undated). In 1969 Dow Chemical conducted a survey of nine in-
plant chloromethane-containing manufacturing operations using
continuous monitoring devices for four months for 54 job
classifications. Time weighted average concentrations ranged
from 5-78 ppm with an average 30 ppm concentration. Peak concen-
•>
trations were as high as 440 ppm, but the duration of peak con-
centration exposure was not reported (SRI Undated).
DuPont Corporation produces chloromethane and uses it in the
production of tetramethyllead (SRI 1978a). Tetramethyllead is
produced in a closed system by the reaction of chloromethane with
a sodium-lead alloy and aluminum chloride. Unreacted chloro-
methane is pumped to a recovery unit. The duration of exposure
to chloromethane for employees in the production area may be up
to eight hours per work shift. A concentration of 209 ppm was
found in the tetramethyllead compressor room. In three operating
28
-------
areas where chloromethane is used, short-term levels ranging from
undetectable to 71 ppm of chloromethane were found. Chloro-
methane exposure levels (as TWA) were 6 ppm to 57 ppm in the
chloromethane manufacturing facility, 2 ppm to 75 ppm in the
tetramethyllead manufacturing facility, and 1 ppm to 34 ppm in
the chloromethane recovery area.
Continental Oil Company (Conoco) produces chloromethane with
potential exposures in the production area and in the tank-car
loading operations (SRI 1978b). In an industrial hygiene survey
done by SRI at the Conoco Chemicals facility in Westlake,
Louisiana, on October 18-19, 1977, it was determined from sam-
pling data that airborne chloromethane levels in the working
environment ranged from 3 to 36 ppm (as area samples) (SRI
1979a). Simultaneous sampling by Conoco showed chloromethane
concentrations ranging from less than 1 to 58 ppm. Personal
sampling data accumulated by Conoco since 1975 in their quarterly
sampling program showed six-hour TWA concentrations in the
breathing zone ranging from 0 to 67 ppm for all chloromethane job
classifications. (Fourteen employees were working in the chloro-
methane work areas at the time of the SRI survey.) SRI sampling
data showed eight-hour TWA chloromethane concentrations deter-
mined from personal monitoring varying from less than 0.2 to 7.5
ppm. Average eight-hour TWA concentrations in 11 work areas
ranged from 0.7 to 55.7 ppm. The highest concentrations were
found in the compressor areas (SRI 1979a).
Chloromethane and methyl chlorosilane manufacturing facil-
ities are located at a General Electric plant site (SRI 1978c) .
29
-------
Workers were reported to be exposed to chloromethane levels of
0.8 ppm to 75 ppm in the manufacturing facility, recovery unit,
and compressor room from three to five hours each day.
Chloromethane is also used in the manufacturing process of
four herbicides: paraquat (1,1'-dimethyl-4,4'-bipyridinium);
DSMA (disodium methylarsonate); MSMA (monosodium methylarsonate);
and cacodylic acid (dimethylarsinic acid) (Sittig 1977).
Paraquat is made by the reaction of 4,4'-bipyridyl and chloro-
methane in water. MSMA and DSMA are final products after sodium
arsenite is treated with gaseous chloromethane. This reaction
takes place in a closed system; additional chloromethane is con-
sumed in a side reaction with sodium hydroxide. In the produc-
tion of cacodylic acid, chloromethane is added to the reaction
chamber throughout the reaction, then the excess is bled off. No
data were found on the occupational exposure to chloromethane in
these four herbicide manufacturing processes, although the
possibility of low-level, constant air concentrations or high-
level, intermittent concentrations exists, as in other manu-
facturing processes using chloromethane. No information was
found on possible chloromethane contamination of these pesti-
cides .
All of the above direct exposure cases illustrate occupa-
tional exposure to chloromethane during its manufacturing, pro-
cessing, and use. The EPA believes that occupational exposure to
chloromethane presents the most substantial risk of exposure.
30
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C. General Population Exposure^
Chloromethane appears to be the most abundant halocarbon
present in the atmosphere (Lovelock 1975, Singh et al. 1977).
SRI (1979c) have reported that Chloromethane has an average
background tropospheric concentration of 611 parts per trillion
(ppt) (0.0006 ppm) in the northern hemisphere, and 615 ppt
(0.0006 ppm) in the southern hemisphere. It is believed that
this similarity indicates that the anthropogenic sources are
relatively unimportant contributors to the atmosphere, rather
than that extensive mixing occurs in the upper levels. Lower
stratospheric levels are approximately 5 percent less (Cronn et
al. 1977).
Chloromethane is decomposed when it reacts with hydroxyl
radicals in the troposphere, with a small fraction reaching the
upper stratosphere, where it is destroyed by photolysis (NAS
1976). The National Academy of Sciences (NAS 1976) estimated
that the residence time of Chloromethane in the atmosphere is
about one year. More recently, SRI (1979c) estimated the
residence time in the atmosphere to be 231 days.
The National Academy of Sciences (1976) reported an esti-
mated total global emission rate (that is, both natural and
anthropogenic emissions) of 14.7 billion pounds per year, based
on an average global concentration of 750 ppt (0.00075 ppm). Two
The level of detail in this subsection is unnecessary for the
EPA's conclusions regarding the need to require health effects
testing for Chloromethane. However, the EPA is also evaluating
the possible environmental effects of Chloromethane (to be
published later), and the General Population Exposure subsection
is applicable to that assessment as well.
31
-------
years later they estimated that the worldwide industrial
emissions of chloromethane were 17.4 million pounds in 1973, only
about 0.1 percent of the total emissions (NAS 1978). The
estimated intentional and unintentional U.S. chloromethane
release to the atmosphere from its production, transport,
storage, use, and presence as an impurity in other products,
amounted to 11.4 million pounds in 1973, approximately 2 percent
of the annual U.S. production volume (NAS 1978). The EPA (1975)
estimated that the United States accounted for approximately 60
percent of worldwide chloromethane production in 1973 and that
approximately 10.5 million pounds of chloromethane was released
from industrial activities in the United States in that year.
Singh et al. (1979) estimate that 5-10 percent of annual U.S.
production is emitted to the atmosphere. For example, Dow
Corning vents escaping chloromethane from the manufacturing area
through a stack to the outside air (SRI 1979b). However,
industry responses to questionnaires indicated that the fraction
of total annual production that escapes from the plant site to
the atmosphere during manufacture of chloromethane is 0.0011-
0.005 (NSF 1975). Assuming that the 2 percent release rate
applies worldwide, the total release of chloromethane would not
exceed 20 million pounds. However, using Singh et al.'s (1979)
estimated 5-10 percent release rate, total release could be as
high as over 50 million pounds annually. Either way it appears
that industrial emissions of chloromethane are only a small
fraction of the total of the more than 11-15 billion pounds
estimated to be entering the atmosphere annually.
32
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It is believed that the oceans constitute a major natural
source of chloromethane. Singh et al. (1979) reported that the
average surface concentration of chloromethane in the Pacific
Ocean is 26.8 x 10 g/liter (26.8 ppt). It has been suggested
that iodomethane, found ubiquitously in ocean water, reacts with
chloride ion in the ocean surface water to form chloromethane,
which then diffuses into the atmosphere (NAS 1976). Singh et al.
(1979) have calculated that 6.6 billion pounds of chloromethane
enters the atmosphere annually from the oceans.
It has also been suggested that burning vegetation is
another important natural source of chloromethane. Palmer (1976)
calculated that forest fires in the United States are responsible
for about 252 million pounds per year of chloromethane released
(average for 1972-1974). An additional 5.4 million pounds per
year was calculated by Palmer to have been released from agri-
cultural burning.
Another possible source of chloromethane is from photolytic
decomposition of higher alkyl halides in the environment. The
photolysis of gaseous chloroethane (a solvent) gives rise to
chloromethane is one of the products (Cremieux and Herman 1974),
which suggests that levels in the atmosphere may be less static
than is implied by the relatively long residence time estimated
by either the National Academy of Sciences (1976) or SRI (1979c) .
Although it is clear from the above information that major
sources of atmospheric chloromethane are natural, anthropogenic,
i.e., resulting from human activities, sources may be responsible
33
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for significantly elevated local concentrations. For example,
Singh et al. (1979) reported that they found elevated urban
concentrations of chloromethane in Lisbon [2.20 ppb (0.0022 ppm)]
and near Los Angeles [average 1.50 ppb (0.0015 ppm); maximum 3.80
ppb (0.0038 ppm)]. They have suggested that automobile exhaust
may be an important source of chloromethane. Palmer (1976)
estimated that about 120 million pounds of chloromethane is
released annually from building fires and 40 million pounds from
the burning of polyvinyl chloride (PVC) in wastes (average for
1972-1974). The latter source was recognized by Palmer to be
decreasing as the burning of such wastes was declining. The
National Academy of Sciences (1978) estimated that tobacco
smoking worldwide results in about 44 mil ion pounds of
chloromethane entering the atmosphere annually. Based on average
air intake of 23 m /day, and mean chloromethane concentrations
over Los Angeles, Phoenix, and Oakland of 3.00 ppb (0.003 ppm),
2.39 ppb (0^0024 ppm), and 1.07 ppb (0.0011 ppm), respectively,
the average human dose of chloromethane was calculated to be 140
ug/day, 109 ug/day, and 60 ug/day at the three sites respectively
(SRI 1979c).
Elevated levels of chloromethane can occur in indoor air.
Measurements of chloromethane in various contained atmospheres
showed between 0.65 ppb (0.00065 ppm) and 8.00 ppb (0.008 ppm) by
volume in various automobiles, 1.4 ppb (0.0014 ppm) in a
restaurant, and over 20 ppb (0.02 ppm) in an apartment after a
cigarette was smoked (Harsch 1977). Chloromethane was generally
the predominant halomethane found in indoor air, and was
34
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typically present at between two and ten times the ambient
outdoor level (Harsch 1977, NAS 1978). It was suggested by the
National Academy of Sciences (1978) that these elevated indoor
levels may be due to cigarette smoking.
Chloromethane is found primarily in the air and ocean
surface water, although it has also been qualitatively detected
once in United States river water, three times in effluents from
chemical plants, twice in effluents from sewage treatment plants,
and eight times in drinking water (EPA 1979d), very possibly from
the chlorination treatment of drinking water (EPA 1977, OWPS
1979). For the protection of human health from the toxic
properties of Chloromethane ingested through water and through
contaminated aquatic organisms, the ambient water criterion level
for Chloromethane is 2 ug/1 (OWPS 1979).
Although Chloromethane is present in the atmosphere at a
background parts per trillion level from natural sources (e.g.,
ocean waters) and at a parts per billion level in urban atmos-
pheres from anthropogenic (e.g., cigarette smoke) sources, the
EPA believes that the local, high concentrations of Chloromethane
in the parts per million levels found in occupational settings
present the greatest risk of health effects resulting from
exposure to Chloromethane.
D. Existing Guidelines and Standards for Chloromethane
1. Threshold Limit Value
The TLV for Chloromethane, 100 ppm, is based in part, upon
the studies performed by Smith and von Oettingen (1947a,b) in
35
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which the exposure of a number of different species of mammals to
300 ppm chloromethane for 64 weeks at 6 hours per day, 6 days per
week, caused no observable detrimental effects to the animals.
In addition, 9 unpublished in-plant surveys by Dow at their
chloromethane manufacturing plants indicated that human illnesses
were associated with exposures of 195 ppm and above, whereas
exposures between 15 ppm and 195 ppm were not associated with
reports of ill effects (ACGIH 1971).
The American Conference of Governmental Industrial
Hygienists (ACGIH) is proposing to drop the present TLV to 50
ppm, on the basis of some of the- literature discussed in later
sections (see Section III) (ACGIH 1979).
2. Warning Label Required by Federal Insecticide,
Fungicide, and Rodenticide Act (FIFRA)
Chloromethane is classified as an economic poison. Inter-
pretation with respect to warning, caution, and antidote
statements is required to appear on the label of technical
chloromethane (USDA 1962).
3. Food Tolerance Requirement of Federal Food, Drug
and Cosmetic Act
Chloromethane is regulated as a food additive permitted in
food for human consumption as follows:
36
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a. Its use as a propellant in pesticide
formulations in an amount not to
exceed 30 percent of the finished
formulation is permitted.
b. It is restricted to use in food
storage and processing areas where
spray applications do not contact
fatty foods.
c. To assure safe use of the additive,
the label and labeling of the
pesticide formulation containing the
food additive shall conform to the
label and labeling registered by the
United States Department of
Agriculture (FDA 1962).
4. Other
a. Chloromethane is one of the
chlorinated hydrocarbons under
consideraton for addition to the list
of compounds for Toxic Effluent
Standards (U.S. EPA Water Program
Proposed Toxic Pollutant Effluent
Standards) (EPA 1973).
37
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b. It is listed on EPA Consent Decree
Priority II List as one of the 129
priority Pollutants (OWPS 1979).
III. Health Effects
A. Systemic Effects
Chloromethane exposure has been reported to result in a wide
range of systemic toxicity following both acute and chronic
exposure. Although effects on the liver, kidney, heart and
hematopoietic system have been demonstrated in both humans and
animals, the most sensitive organs seem to be the central nervous
system (CNS). The available animal studies appear to be adequate
for determining chronic toxicity in systems other than the CNS.
1. Human Studies
The EPA is not aware of any epidemiology study which evalu-
ates the systemic effects of Chloromethane on humans exposed
chronically. However, there is a substantial case history
literature of poisoning in humans, beginning with Gerbis1 paper
in 1914. Smith and von Oettingen (1947a) tallied the number of
published Chloromethane intoxication cases. By 1947 there had
been 210, and 15 were fatal. The majority of poisonings before
1960 occurred from exposure to its use as a refrigerant (see
e.g., Kegel et al. 1929, Schwarz 1926), while present day
poisonings in this country appear to occur mainly in the rubber
38
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and plastics industries (see e.g., Hansen et al. 1953, MacDonald
1964, Scharnweber et al. 1974).
Most of the case histories are believed to have involved
acute exposures to levels of the chemical well in excess of the
currently accepted TLV of 100 ppm (see e.g., Gerbis 1914, Kegel
et al. 1929, Laskowski et al. 1976, Schwarz 1926) although
idiosyncratic responses to low levels could conceivably account
for some known instances of poisoning. In mild cases of acute
poisoning, the toxic manifestations are primarily neurologic in
character, as are those in chronic intoxications (see Section
III.B.). However, gastrointestinal effects such as nausea,
vomiting, and diarrhea are also prominent (see e.g., Mackie 1961,
Sharp 1930, van Raalte and van Velzen 1945, Wiernikowski et al.
1974). Elevated body temperatures, pulse rate and heart rate are
commonly reported (see e.g., Hansen et al. 1953, Kegel et al.
1929, Laskowski et al. 1976), while depressed blood pressure (see
e.g., McNally 1946, Suntych 1956, Trubecka and Brzeski 1968,
Weinstein 1930), and abnormal EKG readings (see e.g., Gaultier et
al. 1965, Gummert 1961, Noro and Pettersson 1960, Walter and
Weiss 1951) also indicate cardiovascular involvement.
The other organs or systems primarily influenced by chloro-
methane are the liver, kidney, and blood. Hepatic damage occurs
in acute cases (see e.g., Saita 1959, Spevak et al. 1976, Wein-
stein 1930), and in long-term exposures (see e.g., Del Zotti and
Gillardi 1954, Mackie 1961, Wood 1951), while kidney damage
manifests itself as renal insufficiency and anuria in the more
severe cases (see e.g., Borghetti and Gobbato 1969, Hayhurst and
39
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Greenburg 1929, Kegel et al. 1929, Suntych 1956), and proteinuria
which is representative of less severe damage (see e.g., Birch
1935, Mackie 1961, McNally 1946).
The hematologic picture is not as clear. Although some
investigators have seen anemia (see e.g., Hayhurst and Greenburg
1929, Kegel et al. 1929, Mackie 1961, Milkov et al. 1965) and
others leukocytosis (see e.g., McNally 1946, Noro and Pettersson
1960, Suntych 1956, Wiernikowski 1974); in some instances, even
following severe poisonings, the blood cell counts remain within
normal levels (see e.g., MacDonald 1964, Spevak 1976, van Raalte
and van Velzen 1945, Weinstein 1937).
Although most exposures to chloromethane are assumed to be
by inhalation, the lung apparently is relatively insensitive to
the chemical.
2. Animal Studies
There have been few studies on the effect of repeated
exposure to chloromethane in animals. Details of four of the
most relevant of these studies follow.
An experiment was undertaken by White and Somers (1931) for
the purpose of determining the minimal concentration of chloro-
methane which would cause death in average-sized guinea pigs when
the exposure via inhalation covered a 72 hour period. After the
exposure period, the animals were observed for an additional
thirty days. Each of three groups of animals (18 animals per
group) was exposed to an average concentration of 49, 77, or 140
ppm. In the group exposed to an average of 49 ppm, none of the
40
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animals died within the 30 day observation period; in the group
exposed to an average of 77 ppm, 50 percent of the guinea pigs
died; and in the group exposed to an average of 140 ppm, all of
the animals died within a few days after exposure. The
pathologic changes in the guinea pigs dying from the effects of
chloromethane indicated widespread systemic poisoning
characterized mainly by severe circulatory disturbances and
congestion of the lungs and meninges.
Smith, von Oettingen, and Dunn conducted an extensive study
of the acute and chronic toxicity of chloromethane (Smith and von
Oettingen 1947afb, Smith 1947, Dunn and Smith 1947). They
studied the mortality, symptomatology, effects on hematopoietic
and biochemical parameters, and the histopathologic changes
resulting from exposure to chloromethane. In these studies, the
chemical was administered to 10 species of animals via inhalation
6 hours/day, 6 days/week for up to 64 weeks, at concentrations of
300-4000 ppm.
Table 1 summarizes the mortality data at 500 and 2000 ppm in
terms of the number of days from first exposure to death of 50
percent of the experimental animals (LT50) (Smith and von
Oettingen 1947a). The most sensitive species at the 500 ppm
concentration was the dog, the least sensitive was the rat. No
apparent effect was noted in guinea pigs, mice, dogs, monkeys,
rabbits, and rats exposed to 300 ppm, 6 hours/day, 6 days/week
for 64 weeks. The other four species were not exposed to this
concentration.
41
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TABLE 1
Mortality of Animals Exposed to Chloromethane
6 Hours/Day, 6 Days/Week
LT50 (Days)
Species 500 ppm 2000 ppm
Guinea pig
Mouse
Goat
Dog
Monkey
Rat
Rabbit
Cat
Chicken
Frog
71
143
NSa
23
11 °b
NED
192
NS
NS
NE
3
3
3
4
10
15
23
27
38
NE
aNS—not studied
bNE—not lethal
Smith and von Oettingen (1947a) also found that several
factors influenced survival time within a species. These factors
included exposure frequency, age, and certain dietary constit-
uents. As shown in Table 2, the interval between exposures
(i.e., exposure-free period) greatly influenced the mortality
rate. Allowing exposure-free periods apparently decreases the
cumulative effects of chloromethane. The work of White and
Somers (1931) also supports this thesis. In their study, the
LDj-Q for guinea pigs exposed to chloromethane continuously for 72
hours was only 77 ppm, indicating that uninterrupted contact is
much more lethal.
42
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TABLE 2
Mortality of Animals Exposed to Chloromethane
for Different Exposure Intervals
Species
Mouse
Guinea
Pig
Concentration
(ppm)
1,000
2,000
2,000
2,000
2,000
Exposure Time
(hours/day)
6
3
6
6
6
Frequency
(days/week)
6
6
6
6
3a
LT50
(days)
5
131
3
3
201
Three alternate days a week
Variation in age also influenced mortality. Younger animals
appeared to be more resistant than older animals. For example,
when adult and weanling rats were exposed to 2000 ppm, 6
hours/day, 6 days/week, the LT50 for the adult animals was 15
days, while that for the weanlings was 27 days.
Supplementing the diet of guinea pigs exposed to 1000 ppm
with ascorbic acid, or the diet of rats exposed to 2000 ppm with
thiamine hydrochloride, nicotinic acid, or calcium pantothenate
did not increase resistance to the lethal effects of chloro-
methane. However, increasing dietary casein by 20 to 35 percent
or supplementing moderate to low casein diets with cystine or
methionine led to an increase in the time before 50 percent of
the rats died.
No differences in LT50 could be attributed to differences in
sex.
43
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Smith and von Oettingen (1947b) also studied the symptoma-
tology of the animals poisoned by chloromethane. Commonly seen
were anorexia, discharge of fluid from the respiratory tract,
hyperactive reflexes, disturbances in ability to correct
position, and extreme spasticity. The neurologic behavior of
monkeys (tonic-clonic convulsions and periods of unconscious-
ness), was different from that of dogs (sustained tonic spasms
without remission). However, both types of neurotoxicity have
been reported in humans (see Section III.B.).
Development of symptoms varied with concentration and
frequency of exposure. Symptoms were delayed or gradual with low
concentrations or with high concentrations separated by longer
exposure-free intervals. Young animals responded with a slow
development of symptoms at some concentrations where older
animals developed symptoms acutely. Although animals generally
recovered from acute symptoms if exposure was discontinued when
symptoms first\appeared, symptoms acquired over a long period of
time were sometimes irreversible.
Smith (1947) reported hematology and biochemical results on
certain of the animals studied by Smith and von Oettingen
(1947a,b). No hematologic or biochemical test was purported to
be useful in the diagnosis of chloromethane poisoning in the
species studied. No evidence of liver dysfunction, of renal
failure, or of a primary effect upon the formed elements of the
blood were detected without severe neuromuscular disturbances
having preceded the detected hematologic or biochemical
44
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changes. These data indicate the CNS to be the system most
sensitive to chloromethane toxicity.
Histopathologic examination on the same group of experi-
mental animals was reported by Dunn and Smith (1947). Morpho-
logic changes that appeared to be a direct result of inhalation
of chloromethane 6 hours/day, 6 days/week, were variable degrees
of necrosis of the convoluted tubules of the kidney in mice and
rats (2,000 ppm) and fatty metamorphosis of the liver in the
smaller species. Pulmonary edema appeared to be a direct result
of the irritation due to inhalation of chloromethane. No
morphologic changes were found in the brains of those dogs and
monkeys examined which showed severe neuromuscular disturb-
ances. No morphologic changes were observed in the rats exposed
to 500 or 1000 ppm, 6 hours/day, 6 days/week for nine months.
However, the tissues examined were not specified. Guinea pigs
surviving 9 months of exposure at this level also demonstrated no
histopathologic changes, although guinea pigs were the second
most sensitive species (LT50) at 500 ppm while rats showed no
lethality at 500 or 1000 ppm (Smith and von Oettingen 1947a).
The major limitations of the four studies done by Smith, von
Oettingen, and Dunn are:
(1) small numbers of animals were used in certain test
groups (e.g., two goats at 2000 ppm, four rabbits at
1000 ppm, two monkeys at 500 ppm);
45
-------
(2) histopathologic examinations were not reported on
animals exposed to 300 ppm for 64 weeks for species
which showed effects at 500 ppm; and
(3) no indication of the animals or tissues routinely
examined was given.
Therefore, while the studies indicate at what levels major
effects of concern might appear, they are inadequate for
determining a no-effect level.
Yevtushenko (1966) studied the chronic effects of chloro-
methane on rats (10 animals/group) and rabbits (4 animals/group),
exposed to 40 or 240 mg/m (i.e., approximately 20 and 120 ppm,
respectively), 4 hours/day, daily for 6 months. In both groups
of rats hematologic examination revealed consistent decreases in
erythrocyte number. In both rats and rabbits exposed to 240
mg/m , excretory function of the liver was disturbed while no
effect was observed in the animals exposed to 40 mg/m . In rats
of both groups, kidneys functioned normally, but microscopic
examination of the blood-forming organs indicated depletion of
lymphoid elements and proliferation of the reticular base of the
organs examined (spleen, lymph nodes). Changes in parenchymal
tissues were unpronounced. The rabbits were also used to observe
effects on the eyes. These were observed in both exposure groups
and included discoloration of the optic disc and histopathologic
disturbances of the retina and optic nerve. The most significant
changes occurred in the CNS (see Section III.B.2.b.).
46
-------
CUT (1979a) sponsored a 90-day inhalation study using rats
and mice. Groups of animals (ten animals/sex/dose) were exposed
to 375, 750, or 1500 ppm of chloromethane 6 hours/day, 5 days/
week for 13 weeks. The purpose of this study was to select
appropriate exposure levels for a subsequent chronic toxicity
study. All three groups of treated male rats showed a
significant decrement in body weight compared to controls, while
female rats treated with 750 and 1500 ppm also showed significant
decrements in body weight compared to controls. There was a
significant difference in final body weight between treated and
control mice at only the 1500 ppm dose level in females,
however. Weight loss, or a lag in weight gain is accepted as a
sensitive and easily measurable method for observing toxicity.
The top dose level in a combined chronic-oncogenicity test should
be the maximally-tolerated dose, (MTD) and is defined to be the
highest dose that causes no more than 10 percent weight decrement
and is predicted to produce no lessons (other than those related
to a neoplastic response) (EPA 1979). It is more difficult to
decide what is the proper MTD in a species in which in the
highest dose used caused little or no toxicity in preliminary
testing.
The group of male mice treated with 1500 ppm had
significantly higher serum glutamic-pyruvic transaminase
(SGPT) activity than controls. However, this activity was
increased in only two of the mice. One was found to have a liver
infarction while the other had severe hepatic changes. One of
the major problems with this study was the fact that although
47
-------
7/10 male mice at the high dose died of trauma, 6 males are
listed as the total on which final studies were done, without any
indication as to dates of death, or which animals were showing
which effects. Other clinical and hemotologic parameters
measured were reported to be within the normal clinical range.
The major histopathologic finding was cytoplasmic vacuolar
changes of the hepatocytes in mice. Sixty-four percent (9/14) of
the mice treated with 1500 ppm exhibited the effect, while 37
percent (7/19) of controls exhibited the effect. The effect was
highest in females and was more pronounced in the treated group
than in the control group. One female not in the high dose group
had a massive liver infarction. Thirteen of the 60 treated mice
had eye lesions, but it was concluded that this effect was not
compound related. However, deficiencies in the design and
conduct of this study, have lead the EPA to decide that the
findings from it cannot be used as the sole determinant of the
chronic toxicity of chloromethane (see Section IV).
Several major conclusions about the chronic toxicity of
chloromethane can be drawn from the human studies and from the
animal studies of White and Somers (1931), Smith and von
Oettingen (1947a, b), Smith (1947), Dunn and Smith (1947), and
Yevtushenko (1966). These conclusions are:
(1) chloromethane is toxic to a variety of species
including humans;
48
-------
(2) the major systems affected include the CNS, liver,
kidney, blood forming elements, and ocular tissue;
(3) the most sensitive system affected in humans and
animals appears to be the CNS; and
(4) the level of toxicity is not only affected by the
exposure concentration but also by the length of the
exposure-free period and the amount of cystine or
methionine in the diet.
B. Neurotoxicity
Chloromethane is a non-specific central nervous system (CNS)
depressant. There are human case reports, several animal
studies, and controlled human laboratory studies that document
its acute and chronic neurotoxicity. Chloromethane intoxication
produces neurological signs, mood changes, and cognitive and
intellectual deficits, as well as other symptoms. Neurological
signs include ataxia, tremor, motor reflex changes, and signs of
cranial nerve involvement such as blurred vision, weakened con-
vergence, mydriasis, and vertigo. Mood changes such as apathy,
irritability, euphoria in earlier stages of acute exposure and/or
depression in later stages also occur. Cognitive deficits relate
to difficulties in concentration and memory loss. More severe
CNS alterations also occur in acute poisoning. Convulsions of
both the tonic-clonic type (Hartman et al. 1955, McMally 1946)
49
-------
and that characterized by sustained tonic contractures (Kegel el
al. 1929, Schwarz 1926) have been seen. Other major symptoms are
headache, fatigue, and sleep disturbances (Table 3). The onset
of these signs and symptoms may be delayed by several hours
following exposure and can persist indefinitely (see e.g.,
Gudmundsson 1977, Walter and Weiss 1951). While the information
available is insufficient to completely characterize chloro-
methane for acute effects, the Agency deems it unnecessary at
this time to require further testing for systemic effects
resulting from acute exposure. However, chronic neurotoxicity,
with a potential for permanent effects, is definitely indicated
by the evidence as a risk to human health that is impossible to
assess at this time without additional data, acquired by testing.
1. Acute and Sub-Acute Neurobehavioral Effects
a. Human Reports and Studies
There have been numerous human case reports of acute intoxi-
cation (see e.g., Noro and Pettersson 1960, Spevak et al. 1976,
Thordarson et al. 1965, Wiernikowski et al. 1974). The first
column of Table 3 shows the frequency and nature of reported
signs and symptoms. These studies generally lack any quanti-
tative estimates of levels or duration, which generally makes it
difficult to use the information for hazard assessment purposes,
or the establishment of a no-effect level.
While victims of acute exposure generally show complete and
rapid recovery, very long lasting changes have also been
reported. Gudraundsson (1977) did a follow-up study 13 years
50
-------
Table 3. Neurologic Symptoms Seen in Man
Acute
Acute & Chronic
Chronic
References
Clinical Symptoms
visual disturbances
dlplopia
ptosis
mydrlasis
anlsocoria
nystagmus
weakened
convergence
strabismus
photophobia
dysphagia
hiccup
paresis of facial
nerve
t.witchinij muscles
tremors
pyramidal signs
i
r~i ,*? r~> id *d ^* • f^~ in r*
id1" a — C ^~ w m — m got v in
~' i>i fl W X "" ^-v^.w, .^- at ^ fl) 0
^~4J^^(rt ^-i ^-Jfl'^'4l»-J(4^C" p-H-
>-icniAi^-^>{>m<-lu> oio(jHidu> n r^ p.cJ a) n -H H Q ^ C^ • ia > c-i
QJ C4 £4 f*^ "vf A| *4* O -4^ ^^ ( 1 id ^ r^ 0) fH ( 1 f*l M Ul _y* ' 10 £) i^ At fll (4 t?^ O Al
D'0> -Hoi cqov JSo^ M 4-1 at nl^flJm J
-------
Table 3. Neurologic Symptoms Seen in Man
Acute
Acute & Chronic
Chronic
References
Clinical Symptoms
visual disturbances
dlplopia :
ptosis
mydrlaaia
anisocoria
nystagmus
weakened
coaveryence
strabismus
photophobia
dysphagia
hiccup
paresis of facial
nerve
twitcliinij muBcles
tremors
pyramidal signs
H •* x r- i fl w >j "~~ "
-- tA«-lU) 01 O R i-i BI U> 1-1 f"^
b«n -ri 01 c q X en
Otr-l 01 rl lda>— S — ZA.^ SfttCiW1-- j (0 ^
5b 12^°+ 16
1 + 2 4
H ^ ^i ^
*^* *"^ ri' 3t ^^ U 0
r-J (1 r< OJ r-4 H ^ C * P -H •
•d 01 n oi (i-ot u oi £ ^^ o^-^ c 5 tst
_/l f-| |^ •— | jrt ir^ |Q r~ \ fj 4^IX) d 1 Itf 4^ 4^ CM
W *^* DC "^ fL v^ £ ^-^ in 01*—* tb-^ *^ i\ 0)^^
6 1 5 3 2
2 3
2 21 t
624
2 4
1 4
2
\
1
1
3
1 2
4
3
3 +1
1 4
31 4
2 11 +4 5
2 1
2
3
1
1 1 3
^ 4 n
-------
^a
Iteferences
Clinical Symptoms
itaxia
tombergism'
/ertlgo
itaggera
Loss of balance
lizzinees
tdiadochokinesiu
>aresthesia
lensitivity
teakness in limbs
leneralized
weakness
ipathy
liredness,
sleepiness
:uphoria
lepression
Table 3 . Continued
Ir
Acute Acute & Chronic Chronic
r\ X r~ *l o| t-* in i^
«!-, ~ h|~- nd Olfn-rlrHOM-Rr-l -«SH
«J«NCn -j-n(r- alcslHdWuiXuJQiOMnJ C &> O «J
O>tn -HOI c fi a» S 01 M4Jen 4l'-4)a> X«n Hoi floi UOt uai ^ o-^ C 5 ~
ttlH 4IH g«lH 0 rj OOI.H -H +J[2 O.H dH fir* rtH «H «H ^+J^ J«;H S J ^^
21 81
14
12 21 1 1 +
1 21 2 6 3
I +
1 1 + 11202. 1^
1
1 i f
3 ! v
2 + 1 3
4 11* 913 1.+
1 1
5 1 4 21 1 12 1 1 +
2 1
1 4
-------
Pnble 3 j Continued
References;
Clinical Symptoms
confusion
nervoue, worried
irritable
loss of memory
loss of libido
psychosis
insomnia
anorexia
nausea
speech disturbance
headaches
hearing
difficultieu
difficulty in
concentration
heavy hoad
convulsions
H m
4J f- -rl *N •*-* N*-^ CO «g
01 *- 01 "- >-\ H H~ K
~ 4J~ % Ol~ H~ Id
r-l sn i/i r- !"» ^ in ri vf<
0) (N £ ri « v nt f o
ti> en -H ut £ a ui ?. a% M
4)H qJH ^P^ UH °
2
1
h? . H ', ., H ^ ^ i;
X i^ d i 1 a) uf- uir-
K-— Wcn|l •-» *^ m0* M-O>
W X "1 ;i "^ ^* "~" *" d"^ §J --' 01 U
0) 0 C H d IO 1 1 ^ 1- P.CJ Olrl-HrlO-'T^fH • W ^ rH
•PvDHaipf^i i 01O4Mf*llAlAX^O^idd pbtO Al'
4J£riftl^^O)£nl ' XtnnlaidolUotOchx'V- O ^> C S in
0 f~^ ti -M[r| Pl*H 1 A| pf jj r^ CO f~l A t~i A i~i O 4^1Q C r~i tl 0^ -{-T^
A« *^* &f 0)^^ W ^^* 1 M *-** I/) *-* |z] *^ £ *"•* £ *~^ I/J fl)*-' rf ^-^ J L.J flj^^
+ I 1 4 2
\ 123
\
1
+ 113 1 +
1 I
2
1 1
1 1
5211
2 1
2
15
21 1 1
+ 11 2 5 1 6 1
t 4 324
t t ' 1171 +
*
1
1
+ ^ 1
I
2
2 1-| .
a) The number in parentheses ' following the year of the reference is the number of persons
exhibiting chloromethane intoxication in the paper.
b) The number of patients exhibiting this particular sign.
c) If the number of persons exhibiting the sign was not mentioned, but the sign was
reported as occurring, a (+) is indicated.
-------
after 15 people were exposed to chloromethane from a leaking
refrigerator on a fishing boat (Thordarson et al. 1965). One
patient died within 24 hours of the incident, two suffered severe
depression and committed suicide within 2 years of the incident,
and one man who had been seriously disabled, died ten years later
of a coronary occlusion. Ten of the eleven survivors were
examined. Nine patients reported a reduced tolerance to alcohol
and six had chronic fatigue and depression. Five patients showed
neurological signs: 3 with tremor, 2 with paralysis of
accommodation, and 2 with peripheral neuropathies complicated,
however, by a history of alcohol abuse. Hartman et al. (1955)
reported that 1.5 years following a severe acute exposure, a
woman still displayed intention tremor, headaches, insomnia, and
"nervousness". Few of the other studies in the literature have
reported any long term follow-up.
There have been 2 recent laboratory studies of acute
exposure in humans. Putz et al. (1979) reported behavioral per-
formance deficits in a complex visual vigilance task during and
after 3 hours of exposure to 200 ppm, but no effects at 100
ppm. Stewart et al. (1977) exposed 4 humans to 100 ppm for 5
days for 7.5 hours a day. Analysis by the authors revealed no
impairment on a battery of neurologic and behavioral tests,
including 2 timing tasks, one with no cues and one with auditory
cues. But their analysis of variance revealed a significant
impairment in a timing task that relied on visual cues. Although
> investigators concluded that there was no cognitive
irment of timing behavior, the EPA believes that the
51
-------
demonstration of a visual system-related decrement in such a
controlled study seems significant when considered in light of
Putz's visual task deficits after 3 hours at 200 ppm.
b. Animal Studies
Yevtushenko (1966) reported that the four hour LC^Q (lethal
concentration in 50 percent of the animals) for rats was roughly
11,000 ppm. Depression of motor activity occurred, as well as
widespread edema and vascular congestion of the brain and other
organs. A four hour exposure to 114 ppm was reported to produce
a behavioral deficit, namely, an, increase in the time required to
develop a conditioned reflex.
2. Subchronic and Chronic Neurobehavioral Effects
Signs and symptoms of chronic toxicity, based on case
reports, do not differ qualitatively from those described above
(see introduction to III.B.).
a. Human Reports and Studies
Based on a study of refrigerator workers, Klimkova-
Deutschova (1957) suggested that fatigue, headache, sleep dis-
turbances, and difficulty in concentration are among the earliest
symptoms of chronic intoxication, and that cerebellar neuro-
logical signs predominate early while extrapyramidal signs are
more frequent later on. In addition, onset of toxicity was
insidious and once signs and symptoms appeared they were
sometimes permanent. In many reports it appears that signs and
52
-------
symptoms were reported in workers exposed both chronically at low
levels and acutely at much higher levels from accidental spills
or leaks (see e.g., Baker 1927, MacDonald 1964, Scharnweber et
al. 1974; see also the second column of Table 3). This makes
these studies difficult to evaluate in relation to separating
chronic from acute effects. As in acute case reports, quanti-
tative exposure data or correlation with employment duration are
generally absent for both mixed and chronic exposure studies.
Belova and Yevtushenko (1967) performed detailed exami-
nations of the visual system of chronically exposed workers. In
those exposed for 2 to 3 years, roughly one-third showed a
decline in corneal sensitivity and in some there was slight
discoloration of the optic disc. Two-thirds of those exposed for
5 to 8 years showed a decline in corneal sensitivity. In
addition, half of the workers displayed a complex group of visual
changes.
Repko et al. (1977) performed a behavioral, neurological,
and psychological study of chronically exposed workers (1-311
months, mean=84 months) in comparison with a control group. The
exposed cases consisted of xL71 "physically normal" paid
volunteers from eight different plants at seven locations in six
states (11 female/160 male, 10 black or minority/161 white). The
controls (comparisons), who were matched (attempted) by sex, age,
and race to the cases, consisted of 49 workers who were not known
to be exposed to chloromethane or other neurotoxicants (3
female/46 male, 3 black or minority/46 white). Regardless of
matching, the differences in mean age and level of education
53
-------
and increases in resting tremor, but could only establish a sug-
gestive relationship between these effects and airborne chloro-
methane levels.
The data analysis performed by Repko et al. to determine
this relationship exemplifies the limitations of the study.
Overall, the statistical comparisons are not sensitive to
cumulative exposure simply because this information is not
contained within the data. However, an attempt was made to
establish a relationship between duration of chloromethane
exposure (months employed) and physiologic effects of exposure.
Duration of exposure was compared with measurements of breath
chloromethane, urine pH, hematocrit, and ambient air concentra-
tions. A statistically significant positive correlation was
found between breath and air levels, and a negative correlation
was found between air concentration and hematocrit levels. In
subsequent analyses, correlations between neurologic effects and
these physiologic variables were investigated. For the
behavioral data, means from cases and controls were compared.
Such a technique does not distinguish levels of exposure among
cases. Behavioral data was investigated via scatter plots of
ambient air concentration versus factors related to behavioral
tasks. This comparison investigates the responses to various
doses measured in the workplace. Repko et al. (1977) concluded
that performance levels were reduced among workers exposed to
chloromethane. Due to the aforementioned limitations of the
study design, this reduction is only suggested rather than
conclusively supported. No measurements of performance previous
55
-------
Table 4.. Neurologic Signs Occurring Following Continued Exposures
Air
Concentration
in ppm
500
Species
Mice
Dogs
1,000
Guinea Pigs
Dogs
Effects
Convulsive activity occasionally occurred during the first week.
After a week or more of exposure, mice developed a syndrome which
began with a clamping of the hind legs to the body when the mouse
was held up by the tail. This syndrome worsened with continuing
exposure. Mice surviving 15 weeks of exposures retained the
clamping response 6 months following termination of exposure.
Three of four dogs exhibited spasticity and staggering by the end
of the second week. Two days later two were no longer able to
stand and the third was shaking violently. Two died within the
next 8 days and the third showed maximum extensor rigidity and
opisthotonus when it was held off the floor. The fourth animal
developed a slight ataxia after 4 weeks, lack of neuromuscular
fine control following 2 months exposures, prominent tremors,
ataxic gait and abducted hind legs when standing at 6 months.
Tendon reflexes were hyperactive and spasticity increased when
the dog was lifted off the floor. Exposures were discontinued
after 29 weeks, and during the following 17 weeks there was no
notable amelioration of the neuromuscular symptoms.
Loss of righting reflex; hind leg displacement reflex retarded;
later development of convulsions and opisthotonus within the
first week. Following exposure to 3 weeks of chloromethane,
neuromuscular signs first appeared and progressed until the
guinea pig was unable to walk. At 12 weeks a regular flicking
of the ears and a fine tremor was noticeable, but running a
pencil along the mesh of the cage would initiate a convulsive
episode. At 14 months, although many neuromuscular effects had
disappeared, it still could not right itself.
Generalized tonic spasm with powerful opisthotonus and risus
sardonicus; hyperactive reflexes and coarse tremors accompanied
spasticity occasionally in the first week of exposure.
-------
ConoWTtr/at jon
in ppm Species
,«. Puppies
2,QOQ
Rabbits
Chickens
Cats
Rats
Goat
Monkeys
Effects
First symptoms appeared in second month of exposure when
alternate gait was replaced with gamboling gait with frequent
tumbling. In the third month the more severely affected pup
showed tremors, and after 11 weeks intermittent convulsive
seizures, attacks of hiccups, audible grinding of the jaws and
risus sardonicus, and sustained contraction of tongue and jaw
muscles, Exposures were discontinued at 12 weeks, and the
general condition of the more severely affected animal improved
for a,bout one month, but after the fourth month, it grew worse and
was sacrificed three months later. The other pup was observed
for 1Q 1/2 months and the general condition was excellent, though
the pup could not stand without sagging or swaying of the posterior
trunk and legs and there was a tendency for the hind legs to remain
displaced posteriorly.
After several weeks exposure to methyl chloride rabbits were first
unable to bring the hind legs to the normal position for hopping, and
later the hind legs gradually became permanently adducted.
After three weeks the legs became weak and abducted and the chickens
unable to walk. Debility and paralysis increased until the entire
body except head and neck were paralyzed and cold to touch.
After a week, cats became weak, ataxic, lost righting reflex.
Symptoms progressed until cats unable to walk and had frequent
extensor spasm. Hyperactive reflexes.
Rats on stock diet occasionally, showed opisthotonus. On semi-
synthetic diet, in which survival times were prolongedf the
clamping syndrome seen in mice appeared after about 5 weeks of
exposure, culminating in paralysis of the hind legs several
weeks later. Residual abnormalities observed after 4 months
in one rat.
Following removal from the exposure chamber after the fourth
exposure, the legs became rigidly extended and 20 minutes later
spastic activity became general.
Ataxia; poor hand to mouth coordination; one monkey developed
convulsions followed by unconsciousness within the first week
of exposure.
-------
to exposure (pre-employment) were obtained. Therefore, there is
no certain means of knowing whether workers' performance levels
were affected by exposure.
b. Animal Studies
As noted above, the major study of chronic toxicity in
animals (rats, mice, guinea pigs, rabbits, dogs, monkeys, others)
was performed by Smith and von Oettingen (1947a,b) (see also
Section III.A.2.). Neurological effects seen in the 500, 1000
and 2000 ppm groups exposed for 6 hours/day, 6 days/week are
summarized in Table 4. In general, they found that 300 ppm for
64 weeks "had no apparent effect on any species tested", but that
500 ppm produced serious toxicity in most species, with parti-
cularly pronounced neurological signs in dogs and monkeys.
A Russian study (Yevtushenko 1966), which the author cited
as one basis for the 1965 Soviet TLV of 2.5 ppm, reported behav-
ioral and pathologic effects in rats and rabbits exposed for 4
hours/day to either 120 ppm or 20 ppm for 6 months. Development
time of a food conditioned reflex to a bell increased in both
groups of exposed rats compared to controls, while the natural
conditioned reflex to the sight and smell of food was signifi-
cantly delayed in rats exposed to 240 mg/m . Microscopic
examination established that the brain was significantly affected
in both groups of rats with vacuolization of protoplasm being the
predominant kind of change noted in nerve cells.
Belova and Yevtushenko (1967) performed visual pathologic
examination of the rabbits exposed in the same study. In the
56
-------
initial weeks of the experiment, slight hyperemia of the con-
junctiva and the appearance of a small amount of discharge from
the eyes were observed. In animals of both groups (20 and 120
ppm) the optic disc was pale or grayish and frequently had dif-
fused edges. Edema of the optic disc was noted in rabbits
exposed to the higher level. Blood vessels of the eye were of
uneven caliber, arterial vessels were primarily constricted, and
small hemorrhages were noted in the retina of some animals. His-
tologic examination of the retina and optic nerve indicated mor-
phologic changes, as well as increased vascularization,
plasmorrhagia and hemorrhaging. No information on the optic
system of the rats exposed at the same dose was given.
In the 90-day inhalation study of chloromethane sponsored by
CUT (see Section IV), rats exposed to 375, 750, or 1500 ppm
exhibited no gross pathologic alterations of the eye. However,
mice exposed to the 375 and 750 ppm dose had a high incidence of
eye lesions that began as a mucopurulent conjunctivitis and
progressed until some animals' eyes were totally destroyed (CUT
1979a).
The following conclusions on the neurotoxicity of chloro-
methane can be made:
(1) adult dogs and monkeys appear to be the species most
sensitive to the neurologic motor effects of
chloromethane; and
57
-------
(2) humans appear to show both the type of neurologic and
behavioral toxicity expressed in monkeys and that
expressed in dogs.
C. Mutagenicity
Chloromethane has been reported to possess mutagenic
activity in bacterial systems that detect gene mutations (Andrews
et al. 1976, DuPont 1978, Simmon et al. 1978), and to cause
chromosomal aberrations in higher plants (Smith and Lotfy 1954).
However, the evidence for Chloromethane mutagenicity from this
series of experiments is insufficient to permit a mutagenicity
hazard assessment for Chloromethane. To perform such a hazard
estimation for humans, scientists must first demonstrate that a
substance and/or its metabolite(s) does or does not 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) in a higher system, and whether
or not the mutagenically active form can reach the genetically
significant target molecules in mammalian germinal tissue.
1. Gene Mutation
A review of the data available shows that Chloromethane is a
direct-acting mutagen. This means that Chloromethane does not
have to be metabolized by mammalian enzymes to an active form.
In bacterial systems capable of detecting gene mutations,
Chloromethane produces a strong, positive, reproducible dose-
response curve of chemically induced mutations in Salmonella
58
-------
typhimurium strains TA 1535 and TA 100. These strains normally
v
cannot synthesize the amino acid histidine and this must be added
to the nutrient medium to support their growth. When the proper
mutation occurs in the specific portion of deoxyribonucleic acid
(DNA) of these organisms that regulates this effect, they are
able to synthesize histidine and are then capable of growth in
histidine-free medium. The bacterial strains (TA 1535 and TA
100) which demonstrate chloromethane mutagenicity are mutated by
agents which cause changes in a specific guanine-cytosine base pair as
well as others in the DNA molecule. Agents that cause such
changes are called base pair mutagens. _S_. typhimurium TA 1535
and TA 100 are the same basic strain; TA 100 is TA 1535.with the
addition of a resistance factor, pKM 101, which confers
resistance to the antibiotic ampicillin and, at the same time,
increases the sensitivity to mutagenic agents.
After exposure to chloromethane, both with and without
metabolic activation, increased numbers of bacteria of strains TA
1535 and TA 100 were capable of growth in histidine-free
medium. On a quantitative basis, increasing concentrations of
chloromethane caused the mutation of greater numbers of
g
bacteria. For example, in a population of approximately 1 x 10
strain TA 100 bacteria, there will ordinarily be approximately
100 bacteria capable of growth in histidine-free medium.
Exposure to 2.5 percent chloromethane increased this number to
g
approximately 400 in 1 x 10 ; exposure to 20 percent chloro-
methane increased the number to approximately 1,100 in a total
g
population of 1 x 10 bacteria (Simmon 1978). Similar results
59
-------
were reported by Andrews et al. (1976), and DuPont (1978). In
this test system, therefore, chloromethane is a base pair mutagen
which causes an alteration in at least one guanine-cytosine
portion of the DNA molecule.
DuPont (1978), however, tested chloromethane in two strains
of S. typhimurium that detect frameshift mutagens, TA 98 and TA
1537, and reported it to be inactive. It is not uncommon for a
chemical which is a positive base pair mutagen to be inactive in
a frameshift strain and vice versa. This is one reason that a
good Ames test will include strains of both types of S.
typhimurium. However, the EPA believes that positive results in
only one strain are considered adequate to determine that a
chemical may pose a risk of human mutagenicity and should be
tested further. The tests the EPA proposes for assessing hazard
(see Summary B. 2. a) do not specifically measure base pair or
frameshift mutations, but are valid for both types.
Given the universality of the structure of DNA, it is rea-
sonable to assume that chloromethane may also cause base pair
alterations in the DNA of higher organisms, including man.
The Diamond Shamrock Corporation (1978a) has submitted a
series of test results in which chloromethane is reported to be
non-mutagenic for S. typhimurium strains TA 1535 and TA 100 and
Escherichia coli ATCC 23221 and ATCC 23233 and inactive in a
host-mediated assay in mice with strain TA 100 as the tester
strain. However, the significance of these negative test results
is questionable because of the experimental techniques reported.
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Chloromethane is a gas under conditions of normal
temperature and pressure. To adequately test such substances in
bacterial mutation systems requires special test methods and
procedures that were not mentioned in the reported study. The
test as described in the Diamond Shamrock submission is a spot
test. In a spot test, bacteria are incorporated into top agar
and poured over a base plate of minimal medium. The test agent
is then placed on the plate (either in crystalline form or on a
liquid saturated filter paper disc) and allowed to diffuse into
the medium. The formation of a ring or concentrated zone of
mutant colonies in the vicinity of the test sample is generally
considered to be a positive result. The report submitted by
Diamond Shamrock states that 1 to 5 ug of test chemical
(chloromethane was one of a series tested) were added to the
plate with a spatula. No discussion of the method of testing
chloromethane, a gas at normal temperature and pressure, is
i
made. The results of this assay are open to question because the
spot test, as described, is inappropriate for testing chloro-
methane. In addition, the EPA feels that the evidence of a
single negative test result conducted under less than optimal
conditions is outweighed by the positive results obtained in
three independent studies. The EPA, therefore, considers
chloromethane to be mutagenic for S. typhimurium strains TA 1535
and TA 100.
Chloromethane was also reported to be inactive in a host-
mediated assay in which S. typhimurium strain TA 100 was used as
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an indicator organism (Diamond Shamrock 1978a). The host-
mediated assay employs an intact mammalian host as the activation
system for a microbial mutagen. The test chemical is adminis-
tered to animals over a period of time which may range from
several hours to several days. At the end of the treatment
period, the indicator organism is administered to the host animal
and allowed to incubate, presumably in the presence of the test
agent and/or its metabolites, over a period of several hours. At
the end of the incubation period, the indicator organisms are
removed and plated on a selective medium to determine mutation.
The test chemical may be administered by one of several routes;
e.g., intraperitoneally, intramuscularly, intratesticularly, or
orally. The indicator organism is generally administered by a
different route than the test chemical. The indicator organisms
are most often administered by intraperitoneal and intravenous
injection. Indicator organisms include bacteria, yeast and some
mammalian cells capable of growth in culture. In the study sub-
mitted by Diamond Shamrock, the test chemical was administered
orally and the indicator organism was administered intraperi-
toneally. The report states that the test chemical was dissolved
or suspended in 10 percent ethanol or peanut oil, without
specifying which was used for chloromethane and without
specifying how gaseous chloromethane was added to the solvent.
Concentrations are given in mg/kg but with no indication of how
this was determined. No positive control data were presented;
test data which are presented.are inadequate and not subject to
critical review and evaluation. In addition, the variables
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inherent in this system, e.g., concentration of test agent in the
animal, animal strain insensitivity, less than optimal amounts of
test substance administered, failure of the test chemical or its
active metabolites to reach the bacteria in effective amounts, or
administration of either test agent or bacteria by the least
effective route, may have resulted in false-negative or seemingly
incongruous results with chloromethane in this assay. For these
reasons, the EPA considers the lost-mediated assay particular
test results to be of questionable value in assessing the
mutagenic potential of chloromethane.
The EPA believes that in any instance where contradictory
data is received on mutagenicity tests, even if all tests are
well-conducted, further testing is necessary to determine if a
potential human risk exists.
2. Heritable Translocation
Chloromethane has also been reported to cause chromosome
breaks in pollen grains of Tradescantia paludosa (Smith and Lotfy
1954). At optimal levels for each, chloromethane (9231 ppm)
caused a higher percentage of chromatid breaks (240 breaks/5,932
chromosomes, or 4.04 percent) than did ethylene oxide at 7692 ppm
(24 breaks/2,150 chromosomes, or 1.12 percent). At equivalent
ppm (10,769) chloromethane was also more potent than ethylene
oxide in causing chromatid breaks (3.09 percent vs. 0.65
percent). Chloromethane produced only chromatid breaks, however,
while ethylene oxide also induced erosions and contractions,
leading to a higher level of total chromosomal abnormalities than
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chloromethane at optimal levels (5.21 percent vs. 4.04
percent). There were six breaks per 6,590 chromosomes, or 0.09
percent, in untreated control pollen grains. Ethylene oxide is
one of the best-studied mutagens known and has demonstrated
positive mutagenicity in every system it has been tested in (EPA
1978a).
Diamond Shamrock (1978b) also submitted the results of a rat
dominant lethal study in which chloromethane proved to be
inactive. A dominant lethal mutation is a change in the germ
cell, either egg or sperm, which is lethal to zygotes produced by
the mutated germ cell. In mammals, dominant lethal mutations
will reduce litter size. This reduction in litter size can be
due to the failure of the fertilized egg to implant or to develop
after implantation has taken place. Brewen et al. (1975) have
shown that dominant lethality results from chromosome breakage,
and that the incidence of broken chromosomes at metaphase of the
first cleavage of the fertilized egg corresponds to the incidence
of dominant lethal eggs. From the tenth day of pregnancy onward
in rats and mice, uterine contents can be recognized and
classified into living embryos and early and late fetal deaths.
Dominant lethal tests can be performed by exposing either male or
female animals to the test substance and mating them with
untreated members of the opposite sex. The test is most commonly
performed by treating male animals and mating them to untreated
females. The most common route of administration of the test
substance is intraperitoneally; other routes include ingestion,
gavage and inhalation. In the test as described by Diamond
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Shamrock (1978b), chloromethane was administered by oral
intubation as a saturated solution in dichloromethane. Given the
gaseous nature of chloromethane, exposure by inhalation is
considered to be more appropriate and would have eliminated the
need to use a solvent such as dichloromethane which is itself a
biologically active material (see Section III.D.3., III.E., and
III.P.4.). In any case, a dichloromethane control should have
been included in the study and this was not reported. The assay
is also difficult 'to evaluate because of apparent inconsistencies
in the data and because of the manner in which the data are
presented (Diamond Shamrock 1978b) . The test as reported is
divided into two parts: an acute and a subacute study. Survival
rates of the animals used for the positive control are shown in a
table that presents survival data only and are presented later in
a table which shows fertility data. The data in the two tables
do not agree. The narrative text of the study and the table
which presents survival data imply that separate groups of
animals were used as negative controls in the acute and subacute
parts of the study. The tables which present fertility data
imply that the same animals served as negative controls for both
parts of the study. Data on such aspects of the study as corpora
lutea counts and preimplantation loss, necessary to properly
evaluate the results, are not presented and a table listing
average implants fails to specify whether it is referring to
total implants (live plus dead embryos), or living embryos
only. The data as presented are difficult to interpret, and do
not lend themselves to statistical evaluation and critical
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review. As a result, the validity of the dominant lethal study
as presented is open to question.
D. Oncogenicity
Neither epidemiology or other systemic human studies nor any
animal assays sufficient to evaluate the oncogenicity of
chloromethane have been reported. However, there is substantial
information suggesting that this chemical may possess oncogenic
potential. This information includes evidence on its mutagenic
activity, on its in vitro and in vivo alkylating capabilities,
and on its structural relationship to known or suspected
oncogens.
1. Mutagenic Activity
As described earlier, chloromethane has been reported to
possess mutagenic activity in bacterial systems that detect gene
mutations and to cause chromosomal aberrations in higher plants
(see Section III.C. for detailed discussion and evaluation of
each of these studies). In assays employing S. typhimurium test
strains TA 1535 and TA 100, the chemical induced a strong,
positive dose-dependent mutagenic response, both with and without
metabolic activation (Andrews et al. 1976, DuPont 1978, Simmon
1977). These tester strains detect base pair mutagens. In
Tradescantia paludosa pollen tubes, chloromethane increased the
chromatid breakage rate about forty-fold (Smith and Lotfy
1954). Considering chloromethane's activity in S. typhimurium
strains TA 100 and TA 1535 and in Tradescantia paludosa pollen
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tubes, the EPA considers chloromethane to be a direct acting
mutagen (i.e., it does not have to be metabolized to be active).
The concept that neoplasms arise from mutations in somatic
cells was originally postulated by Boveri in 1914 to account for
the unlimited variety of tumor types, and the fact that, on cell
division, the daughter cells maintain their neoplastic properties
(Boveri 1914, Chu et al. 1977, Trosko and Chang 1978). Oncogens
and mutagens have two properties in common:- 1) the ability to
induce new properties in cells that can be transmitted 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 the
majority of oncogens need to be metabolized in order to be active
(Miller and Miller 1974, MiJller 1978, Miller 1979); second,- in
vitro metabolic activation systems which could be incorporated
~~"~~~~~ A.
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 whose atoms have an electron deficiency
that enables them to react with electron-rich sites 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
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mutations including gene mutations (both base pair substitution
and frameshift alterations), chromosomal aberrations, and non-
disjunctions. The oncogenic potential of a chemical has also
been correlated with its ability to interact with and modify DNA
(Rosenkranz and Poirier 1979).
A wide variety of assay systems have been developed to
detect effects on genetic material, including gene mutations and
chromosomal aberrations. Gene mutations are alterations in
part(s) of the DNA and may be due to substitutions, loss, or
acquisition of one or more pairs of nucleotides. Such mutations
may be detected as forward (i.e., a change from the wild type) or
backward (i.e., reversion to wild type in a mutant strain)
mutations.
The Ames Test is an assay that measures gene mutation at
histidine loci in Salmonella typhimurium (a bacterium) (Ames et
al. 1975). Five mutant strains, which can measure backward base
pair substitutions or frameshift mutations, are treated with the
test chemical, and histidine revertants are selectively cultured
from a population of histidine requiring cells by their ability
to grow on media without histidine. Tests are performed both
with and without metabolic activation.
The Ames test is a relatively easy test for detecting muta-
gens. Advantages of the Ames test are that the tester strains
are well characterized, thus ensuring that gene mutation is the
characteristic being measured and indicating the type of
mutational event being caused. The disadvantages, however, are
that reverse rather than forward mutation is measured, a
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prokaryotic, not a eukaryotic system is measured, and the test is
in vitro.
The particular value of the Ames test to the EPA's work is
that it can be used as a indicator of oncogenic potential. An
excellent correlation between mutagenic activity and oncogenic
activity has been demonstrated (Bartsch 1976, Brusick 1979).
Eighty to ninety percent of the known oncogens tested in this
system have been positive. The number of false positives is also
low in this system, ranging from 10 to 15 percent.
Chromosomal aberrations are alterations in the structure or
number of chromosomes. Structural alterations are mainly the
result of breaks in chromosome strand(s) and may lead to unstable
nontransmissible changes (achromatic breaks, achromatic gaps,
chromatid breaks, chromatid interchanges, acentric fragments,
ring chromosomes or dicentric chromosomes) or stable transmis-
sible changes (inversion, translocation, or deletion). Varia-
tions in the number of chromosomes, polyploidy or aneuploidy, are
mainly the result of anaphase delay, metaphase arrest, endoredup-
lication, or non-disjunction during mitosis and meiosis. Such
chromosomal changes may be detected by cytologic or genetic
methods in micro-organisms, plants, insects, mammalian cells in
vitro, and mammals in vivo. While tests on microorganisms
largely detect changes in chromosome number, tests on plants,
insects, mammalian cells in vitro and mammals in vivo will detect
changes in both chromosome number and structure.
Although chloromethane has been shown to cause chromatid
breakage in T. paludosa pollen tubes, there are no correlation
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studies showing the exact relationship between mutagenic activity
in this system and activity as an oncogen.
2. Alkylating Capabilities
Alkylating agents belong to the larger class of reactive
compounds called electrophiles (electron seeking). Representa-
tive animal tests show that some members of virtually all classes
of alkylating agents are oncogenic (Lawley 1976). The chemical
basis for their biologic effect is the chemical modification of
cellular DNA by these agents (Singer 1975). Lawley (1976) has
reviewed oncogenesis by alkylating agents, while Singer (1975)
and Pegg (1977) have reviewed the effects of these chemicals on
nucleic acids and the relationship of these effects to
oncogenesis and mutagenesis.
Alkylation is the most common reaction of chloromethane. In
this reaction, the methyl group is transferred to a nucleophilic
(electron donating) atom of another molecule with simultaneous
elimination of chloride ion, to form a new, stable covalent
carbon-heteroatom bond; that is, the nucleophilic reactant is
alkylated, or in this case methylated. Chloromethane is a strong
enough alkylating agent to be used commercially for this purpose,
e.g., in the production of tetramethyllead (von Oettingen
1964). The chemical also has been shown to have alkylating
activity in both human (in vitro) and rat (in vivo) tissues
(Redford-Ellis and Gowenlock 1971a, Reynolds and Yee 1967),
forming primarily S-methylglutathione and S-methylcysteine.
Although some compounds are biologically active only after
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metabolic conversion to electrophiles, chloromethane can react
directly with genetic material, as illustrated by its ability to
induce mutagenesis in S. typhimurium without metabolic activation
(Section III.C.). In addition, chloromethane may be metabolized
to formaldehyde (see Section III.F.4.), another reactive
electrophilic compound.
3. Structure-Activity Relationships
Known chemical oncogens comprise a structurally diverse
group of synthetic and naturally occurring organic and inorganic
chemicals (Miller and Miller 1974, Miller 1979). Although
knowledge of the chemical structures of known oncogens currently
provides in itself no way of definitively assessing molecular
structures of unknown oncogenicity (Fishbein 1977), certain
structural criteria for suspecting chemicals of oncogenic
activity have been determined (Arcos 1978). Meeting these
criteria are halogenated hydrocarbons and alkylating agents.
Chloromethane falls into both categories. Its alkylating
properties have been discussed in the preceding subsection.
Chloromethane is structurally very similar to other
chlorinated methanes (i.e., dichloromethane, CI^C^; chloroform,
CHC13; and carbon tetrachloride, CC14) and of other
monohalomethanes (i.e., fluoromethane, CH-,F; bromomethane,' CH-,Br;
and iodomethane, CH^I). Of these structural analogues,
chloroform and carbon tetrachloride have been shown to be
oncogenic in animals while iodomethane and dichloromethane are
suspect oncogens. Another suspect oncogen, formaldehyde (CUT
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1979b), is a known metabolite of chloromethane (see Section
III.F.4.). The EPA is not aware of any oncogenicity studies on
the other two monohalomethanes, fluoromethane and bromomethane.
Carbon tetrachloride produces liver tumors in the mouse,
hamster, and rat following several different routes of
administration. The International Agency for Research on Cancer
(IARC) determined in 1972 that evidence of the oncogenicity of
carbon tetrachloride in experimental animals is sufficient to
regard the chemical for practical purposes as if it were
oncogenic to humans (IARC 1972, Tomatis 1979).
The oncogenicity potential of chloroform has been reported
by NCI (NCI 1976). In this study, the chemical was administered
in corn oil via stomach tubing five times per week for 78 weeks
to female mice at 7 and 4 mg/day and to male mice at 14 and 8
mg/day. Ninety-five percent of the female mice and 98 percent of
the male mice in the high dose groups developed hepatocellular
carcinomas while 80 percent and 36 percent, respectively, in the
low-dose group developed such tumors (controls: females 1
percent and males 6 percent). Male Osborne-Mendel rats receiving
80 mg/day of chloroform for 78 weeks developed a 24 percent
incidence of kidney tumors, 70 percent of which were malignant.
The EPA has accepted the study as adequate support for the
regulation of chloroform in water on the basis of its oncogenic
potential (EPA 1978b).
lodomethane has been shown to induce lung adenomas in strain
A mice following intraperitoneal injection (Poirier et al. 1975)
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and to cause local sarcomas with lung metastasis in rats follow-
ing subcutaneous injections (Druckrey et al. 1970). The develop-
ment of lung adenomas in strain A mice is considered to be a
sensitive indicator of the oncogenic activity of alkylating
agents (such as iodomethane) (Poirier et al. 1975, Weisburger
1978). In the case of iodomethane, 0.31 mmole/kg (44 mg/kg)
given over a 24-week period (3 times per week) induced a
significant increase in the average number of lung adenomas per
mouse. In fact, iodomethane on a mmole basis was more active
than urethane, which is the usual positive control used in this
assay system. In the Druckrey et al. studies (1970), all 6 rats
receiving 20 mg/kg of iodomethane once a week for a year
developed sarcomas at the site of injection. Of 12 rats
receiving 10 mg/kg once a week for a year, 11 developed sarcomas
at the site of injection. Another important finding was that in
most cases the tumours had metastasized to the lungs. The latter
information indicates the malignant nature of the induced
tumours. Although neither of these studies provides sufficient
information on iodomethane oncogenicity to do an adequate hazard
assessment, they do indicate that it has oncogenic potential.
While evidence on the oncogenic potential of dichloromethane
is inconclusive because of the inadequacy of the data reported
(Theiss et al. 1977, Rampy et al. 1979), it nevertheless has been
demonstrated that dichloromethane can cause a statistically
significant increase in the numbers of benign mammary tumors in
female Sprague-Dawley rats at levels of 500-3500 ppm and in males
at 3500 ppm (Rampy et al. 1979).
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The EPA has recently received from CUT a notice of the
development of nasal squamous cell carcinomas in rats exposed to
formaldehyde (a metabolite of chloromethane), at 6 and 15 ppm
(CUT 1979c). Although the study is not complete and signficance
of these findings cannot be fully assessed, the discovery of such
rare tumours in this species leads the EPA to consider the
preliminary notification of great import. Exposure to
formaldehyde was by inhalation and the carcinomas were found in
the nose, so that the irritant effect and localized high levels
may play an important part in the oncogenicity, whereas the
production of formaldehyde as a- metabolite of chloromethane might
lead to different results, since concentrations would be expected
to be diffuse.
E. Teratogenicity
Little direct information is available regarding the terato-
genic potential of chloromethane. There has been one report
associating the birth of a severely deformed child with maternal
exposure to chloromethane and ammonia vapors (Kucera 1968), but
no details as to dose, length, or time of exposure are
presented. To date no animal studies to evaluate the effects of
chloromethane on the fetus have been published. However, Smith
and von Oettingen (1947a) reported that "a rabbit conceived and
born during exposure to 500 ppm grew at a normal rate during 33
weeks of exposure," but developed slight neuromuscular symptoms
like those seen in adults. Because exposures continued after
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birth, and only one rabbit was so exposed, no conclusion can be
drawn from this study.
Chloroform (trichloromethane) has been found by Schwetz et
al. (1974) to be both embryotoxic and fetotoxic. Serious
malformations, as well as retarded fetal development, were
reported. In addition, Murray et al. (1979) observed a
statistically significant increase in cleft palate in mice due to
chloroform exposure. Although cleft palate was not observed in a
longer dosing regimen, embryotoxicity has now been observed in
both the mouse and the rat exposed to chloroform.
Chloromethane, as a gas, would be expected to cross the
placenta readily (Villee 1971, Nishimura and Tanimura 1976).
Although no direct evidence of chloromethane induced fetal
toxicity has been found, Hartman et al. (1955) tell of a seven-
month pregnant woman severely poisoned by chloromethane. When
found, the fetus had been aborted and was still attached, dead,
to the undelivered placenta.
Additional concern for the teratogenic potential of
chloromethane is based on its documented neurotoxicity. 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 a
neurotoxicant such as chloromethane warrants its evaluation as a
teratogen. Evidence has been presented that suggests that both
structural and behavioral deficits in adult and developing
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systems are associated with exposure to other nonspecific 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).
Recent evidence from Bornschein et al. (In Press) indicates
behavioral defects in rats exposed in utero to dichloromethane at
a dose which caused no morphologic defects (Hardin and Manson In
Press).
On the basis of chloromethane1s neurotoxicity in adults,
accessibility to the fetus, the embryo-fetal effects of its
congeners, dichloromethane and chloroform, and in agreement with
the concept that anatomical and behavioral evaluations are
complementary approaches to CNS toxicity (Barlow and Sullivan
1975, Langman et al. 1975), the EPA concludes that chloromethane
has a potential risk for teratogenicity in the human for both
behavioral and structural malformations.
F. Metabolism
Although fragmentary research has been accomplished in
several areas of chloromethane metabolism, insufficient
information exists to give a complete characterization. It is
known that chloromethane is absorbed through the lungs, that
radioactivity can be detected to varying degrees in all tissues
1 4
tested following inhalation of x C-chloromethane, and that
excretion of unchanged compound is through the lungs, urine and
feces, while possible metabolites also appear in the expired air
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and urine. Some percentage of inhaled radioactivity is retained
14
by the organism following administration of CH^Cl, and is
apparently primarily bound to tissue sulfhydryl groups. Other
known metabolic products include methanol, formaldehyde, and
formate. Although the EPA feels that metabolism studies on
chloromethane are not complete, the Agency believes that the data
available are sufficient at this time to assist in evaluating the
risk of exposure to chloromethane.
1. Absorption
Although it is generally believed that the principal route
of human exposure to chloromethane is almost certainly by
inhalation, most inhalation experiments in both man and animals
are really whole-body exposure experiments and possible skin and
GI absorption cannot be wholly ruled out (CUT 1979d, Smith and
von Oettingen 1947a, Stewart et al. 1977, Yevtushenko 1967). It
has been demonstrated, moreover, that chloromethane can be
absorbed through the skin (NIOSH 1977). However, in one
experiment (Morgan et al. 1970) the human volunteers inhaled the
radiolabeled chloromethane directly through a tube placed in the
mouth (which does not eliminate the possibility of GI or mucous
membrane absorption, but does eliminate that through the skin)
and showed that absorption through the airways probably does
occur.
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2. Distribution
Several different experimenters have followed blood and
tissue levels of chloromethane over time. The experiments can
usually be divided into two types: 1) those in which after a
single brief exposure, either by injection or inhalation, the
disappearance of chloromethane from the tissue is followed; and
2) those in which the subject has been exposed for a considerable
period of time (i.e., the condition is more or less stabilized)
and the levels of the chemical are followed after cessation of
exposure.
Sperling et al. (1950) injected chloromethane into dogs
intravenously (i.v.) and measured blood and tissue chloromethane
at various times. At time zero, the percentage of chloromethane
present in the blood varied between 4.5 and 13.1 percent (of
1680 mg injected), at 30 minutes, the values were 1.5 to 2.7
percent, and at 60 minutes they ranged from 0.6 to 1.3 percent.
However, between eight and thirty minutes were required to inject
the total amount of chloromethane (as a gas), thus allowing quite
a bit of time for inital redistribution to tissues and biotrans-
formation. This group did another series of experiments where
they measured blood and tissue levels in one group of animals at
zero time following injection of various amounts of chloromethane
(173-206 mg/kg), and another group (154-228 mg/kg) after one
hour. The blood chloromethane concentration initially varied
between 0.119 to 0.135 mg/cc, while at one hour, the range was
0.035 to 0.041 mg/cc. Neither of the groups showed a dose-blood
level relationship, however. Various tissues were measured for
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chloromethane content during the same experiments with similar
results occurring. Levels at 60 minutes were on the average
lower than those at the beginning, although there was
considerable variation among dogs.
Soucek (1961) used subcutaneous (s.c.) injection in rats to
measure the disappearance of chloromethane from the blood. At
two minutes following a single 1200 ug injection of chloromethane
in H20, 1.4 percent of the dose appeared in the blood, at 10
minutes 0.7 percent, while at 25 minutes, chloromethane
concentrations were below the level of detection. However, it is
difficult to compare the two experiments, as Soucek could not or
did not measure the rate of chloromethane1s entrance into the
blood, and measurements made after s.c. injection are the result
of a two-way flow, both into and out of the blood. Although
Soucek was unable to measure chloromethane in the blood beyond 10
minutes, he was still able to detect unaltered chemical in the
expired air at 120 minutes, as were Sperling et al. (1950).
CIIT's study (1979d) is of the second type, but with an
additional change. Instead of measuring chloromethane, CUT
1 4
administered CH~C1 and measured radioactivity, which enabled
the investigators to pick up metabolites and bound compound as
well as free compound dissolved in the plasma. Blood C-levels
were measured at intervals following a 6-hour exposure to 1500
1 4
ppm in rats. At time zero, the C-content of the blood was
0.93±0.02 umole of CH3C1 equivalents/ml, after which the level
steadily dropped until at 24 hours the value was 0.17+0.02 umole
CH-jCl equivalents/ml. Levels in all other tissues measured
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(liver, fat, kidney, spleen, lung, heart and brain) acted in a
similar manner. However, some tissues lost radiolabel much more
quickly than others. At 24 hours the amount of c in fat was
12.1 percent of the initial value, while the heart still carried
38.9 percent of its initial load. At time zero, liver was
highest with 2.63, while brain had the lowest concentration, 0.55
umole CI^Cl equivalents/ml, but at 24 hours levels in all the
tissues were closer to each other, from liver with 0.45 to brain
with 0.12 umole CHjCl equivalents/g wet weight. There is
apparently little or no redistribution to other tissues nor are
the organs which appear to be most affected (i.e., brain, liver,
kidney) those which retain the greatest amounts of the
radioactivity.
3. Excretion
Some portion of the gas is excreted unchanged, not only
through the lungs in man (Morgan et al. 1970, Stewart et al.
1977), dogs (Sperling et al. 1950), and rats (Soucek 1961), but
in the urine and bile following i.v. injection in the dog
(Sperling et al. 1950). CUT (1979d) also claimed that a portion
of the administered chemical was found in the expired air
(radiolabel trapped in a charcoal filter but not chemically
identified). Under the conditions of the study (6-hour
inhalation of different dose levels by rats and mice, 48 hour
observation period) the amount excreted unchanged was fairly
small and did not appear to be strictly dose-dependent. In rats,
at 100 ppm, 2.4 percent of the retained material was found in the
80
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expired air, at 375 ppm, 1.8 percent, and at 1500 ppm, 6.3
percent; in mice, at 1500 ppm, 4.4 percent was exhaled (CUT
1979d).
Sperling et al. (1950), following injections of various
amounts of chloromethane into dogs, observed that about 5 percent
of the total injected was found unchanged in the expired air
within the first hour. However, Morgan et al. (1970) found that
-}Q
one hour after inhalation of CF^J Cl in man, 29 percent of the
administered radioactivity was excreted through the lungs. The
EPA believes that all the radioactivity measured in the expired
air was chloromethane, rather than a metabolite, even though this
was not verified chemically, as all the known or postulated
biotransformation mechanisms produce chloride ion, a non-volatile
product. The rather large difference between these two results
may be due to the mode of administration, to the species, or to
what was measured in the expired air: chloromethane in the first
•J Q
case (Sperling et al. 1950) and Cl in the second (Morgan et al.
1970) .
Morgan et al. (1970) compared the pulmonary excretion of
•3 Q
CH3 Cl with that of the higher chlorinated methanes, and
concluded that chloromethane acted in a different manner. When
excretion rate versus time was plotted (retention curve), the
di-, tri- and tetrachlorinated methanes had parallel slopes,
while monochloromethane's slope was steeper, cutting across the
others. This may be due to a number of reasons: 1) the compound
is more reactive; 2) a greater percentage is excreted by
alternate routes; 3) it is more fat-soluble. The authors felt
81
-------
that chloromethane behaved like iodomethane, which reacts rapidly
with sulfhydryl groups in the erythrocyte in an enzyme-catalyzed
methylation process.
Stewart et al. (1977) discovered two populations among their
human subjects. Four of their subjects, as well as two from a
previous study, had considerably elevated post-exposure breath
and blood chloromethane levels. The rest of the volunteers
carried a two to six times lower body burden than these. Stewart
and his coworkers postulated that the worker who carries a lower
body burden than the majority may be at greater risk from chloro-
methane exposure. This would appear to indicate that a larger
portion of the exposed population would be at greater risk. The
data can be interpreted to mean that those people with greater
amounts of unaltered compound in their bloodstream and airspace
might be metabolizing less of the material rather than absorbing
or carrying more. And as the metabolized material is probably
responsible for toxicity, the subjects excreting more compound
unchanged would be at a lower risk rather than a higher one. If,
on the other hand, chloromethane per se is the toxic compound,
those persons with higher blood and breath levels may be more
susceptible to overexposure. Of course, the possibility exists
that neither of these theories is important, and the different
populations are at equal risk.
82
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4. Biotransformations
The earliest theories about chloromethane's biotransforma-
tion (Flury 1928) dealt with its probable conversion to formalde-
hyde through methanol. Formaldehyde has been found in the blood
of rats (Yevtushenko 1967) and mice (Sujbert 1967) following
inhalation of chloromethane, and in mice (Sujbert 1967) following
intraperitoneal (i.p.) injection. Sujbert (1967) also was able
to detect methanol in the bloodstream of mice following
inhalation or i.p. injection as did Hayhurst and Greenburg (1929)
who detected methanol, formaldehyde and formates in the organs of
victims of chloromethane poisoning. Smith (1947), on the
contrary, was unable to find any methanol in the blood of dogs
that had been exposed to chloromethane by inhalation for 23 or 25
days. Other researchers have tested for formate in the urine or
tissues of subjects exposed to chloromethane, with variable
results. Baker (1927), Kegel et al. (1929) and Hayhurst and
Greenburg (1929) found formates in human tissues and urine fol-
lowing accidental exposure to the compound, whereas neither
Lundgren (1947) nor Hansen et al. (1953) were able to demonstrate
increased formate in their human subjects correlating with levels
of chloromethane in the ambient air. Lundgren could not estab-
lish a correlation between excretion of formate in rabbits, rats,
or guinea pigs, and extent of exposure to chloromethane, but
guinea pigs in the apartment of a person who died of chloro-
methane toxicity when her refrigerator sprang a leak, showed
formate in their tissues (Hayhurst and Greenburg 1929).
83
-------
The formation of formaldehyde from chloromethane is probably
analogous to that proposed for the biotransformation of
chloroform (CHCl-O to phosgene, a reaction that has recently been
confirmed experimentally (Pohl et al. 1977, 1979, Pohl and
Krishna 1978).
H
Cl
I
-C -
1
Cl
P-450
H -
Cl
1
0 - C -
1
Cl
Cl
•
NE
Cl
0 = C
\
-I- HC1
Cl
NE = nonenzymatic
It has been suggested that a cytochrome P-450 monooxygenase
oxidizes CHC^ to unstable trichloromethanol, which spontaneously
dehydrochlorinates to yield the reactive phosgene. Dichloro-
methane seems to follow a similar initial pathway (Kubic and
Anders 1978) to eventually yield CO through a formyl halide
intermediate:
Cl P-450
1
C — Cl >
I
H
Cl
1
H - 0 - C - Cl
1
H
NE
'^ ^
^\
\
*
Cl
H
NE
CO + HC1
HC1
84
-------
The formation of formaldehyde from chloromethane probably occurs
as follows:
H
H
I
C
I
H
P-450
H - 0 -
H
I
I
H
NE
0 = C
H
H
+ HC1
Additional reactions can occur after formaldehyde production:
(1) aldehyde reduction, with formaldehyde going to methanol;
(2) aldehyde dehydrogenation, with formic acid and/or formates as
the ultimate product.
Ahmed and Anders (1978) have proposed an additional route
for metabolism of dihalomethanes, which involves alkylation and
dealkylation of glutathione (GSH). This pathway yields
formaldehyde, formic acid and inorganic halide. As it is known
that chloromethane binds very specifically to GSH in erythrocytes
(Redford-Ellis and Gowenlock 1971a), this alternative route may
also be important for chloromethane.
H
I
H — C — H + GSH
I
Cl
GS -
P=450
CH3 >GS - CH2OH
NE
HC1
1
GSH
Formaldehyde dehydrogenase/NAD+
GS - C(=0)H
I S-Formyl glutathione hydrolase
GSH + HCOOH
= enzymatic
85
-------
CUT (1979d) measured radioactivity in the expired air for
48 hours following a 6-hour exposure to chloromethane in rats and
mice. That percentage of radioactivity trapped by a charcoal
filter they designated as chloromethane, while that trapped by
ethanolamine in methoxyethanol was considered to be CCU • No
mention was made as to whether such possible alternative volatile
metabolites as carbon monoxide or formaldehyde would be trapped
and measured by their methods.
CUT (1979d) found that in rats in the 48 hours following a
i 4
6-hour exposure to 1500 ppm A CH^Cl, more than 41 percent of the
total recovered radioactivity was CCU from the expired air,
while in mice given the same dose, less than 18 percent was
excreted as CO-. In mice, the- largest percentage of recovered
radioactivity (60 percent) was excreted in the urine, while the
rat excreted only 40 percent of the retained radioactivity as
urinary components. It is possible that a portion of the urinary
radioactivity occurs as bicarbonate, for following acid
hydrolysis of the mouse urine, a small portion (9 percent) of the
urinary radioactivity was found in the headspace of the vial,
presumably as C0<
5. Tissue Retention
In addition to measuring radioactivity in the various
excreta, CUT (1979d) measured radioactivity retained in some
organs and the carcass following exposure to CH^CI. in rats at
the end of a 48 hour period following 6 hours of exposure to 100,
86
-------
375 or 1500 ppm of the gas, there was no increase in the amount
of associated radioactivity between the 375 and 1500 ppm groups
(e.g., for liver at 100 ppm, tissue radioactivity equalled 52.3
umole of C-chloromethane equivalents/g wet weight, while at 375
ppm, the radioactivity was 325.2, and at 1500 ppm, it was 265.2),
which may indicate a saturation of available binding sites. Of
the amount retained following a 6 hour exposure, 22.5 percent was
associated with the tissues at 100 ppm, 21.4 percent at 375 ppm
and 17.3 percent at 1500 ppm. Although neither the form nor the
type of binding in the tissues was specified by CUT, the
retention of such a high proportion of radioactivity after two
days indicates a fairly strong binding capacity. At all dosages,
retention was lowest, by a factor of three, in the brain, highest
in the liver at 375 and 1500 ppm and highest in fat at 100 ppm.
In mice following a similar exposure regimen at 1500 ppm, only
8.3 percent of the total recovered was associated with the
tissues, and while the brain level had again the lowest value,
liver and kidney had the highest.
6. Binding
Morgan et al. (1970) postulated that chloromethane acts like
iodomethane, reacting rapidly with sulfhydryl groups in an
enzyme-catalyzed methylation process. Redford-Ellis and
Gowenlock (1971a,b) studied the reaction of 1 C-chloromethane
with human blood in vitro. In serum or plasma, about 65 percent
of the radioactive uptake was associated with plasma protein but
only about 2-3 percent covalently bound to the plasma protein
87
-------
(specifically albumin), producing primarily S-methylcysteine,
although minor radioactive components of 1-methyl and 3-methyl-
histidine were also found. In erythrocytes, uptake was
independent of dose over the range used (600-1000 mg/ml
erythrocyte), being 357 mg/ml erythrocyte after 80 minutes, of
which 58-130 mg was bound covalently to glutathione (GSH') .
However, in studies on red cells, after uptake was complete, no
radioactivity was lost by hemolysis or by washing and no
radioactivity could be detected bound to any other components of
the erythrocyte, so there appears to be some discrepancy between
uptake and binding. Heating the blood before adding the
chloromethane reduced binding by over 90 percent, indicating a
probable enzyme-catalyzed reaction. Redford-Ellis and Gowenlock
(1971b) continued their researches by studying chloromethane1s
binding to rat brain, liver and kidney homogenates in vitro, as
these are the organs primarily associated with chloromethane
toxicity. In all these tissues, the primary products are c-S-
1 4
MeCys and C-S-MeGSH, while in the kidney additional traces of
radioactivity were found in methionine. The formation of these
compounds in tissue homogenates also appears to be partially
enzyme-dependent, as heating the tissues reduced the level of
binding.
As part of the CUT study, Dodd et al. (1979) looked at
alterations in tissue sulfhydryl concentrations in rats after
acute inhalation exposure to 1500 ppm chloromethane for 6
hours. They found that although changes in total tissue sulfhy-
dryl groups were minimal at all times (0,1,2,4,8,18 hours) after
88
-------
exposure, non-protein sulfhydryl content was reduced in liver,
kidney, lung and blood (most to least) indicating a decrease in
free reactive SH groups. At eighteen hours after exposure non-
protein sulfhydryl content had returned to control values.
However, earlier work by the same group (CUT 1979d) reported
that radioactivity was still present in these tissues 48 hours
after exposure. It appears to the EPA that either: 1)
significant amounts of CH^Cl or a metabolite are reacting with
non-sulfhydryl groups or 2) rearrangement is occurring.
IV. Current and Planned Testing
The Chemical Industry Institute of Toxicology (CUT) has in
progress a toxicologic evaluation of chloromethane in laboratory
animals. The major components of the CUT program are a pharma-
cokinetics study, a 90-day preliminary study, teratogenesis-
reproduction studies, and a 24-month chronic inhalation toxicity
study. Thus far, the pharmacokinetics and 90-day probe study
have been completed, and the 24-month chronic toxicity study was
initiated in June 1978. The teratogenesis-reproduction studies
have not yet begun.
The pharmacokinetics study involved the dosing of C-
labeled chloromethane to rats (F-344 albino) and mice (BgC.,F,,
hybrid) by inhalation (see Section III.F.) (CUT 1979d) .
The 90-day probe study (CUT 1979a) involved the inhalation
exposure of F-344 albino rats and BfiC~F, hybrid mice to various
levels (300, 750, 1500 ppm) of chloromethane, 6 hours per day, 5
89
-------
days per week for 13 weeks. There are certain deficiencies
associated with this study including the following:
(1) The rat is not the most appropriate test species for
systemic chronic effects at the dose levels used.
This species was previously shown to be unaffected by
exposure to 1000 ppm chloromethane, 6/hours/day, 6
days/week for 64 weeks (Smith and von Oettingen 1947
a,b). Also, since the toxicity of chloromethane
decreases as the exposure-free period is increased,
one would anticipate that decreasing the exposure
frequency to 5 days/week over 90 days would -lead to
little, if any, toxicity even at 1500 ppm. Dogs or
monkeys may be more appropriate since they showed
signs of toxicity even at 500 ppm, 6 hours/day, 6
days/week and exhibited neurologic and behavioral
effects seen in humans exposed to chloromethane
(Smith and von Oettingen 1947a, Smith 1947).
(2) Although the protocol required twice daily obser-
vations of the animals to detect signs of toxicity
and behavioral changes, no information on these
observations, if actually done, was reported.
(3) Of the 80 mice used in the study, 19 died during the
13-week study; 14 of which died due to stated
problems with new cages. Seven of those dying of
90
-------
trauma were in the high dose group of male mice.
Although there was no statement of time of death,
three of the male mice in the high dose group dying
of trauma were necropsied and the results included in
the overall histopathologic findings.
(4) Although there were eye lesions in 13 of the treated
mice and although chloromethane is known to induce
eye lesions (see Section III. B.)f there was no clear
explanation of the nature of the lesions or why the
results were concluded not to be compound related.
(5) There was a wide variability of response in the
control groups. A large standard deviation in a
control group means that the difference between a
treated group and the control group needs to be
larger in order to detect a significant difference.
Therefore, in a better controlled study, perhaps more
significant differences would have been detected.
CUT (1979b) also has in progress a 24-month chronic inhala-
tion study in mice and rats. This study was initiated in June
1978; to date, the EPA has received a 6 month interim report
(CUT 1979e) . The exposure levels being administered are 50,
225, and 1000 ppm. The frequency of administration is 6
hours/day, 5 days/week for 18 months. As planned, during the
last 6 months of the study, the animals will not be exposed to
91
-------
chloromethane but will be held for observation. (CUT may choose
to extend the dosing period to the full 24 months.) There are
120 animals of each sex in each of the three exposure groups and
in each of two control groups. Interim sacrifices are scheduled
at 6, 12f and 18 months. In the interim report submitted to EPA
(October 1979), it was revealed that female mice in all treated
groups and male mice treated with 1000 ppm showed significant
body weight decrements compared to controls. This is in contrast
to the results reported in the 90-day probe study. Chronic
inhalation of chloromethane in mice (1000 ppm) was reported to be
associated with focal acute scleritis (3/10 males, 1/10 females),
hepatocellular degeneration (7/10 male's, 7/10 females), splenic
lymphoid depletion (8/10 males, 4/10 females) and thymic lymphoid
necrosis (4/10 males, 1/10 females). In rats chronic adminis-
tration of the chemical was reported to be associated with sperm
granuloma (2/10), interstitial pneumonia (1/10 males, 4/10
females) and subacute tracheitis in females (5/10). No
significant histopathologic findings were discovered in the liver
of rats or in the kidneys of rats or mice.
There are several difficulties with both the design and the
execution of the 24 month study that CUT has started:
1. In the six-month interim report, CUT reported a
significant death rate due to trauma, especially
within the first 6 months in male mice. There was an
overall death rate due to trauma of 4.5 percent. For
male mice this rate was almost 9 percent. If this
92
-------
rate continues over the next 18 months of the study,
there will not be sufficient animals surviving to
evaluate.
2. In the 90-day study (CUT 1979a) , there was no signi-
ficant weight decrement in treated male mice even at
1500 ppm, while female mice showed such a decrement
only at 1500 ppra. Under these circumstances, the
reason for the choice of 1000 ppm as the high dose in
this species for determining either oncogenicity
potential or chronic toxicity is unclear. However,
the 6-month interim report weight loss has been
recorded in all groups of treated female mice. The
EPA believes that in an oncogenicity study, no toxic
effects should be seen in the two lower dose groups
other than tumors, in order to ensure the survival of
enough of the animals to demonstrate the production
of tumors as a late effect. On the other hand, if
considered as solely a straightforward chronic
toxicity test, the EPA prefers that the lowest dose
represent a no-effect level, i.e., demonstrate no
toxicity at such an early stage, even weight loss.
Regarding chronic toxicity specifically, various differences
from the EPA's proposed protocol (EPA 1979c) raised questions
concerning the usefulness of the results:
93
-------
1. As discussed previously in Section III.A.2., use of
rats have been exposed at levels for which rats had
previously been shown to exhibit no effects on the
liver, kidney, cardiovascular or hematopoietic
systems. CIIT's original protocol (CUT 1979b)
indicated that it was originally planning on using
dogs as well as rats and mice for the 24-month
inhalation study. CUT has not given the EPA any
reasons for this change in protocol. In this
particular case the lack of a non-rodent species as
required by the EPA for chronic toxicity testing is
more than usually important. Because non-rodents
such as dogs and monkeys appear to be more sensitive
to chloromethane, the use of rats and mice may give
spurious no-effect levels when used to evaluate the
risk to humans.
2. There is no indication that the animals were
adequately observed during the first 6 months for
general toxicity or for neurologic or behavioral
toxicity.
3. No tests will be conducted to determine the more dis-
criminating aspects of behavior and performance.
The Agency believes that because the information gathered as
a result of this study, along with the previous data available on
94
-------
chronic systemic effects other than neurologic, will be adequate
for hazard assessment purposes for systemic effects other than
neurotoxicity, it is not deemed necessary to require further
general chronic toxicity testing. The EPA will be requiring
neurobehavioral studies to evaluate risk from the apparently most
sensitive system.
Regarding oncogenicity, the EPA has concluded that the
eighteen-month exposure period may not be sufficient to indicate
a lack of oncogenicity. If no tumours are detected in the test
animals, the Agency has decided that a minimum of 24 months in a
rodent is necessary to demonstrate a lack of oncogenicity for the
Agency's p.urpose (EPA 1979e) .
This deficiency, in addition to those discussed earlier
under general problems, is severe enough to warrant additional
testing, as there is at present no way to determine the oncogenic
potential of a chemical other than by adequate long-term testing.
Although CUT has not yet initiated its teratology testing,
they have proposed a protocol for teratologic evaluation of
chloromethane (CUT 1978f). Using this protocol, CUT intends to
collect data on anatomical abnormalities, neurofunctional
deficits, and acquisition of developmental landmarks in rats
exposed to chloromethane in utero.
The CUT protocol differs significantly from that proposed
by the EPA (EPA 1979b) in several ways:
95
-------
(1) It specifies the use of a single species, the rat, to
evaluate teratogenic effects. The EPA has proposed
teratology testing in a minimum of two mammalian
species. A study in one with negative results would
be considered inadequate, although findings of mal-
formations in a single species would be highly
suggestive of teratogenesis.
(2) The dosages proposed are arbitrary and do not conform
to suggested criteria for dosage levels as published
in the proposed Test Standards (EPA 1979b). The
intermediate dose should be related to the high dose
(which should cause toxicity) not with the lower
dosage, as indicated in the protocol. This is
particularly the case where extensive embryonic or
fetal death occurs at the high dose. If maternal
toxicity occurs at the high dose but no fetal
toxicity is demonstrated, then no fetal effects need
to be demonstrated in the intermediate dosage
range. The EPA believes that the low dose of 100 ppm
or some multiple thereof is satisfactory.
(3) Although the battery of tests for the evaluation of
neurofunctional deficits and the acquisition of
developmental landmarks proposed by CUT (1979b) may
not be completely appropriate, standards for the
testing of behavioral alterations have not yet been
96
-------
proposed by the EPA and the Agency will consider
CIIT's proposed battery as the basin for such
testing. Guidance will be proposed for public
comment in an Advanced Notice of Proposed Rule Making
(ANPRM).
(4) Because the rat has demonstrated less sensitivity to
chloromethane intoxication than other species (Smith
and von Oettingen 1947a), such other species should
be considered.
97
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