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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