United States
                 Environmental Protection
                 Agency
Office of Health and
Environmental Assessment
Washington DC 20460
                 Research and Development
EPA/600/S6-85/001 Sept. 1985
SERA        Project Summary
                 Assessment  of the Mutagenic
                 Potential  of Carbon  Disulfide,
                 Carbon  Tetrachloride,
                 Dichloromethane,  Ethylene
                 Dichloride, and  Methyl  Bromide:
                 A Comparative Analysis in
                 Relation to Ethylene  Dibromide

                 Vicki Vaughan-Dellarco, John R. Fowle III, and Sheila Rosenthal
                  The Reproductive Effects Assessment
                 Group was requested by the Haiard
                 Evaluation Division of the Office of
                 Pesticide Programs (OPP) to prepare a
                 mutagenicity assessment of proposed
                 pesticide alternatives to the fumigant,
                 ethylene dibromide. These alternatives
                 included carbon disulfide, carbon tetra-
                 chloride, dichloromethane,  ethylene
                 dichloride, and methyl bromide. This
                 mutagenicity assessment is to serve as
                 a "source document" for OPP's use.
                  In the development of this document,
                 the scientific literature has been inven-
                 toried, and key studies have been crit-
                 ically evaluated. The Environmental
                 Mutagen, Carcinogen, and Teratogen
                 Information Department at the Oak
                 Ridge relational Laboratory identified
                 the published literature.
                  Three sections of Chapter 4 in the full
                 report were taken  from the  health
                 assessment documents prepared by the
                 Office of Health and Environmental
                 Assessment (OHEA) for the Office of
                 Air Quality Planning and Standards.
                 These sections include data evaluations
                 of carbon tetrachloride, dichlorometh-
                 ane, and ethylene dichloride. The Health
                 Assessment Document for Carbon Tet-
                 rachloride has received full administra-
tive and peer review. The Health As-
sessment Documents for Dichlorometh-
ane and for Ethylene Dichloride are
undergoing public review and comment
and EPA Science Advisory Board re-
view. The reader is referred to the health
assessment  documents  (U.S. EPA,
1983a. 1984b, 1984c), if additional
information is needed regarding health
effects other than mutagenicity or back-
ground information such as physical-
chemical properties.
  This Project Summary was developed
by EPA's Office of Health and Environ-
mental Assessment, Washington, DC,
to announce key findings of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).

Introduction

  This Summary provides a brief evalua-
tion of the mutagenic potential of five
proposed alternatives to the use of the
fumigant, ethylene dibromide. The alter-
native compounds are carbon disulfide,
carbon tetrachloride, dichloromethane,
ethylene dichloride, and methyl bromide,
Figure 1. The evaluation involved a survey

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       Chemical
Empirical
Formula
                                                             Structure
  Carbon Disulfide
  Carbon Tetrachloride
  Ethylene Dibromide
  (1,2 -Dibromoethanei
  Ethylene Dichloride
  (1,2 -Dichloroethane)
  Methyl Bromide
  (Bromomethane)
  Methylene Chloride
  (Dichloromethane)
  CSZ
                                    ecu
 CH3Br
    Cl
     I
CI — C — CI
     I
    Cl

     H  H

Br — C — C—Br

     H  H

     H  H
     I   I
CI — C — C—Cl
     I   I
    H  H

     H
     I
fir — C —H
     I
     H

     H
     I
CI — C — CI
     \
    H
figure 1.    Chemical structures of ethylene dibromide and proposed alternatives.
and critical analysis of relevant studies. A
separate analysis of the mutagenicity of
each proposed alternative is found in the
individual sections of Chapter 4 in the full
report. The evaluation of the five proposed
alternative fumigants  included a deter-
mination of the intrinsic mutagenic po-
tential of each agent  and its ability to
reach germinal tissue in intact mammals.
Ethylene dibromide  is  not included  as a
separate section in the full report because
it has been evaluated previously by OPP.
  A comparative analysis of mutagenicity
between each of the proposed altenatives
and C2H4Br2 is presented. The spectrum
of genetic damage induced by each agent
is discussed and mutagenic potencies are
compared whenever  appropriate.  Be-
cause judgments cannot be reached due
to gaps in current  knowledge,  recom-
mendations are made for additional stud-
ies that could be conducted to determine
if a potential mutagenic risk exists.

Comparative Analysis of
Ethylene Dibromide and
Proposed Alternatives
  This comparison considers the genetic
      damage that is induced by each agent and
      mutagenic potencies in selected tests.
        Of the five proposed alternative fumi-
      gants,  there is sufficient  evidence on
      ethylene  dichloride, dichloromethane,
      and methyl bromide, in addition to ethyl-
      ene dibromide  itself, to classify them as
      mutagens. These chemicals  have  been
      reported as positive in two or  more gene
      mutation tests  in phylogenetically differ-
      ent organisms. Table  1. There  is also
      ancillary  information regarding  their
      DNA-damaging potential (e.g., SCE, DNA
      repair, DNA alkylation). The evidence that
      ethylene dichloride is a presumed mam-
      malian mutagen is stronger than that for
      dichloromethane or  methyl bromide be-
      cause of (1) the larger number of positive
      tests conducted in different laboratories,
      (2) the suggestive evidence that ethylene
      dichloride causes somatic gene mutations
      in  whole  mammals, and  (3) a  study
      demonstrating  the alkylation  of DNA in
      somatic tissues of whole mammals. Al-
      though the data on  the ability of these
      agents to cause chromosomal aberrations
      are limited, none of the chemicals appear
      to  be strong clastogens.  It is uncertain
whether these agents produce a similar(
array of other types of genetic damage
(e.g.,  nondisjunction,  SCEs,  mitotic re-
combination), because they have not all
been sufficiently evaluated for the induc-
tion of other types of genetic alterations.
Furthermore, it is uncertain whether the
proposed alternatives reach and interact
with  mammalian germ-cell DNA, but
ethylene dibromide is known to do so and
this is presumed to be a human germ-cell
mutagen.  Ethylene  dichloride, methyl
bromide, and dichloromethane are posi-
tive in the Drosophila sex-linked recessive
lethal test. This test organism has germ-
cell stages  analogous to those in mam-
mals  and  provides  some  information
regarding germ-cell risk in intact animals.
Although the data bases are not equally
complete for each of the  compounds,
none of the proposed alternatives appear
to be as mutagenic as ethylene dibromide
when  results from  similar  tests are
compared.
  The mutagenic  potential of the other
two  alternatives,  carbon disulfide and
carbon tetrachloride, could not be judgec
because of insufficient information. The
available studies suggest, however, thai
if they are mutagenic, they are weakly so
This conclusion does not necessarily appl^
to chromosome non-disjunction because
carbon tetrachloride has not been evalu
ated for its  ability to  disrupt spindle
structures  or function, and inadequate
evidence is available for  the induction o
numerical chromosomal aberrations bi
carbon disulfide. The reader is referredt<
Chapter 4 of the full  report for a critica
analysis of  the data  pertaining  to th<
mutagenicity  of  these five  propose!
alternative  fumigants and for  a detailet
summary for each chemical.

Mutagenic Potencies
  The mutagenic potencies of each of th>
five proposed alternative fumigants wer
compared with those of ethylene dibro
mide using results from the Salmonell
assay. This test was the only one in whic
all  chemicals have  been evaluatec
Several criteria were imposed to selec
appropriate experiments for this analysis
Only experiments using the  desiccate
procedure were included because all c
the chemicals are volatile, and testing i
sealed containers is more appropriat
than in the standard plate assay in whic
the volatile test material evaporates an
escapes. In addition, results were consic
ered  only  if there  were at  least tw
nonzero dose points; spontaneous coum
were reported, and revertant  data wet
given in the report.  Results on tests

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Table 1.    Qualitative Comparison of the Mutagenicity of Ethylene Dibromide and Proposed Alternatives
Chemical   Bacteria
                       Fungi
                                 Gene Mutation	      Clastogenicity     Numerical   Other
                                                                                           Chromo-  Indicators of
                               Higher            Mammalian   Whole                         some     DNA       DNA
                               Plants   Drosophila   Cells    Mammals    In Vitro     In Vivo    Mutation   Damage   Binding
 Carbon
 disulfide
            -d)
            K3)
                                           K2)
                                                                                            1(2)
                                                                                   -12)
                                                                                                    1(1), UDS
Carbon
tetra-        1(4)
chloride     -(2)
 Dichloro-
 methane
 Ethylene
 dibromide
Ethylene
dichloride
 Methyl
 bromide
            +(12)
            1(2)'
             -(4)
             W(7f
             1(3)
            +(4)
                       W(J)
                                                                         -d)
                      +(3)
                                 +(3)
                                           +(1)
                                           KD
+(3)
          KD
                                        -d)
                                 KD
                                                     +(2)
                    Wft)
                                           KD
                                                                                   -(2)
-12)
                                                                                   K2)
                 W(1), YMR
                 -(3), UDS

                 (I). YMR
                 /+). YMR
                 w(2), see
                 -(2), UDS

                 +(1). YMR
                 +(1). UDS
                 -d). UDS
                 +d). Pol
                 +(1). SCE
                                                         1(1). UDS
                                                         Id). Pol

                                                         1(2). UDS
                                                                                                                  +(2)
Information on ethylene dibromide is based on OPP documents (U.S. EPA, 1983a; Mauer, 1979; Lee. 1980).
"+" designates a positive result; "-" a negative result; "I" an inconclusive study; "W" a weak, borderline, or suggestive result.
Numbers of studies are indicated in parentheses.
UDS = unscheduled DNA synthesis; YMR = yeast mitotic recombination; SCE = sister chromatid exchange; Pol = bacteria.
"Plants only.
tStronger response with an S9 metabolic activation system.
^Drosophila only.
strain TA100 in the presence or absence
of metabolic activation were used  be-
cause this was the only strain for which
data were available on all compounds. A
simple  linear regression analysis  was
used on the linear portion of the dose-
responses; linear regression calculations
with correlation coefficients  less than
0.90 were not accepted.
  It was clear from these tests that eth-
ylene dibromide is a much more potent
mutagen than the  corresponding chlori-
nated compounds  and more  mutagenic
than methyl bromide based on structural-
activity relationships. Ethylene dibromide
is a  bi-functional agent, and thus  more
biologically reactive. Carbon tetrachloride
and  carbon  disulfide are predominantly
negative in bacterial tests.
  Potencies were  also examined in  the
Drosophila  sex-linked recessive lethal
test. Data were available  for ethylene
dibromide, ethylene dichloride, dichloro-
methane, and methyl bromide. Although
certain  germ-cell  stages appear to be
                                         more  sensitive than  others,  the total
                                         lethal frequencies were compared and it
                                         was found that ethylene dibromide is a
                                         more  potent mutagen than the alterna-
                                         tives,  whether data from inhalation or
                                         feeding experiments are compared. It is
                                         uncertain if ethylene dichloride is  more
                                         active than  methyl bromide.  There  is
                                         some  overlap of the lethal frequencies
                                         per unit of exposure for these alternatives.
                                         Different strains of Drosophila were used,
                                         which could account for differences  in
                                         lethal  frequencies. Although carbon di-
                                         sulfide has been reported as negative, the
                                         possibility of weak effects cannot be
                                         excluded. In feeding experiments, ethyl-
                                         ene dichloride seems to be more active
                                         than dichloromethane. However, these
                                         experiments were conducted in different
                                         laboratories, and factors such as stocks of
                                         Drosophila and solvents differed; these
                                         variations could contribute observed dif-
                                         ferences among the alternatives in lethal
                                         frequencies. Nevertheless, in their total-
                                         ity, the data show that ethylene dibromide
                                                                                  is more  mutagenic  than the proposed
                                                                                  alternatives in the Drosophila sex-linked
                                                                                  recessive lethal test.  The  Drosophila
                                                                                  results  are therefore  consistent  with
                                                                                  those in Salmonella.
                                                                                    The  mutagenicity of  the  alternative
                                                                                  compounds in cultured mammalian cells
                                                                                  cannot readily be compared to that of eth-
                                                                                  ylene dibromide because test results are
                                                                                  based on different cell lines and different
                                                                                  loci. However, in one study using human
                                                                                  lymphoblasts  and another study  using
                                                                                  Chinese  hamster ovary cells,  ethylene
                                                                                  dibromide was a more potent  mutagen
                                                                                  than ethylene dichloride.
                                                                                    Ethylene  dibromide,  ethylene dichlo-
                                                                                  ride,  and  methyl bromide  have been
                                                                                  examined for DNA adduct formation in
                                                                                  whole mammals after  inhalation  expo-
                                                                                  sure. Although there is DNA binding data
                                                                                  with carbon tetrachloride, it is derived
                                                                                  from  intraperitoneal  injection  experi-
                                                                                  ments and  therefore were not used in
                                                                                  comparison with the other compounds.
                                                                                  Liver was the only organ in which DNA

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   alkylations  could be  compared for the
   three chemicals. It was found in these
   experiments that methyl bromide binds
   DNA to a much lesser extent than does
   ethylene  dibromide; approximately five
   orders  of magnitude difference were
   observed.
     Ethylene  dichloride appears to bind to
   DNA to a  greater extent  than  methyl
   bromide but to a lesser extent than eth-
   ylene dibromide. It should be cautioned,
   however, that  the  measurements for
   ethylene dichloride were derived from a
   different rodent species (rat) than those
   for ethylene dibromide and methyl bro-
   mide (mouse). Although there were two
   orders  of magnitude difference in the
   alkylations, a species difference could
   conceivably account for the amount of
   binding.
     Given the available data, few conclu-
   sions can be drawn about DNA alkylation
   by methyl bromide and ethylene dichlo-
   ride, except that both agents interact with
   DNA. Methyl bromide appears to do so to
   a lesser extent than does ethylene di-
   bromide.  Information from experiments
   that involve measurements at different
   time intervals to determine the stability of
   various DNA adducts formed by these
   compounds would be a valuable addition
   to current knowledge. If stable adducts
   are formed in testicular DNA, which is a
   target tissue for heritable risk, then the
   induced genetic damage could accumu-
   late during the  cellular life cycle of the
   gonial cells. The gonia are an important
   cell type relevant for human genetic risk
   assessment. In the  case  of ethylene
   dibromide,  it is known that adducts are
   formed in testicular DNA. Data regarding
   the degree of alkylation is more useful if it
   includes  information on the type of ad-
   ducts  formed and the stability of these
   adducts. Such information is needed for
   ethylene dichloride, methyl bromide, and
        dichloromethane in  germinal tissue of
        intact mammals.
        Conclusions and
        Recommendations
         The five proposed alternatives do not
        appear to be as mutagenic as ethylene
        dibromide. Two alternatives, carbon tet-
        rachloride and  carbon disulfide,  have
        been primarily negative in mutagenicity
        testing, however it cannot be stated that
        they do not pose a mutagenic risk because
        the available information is limited and
        sometimes inadequate. Additional testing
        would be necessary  for them to be
        classified. It should be noted that even if
        these agents do not pose a mutagenic
        hazard, they do pose other health hazards;
        for example, carbon disulfide is extremely
        toxic and carbon tetrachloride is extreme-
        ly toxic and carcinogenic in mice and rats.
         The alternative  compounds that are
        mutagenic in several  short-term  gene
        mutation assays are ethylene dichloride,
        dichloromethane, and methyl bromide. It
        cannot be concluded  that one of these
agents is more mutagenic than the other
because of limited data. It  does appear,
however, that these agents are not strong
mutagens,  because  rather  large,  and
often toxic, doses are required to elicit
mutagenic responses. Delineation of dif-
ferences in mutagenic  activity among
these agents will require dose-response
data that are  generated  in the same
laboratory so as to minimize technical
and  biological  variation. The proposed
alternatives are all volatile chemicals and
precautions are  therefore essential to
prevent excessive evaporation  of  test
material. Several different assay systems,
including mammalian systems, should be
used to determine  a rank order for
mutagenic potency. If these experiments
were coupled with molecular dosimetry,
the relationship  of mutation frequency
could be compared to target dose rather
than to exposure. If a similar rank order of
potency is observed in different species, it
might be reasonable to assume that a
similar  ranking  may exist in  humans.
After these determinations, whole mam-
mal germ-cell  studies would have to be
conducted to estimate heritable risk.
          The EPA authors. Vicki L. Vaughan-Dellarcofalso the EPA Project Officer, see
            below), John R. f ovule III, and Sheila Ftosenthal are with Office of Health and
            Environmental Assessment, Washington, DC 20460.
          The complete report, entitled "Assessment of the Mutagenic Potential of Carbon
            Disulfide.'Carbon Tetrachloride, Dichloromethane, Ethylene Dichloride, and
            Methyl Bromide: A Comparative Analysis in Relation to Ethylene Dibromide,"
            (Order No.  PB 85-241 800/AS; Cost: $16.00, subject to  change) will be
            available only from:
                  National Technical Information Service
                  5285 Port Royal Road
                  Springfield, VA22161
                  Telephone: 703-487-4650
          The EPA Project Officer can be contacted at:
                  Office of Health and Environmental Assessment
                  U.S. Environmental Protection Agency
                  Washington, DC 20460
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
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