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
                                22

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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

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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

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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

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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

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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

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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

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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

-------
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

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       (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

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     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

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     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

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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

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       (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

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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

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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
                                61

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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
                                62

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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
                                63

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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
                                64

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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
                                65

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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
                                66

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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
                                67

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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
                                68

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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
                                69

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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
                                70

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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
                                71

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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)
                                72

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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).
                                73

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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
                                74

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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
                                75

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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
                                76

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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.
                                77

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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
                                78

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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
                                79

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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

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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

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     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

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     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

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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

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(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

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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

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            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

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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

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            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

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       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

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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

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(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

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     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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