EPA/600/6-85/001
August 1985
ASSESSMENT OF THE MUTAGENIC POTENTIAL OF CARBON DISULFIDE,
CARBON TETRACHLORIDE, D1CHLOROMETHANE, ETHYLENE DICHLORIDE, AND METHYL BROMIDE:
A COMPARATIVE ANALYSIS IN RELATION TO ETHYLENE DIBROMIDE
by
Vickl L. Vaughan-Dellarco
John R. Fowle III
Sheila Rosenthal
Reproductive Effects Assessment Group
Office of Health and Environmental Assessment
U.S. Environmental Protection Agency
Washington, DC 20460
OFFICE OF HEALTH AMD ENVIRONMENTAL ASSESSMENT
OFFICE OF RESEARCH AM) DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
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DISCLAIMER
This document has been reviewed in accordance with the U.S. Environmental
Protection Agency's administrative review policies. Mention of trade names or
commercial products does not constitute endorsement or recommendation for use.
ii
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CONTENTS
Page
PREFACE v
AUTHORS, CONTRIBUTORS, AND REVIEWERS vi
1. SUMMARY . . . . • 1-1
2. INTRODUCTION 2-1
3. COMPARATIVE ANALYSIS OF EDB AND PROPOSED ALTERNATIVES . 3-1
3.1. QUALITATIVE. 3-1
3.1.1. Gene Mutation 3-1
3.1.2. Chromosome Mutation ..... 3-5
3.1.3. Other Indicators ....... 3-6
3.1.4. DNA Alkylation. 3-7
3.1.5. Summary and Conclusions 3-8
3.2. MUTAGENIC POTENCIES .......... . 3-10
3.3. RECOMMENDATIONS 3-17
4. MUTASENICITY OF THE PROPOSED ALTERNATIVES. .......... 4-1
4.1. CARBON DISULFIDE 4-1
4.1.1. Gene Mutation Studies. ...... 4-1
4.1.1.1. Bacteria. . 4-1
4.1.1.2. Drosophila ............. 4-5
4.1.2. Cytogenetic Studies ..... 4-7
4.1.3. Other Studies Indicative of DNA Damage ....... 4-10
4.1.4. Gonadal Studies. ....... 4-11
4.1.5. Summary and Conclusions . ...... 4-13
4.2. CARBON TETRACHLORIDE. ............... 4-14
4.2.1. Metabolism and Covalent Binding to Macromolecules . , . 4-14
4.2.1.1. Metabolism 4-14
4.2.1.2, Covalent Binding . 4-15
4,2.2. Sene Mutation Studies. ............ 4-20
4.2.2.1. Bacteria. ....... . 4-20
4.2.2.2. Yeast. .............. 4-24
4.2.3. Cytogenetic Studies .... 4-25
4.2.4. Other Studies Indicative of DNA Damage ....... 4-25
4.2.5. Suggested Additional Testing .... 4-29
4.2.6. Summary and Conclusions ..... 4-30
4.3. DICHLOROMETHANE (METHYLENE CHLORIDE) .... 4-33
4.3,1. Gene Mutation Studies. ... 4-33
4.3.1.1. Bacteria 4-33
4,3.1.2. Yeast. ...... 4-43
iii
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CONTENTS (continued)
4.3,1.3, Drosophila ... 4-46
4.3.1,4. Nematodes ....... 4-49
4.3.1.5. Mammalian Cells in Culture. ....... 4-50
4.3.2. Cytogenetic Studies . , 4-52
4.3.3. Other Studies Indicative of DNA Damage ....... 4-55
4.3.3.1. Sister Chromatid Exchange 4-55
4.3.3.2. DNA Repair Assays. .......... 4-58
4.3,4. Summary and Conclusions ....... 4-60
4.4. ETHYLENE BICHLORIDE ................ 4-62
4.4.1. Gene Mutation Studies 4-62
4.4.1.1. Bacteria and Fungi .... 4-62
4.4.1.2. Higher Plants .4-79
4.4.1.3. Insects 4-81
4.4.1.4. Mammalian Cells in Culture. ....... 4-87
4.4.1.5. Whole Mammals 4-87
4.4.2. Cytogenetic Studies ........ 4-89
4.4.3. Other Studies Indicative of DNA Damage 4-95
4.4.3.1. Bacteria. . 4-95
4.4.3.2, Eucaryotes ..... 4-97
4.4.4. DNA Alkylation Studies 4-98
4.4.5. Summary and Conclusions ... 4-100
4.5. METHYL BROMIDE (BROMOMETHANE), . . 4-102
4.5.1. Gene Mutation Studies 4-102
4.5.1.1. Bacteria 4-102
4.5.1.2. Drosophila ...... 4-107
4.5.1.3. Mammalian Cells in Culture 4-108
4.5.2. Chromosomal Aberration Studies 4-109
4.5.3. Other Studies Indicative of DNA Damage ....... 4-109
4,5.4, DNA Alkylation Studies ............ 4-110
4.5.5. Sonadal Effects. .... ..... 4-111
4.5.6. Summary and Conclusions , ..... 4-111
5. REFERENCES . 5-1
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PREFACE
The Reproductive Effects Assessment Group was requested by the Hazard
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
tetrachloride, 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
inventoried, and key studies have been critically evaluated. The Environmental
Mutagen, Carcinogen, and Teratogen Information Department at the Oak Ridge
National Laboratory identified the published literature.
Three sections of chapter 4 in this document have been 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, dichloromethane,
and ethylene dichloride. The Health Assessment Document for Carbon Tetrachloride
has received full administrative and peer review and is being submitted to the
National Technical Information Service for publication. The Health Assessment
Document for Dichloromethane and the Health Assessment Document for Ethylene
Dichloride are still draft documents that are undergoing public review and
comment and EPA Science Advisory Board review. 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
background information such as physical-chemical properties.
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AUTHORS, CONTRIBUTORS, AND REVIEWERS
AUTHORS
The principal author of this document is:
Dr. Vicki Vaughan-Dellarco, Geneticist
The sections on individual chemicals were prepared by the following
members of the Reproductive Effects Assessment Group:
Dr. Sheila Rosenthal, Toxicologist : Carbon tetrachloride
Dr. John R. Fowle III, Geneticist : Dichloromethane
Ethylene dichloride
Dr. Vicki Vaughan-Dellarco, Geneticist: Methyl bromide
Carbon disulfide
The following consultants have reviewed this document or parts of
earlier drafts.
Dr. George R. Hoffmann, College of the Holy Cross, Worcester, MA
Dr. Stanley Zimmering, Brown University, Providence, RI
Dr. Daniel S. Straus, University of California, Riverside, CA
Dr. Gary Williams, Naylor Dana Institute, Valhalla, NY
Dr. Elizabeth Von Halle, Oak Ridge National Laboratory, Oak Ridge, TN
The members of the Reproductive Effects Assessment Group also reviewed
this document or parts of earlier drafts. Participating members are indicated
by an asterisk.
Peter E. Voytek, Ph.D., Director*
John R. Fowle III, Ph.D., Geneticist*
David Jacobson-Kram, Ph.D., Geneticist*
Carole Kimmel, Ph.D., Developmental Toxicologist
Gary Kimmel, Ph.D., Developmental Toxicologist
K.S. Lavappa, Ph.D. Toxicologist*
Sheila Rosenthal, Ph.D., Toxicologist*
Carol Sakai, Ph.D., Reproductive Biologist
Vicki Vaughan-Dellarco, Ph.D., Geneticist*
Lawrence R. Valcovic, Ph.D., Geneticist*
VI
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1. SUMMARY
One objective of this mutagenicity evaluation is to determine if five
proposed alternatives to ethylene dibromide have the potential to cause mutations
in humans. If mutations occur in germ cells, they may be passed on to future
generations and increase the incidence of genetic disease in the human population;
if mutations occur in somatic cells, they may lead to the onset of cancer and
possibly other diseases. A second objective is to determine whether there are
differences in the types of genetic damage induced or in the potency of mutagenic
activity among these proposed alternatives.
Of the five proposed alternatives, three agents (dichloromethane, ethylene
dichloride, and methyl bromide) cause gene mutations in several diverse organisms
ranging from bacteria to mammalian cells in culture. These agents have not
been sufficiently studied for their potential to cause chromosomal aberrations.
Except for ethylene diehloride, there are no available data on the ability of
these agents to cause gene mutations in whole mammals. The data for ethylene
dichloride are limited, but they suggest the induction of mutation in somatic
cells. The potential of the alternative fumigants to reach germ-cell DNA in
intact mammals has not been sufficiently studied, but dichloromethane, ethylene
dichloride, and methyl bromide do cause heritable effects in male Drosophila.
Results of carbon disulfide and carbon tetrachloride in mutagenicity tests
have been predominately negative, but additional studies are needed before a
definitive judgment is reached with respect to their mutagenic potential.
Ethylene dibromide is a stronger mutagen than its structural analog,
ethylene dichloride, in several different organisms (bacteria, Drosophila,
mammalian cells in culture). This finding is in keeping with the relative
electrophilicities of bromine and chlorine atoms. Ethylene dibromide also
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appears to be a stronger mutagen than dichloromethane and methyl bromide, but
this conclusion is based on limited information. Differences among the
alternatives cannot be readily delineated because their mutagenic activities
generally fall within the same order of magnitude and this was further complicated
by the limited data available and the lack of comparability in studies from
different laboratories.
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2. INTRODUCTION
This document provides an evaluation of the mutagenic potential of five
proposed alternatives to the use of the fumlgant, ethylene dibromide. The
alternative compounds are carbon disulfide, carbon tetrachloride, dichloromethane,
ethylene dichloride, and methyl bromide (Figure 2-1). The evaluation involved
a survey 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. The evaluation of the five proposed alternative fumigants included
a determination of the intrinsic mutagenic potential of each agent and its
ability to reach germinal tissue in intact mammals. Ethylene dlbromide Is not
included as a separate section because it has been evaluated previously by
OPP.
A comparative analysis of mutagenicity between each of the proposed
alternatives and ethylene dibromide is presented. The spectrum of genetic damage
induced by each agent is discussed and mutagenic potencies are compared whenever
appropriate. Because judgments can not be reached due to gaps in current
knowledge, recommendations are made for additional studies that could be conducted
to determine if a potential mutagenic risk exists.
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CHEMICAL STRUCTURE
s
CARBON DiSULFIDE cf
CI
CARBON TETRACHLOR1DE CI—C-CI
CI
H H
ETHYLENE DIBROMIDE Br c c—Br
(1,2-DiBROMOETHANE)
H H
ETHYLENE DICHLORIDE CI—C C—CI
(1,2-DlCHLOROETHANE)
H
METHYL BROMIDE Br—C—H
(BROMOMETHANE) |
H
H
METHYLENE CHLORIDE CI-C-CI
(DICHLOROMETHANE) I
H
Figure 2-1. Chemical structures of ethylene dibronvide and proposed alternatives.
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3. COMPARATIVE ANALYSIS OF ETHYLENE DIBROMIDE AND PROPOSED ALTERNATIVES
In this chapter, data on the mutagenicity of the five proposed alternative
fumigants are reviewed and compared with data on ethylene dibromide. The
comparisons consider the spectrum of genetic damage that is induced by each
agent and mutagenic potencies in selected tests.
3.1. QUALITATIVE
The evaluation of data to judge whether a chemical 1s mutagenic and
has the potential to cause mutations in human germ cells can be referred to as
a qualitative assessment. A qualitative assessment uses a weight-of-evidence
approach that considers such factors as the types of genetic damage induced
(e.g., gene mutation, structural or numerical chromosome mutation), the type
and number of tests used, and the quality and adequacy of the testing. The
U.S. Environmental Protection Agency's (EPA) Proposed Mutagenicity Risk Assessment
Guidelines (U.S. EPA 1980, 1984d) considers, for example, that two positive
gene mutation tests in phylogenetically different organisms (e.g., mammalian cells
in vitro and bacteria) provide sufficient evidence to judge a chemical to be
mutagenic. When such evidence is coupled with data from whole mammals regarding
the ability of the agent to reach germ-cell DNA, human germ-cell mutagenicity
is presumed. This approach is consistent with that outlined by the National
Academy of Sciences (NAS) Committee on Chemical Environmental Mutagens (NAS, 1982),
3.1.1. Gene Mutation
Mutagenic endpoints that have been evaluated for the proposed alternative
fumigants in various organisms and the numbers of studies conducted are listed
in Table 3-1. Information from abstracts or review articles that do not provide
original data are not included. Based on the evaluations in Chapter 4, the
results were designated as 'V for a positive result, "-" for a negative result,
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TABLE 3-1. QUALITATIVE COMPARISON OF THE MUTAGENICITY OF ETHYLENE DIBROMIDE AND PROPOSED ALTERNATIVES
Chemical
Carbon disulfide
Carbon
tetrachloride
Dichloromethane
Ethylene
di bromide
Ethylene
dichloride
Methyl bromide
Gene mutation
Higher Mammalian Whole
Bacteria Fungi Plants Drosophila cells mammals
-(1) K2)
1(3)
W(l)
1(4) W(l)
-(2)
+(12) +(1) +(1) 1(1)
K2) 1(1)
+(5) +(3) +(3) +(3) +(2)
-(4)
W(7)f +(1) +(4) +(2) W(l)
1(3) 1(1)
+ (4) +(D +(D
KD
aThe results are based on the evaluations presented in chapter 4 of t
Clastogenicity
in vitro in vivo
W(D
-(2)
-(1)
+ (D W(l)
-(1)
+ (D +(2)e
-(2)
-(2)
1(2)
Numerical
chromosome
mutation
1(2)
+ (1)9
W(l)9
Other
Indicators of
DNA damaged
1(1), UDS
W(l), YMR
-(3), UDS
(I), YMR
( + ), YMR
W(2), SCE
-(2), UDS
+(1), YMR
+(1), UDS
-(1), UDS
+ (D, Pol
+(1), SCE
1(1), UDS
KD, Pol
1(2), UDS
DNA
binding
+(2)
+(D
+(D
+(D
his document.
Information on ethylene dibromide is based on OPP documents (U.S. EPA, 1983a; Mauer, 1979; Lee, 1980).
b"+" designates a positive result; "-" a negative result; "I" an inconclusive study; "W" a weak, borderline, or suggestive
result.
cNumbers of studies are indicated in parentheses; abstracts and review articles are not included.
dUDS = unscheduled DNA synthesis; YMR = yeast mitotic recombination; SCE = sister chromatid exchange; Pol = bacteria
Pol A assay
ePlants only.
fStronger response with an S9 metabolic activation system.
QDrosophila only.
0*
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"W" for a weak or suggestive response, and "I" for an Inconclusive study. The
information pertaining to the mutagenicity of ethylene dibromide was derived
from OPP's documents (U.S. EPA, 1983a; Matter, 1979; Lee, 1980). Similar to
ethylene dibromide, the compounds dichforomethane, ethylene dichloride, and
methyl bromide cause gene mutations in two or more unrelated organisms (see
Table 3-1). Ethylene dibromide is active in a wide range of organisms; bacteria,
Drosophila, fungi, mammalian cells, and higher plants (for review see Mauer,
1979 and Rannug, 1980). Dichloromethane, ethylene dichloride, and methyl bromide
are mutagenic in bacteria and Drosophila (see chapter 4). Dichloromethane is
also mutagenic in yeast (Callen et al., 1980), and ethylene dichloride is active
in plants (Ehrenberg et al., 1974). Ethylene dichloride and methyl bromide are
both mutagenic in mammalian cells in culture (Tan and Hsie, 1981; Gentese
Limited Partnership, 1984; Kramers et al., 1984), whereas dichloromethane has
not been adequately evaluated. Ethylene dichloride is the only agent for
which results are available on effects in whole mammals (the mouse spot test;
Socke et al., 1983); the findings are suggestive of a positive response. Carbon
disulfide and carbon tetrachlorlde do not appear to cause gene mutations, but
available data are not sufficient to support definitive conclusions as can be
seen in Table 3-1.
In Drosophila, there are differences among the compounds in the pattern
of germ-cell stage sensitivity. The mutagenic effects of ethylene dibromide
and its structural analog, ethylene dichloride, are greatest in spermatogonia,
spermatocytes or spermatids (Kale and Baum, 1979; King et al., 1979), whereas
the postmeiotic germ-cell stages appear to be most sensitive to methyl bromide
and dichloromethane (Kramers et al., 1984; Socke et al., 1981). The higher
frequency of lethals in mature sperm and lower frequencies in earlier germ-cell
stages may suggest that methyl bromide and dichloromethane are repaired efficiently
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or that labile DNA adducts are formed. On the other hand, there is evidence
from the Drosophila sex-linked recessive lethal test and DNA alkylation studies
that ethylene dibromide forms stable adducts that are apparently not efficiently
repaired in testicular DNA (Lee, 1980). Thus, ethylene dibromide-induced
damage would be expected to accumulate in germ cells. It is unknown whether
the structural analog, ethylene dichloride, forms stable adducts. DNA binding
studies using different dosage levels and sampling times are needed to determine
whether or not stable adducts are formed after dlchloromethane, ethylene dichloride,
or methyl bromide exposure. Such data are an important issue because a mutagenic
agent producing damage that accumulates might be expected to pose a greater
hazard than one producing damage that forms labile adducts or is repaired
efficiently.
In bacterial tests, ethylene dibromide and the alternative fumigants,
dichloromethane*, ethylene dichloride, and methyl bromide, do not require an
exogenous source of metabolic activation for the induction of mutagenieity
(see Barber et a!., 1981; Moriya et a!., 1983; Jongen et al., 1978; Green, 1980;
Snow et al., 1979). Although S9 activation has no enhancing effect on the
mutagenic response of methyl bromide, it enhances the responses of dichloromethane,
ethylene dibromide, and ethylene dichloride. Therefore, all of these agents
are direct-acting mutagens with the latter three compounds apparently metabolized
to stronger mutagenic intermediates.
Dichloromethane undergoes oxidative dechlorination by the microsomal
P-450 mixed-function oxidase system, and formyl chloride is believed to be an
Intermediate in this pathway (U.S. EPA, 1983b). A second pathway involves a
glutathione transferase system which dehalogenates dichloromethane to produce
^Metabolism by the bacteria is occurring; see section 4.3. for discussion.
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formaldehyde (a known mutagen), which is further oxidized to carbon dioxide.
S-chloromethyl glutathione is thought to be an intermediate in this pathway.
Although both formyl chloride and S-chloromethyl glutathione are highly unstable,
they are very reactive alkylating agents. Thus, they are likely to be mutagenic
if they reach DNA. At least two metabolic pathways also exist for both ethylene
dibromide and ethylene dichloride (Rarmug, 1980). One involves the microsomal
mixed-function oxidases producing a haloacetaldehyde, and the other involves
conjugation with glutathione, giving rise to a highly reactive half sulfur-mustard
[S-(2-haloethyl)-L-cysteine], Both of these metabolites are likely to be strong
bacterial mutagens. Methyl bromide is an alkylating agent that directly reacts
with nucleophilic sites in DNA. Although carbon tetrachloride has been predominantly
negative in bacterial tests, it is expected to produce the free radical -CClg
and phosgene (U.S. EPA, 1984a). There is insufficient information regarding
the mutagenic potential of phosgene (U.S. EPA, 1984b), It is possible, however,
that it is too short-lived to produce a detectable effect.
3.1.2. Chromosome Mutation
The ability of ethylene dibromide and the proposed alternative fumigants
to cause chromosomal damage have not been sufficiently studied. Among the few
studies on ethylene dibromide, those in whole mammals have been negative, (Epstein
et a!., 1972; Generoso, 1978), whereas positive results have been reported in
cultured mammalian cells (Tezuka et a!,, 1980), and positive and negative results
have been reported implants (Ma et a!., 1978; Ehrenberg et al.» 1974). Two
negative tests in whole mammals (which are considered inconclusive) are reported
for methyl bromide (McGregor, 1981), and there is a single negative test of
carbon tetrachloride in cultured mammalian cells (Dean and Hodson-Walker, 1979).
Dichloromethane is positive in cultured mammalian cells (Thilager and Kumaroo,
1983) and there is a micronucleus test that suggests that it also induces
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chromosomal damage in intact mammals (Gocke et al., 1981). Only two studies
in whole mammals were available for ethylene dichloride, and both were negative
(King et al., 1979; Jenssen and Ramel» 1980); ethylene dichloride, however,
has been shown to cause meiotic nondisjunction (i.e,» numerical chromosome
mutations) in Drosophila (Shakarnis, 1970). Except for carbon disulfide which
gave results that are merely suggestive of a positive response (Vasil'eva,
1982), the other agents have not been evaluated for nondisjunction.
The available results on the capacity of these chemicals to cause chromosomal
damage are inadequate to draw clear comparisons. The available data are not
extensive and in some cases appropriate exposure levels and concurrent controls
were not used. Based upon these limited data, it appears that none of
these agents is a strong clastogen.
3.1.3. Oth_e_r _I_ndicators
Ethylene dibromide and some of the proposed alternatives have been evaluated
using other indicators of DNA damage. Ethylene dibromide has been reported to
induce sister chromatid exchange in mammalian cells in vitro, mitotic recombination
in yeast and differential growth inhibition of DNA repair-deficient and repair-
proficient strains of bacteria (i.e., pol A assay) (Tezuka et al., 1980; Fahrig,
1974; Brem et al., 1974). The induction of unscheduled DNA synthesis (UDS) has
been reported for mammalian lymphocytes in vitro (Meneghini, 1974), but negative
findings are reported for mammalian germ cells in vivo (Lee, 1980). The structural
analog, ethylene dichloride, was reported to produce positive results in the
bacterial pol A assay {Brem et al., 1974) and in a UDS assay using mammalian
cells in vitro (Perocco and Prodi, 1981) but these data are equivocal.
Dlchloromethane has been shown not to stimulate UDS (Jongen et al., 1981;
Perocco and Prodi, 1981) but produces a weak increase in sister chromatid
exchanges in mammalian cells in vitro (Jongen et al., 1981; Thilagar and Kumaroo,
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1983) and Induces mitotic recombination in yeast (Caller) et al.» 1980). Carbon
tetrachloride has not been adequately evaluated for other indicators of DNA
damage; there are positive results for the induction of mitotic recombination
in yeast (Callen et a!., 1980) and negative results for the induction of UDS
in mammalian systems (Mirsalis et a!., 1982; Mirsalis and Buttersworth, 1980;
Craddock and Henderson, 1978). Tests of methyl bromide and carbon disulfide
for the induction of UDS in mammalian cells in vitro are reported as negative
(McGregor, 1981; Kramers et al.» 1984; Beliles, 1980) but are not considered
conclusive.
In summary, ethylene dibromide, dichloromethane, and ethylene dichloride
have been shown to be positive in tests for other indicators of DNA damage,
albeit the data are limited. It is not clear whether the five proposed
alternative fumigants elicit UDS. It should be stressed that certain kinds
of DNA alterations that lead to mutations may not stimulate UDS or may do so
to such a small extent (e.g., short patch repair) that UDS detection is not
practical.
3.1.4. DNA Alkylation
Ethylene dibromide, dichloromethane, ethylene dichloride, and methyl
bromide are alkylating agents. As shown in Table 3-1, three of the proposed
alternative fumigants to ethylene dibromide have been shown to form adducts in
ONA. Ethylene dibromide alkylates both mammalian germ- and somatic-cell DNA
in vivo and Drosophila germ-cell DNA (Lee, 1980); for methyl bromide and ethylene
dichloride, only mammalian somatic tissues (liver, spleen, kidney) have been
studied (Reitz et a!., 1982; Djalali-Behzad et a!., 1981). Although carbon
tetrachloride has not been demonstrated to be mutagenic, it has been found to
bind to DNA in mammals (Rocchi et al., 1973; Diaz Gomez and Castro, 1980a),
No studies are currently available for dichloromethane. The degree of alkylating
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activity exhibited by these agents will be discussed later.
3.1.5. Summary and_ Conclusions
Of the five proposed alternative fumigants, there is sufficient evidence
on ethylene dichloride, dichloromethane, and methyl bromide, in addition to
ethylene dibroniide itself, to classify them as mutagens (Table 3-2). These
chemicals have been reported as positive in two or more gene mutation tests in
phylogenetically different organisms (see Table 3-1). 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 mammalian
mutagen is stronger than that for dichloromethane or methyl bromide because
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. Although 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 recombination), because they have not all been sufficiently evaluated
for the induction 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 positive in the Drosophila sex-linked recessive lethal
test. This test organism has germ-cell stages analogous to those in mammals
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 dibroniide
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TABLE 3-2. MUTAGENIC POTENTIAL OF ETHYLENE DIBROMIDE AND PROPOSED ALTERNATIVES
Mutageriic Activity Chemical
Presumed human germ-cell mutagen Ethylene dibromide3
Confirmed mutagenic activity but Dichloromethane
insufficient information on chemical Ethylene d1chlorine
interaction with mammalian germ-cell DNAb Methyl bromide
Insufficient information to reach any Carbon disulfirie
judgmentc Carbon tetraehloride
Evidence of DMA binding in whole mammal germ cells.
data on ability to reach mammalian germinal tissue, but these
chemicals have effects on germ cells in Drosophila.
cAvailable studies for these chemicals generally negative or weakly positive,
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when results from similar tests are compared. (Mutagenic potency is discussed
later in this chapter in section 3,2).
The mutagenic potential of the other two alternatives, carbon disulfide and
carbon tetrachloride, could not be judged because of insufficient information.
The available studies suggest, however, that if they are mutagenic, they are
weakly so. This conclusion does not necessarily apply to chromosome non-
disjunction because carbon tetrachloride has not been evaluated for its ability
to disrupt spindle structures or function, and inadequate evidence is available
for the induction of numerical chromosomal aberrations by carbon disulfide.
The reader is referred to chapter 4 of this document for a critical analysis
of the data pertaining to the mutagenicity of these five proposed alternative
fumigants and for a detailed summary for each chemical.
3.2. MUTAGENIC POTENCIES
The mutagenic potencies of each of the five proposed alternative fumigants
were compared with those of ethylene dibromide using results from the Salmonella
assay. This test was the only one in which all chemicals have been evaluated.
Several criteria were imposed to select appropriate experiments for this analysis,
Only experiments using the desiccator procedure were included because all of
the chemicals are volatile, and testing in sealed containers is more appropriate
than 1n the standard plate assay in which the volatile test material evaporates
and escapes. In addition, results were considered only if there were at least
two nonzero dose points; spontaneous counts were reported, and revertant data
were given in the report. Results on tester strain TA100 in the presence or
absence of metabolic activation were used because this was the only strain for
which data were available on all the 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.
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TABLE 3-3. DOUBLING DOSE OF THE PROPOSED ALTERNATIVES AND ETHYLENE OIBROMIDE
IN THE SALMONELLA TA100 USING A DE SSI CAT OR PROCEDURE
Chemical
A.c Ethylene
di bromide
Ethylene
dichloride
Dichloro-
raethane
B.d Dichloro-
methane
Methyl
bromide
Duration
of exposure
(hours)
48
48
48
7
not reported
up to 72
up to 72
48
48
21
Doubling dose
+S9
93 (Aroclor rat)
7286 (Aroclor rat)
961 (Aroclor rat)
—
0.2 (DCM hamster)
267 (Aroclor rat)
1012 (Aroclor rat)
748 (Aroclor rat)
587 (Phenobartital
rat)
—
(ug)a
-S9
83
8296
1641
2.8
0.95
357
1301
—
872
2.4
Reference^
Barber et al .
Barber et al .
Barber et al .
Simmon, 1978
Snow et al .,
Green, 1980
Green, 1981
Jongen et al .
Jongen et al .
Simmon, 1978
, 1981
» 1981
, 1980
1979
, 1980
, 1978
aSlope of dose response determined by simple linear regression analysis except for
ethyl ene di bromide where slope was calculated by the following equation _[xy]
"Discussion of these references can be found in chapter 4 for each
proposed alternative fumigant.
cAgar concentration of test chemical measured by GC/MS in aqueous phase of petri dish.
concentration of test chemical was based on theoretical calculations of
concentration of test agent in the gas phase: Concentration (umole) in agar = volume
of gas in dessicator x solubility in agar x 760/vapor presssure of test chemical at
solubility x lO^/MW x density of agar x amount of agar added per plate.
3-11
-------
As shown in Table 3-3, the doubling dose was then determined for each chemical
(i.e., the dose producing a twofold increase in the spontaneous frequency).
When studies are compared from the same laboratory (Barber et al., 1980; 1981)
ethylene dibromide is a stronger mutagen than ethylene dichloride and dichloro-
methane (Table 3-3A). Although the reported mutagenic activities for ethylene
dichloride and dichloromethane overlap and fall within one order of magnitude,
it appears that dichloromethane is slightly more active than ethylene dichloride.
It should be cautioned that although these studies were performed by the same
laboratory group, they were not done concurrently, and thus technical and
biological variation could account for the differences. It does appear, however,
that these proposed agents are weak mutagens when considering the amount of
material needed to cause a doubling in the spontaneous mutation frequency.
Simmon (1978) concurrently tested methyl bromide, dichloromethane, and carbon
tetrachloride in desiccators using Salmonella TA100 without S9 activation.
Carbon tetrachloride was reported as negative under conditions which elicited
positive responses for methyl bromide and dichloromethane. It should be pointed
out that the exposure time was much longer for methyl bromide (21 hours) than
for dichloromethane (7 hours), and testing was conducted only in the absence
of S9. Other studies used to develop the data in Table 3-3B were not performed
in the same laboratory, and as can be seen for dichloromethane, a difference
in doubling dose ranged from 0.95 to 1301 ug with S9 and 0.2 to 1012 ug without
S9. Therefore, inter!aboratory variations can account for the observed
differences (e.g., type of S9 and length of exposure times varied).
It is clear that ethylene dibromide is a much more potent mutagen than
ethylene dichloride and dichloromethane. This is not surprising because
bronrinated compounds are generally more mutagenic than the corresponding
3-12
-------
chlorinated compounds (Rannug, 1980). Although there are no data from the
same laboratory, ethylene dibromide is expected to be more mutagenic than
methyl bromide based on structural-activity relationships. Ethylene dibromide
is a bi-functional agent, which are generally 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 (Table 3-4). Data were available for ethylene dibromide, ethylene
dichloride, dichloromethane, and methyl bromide. Although certain germ-cell
stages appear to be more sensitive than others, the total lethal frequencies
)
were compared. As can be seen in Table 3-4, ethylene dibromide is a more
potent mutagen than the alternatives, whether data from inhalation or feeding
experiments are compared (Table 3-4). 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 disulfide has been reported as negative, the possibility of
weak effects cannot be excluded. In feeding experiments, ethylene dichloride
seems to be more active than dichloromethane. However, these experiments were
also conducted in different laboratories, and factors such as stocks of Drosophila
and solvents differed; these variations could contribute observed differences
among the alternatives in lethal frequencies. Nevertheless, in their totality,
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 ethylene dibromide because test results
3-13
-------
TABLE 3-4. MUTA6ENIC ACTIVITY OF ETHYLENE DIBROMIDE AND CERTAIN PROPOSED ALTERNATIVES
IN THE DROSOPHILA SEX-LINKED RECESSIVE LETHAL TEST
I
I—"
.o
Ratio of lethals
Chemical Strain Dose (ppm-h) (% treated/% control) References3
A. Inhalation Exposure
Ethyl ene dibromide Canton-S 125
Ethylene dichloride: Canton-S 2,800
and D-32 2,800
4,200
5,600
Methyl bromide Berlin-K 3,446
2,653
2,031
1,415
1,061
Carbon distil fide Not 280
reported 19,200
(highest ineffective dose)
16 Kale and Baum, 1979
11 Shakarnis, 1969 and 1970
40
66
20
14 Kramers et al., 1984,
8 unpublished
2.2
3.6
1.4
Beliles et al., 1980;
Donner et al ., 1981
Dose (mM)
B. Feeding Exposure
Ethylene dibromide Berlin-K 2.7 (507 ppm)
5.4 (1014 ppm)
Ethylene dichloride Berlin-K 50 (4948 ppm)
Dichloromethane Berlin-K 125-625 (10,617-33,088 ppm)
6 Vogel and Chandler, 1974
5
9 King et al., 1979
1-2 Gocke et al . , 1981
^Discussion of references can be found in individual chapters on the respective proposed alternatives.
-------
are based on different cell lines and different loci. However, in one study
using human lymphoblasts (Gentes Limited Partnership, 1984) and another study
using Chinese hamster ovary cells (Tan and Hsie, 1981), ethylene dibromide was
a more potent mutagen than ethylene dichloride.
Ethylene dibromide, ethylene dichloride, and methyl bromide have been
examined for DNA adduct formation in whole mammals after inhalation exposure.
Although there is DNA binding data with carbon tetrachloride, it is derived from
intraperitoneal injection experiments and therefore were not used in comparison
with the other compounds. Liver was the only organ in which DNA alkylations
could be compared for the three chemicals (Table 3-5). As can be seen in
Table 3-5, methyl bromide binds DNA to a much lesser extent than does ethylene
dibromide; approximately five orders of magnitude difference were observed for
the alkylations per ppm-h. Although the binding of methyl bromide to DNA was
measured by adduct formation on N7 guanine and not by total adduct formation,
methyl bromide has a high s value (1). Compounds with a high s value have
caused almost exclusively more N-alkylations, rather than mixtures of
N-alkylations and 0-alkylations. Therefore, it is assumed that the extent of
N-alkylation for methyl bromide approximates the amount of total alkylation.
Even if this assumption is inaccurate, it is highly unlikely that five orders
of magnitude difference could have accounted for this difference 1n measurements,
Ethylene dichloride appears to bind to DNA to a greater extent than methyl
bromide but to a lesser extent than ethylene dibromide. It should be cautioned,
however, that the measurements for ethylene dichloride are derived from a
different rodent species (rat) than those for ethylene dibromide and methyl
bromide (mouse). Although there were two orders of magnitude difference in
the alkylations per ppm*h, a species difference could conceivably account for
this difference in the amount of binding.
3-15
-------
TABLE 3-5. PNA ALKYLATION IN LIVER IN WHOLE RODENTS
Dose Total Alkylations
Chemical Species (inhalation) alkylations/nucleotide per ppm.h x 10~6 Reference
Ethylene dibromide mouse 30.5 ppm.h3
Ethylene dichloride rat 900 ppm.h
Methyl bromide mouse 144 ppm.h
R x 10-6
4 x lO-6
3 x 10-9b
0.3 Lee, 1980
0.004 Reitz et al .,
0.00002 Djalali-Behzad
al., 1981
1982
et
aData are derived from i.p. experiments. The ppm-h equivalent was determined on the basis of equal alkylation
of liver DNA in inhalation exposure 6 days post-treatment. It has been experimentally determined for ethylene
dibromide that 1 ppm.h is equivalent to an i.p. injection of approximately 0.17 mg/kg.
bAlkylations at N7 guanine were determined in this study. Because methyl bromide has a high s value (1);
it is assumed that the number of N7 alkylations will approximate the number of total alkylations.
-------
Given the available data, few conclusions can be drawn about DNA alkylation
by methyl bromide and ethylene dichloride, except that both agents interact
with DNA. Hethyl bromide appears to do so to a lesser extent than does ethylene
dibromide. 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 accumulate 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 adducts
formed and the stability of these adducts. Such information is needed for
ethylene dichloride, methyl bromide, and dichloromethane in germinal tissue of
intact mammals.
3.3. RECOMMENDATIONS
The five proposed alternatives do not appear to be as mutagenic as ethylene
dibromide. Two alternatives, carbon tetrachloride 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 even provisionally as nonmutagens. (Research needs for
each of these chemicals are identified in Chapter 4.) 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 extremely toxic and carcinogenic in mice and rats (U.S. EPA, 1984c).
3-17
-------
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 differences 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 mammal germ-cell studies
would have to be conducted to estimate heritable risk.
3-18
-------
4. MUTAGENICITY OF PROPO^D ALTERNATIVES
The following sections provide a critical analysis of data pertaining to
the potential mutagenicity of five proposed alternative fumigants to ethylene
dibromide.
4.1. CARBON BISULFIDE
Information on the mutagenic potential of carbon disulfide has primarily
been negative. Most of the testing has been conducted in bacteria. Although
there are results in eucaryotic tests, the data are limited; studies have
been performed by two different laboratories in Drosophila for gene mutations
and in whole mammals for cytogenetic analysis. These results are discussed
below and summarized in Table 4-la.
4.1.1. Gene Mutation Studies
4.1.1.1. Bacteria—Carbon disulfide has been found to be negative in mutagenicity
tests in Salmonel1 a typhimuriurn and in Eschericnla coli. In Salmonella, various
protocols were used: preincubation, liquid suspension, fluctuation, and
desiccator assays, and host-mediated assay.
Haworth et al. (1983) evaluated 250 chemicals, including carbon disulfide
(MC/B, technical purity), in the Salmonella/microsome assay using a preincubation
protocol. With this procedure, the liver activation system, bacteria, and
test chemical are mixed and incubated for 20 minutes at 37°C. Melted top
agar is then added, and the mixture is poured into petri plates and incubated
for 48 hours at 37°C. Two types of S9 mix were employed: Aroclor 1254-induced
rat liver and Aroclor 1254-induced hamster liver. Carbon disulfide, dissolved
in DMSO, was examined at five concentrations by two different laboratories,
SRI-International used concentrations of 0, 33.3, 100, 333.3, 1000, and
3333.3 ug/plate. Microbiological Associates used concentrations of 0, 20.7,
4-1
-------
TABLE 4-la. MUTAGENICITY TESTS ON CARBON DISULFIOE
Test system
Reported
Result
by Authors
Continent
Reference
A. Gene mutation studies: Bacteria
Salmonella typhimurium (prelncubatlon
assay, liquid suspension test,
fluctuation test, desiccator procedure)
EschericMa coli WP2 (liquid suspension,
fluctuation test)
Host-mediated assay (using Salmonella TA98
as indicator and CD-I mice as host)
Gene mutation studies: Eucaryotes
Orosophila sex-linked recessive lethal
test
with/without
S9 mix
with/without
S9 mix
Haworth et al., 1983
Hedenstedt et al., 1979
Donner et al., 1981
Donner et al., 1981
cannot rule out Bellies et al., 1980
mutagenesis by
base-pair substitution,
suggestive Increase
in male-hosted bacteria
sample size not
sufficient to
rule out a
doubling in the
lethal frequency
relative to the
control value
Donner et al., 1981
Bellies et al., 1980
B. Cytogenetlc Effects: Whole mammals
Rat bone marrow cells and embryonic
cells
Rat bone marrow cells
Rat dominant lethal test
0. flfhap Fwfrlopco Tni-Hraflun nf DN4 nam;»n.o-
Unscheduled DNA synthesis in W1-3R human
fibroblasts
suggestive
evidence for
polyploidy and
chromosome
fragments at
0.1 LD50
dosages may
have been
too low
dosages may
have been
too low
a toxic
was not ex-
amined and
positive control
values were weak
BaMlyak and Vasll'eva, 1974
Vasil'eva, 1982
Bellies et al., 1980
Beliles et al., 1980
Rpliles et al., 1980
-------
69, 207, 690, and 2070 ug/plate. In establishing these doses, the test agent
was checked for toxicity in TA100 and the highest dose tested exhibited some
degree of toxicity (i.e., thinning of bacterial lawn). Both laboratories used
tester strains TA98, TA100, TA1535, and TA1537 and repeated experiments at
least twice. Carbon disulfide was not mutagenic either in the presence or
absence of S9 mix. Although carbon disulfide is a volatile compound, it is
dense (vapor density = 2.67; air = 1) and excessive evaporation would not be
expected to occur with this test procotol.
Hedenstedt et al. (1979) found carbon disulfide (purity not reported,
dissolved in acetone) negative in Salmonella TA100 using a desiccator procedure
and a liquid suspension test. In the desiccator procedure, the bacteria were
incubated for 48 hours at 37°C with 8400 ppm gaseous carbon disulfide with or
without Aroclor-induced rat liver S9 mix. In the liquid suspension assay, 0,
0.63, 1.23, and 3.15 ug of carbon disulfide per ml of media were incubated in
a sealed glass tube at 37°C for 1 hour. Insufficient information about procedure
is given to evaluate the liquid suspension results. In addition, no concurrent
positive controls were reported for this study.
Donner et al, (1981) reported carbon disulfide (source and purity not
reported, dissolved in DMSO) as nonmutagenic in bacterial fluctuation tests.
Salmonella typhimurium strains TA98 and TA100, and Escherichia coll WP2 uvrA
were used as indicator organisms. Assays were performed with or without S9
mix (rat liver; inducer not reported). The concentrations used were 300, 600,
and 1000 uH in Salmonella and 20, 100, 300, and 600 uM in E. coli. It should
be noted that no positive control data were reported; thus, it is uncertain If
the test system or 59 was functioning properly.
Beliles et al. (1980) used a host-mediated assay to evaluate the effects
4-3
-------
i
of carbon disulfide (86.97% purity: technical reagent, A.C.S.) in the presence
of whole mammal metabolism. In this study, Salmonella TA98 was used as the
indicator organism, and male and female mice (strain CD-I) were the hosts.
The animals were exposed by inhalation 7 hours per day for 5 days to either 20
or 40 ppm carbon disulfide. Bacteria were injected intraperitoneally into the
mice after the last exposure. The bacteria were retrieved from the mice 3 hours
later. At the 40-ppm-dose level, small increases in the number of revertants
per plate were observed for bacteria hosted in males (approximately twofold
to threefold increases above the negative control) but not for those exposed
in females. It is unusual that this effect was observed only in male host-
mediated bacteria because there is no information that indicates a difference
in response between sexes. Furthermore, a low survival was found in the male-
hosted group and thus the positive result could be spurious because of clumping
or differential cell growth. In addition, replicates and repeat tests were
not conducted. Therefore, the positive finding in male host-mediated bacteria
is considered inconclusive.
Carbon disulfide is reported to inhibit the mutagenic activity of 1,2-
dimethylhydrazine (DMH) and azoxymethane (AOM) in a host-mediated assay.
Moriya et al. (1979) gave male ICR mice oral doses (up to 0.5 mmol/kg body
weight) of carbon disulfide (Wako Pure Chemical Industries Ltd., Tokyo, purity
not reported) diluted in corn oil 2 hours before intraperitoneal injection of
Ar typhinmriurn hisG46 and the subcutaneous injection of DMH (1 mmol/kg) or
AOM (0.5 mmol/kg). Three hours after the treatments, the bacteria were removed
and the number of revertants and survivors were observed. Carbon disulfide
exerted a dose-dependent inhibitory effect on the mutagenieity of DMH and AOM.
The metabolic pathway of DMH in vivo is thought to be as follows: DMH —>
4.4
-------
azomethane —> azoxymethane —> methyl azoxymethanol. It is proposed that
carbon dlsulflde exerts its inhibitory effects on the mutagem'c activity of
DMH by inhibiting the N-oxidation in vivo of azomethane to azoxymethane and
also inhibiting the hydroxylation of azoxymethane to methylazoxymethanol,
Carbon disulfide has been reported to inhibit the carcinogenicity of DMH
(Wittenberg and Fiala, 1978).
4,1.1.2. Drosophila—Donner et al. (1981) evaluated carbon disulfide in the
Drosophila sex-linked recessive lethal test. Negative results were obtained
(Table 4-lb). In this experiment, 1-day-old males (w/Y) were starved for
3 hours prior to feeding carbon disulfide added to Tween 80 and mixed in 5%
sucrose. Flies were allowed to feed on concentrations of 200, 500, 650, 800,
and 1000 ppm (3000 mg/m^) for 24 hours. Concentrations up to 650 ppm were
nonlethal; 80% of the wales died at 800 ppm and all males died at 1000 ppm.
After exposure, 30 males per concentration were each mated with three females
of the strain Y Base mal. A 3 x 3 day brooding technique was used, and females
were allowed to lay eggs for 6 days. The lethal frequency 1n the control
Tween 80 group was 0.09%. Lethal frequencies in the treated were not significantly
increased: 0.08% lethals at 200, 500, and 650 ppm, and 0.12% lethals at 800
ppm. The results from the different broods were pooled. Because chemicals
can be germ-cell stage dependent, these data should have been reported separately,
and the authors' statement that there were no differences among the broods
therefore cannot be interpreted independently. The sample size in each brood
in this study only rules out the possibility of a relatively strong mutagenic
effect (only 4.4 to fivefold increases relative to the control value would
have been detected based on the normal test and Kastenbaum-Bowman test; Margolin
et al., 1983, and Kastenbaum and Bowman, 1970).
4-5
-------
TABLE 4-lb. RESULTS OF SEX-LINKED RECESSIVE LETHAL TEST IN DROSOPHILA
^LANQGASTER TREATED WITH CARBON DISULFIDE (CS2)
Concentration Number of Number of Frequency
of CS2 (ppm) X-chromosomes recessive (%)
tested lethal s
0 (Tween 80) 2343 2 0.09
200 2412 2 0.08
500 2394 2 0.08
650 2576 2 0.08
800 2483 3 0.12
Taken fromDormer et al., 1981.
4-6
-------
Bellies et al. (1980) also reported negative results In the Drosophila sex-
linked recessive lethal test after carbon disulfide exposure. In this study,
the Drosophila melanogaster males carried two repair-deficient mutations:
mej-9a (excision repair) and mei-4l5 (postreplication repair). One-day-old
males were starved 10 hours prior to exposure to 20 ppm (60 mg/m^) and 40 ppm
(125 mg/m3) of carbon disulfide for 7 hours by inhalation. The percent of
survivors at these concentrations was not given; these doses are not high and
may be nonlethal. Four broods were examined to evaluate effects on spermatozoa,
spermatids, spermatocytes, and spermatogonia (2-3-3-2 day mating scheme).
Although a high number of lethals was observed in brood I at the 20 ppm dose,
the majority of these lethils, however, resulted from a cluster in a single
male (Table 4-lc). The cluster is likely to be spontaneous in origin rather
than the result of treatment and there is no dose response. Although the
authors interpreted these data as negative, they should be considered inconclusive
because of the low dosages employed. Moreover, even the largest sample size
(i.e., summing tests from broods 1 and 2) would have been sufficient only to
detect a 3.4 to fourfold increase based on the normal test (Margolin et al.,
1983) or a 4 to fivefold increase based on the Kastenbaum-Bowman test (Kastenbaum
and Bowman, 1970), relative to the control value.
4.1.2. Cytogenetic Studies
Two studies on cytogenetlc damage induced by carbon disulfide in rat bone
marrow and embryonic cells were reported in the Russian literature (Barllyak
and VasiVeva, 1974; Vasil'eva, 1982). In one study (Barilyak and Vasil'eva,
1974), "unpedlgreed" white rats were exposed to carbon disulfide and hydrogen
sulfide at 10 mg/m^. An antimitotic effect and an increase in aneuploid and
polyploid cells were reported. These positive findings are difficult to evaluate
because it appears that exposure was to both carbon disulfide and hydrogen
4-7
-------
TABLE 4-lc.
SUMMARY OF SEX-LINKED RECESSIVE LETHAL RESULTS FOR CARBON DISULFIDE
USING REPAIR-DEFICIENT MALES OF DROSOPHILA
i
00
Number of
chromosomes
Compound Concentration scored
Negative control, 0 ppm
filtered air
Positive control, 0.015M
EMS
Carbon disulfide 20 ppm
40 ppm
Brood
Brood
Brood
Brood
Total
Brood
Brood
Brood
Brood
Total
Brood
Brood
Brood
Brood
Total
Brood
Brood
Brood
Brood
Total
I
II
III
IV
I
II
III
IVD
I
II
III
IV
I
II
III
IV
1788
1.186
918
525
4417
185
131
6
322
1704
1364
1230
568
4866
1347
1016
685
294
3342
Number of
lethals
6 (3)a
8 (8)a
2
0
T6"
52
19
5
76
7 (5)c
3 (l)c
4 (2)C
0
14
2
3
0
1
6
lethals
0
0
0
.34
.67
.22
0.0
0
28
14
83
23
0
0
0
0
0
0
0
0
0
0
.36
.11
.50
.33
.91
.41
.22
.33
.0
.29
.15
.30
.0
.34
.18
Number of
lethals
adjusted for
clusters
3
0
2
0
% lethals
adjusted for
clusters
0
0
0
0
.17
.0
.23
.0
5+l=6d 0.14
Not adjusted
2
2
2
0
6+l=7<*
No change
28
14
83
23
0
0
0
0
0
0
0
0
.11
.50
.33
.91
.12
.15
.16
.0
.14
.15
.30
.0
0.34
0
.18
aLethals occurring in one male and subtracted from total; probably due to a pre-existing lethal
bA 11 males died before mating.
cLethals occurring in one male and subtracted from total; cluster event not attributed to treatment
because they occurred in postmeiotic cells.
Calculations derived as follows: Number of lethals minus lethals in cluster male plus one cluster male =
total adjusted lethals.
SOURCE: Beliles et al., 1980
-------
sulfide. In the other study (Vasil'eva, 1982), carbon disulfide (source and
purity not reported) was evaluated at 1/10 and 1/100 of the LD§o (actual
doses were not reported) in bone marrow and embryonic cells of the rat (Wistar
strain). A solution of carbon disulfide in sunflower seed oil was given
intragastrically to adult females over a period of 15 days. It is not clear if
the administration was daily. Bone marrow cells were examined after treatment.
For examination of embryonic cells, carbon disulfide was administered to female
rats from the 10th to the 13th day of pregnancy. A significant increase in
polyploid cells (P < 0.01, assuming binomial distribution) was found at 1/10
of the LD50. In bone marrow, there were 0.5% polyploid cells +_ 0.2 S.D. in
the controls as compared to 1.6 +_ 0.4 S.D. in treated animals. In embryonic
cells, there were 0.7%jf 0.4 S.D. in the controls compared to 1.8%j^0.6 S.D.
1n the treated group. The type of polyploidy was not defined, and it is possible
that the effect may have resulted from endoreduplication or selection of a
specific subpopulation of polypoid cells in the bone marrow cells. Information
regarding the protocols used is insufficiently reported,
Beliles et al. (1980) tested carbon disulfide for its ability to cause
chromosomal aberrations in rat bone marrow cells. Adult male and female rats
[CRLrCQBS CD (SO) BR] were exposed to 0, 20, or 40 ppm carbon disulfide by inhalation
both acutely and subacutely. Acute exposure consisted of a single 7-hour session
in the exposure chamber. Subacute exposures consisted of 5 daily exposures of
7 hours. Bone marrow cells were then collected, slides were prepared and coded,
and 50 cells per animal were scored. There was no increase in the number of
aberrations or aneuploidy in either study; however, these studies are not
considered adequate tests of the potential of carbon disulfide to cause chromosome
damage or to cause aneuploidy. The positive control, triethylenemelamine
4-9
-------
(TEM), was given by i.p. Injection while carbon disulfide was administered by
inhalation. The concentrations tested in these studies are based on the threshold
limit value in air (20 ppm) and not based on the 1059. The lowest acute
concentration in air reported to cause death in mammals is 2000 ppm for 5
minutes. Thus, the doses may have been too low to detect clastogenic activity.
Beliles et al. (1980) also assessed the ability of carbon disulfide in
air to cause dominant lethal effects in rats [CRL: COBS CD (SD) BR]. The
precise nature of damage causing dominant lethal effects is not known, but
there is a good correlation between chromosome breakage in germ cells and
dominant lethal effects (Matter and Jaeger, 1975). Males (10 animals/dose)
given doses of 0, 20, or 40 ppm carbon disulfide by inhalation 7 hours per day
for 5 days were mated to unexposed females during a 7-week period. The authors
indicated that a dose-related increase in dead implants per total implant
was observed at week 7 but the increase at each dose level was not significantly
different from the controls. These data were not reported. The doses used in
this test may not have approximated the maximum tolerated dose, and even if
the maximum tolerated dose was approached, the dominant lethal test is not a
particularly sensitive test for detecting mutagens in view of the high spontaneous
frequency of fetal wastage (Russell and Matter, 1980).
4.3,1. Other Studies Indicative of DNA Damage
Unscheduled DNA synthesis (UOS) is a measure of the repair of DNA lesions
and is indicative of DNA damage. Beliles et al. (1980) assessed the ability
of carbon disulfide to induce UDS in human (WI-38) lung fibroblasts. UDS is
detected by measuring the incorporation of tritiated thymidine (3H-TdR) into
DNA in the absence of semi conservative replication. Liquid scintillation
counting was used to measure the amount of 3n-TdR incorporation. When cells
were exposed to carbon disulfide at doses ranging from 0.1 to 5.0 ul/ml, no
4-10
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increases In UDS over control values were observed either with or without
metabolic activation (Aroclor 1254-rat liver S9 mix). The positive controls
in this study responded only weakly; N-methyl-N'-nitro-N-n1trosoguanidine
elicited only a 1.8-fold increase over background UDS; and the control requiring
metabolic activation, benzo[a]pyrene, gave only a 1.16-fold increase, which is
an equivocal response. Moreover, the highest concentration of carbon disulfide
tested did not inhibit the incorporation of ^H-TdR; higher concentrations
should have been tested to reach a toxic level. Replicate plates were not
used in this study. Because of these inadequacies, the negative results reported
for carbon disulfide must be considered inconclusive.
4.1.4. Gonadal Studies
If a mutagen reached germinal tissue, it would have the potential to cause
damage that could contribute to the burden of genetic disease.
Lancranjan (1972) and Lancranjan and coworkers (1969) reported a reduction
in sperm counts and altered sperm morphology in workers with occupational
exposure to carbon disulfide. In one study, germinal effects were observed in
140 male workers (average age 30 years) suffering from chronic carbon disulfide
poisoning. Semen samples from these workers showed a high incidence of
asthenospermia, hypospermia, and teratospermia compared with 50 matched controls.
Meyer (1981) studied semen quality of 86 exposed workers for at least
a year. There were three defined exposure levels: high exposure (>10 ppm) in
18 workers, moderate exposure (2-10 ppm) 1n 27 workers, and low exposure (>2
ppm) in 22 workers. A fourth group with uncertain exposure included 19 workers.
No statistically significant effects on sperm counts or sperm morphology were
found in the workers compared with a control group (about 89 controls). The
differences were nonsignificant whether the control group was compared with the
4-11
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exposed group as a whole or with each of the subgroup exposures. Complications
in this study are that the length of employment differed among the subgroups
(workers had to be employed at least 12 months to participate) and some exposures
may have been too brief to cause detectable changes. It is not clear whether
exposure was constant or intermittent, and the basis for conclusions would
have been stronger if more workers had participated.
Carbon disulfide has been shown to cause semen changes in experimental
mammals at high doses, Tepe and Zenick (1984) studied male rats (adult Long
Evans hooded) exposed to 0, 350, or 600 ppm carbon disulfide for 10 weeks
(5 hours/day, 5 days/week). Animals exposed to the high dose (600 ppm) had
slightly lower epididymal sperm counts and significantly reduced plasma
testosterone levels.
Beliles et al. (1980) examined carbon disulfide for its ability to cause
altered sperm morphology in treated rats [CRL: COBS CD (SD) BR] and mice (CD-I).
After treatment with 0, 20, and 40 ppm by inhalation 7 hours per day for 5
consecutive days. Groups of four animals were killed at the end of 1, 4, and
10 weeks to examine effects on various germ-cell stages. Sperm were collected
from the cauda epididymis, and at least 500 cells were examined. Negative
results were reported both in rats and mice. It should be noted, however,
that the positive control TEM proved essentially inactive in the rat assay,
except for a small increase during week 10; this increase indicates the lack
of sensitivity of the test. In the mouse, TEM showed an effect at week 4; but
carbon disulfide and the negative control were also slightly elevated during
week 4. The results of this study are regarded as inconclusive.
Although carbon disulfide affects the male reproductive system in certain
tests, it is not certain that these effects result from carbon disulfide reaching
4-12
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the germinal tissue; the effects on male reproduction can also result from
altered hormone levels and/or general toxicity. A more in-depth analysis of
the potential reproductive effects of carbon disulfide can be found In the REAG
report entitled, "Assessment of the Reproductive and Developmental Toxicity Potential
of Carbon Disulfide" (prepared by Dr. Carole A. Kimmel for OPP, November 14, 1984).
4.1.5. Summary and Conclusions
There is no evidence that carbon disulfide is mutagem'c in bacteria. Two
studies have been performed in Drosophila, and negative results were reported.
Only relatively strong effects, however, would have been detected in these
studies. There is suggestive evidence for the ability of carbon disulfide to
cause polyploidy and chromosome fragments in rat bone marrow cells and embryonic
cells, but this evidence is derived from one study which is inadequate in
several respects. In another study, negative results were found in rat bone
marrow cells; but the dosage may have been too low for the detection of chromosome
abnormalities. Although the results of carbon disulfide have been predominantly
negative, the possibility of weak mutagenicity cannot be ruled out. The inability
to draw a firm conclusion regarding the mutagem'c potential of carbon disulfide
emphasizes the need for additional testing. It is particularly important that
studies be designed to permit the detection of weak mutagenic activity (e.g.,
sufficient sample sizes and concentration range). The focus should be on
the testing of eucaryotes rather than bacteria, and tests for chromosome
nondisjunction should be included.
4-13
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4.2. CARBON TETRACHLORIDE
The mutagenic potential of carbon tetrachloride (0014) has been assessed
by evaluating the results of seven bacterial studies, one yeast study, one
in vitro mammalian chromosome damage study, and three in vivo DNA damage studies
in rodents. The majority of these studies were negative. Information relating
to the metabolism of CC14 and to covalent binding of the metabolites to cellular
macromolecules (including DNA) precedes the sections assessing the
genotoxicity of CC14- This was done to set the stage for the discussion of the
largely negative results obtained in the mutagenicity studies and for the
suggestion that CC14 may be a weak mutagen. Recommendations for additional
testing are presented.
4.2.1. Metabplism and Covalent B1nding to Mac romolecu1es
The evidence described in this section suggests that CC14 is metabolized
in the liver to highly reactive intermediates (the trichloromethyl free radical
and phosgene). The evidence also indicates that metabolically activated CC14
covalently binds to protein, lipid, and DNA, suggesting that CC14 may have
genotoxic potential.
4.2.1.1. Metaboli$m--CCl4 is metabolized in the liver endoplasmic reticulum
by the cytochrome P-450 component of the mixed-function oxidase system (Reynolds
and Moslen, 1980). The available evidence indicates that metabolism of CC14
results in the generation of the trichloromethyl free radical "CClg (Reynolds
and Moslen, 1980; Trudell et a!., 1982) and phosgene (Shah et a!., 1979; Kubic
and Anders, 1980; Pohl et al., 1981). Because of their high reactivity, these
two substances are the most likely metabolites to interact with tissue macromolecules,
By using human cytochrome P-450 reconstituted in phospholipid vesicles, Trudell
et al. (1982) have demonstrated that 'CClg is the major product of the reductive
4-14
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metabolism of CC14 as determined by mass spectral identification of the
adducts formed between 'CClg and the phospholipid dioleoyl phosphatidylcholine.
Phosgene is produced from further metabolism of 'CC^ (Shah et al., 1979).
0
Og "
CC14 - » 'CC13 -»-»-* C1-C-C1
Pohl et al . (1981) measured the amount of phosgene (as diglutathionyl
dithiocarbonate) produced in the aerobic metabolism of CC14, CHC13, and CBrCls
by liver microsomes from phenobarbital -treated rats plus cofactors. The
results indicate that phosgene production from CC14 is only 4% of that produced
from CHC13. Thus, the level of phosgene production from aerobic metabolism of
CC14 is relatively small. Metabolites of CC14 are so reactive that they
bind to and inactivate the cytochrome P-450 enzymes that were responsible for
their generation (suicide mechanism) (Vainio et al., 1976; Sipes et a1.» 1977;
De Groot and Haas, 1980; Cooper and Witmer, 1982).
4.2.1.2. Coval ent Bi ndi ng--Hetabol i cal 1y activated CC14 has been found to
bind to lipid and protein both in vivo (Rocchi et al., 1973; Diaz
Gomez and Castro, 1980a, b) and in vitro (Rocchi et al., 1973; Uehleke et al.»
1977). The amount of label bound was determined after washing and extraction,
indicating that the binding was covalent. Uehleke et al . (1977) measured
covalent binding of 1 mM ^CCl^ (0.25 Ci/mol) to microsomal protein and
lipid in liver microsome suspensions (5 mg protein per ml plus cofactors) from
phenobarbital -treated rabbits. About 10% of the ^C label was covalently
bound to endoplasmic reticulum protein, and greater than 30% was bound to
microsomal lipid. Extramicrosomal binding was evaluated by the addition of 5
mg of bovine serum albumin per ml to the CCl4/microsome mixture. The binding
of metabolically activated CCl to the bovine serum albumin (1.4 nmol/mg
4-15
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in 60 mln) was about 1.5% of that bound to microsomal protein (20.0 nmol/mg)
plus "lipid {76.0 nmol/mg). Thus, It appears that binding of metabolically
activated ^CCl^ to extramicrosomal macromolecules is small compared with
binding to microsomal constituents.
Evidence that the CC14 metabolite, phosgene, reacts with proteins was
obtained by Cessi §t al. (1966) when they measured the in vivo binding of CC14
to rat liver proteins and compared it with the 1n vitro acylation of poly-L-lysine
and serum albumin by phosgene. Similar reaction products were obtained in
both systems, suggesting that phosgene reacts with the £-amino groups of lysine
in proteins, leading to cross-linked carbonyl derivatives:
0 NH? H 0 H
(I i i n i
C1-C-C1 + 2,....lysine,.... > lysine-N-C-N-lysine..,..
phosgene proteins cross-linked proteins
or
0
II
0 NHg NH2 NH-C-NH
C1-C-C1 + .....lysine....lysine... > lysine lysine
phosgene protein cross-linked protein
Such cross-linked proteins would exhibit impaired biological activity. It is
also possible that similar cross-linking reactions of phosgene can occur with
arnino groups in DNA resulting in alterations in DNA structure and function.
Binding of metabolically activated CC14 to DNA was found by two groups.
Rocchi et al. (1973) studied the binding of CC14 to nucleic acids and protein.
^C-labeled CC14 (367 umol/kg) was injected into rats and mice, and the
amount of metabolite(s) of CC14 that covalently bound to liver DNA, RNA,
nuclear proteins, and cytoplasmic proteins was measured. Significant amounts
4-16
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of labeled material were found associated with rRNA, nuclear proteins, and
cytoplasmic proteins in rats. When the rats were pretreated with 3-methylchol-
anthrene (3-MC) (5 mg, 24 hours before treatment with 0014), the amount of label
associated with the macromolecules increased. No label was associated with
DNA in the rat studies, but similar studies in mice indicated that DNA binding
occurs (108 umol CCl4/mol DNA). Binding to DNA was observed only after
pretreatment of the mice with 3-MC (1 mg, 24 hours before treatment with CC14).
In an in vitro experiment, Rocchi et al. (1973) used rat or mouse liver
microsomes to activate labeled CC14 in the presence of calf thymus DNA.
Pretreatment of animals with 3-MC enhanced the amount of label associated with
DNA. Furthermore, pH 5 enzyme preparations containing activating enzymes
(Keller and Zamecnik, 1956) also were found to increase the amount of
label bound to DNA. It therefore appears that metabolites of CC14 can interact
with DNA; for optimal binding, microsomal enzymes required that activation with
3-MC and the binding assay be carried out in the presence of pH 5 enzymes.
Diaz Gomez and Castro (1980a) also measured binding of metabolically
activated CCl^ to cellular macromolecules. ^C from ^CCl4 (specific activity,
27 Ci/mol) irreversibly bound in vivo to liver nuclear DNA, protein, and lipids
in strain A/J mice and Sprague-Dawley male rats. Mouse and rat liver DNA
isolated from animals given 14CC1^ 16 hours before they were killed exhibited
a small but significant labeling (mice, 0.72 pmol/mg; rats, 0.52 pmol/mg).
The count from the assay carried out in the presence of unlabeled DNA was
subtracted from the experimental counts before binding was calculated. In
contrast to the results of Rocchi et al. (1973), induction of liver enzymes by
3-MC was not required for binding of 14C from 14CC14 to DNA. Although
the purified DNA samples contained Q.2% protein, contamination by protein at
4-17
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this low level could not account for all the covalent binding measured in the
DNA sample.
In vitro binding of metabolically activated CC14 to isolated mouse
liver DNA (1.81 pmol/mg) was observed by Diaz Gomez and Castro (1980a) in
anaerobic incubation mixtures containing microsoraes and NADPH. It was also
found that 'CClg, produced by reaction of CCl^ with benzoyl peroxide, interacted
with DNA (826 pmol/mg). This result suggests that *CC13 may be the chemical
species involved in the binding to DNA.
In addition to observing that metabolites of CC14 bind to DNA, Diaz
Gomez and Castro (198Qa) observed binding of metabolically activated
CC14 to rat liver nuclear protein and lipid in vivo. The label bound to nuclear
protein was 47.7 pmol/mg and that bound to nuclear lipid was 113.5 pmol/mg.
Diaz Gomez and Castro suggested that binding to nuclear lipids may be a significant
finding regarding the potential carcinogenicity of CC14, because nuclear
lipid is derived from the nuclear membrane, which contains the cytochrome
P-450 necessary for the activation of CCl^ to the reactive metabolites "CCl^
and phosgene. Since these metabolites are highly unstable and not likely to
exist long enough to travel from the endoplasmic reticulum to the nucleus,
activation by nuclear membrane P-450 enzymes is more likely to allow the metabolites
to react with DNA than is activation in the endoplasmic reticulum.
Diaz Gomez and Castro (1980b) determined the potential of purified rat
liver nuclei to activate CC14 by measuring covalent binding of nuclear-activated
CC14 to nuclear protein and lipid. Binding to DNA was not measured. The
results were compared with those results obtained from similar incubation
mixtures containing microsomes instead of purified nuclei. The incubation
mixtures containing either nuclei (1.3 mg protein/ml) or microsomes (1.56 mg
4-18
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protein/ml) were Incubated for 30 minutes in 37.6 nM CCl^ (6.94 Ci/mol) and
an NADPH generating system in an 02-free N2 atmosphere. The extent of binding
to proteins in the nuclear preparations was 43.5% of that observed for microsomes
(nuclear suspensions, 21.9 pmol/mg; microsomes, 50.3 pmol/mg). Binding to
nuclear lipids was 77.3% of that observed for microsomes (nuclear suspension,
147 pmol/mg; microsomes, 190 pmol/mg). Isolated nuclei were less efficient
than microsomes in metabolizing CC14, but the results were within the same
order of magnitude. This study indicates that metabolism of CC14 to reactive
intermediates can occur in nuclear membranes and suggests that the in vivo
binding observed in the previous study (Diaz Gomez and Castro, 1980a) may have
been due to nuclear rather than microsomal activation of CC14. It should be
mentioned, however, that the nuclear preparations were contaminated with trace
amounts of endoplasmic reticulum, which may have contributed to the nuclear
activation observed.
Diaz Gomez and Castro (1981) have published preliminary evidence that
•CClj, chemically generated from the benzoyl peroxide-catalyzed decomposition
of CC14, reacts with guanine and adenine and to a lesser extent with cytosine
and thymine. This result suggests that *CClg may bind to DNA in vivo by
interaction with the purine and pyrimidine acid bases.
In summary, it has been shown that GC14 is metabolized to the reactive
intermediates *CClg and phosgene and that metabolically activated CC^ binds
to DNA, protein, and lipid. These results suggest that CC14 has genotoxic
potential. The negative results in six of the seven bacterial mutagenicity
studies described in section 4.2.2.1. may be due to inadequate metabolic activation
in the test systems or to scavenging by protein or lipid of any very reactive
metabolic intermediates (e.g., 'CClg and phosgene) formed under conditions
of exogenous activation.
4-19
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4.2.2, Gene Mutations Studies
4.2.2.1. Bacteria--Studies to determine the mutagenic activity of CC14 in
^ne Salmonella typhimurium revertant system have been negative. A review by
McCann et al. (1975) stated that an assay using Aroclor-induced S9 activation
and strains TA100 and TA1535 was negative, but details of the procedure were
not given. In another review article without data, Fishbein (1976) reported
that CC14 was not mutagenic when assayed in a spot test with strain TA1950.
Uehleke et al. (1977) tested the mutagenicity of CC14 in suspension assays
with _S, typhimurium strains TA1535 and TA1538. No mutagenic activity was
detected. About 6-9 x 108 bacteria were incubated for 1 hour under N2 in
tightly closed test tubes with 8 mM CC14 and microsomes (5 mg protein) plus
cofactors. The mutation frequencies (His+ colony forming units/lflS his~ colony
forming units) were less than 10 for both strains, and the spontaneous mutation
frequencies were 3.9 +_ 3.7 for strain TA1535 and 4.4 +_ 3.5 for strain TA1538.
At this concentration of CCl4§ survival of the bacteria was at least 90%.
This negative result for CC14 is questionable because concentrations that
result in less than 90% survival should have been tested. Dimethylnitrosamine,
^
cyclophosphamide, 3-methylcholanthrene, and benzo[a]pyrene were the positive
controls used in this study. Although these chemicals were mutagenic in the
presence of the S9 activation system, they are not ideal controls for €€14
because they are not halogenated alkanes and are not metabolized like CC14.
Uehleke et al. (1977) suggested that any reactive species generated by the
microsomes may not have reached the bacteria, resulting in the negative test.
It is not clear from the description whether rat, mouse, or rabbit microsomes
were used in the mutagenicity studies. It is clearly stated that rabbit microsomes
were used for the binding studies described previously. If mouse or rat microsomes
4-20
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rather than rabbit microsomes were used for the mutagenicity experiments, it
cannot be assumed that CC14 was sufficiently activated, since activation
sufficient for binding of ^CCl^ to macromolecules was shown in this paper
only with rabbit microsomes. Another deficiency in this study is that the
Salmonella strains TA98 and TA100 were not used. These strains contain an R
factor plasmid that Increases the sensitivity of the tester strains to certain
tnutagens. Because of these deficiencies, the negative mutagenicity results in
this paper should be regarded as inconclusive.
The mutagenicity of CC14 was also tested in a study on the mutagenic
potential of chemicals in drinking water (Simmon et al., 1977). No mutagenic
activity was detected with CC14. The authors tested 71 of the 300 chemicals
that had been identified in public water supplies. CC14 *as tested in a
desiccator to assess mutagenicity due to vapor exposure and to avoid excessive
loss of CC14 to the atmosphere. The desiccator contained a magnetic stirrer
that acted as a fan to aid in evaporation of the measured amount of CC14 and
to maintain an even distribution of the vapors. Plates were exposed to the
vapors for 7 to 10 hours and then removed from the desiccators, covered, and
incubated approximately 40 hours before scoring. Mutagenic activity was not
observed and no information on toxicity was provided.
The study by Simmon et al. (1977), although lacking some specific details
of the CC14 assay, clearly identifies certain trihalomethanes (CHBr3, GHB^Gl,
CHBrClg) as mutagens in the vapor assay in desiccators. Methyl bromide, methyl
chloride, methyl iodide, and methylene chloride were also found to be mutagenic
in the desiccator assay. However, these seven halogenated compounds did not
require metabolic activation to exhibit mutagenic activity. It may be that
CC14 itself is not mutagenic and the rat liver 59 does not effectively metabolize
4-21
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CC14 to a mutagen, even though the tnutagenicity of three of the chemicals
tested [bis(2-chloroisopropyl )ether, vinyl chloride, and vinylidene chloride]
required or was enhanced by the rat liver S9 mix. It may also be that a
reactive intermediate was formed but was too short-lived to be detected in a
test system that uses exogenous metabolic activation.
Another negative result for the mutagenicity of CC14 was obtained in a
variation of the Salmonella/microsome assay in which the escape of volatile
compounds was prevented by the use of a specially designed, closed, inert
incubation system (Barber et a!., 1981). Seven of the 10 halogenated alkane
solvents tested gave positive mutagenicity results when the assays were
carried out in the closed incubation system. Under standard conditions (in
which volatilization was not prevented), only 2 of the 10 solvents gave a
positive result. Thus, the closed incubation system allowed for the detection
of five more mutagens than could be detected under standard conditions. CC14
was one of the three solvents that gave a negative result in both the standard
and closed incubation systems. The investigators indicated that CC14 was
tested at concentrations high enough to produce observable toxicity, determined
by the absence of background lawn. The Salmonella strains used were TA1535,
TA1537, TA1538, TA98, and TA100. Levels of CC14 tested were 4.7, 5.7, 10.2,
12.3, and 18,4 umol per plate, and no dose-related response was observed. The
seven solvents that were mutagenic in this closed system did not require metabolic
activation by 59 mix, but the S9 mix did activate the positive control 2-
aminoanthracene. It is also possible that the S9 mix used, although adequate
for activation of the control 2-aminoantnracene, was not adequate to metabolize
CC14. It is also possible that active metabolites, if formed, reacted with
microsomal components or bacterial membrane macromolecules before reaching the
bacterial DNA,
4-22
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Cooper and Witmer (1982) reported in an abstract that exposure of Salmonella
strain TA100 to CC14 for 20 minutes in a 1-ml liquid suspension at low oxygen
tension (3-ml Vacutainer tubes) before preparation of plates resulted in a
twofold increase in revertants above background (background, 72 +_ 9 revertants;
5 umol CC14, 141 j^ 20 revertants). Evidence for a dose-response relationship
was not reported. The weak mutagenic result was observed in two separate
experiments and only when fresh rabbit liver S9 was used as the activation
system. It is unlikely that the rabbit liver S9 alone was responsible for the
mutagenic activity observed, because plate assays with rabbit S9 exhibited no
mutagenic activity. The CC14 was spectrophotometric grade (Spectrar) purchased
from Mallinckrodt (Dr. Charlotte Witmer, personal communication). CC14 was
toxic in this suspension assay; 2 umol/rnl caused 80% toxicity with microsomal
activation and 20% without activation. Addition of 0.1 mM EDTA to inhibit
microsomal lipid peroxidation decreased bacterial toxicity. The investigators
concluded that the suspension assay in Vacutainer tubes may be a suitable
system for testing volatile compounds that undergo reductive metabolism. The
marginal response in this assay suggests that CC14 may be mutagenic in the
Salmonella assay, but only under certain assay conditions (low oxygen tension,
exposure of bacteria in suspension in the presence of the EDTA, and use of
fresh rabbit liver S9). In view of the very weak response and lack of evidence
for dose-dependence, however, the study should be regarded as inconclusive.
In summary, the results of bacterial tests of CC14 are predominantly negative
but inconclusive. False negative results could have been obtained due to a
number of factors, including:
1. The activation systems used may have been inadequate for metabolism
of CC14.
4-23
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2. "CClg and phosgene, the primary reactive metabolites of CCl^, are
unstable and highly reactive. Because exogenous activation systems
were used in many of these studies, any 'CCl-j or phosgene generated
may have been scavenged by microsomal protein or lipid before reaching
the DNA.
3. Adequate exposure to CC14 may not have occurred if appropriate
precautions were not taken to prevent the evaporation of 0014.
4.2.2.2. Yeast—Callen et al. (1980) studied the genetic activity of CC14
in strain D7 of Saccharomyces cerevisiae, which contains an endogenous cytochrotne
P-450 dependent monooxygenase system. By using yeast, Call en and his coworkers
eliminated the need for the exogenous type of metabolic activation system used
in the bacterial studies. Three different genetic endpoints can be examined
in strain D7; gene conversion at the trp5 locus, mitotic crossing over at the
ade2 locus, and reversion at the iIvl locus. The effect of CC14 (Mallinckrodt,
99+% pure) on these endpoints was measured by exposing cells in suspension to
3.23, 4.31, and 5.13 g of CC14 per liter of buffer (21 mM, 28 mM, and 34 mM,
respectively), well above the solubility level of CC14 in water (0.8 g/L at
25°C). A dose-response relationship could not be obtained because the dose
was essentially the same in all cases—the solubility level of CC14 in water
at 37°C. Escape of CC14 was minimal because the incubations were carried
out in screw-capped glass tubes. Although the dose is essentially constant,
amounts in suspension vary. Extracellular or membrane effects may have resulted
in the high toxicity observed at 5.13 g/L.
Results of Callen et al. (1980) are presented in Table 4-2. A 1-hour
treatment of cells with the highest amount of CC14 tested (5.13 g/L) resulted
in significant increases in gene conversion (31-fold) and mitotic crossing-over
(25-fold). Reversion was also increased, but to a lesser extent (threefold
increase). Survival was only 10% at this concentration of CC14. At the
intermediate level (4.31 g/1) of CC14, much weaker effects on the three genetic
4-24
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endpoints were observed (two to threefold increases). Survival at this level was
771. The data of Callen et al. (1980) suggest a positive response, but because
of the solubility problem, additional studies are needed before it can be
stated conclusively that CC14 causes genetic effects in yeast.
4.2.3, Cytogenetic Studies
Negative results have been obtained in an in vitro chromosome assay in an
epithelial-type cell line derived from rat liver (Dean and Hodson-Walker, 1979).
This cell line has sufficient metabolizing activity to activate various chemical
mutagens and carcinogens without the need for exogenous metabolic activation.
Sealed-flask cultures were treated for 22 hours with CC14 dissolved in growth
medium at 0,005, 0.010, and 0.020 mg/1. At these low concentrations, CC14 did
not induce chromatid or chromosomal aberrations, whereas a number of direct-acting
mutagens and several compounds that require metabolic activation produced
chromatid deletions, gaps, and exchanges. No other heavily chlorinated substances
were tested. In addition, most of the compounds were assayed at doses several
orders of magnitude higher than that of CC14. The doses chosen for each substance
assayed were determined from cytotoxicity tests. CC14 was apparently toxic
to rat liver cells. EDTA (0.1 mM) has been found to decrease the cytotoxicity
of CC14 without affecting mutagenicity in bacteria (Cooper and Witmer, 1982);
perhaps addition of EDTA to a mammalian in vitro chromosome assay, such as
that used by Dean and Hodson-Walker (1979), would permit the use of larger
concentrations of 0014.
4.2.4. Other Studies Indicative ofDNA Damage
Mirsalis and Butterworth (1980) measured unscheduled DNA synthesis (UDS)
in primary rat hepatocyte cultures following in vivo treatment of adult male
Fisher 344 rats (200-250 g) with CC14 (certified ACS grade, Fisher Scientific
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TABLE 4-2. GENETIC EFFECTS OF CARBON TETRACHLORIDE ON STRAIN D7 OF Saccharomyces cerevisiae
FOLLOWING 1 HOUR TREATMENT AT 37°Ca
Concentration, g/L
3.23
4.31
5.13
Survival
Total colonies
% of control
t^rpS locus (gene conversion)
Total convertants
Convertants/lO^ survivors
1454 1252 1120 152
100 86 77 10
285 331
2,0 2.6
350 506
3.1 61.7
ade2 locus (mitotic crossing over)
Total twin spots
Mitotic cross-overs/lO* survivors
Total genetically altered colonies
Total genetically altered colonies/
10-3 survivors
ilvl locus (gene reversion)
Total revertants
Revertants/10^ survivors
1
1.6
11
1.7
38
2.6
3
5.3
19
3.4
41
3.3
3
5.8
16
3.1
57
5.1
10
40.1
65
33.3
11
7.2
aThe number of colonies in the different classes represent total counts of colonies
from five plates in the case of survival, conversion, and revertant-frequency estimations.
Mitotic crossing over was estimated from counts of colonies growing on a total of 30
plates, 20 plates containing medium on which all surviving cells grew, and 10 plates
containing medium on which only trp5 convertants grew.
SOURCE: Adapted from Call en et al., 1980.
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Co.) by oral gavage. Control rats received corn oil by gavage. Acetylamino-
fluorine and dimethylnitrosamine were also tested as positive control substances.
At 2 hours after treatment, the livers were perfused in situ and hepatocytes were
isolated. Approximately 6 x 105 viable cells were seeded in culture dishes
and allowed to attach to coverslips for 90 minutes. The coverslip cultures were
washed and then incubated for 4 hours 1n medium containing 10 uCi [3H]thymidine
(42 Ci/mmol) per ml. The cultures were washed again and incubated in medium
containing 0.5mM cold thymidine for 14-16 hours. The extent of UDS was assessed
by autoradiography. Net grains per nucleus were calculated as the silver
grains over the nucleus minus the highest grain count of three adjacent nuclear-sized
areas over the cytoplasm. The area of the silver grains, rather than the
grain number was determined so that UDS could be accurately measured in densely
labeled cells in which silver grains were touching.
Cell counts from control animals had from -2 to -6 net grains per nucleus.
Treatment of rats with dimethylnitrosamine (i.p.) at 10, 1, or 0.1 rag/kg yielded
36.6, 6.4, and -0.9 net grains per nucleus, respectively; dimethylnitrosamine at 10
mg/kg (p.o.) produced 22.2 net grains per nucleus. Oral doses of acetylaminofluorine
at 50 and 5 mg/kg yielded counts of 14.0 and 6.4 net grains per nucleus, respectively,
CC14 at 100 or 10 mg/kg (p.o.) yielded counts of 3.2 and 5.1 net grains per nucleus,
respectively. Thus, dose-related increases in UDS were observed for the positive
control dimethylnitrosamine and acetylaminofluorine, whereas CC14 produced no
such response.
As indicated above, two doses of CC14 were tested: 10 mg/kg and 100 mg/kg. The
oral LD§o for CC14 in rats is 2800 mg/kg. The dose at which hepatic cell
toxicity would occur under the conditions used was not determined and it is not
clear whether adequate doses of CC14 were tested. It is also unclear whether
4-2?
-------
the 2-hour time period between exposure of the rats to CC14 and isolation of
the hepatocytes was sufficient for observation of UDS. In a study by Popp et
al. (1978) in which CCl4-induced hepatocellular changes were noted, the shortest
exposure period studied was 6 hours. If it were shown that the 2-hour exposure
period is sufficient for activation of the CC14 to a reactive intermediate,
for example, by demonstrating alkylation of protein by ^'CCI^, the negative
results would be more convincing. The negative result may reflect the inability
of CC14 to cause UDS or it could be a false negative result due to factors
such as inadequate dose or inadequate exposure time.
Craddock and Henderson (1978) carried out an in vivo UDS study in which
hepatocyte nuclei were isolated and then assayed for radioactivity by
scintillation counting rather than by grain counting. This study used a CC14
dose of 4000 mg/kg, which is significantly larger than the oral 1053 (2800
mg/kg). Negative results were obtained after a 2-hour exposure, but a positive
response was observed after a 17-hour exposure. The investigators suggested that
this result may be attributable to secondary effects such as lysosomal damage,
which may result in release of DNA degradative enzymes.
In their latest study, Mirsalis et al. (1982) used combinations of doses
(up to 400 mg/kg) and exposure times (up to 48 hours) that resulted in liver
toxicity. Criticisms of previous studies relating to inadequate dose and
exposure have been obviated by this study. The results for CC14 were negative
in this study as well. However, benzo[a]pyrene, 7f12-dimethylbenz[a]anthracenef
and N-methyl-N'-nitro-N-nitrosoguanidine were negative in this in vivo UDS
assay, whereas these chemicals tested positive in the in vitro rat hepatocyte
UDS assay (Williams, 1981). This discrepancy suggests that the in vitro test
may be more sensitive than the in vivo assay. CC14 has not been tested in
the in vitro rat hepatocyte UDS assay.
4-28
-------
In summary, the in vivo UDS results provide no evidence that CC14 causes
DNA damage that elicits UDS, However, in vitro UDS studies are needed before
a firm conclusion is reached about CC14 not inducing UDS.
4,2.5. Suggested Additional Testing
Suggested additional testing falls into six categories:
1. The DNA damage studies reported by Craddock and Henderson (1978),
Mirsalis and Butterworth (1980), and Mirsalis et al. (1982), which provide
no evidence that CC14 induce UDS following in vivo treatment of rats and
should be corroborated by the in vitro rat hepatocyte UDS assay of Williams
(1981). The in vitro test may be more sensitive than the in vivo test for
weak genotoxic effects.
2. Studies using sensitive assay procedures for detecting the formation
of chemical adducts in liver DNA after exposure of animals to CC14 are needed
to confirm the low level of DNA binding observed by Diaz Gomez and Castro
(1980a) and by Rocchi et al. (1973).
3. Additional data from yeast studies are needed to confirm the study of
Callen et al. (1980) in the yeast system, which utilizes an endogenous activation
system and is capable of assaying for point mutations, mitotic crossing over,
and gene conversion.
4. Additional cytogenetic testing in mammalian systems is needed before
CC14 can be considered to be adequately tested for chromosome damage. Because
EDTA has been reported to decrease the cytotoxicity of CC14 in bacteria (Cooper
and Witmer, 1982), in vitro mammalian liver cell cytogenetic assays should be
carried out in the presence and absence of EDTA. The EDTA may allow higher
levels of CC14 to be assayed than were used in the study by Dean and Hodson-Walker
(1979). In vivo studies, such as bone marrow cytogenetic analysis or a
micronucleus test, are also needed. Tests for nondisjunction should be included.
4-29
-------
5. A thorough test is needed to corroborate (or refute) the preliminary
evidence for a weak mutagenic response in Salmonella reported by Cooper
and Witmer (1982). The same experimental conditions (fresh rabbit liver S9
and exposure of the bacteria in suspension to CC14 under reduced oxygen tension
1n the presence of EDTA) should be used, and several concentrations of €€14
should be tested to determine if a dose-response relationship exists. If the
bacterial test is positive, in vitro mammalian mutagenicity studies should be
carried out under the same experimental conditions. If the bacterial test is
negative, the cell culture assay should be conducted by a more standard protocol
with provision for the volatility of the chemical.
6. Studies on the ability of CC14 to reach reproductive organs and cause
germ-cell mutation have not been conducted. If results from the tests for
mutation in cultured cells are positive, then studies assessing heritable
risk are needed. The document entitled, Proposed Guidelines for Mutagenicity
Risk Assessment, published in the Federal Register in 1984, is recommended for
guidance on such tests.
4.2.6. Summary an_d ConcUi_sj_o_ns_
CC14 has been tested for its mutagenic potential in bacteria, yeast, and a
mammalian cell line and for its DNA damaging potential in rat hepatocytes when
administered in vivo.
Six of the seven point mutation studies in bacteria were negative. The
remaining bacterial study (Cooper and Witmer, 1982) was a preliminary test that
provided only suggestive evidence of a weak mutagenic response. In none of the
negative studies was it shown that CC14 was activated or metabolized by the
exogenous S9 activation system used. Metabolism of 2-aminoanthracene or vinyl
compounds (used as positive controls) is probably an inadequate indication
4-30
-------
that the activation system can metabolize halogenated alkanes such as CC14-
A better indication that the activation system is sufficient may be to show
that it metabolizes ^CCl^ to intermediates that bind to macromolecules.
It is also conceivable that potentially mutagenic reactive derivatives of CC14
(such as the free radical 'CClj and phosgene) are generated in the presence
of an S9 activation system but are too short-lived to interact with DNA in in
vitro test systems.
The study by Callen et al. (1980) was designed to overcome this problem
by using an in vivo activation system in yeast. Positive results for
mutagenicity and recombinogenicity were reported. In contrast, negative results
for DNA recombinogenicity were reported using in vivo UDS as the assay endpoint.
These negative results should be confirmed, however, by the more
sensitive in vitro hepatocyte UDS assay of Williams (1981). Binding studies
by Rocchi et al. (1973) and by Diaz Gomez and Castro (1980a, 1980b, and 1981}
indicate that metabolically activated CC14 may interact with DNA.
The negative genotoxicity test results that have been reported may be due
to any of four (or more) factors; (1) CC14 is not mutagenic, (2) excessive
volatilization and escape of CC14 if appropriate precautions are not taken,
(3) inadequate activation of CC14 to a metabolite capable of causing mutations
(e.g, *CCl3 or phosgene) by the S9 system, or (4) inability of reactive
derivations of CC14 to reach DNA before being scavenged by lipid and protein
(particularly under conditions of exogenous activation, such as in the Salmonella
test). Additional testing should incorporate appropriate measures to ensure
that (1) volatilization and escape of CC14 does not decrease exposure of the
test organism or cell to ineffective levels, (2) metabolic activation is occurring,
and (3) DNA is exposed to the activated chemical species.
4-31
-------
In its totality, the evidence described in this report is insufficient to
allow firm conclusions concerning the genotoxicity of CC14. The marginal
results for binding of reactive intermediates to DNA (Rocchi et al. 1973;
Diaz Gomez and Castro, 1980a and 1981), the study of Callen et al, in yeast
(1980), and the questionable evidence for mutagenicity in the Salmonella assay
(Cooper and Witmer, 1982) are insufficient evidence for genotoxicity. Since
they do suggest the possibility of weak genotoxic effects, however, further
studies should be done.
4-32
-------
4.3. DICHLOROMETHANE
Dichloromethane (DCM) has been tested for rnutagenlc activity in bacteria,
yeast, insects, nematodes, mammalian cells in vitro, and rodents. These studies
are discussed below and are summarized in Tables 4-3 to 4-8.
4,3.1. GeneMutationStudies
4.3.1.1. Bacteria—There are 14 reports in the literature concerning the
mutagenic potential of DCM in bacteria; the Salmonella histidine reversion assay
was used in all of these studies (Simmon et al., 1977; Simmon and Kauhanen,
1978; Kanada and Uyeta, 1978; Jongen et al., 1978, 1982; McGregor, 1979; Snow et al.,
1979; Green, 1980, 1981; Rapson et al., 1980; Barber et al., 1980; Nestmann et
al., 1980, 1981; Socke et al., 1981). Kanada and Uyeta (1978) also tested DCM
in the BaciVKis subtil is rec assay. DCM was positive in all studies in Salmonella
without or with metabolic activation in strains TA100, TA1535, or TA98 when
assays were performed in sealed, gastight exposure chambers. Negative responses
were reported by Rapson et al. (1980) and Nestmann et al, (1980) in standard
assays, but these tests are inadequate because DCM was added directly to the
agar medium and no precautions were taken to prevent excessive evaporation of
the test material. The tests were carried out at 37°C, which is close to the
boiling point of DCM (39°C), and it is very likely that excessive evaporation
occurred. Data were presented in many of the reports, and a clear dose-related
response is apparent for each. Tenfold or greater increases in numbers of
revertants per plate were observed at the highest doses compared with negative
controls. The doses employed and the responses observed are summarized in
Table 4-3.
The purity of the test material was not given in any report. Because of
this, most positive responses must be viewed with caution, because substances
other than DCM may contribute to the observed mutagenicity. For instance,
4-33
-------
TABLE 4-3. MUTAGENICITY TESTING OF DCM IN BACTERIA
Reference
Test
system
Strain
Activation
system
Concentration/Result
Comments
Simmon et
1977
al.,
Salmonella/
S9 vapor
exposure
TA1535
TA1537
TA1538
TA98
TA100
None
(Extrapolated from
Fig. 17) 0, 50, 100,
200, 400, and 800
ul/9 L
desiccator
(Extrapolated from
Fig. 17)
TA100
Dose (ul) Revertants/plate
0
50
100
200
400
800
170
210
300
400
650
1350
1. Toxicity not
reported.
2. Number of
revertants
observed for TA100
not specified
numerically.
3. Data not
presented for
strains other than
TA100.
4. Purity and
source of compound
not provided.
5. Positive
response.
TA100
revertants
Simmon and
Kauhanen,
1978
Salmonella/ TA100 Aroclor- 0 and 1 ml/9 L
S9 vapor 1254 desiccator for
exposure induced 6.5 and 8 hours
rat liver
microsome
S9 mix
No.h S9 Treated
6.5 - 688
+ 1344
8 - 830
+ 912
Control
133
130
174
158
_ — _ : frnntinued on
1. Toxicity not
reported.
2. Purity and
source of compound
not provided.
3. Used as a
positive control
in the testing of
2-chloroethyl-
chloroformate.
4. Positive
response.
-the following pace)
-------
TABLE 4-3. (continued)
Reference
Kanada and
Uyeta, 1978
Abstract
Jongen et
al., 1978
S^
tf
Test
system Strain
Salmonella/ TA98
S9 and B. TA100
subtilis
rec assay
testing
Salmonella/ TA98
S9 vapor TA100
exposure
Activation
system
PCB-induced
rat liver
microsome
S9 mix
Phenobarbital-
induced rat
liver microsome
S9 mix
Concentration
Not reported
(ppm x 103)
0
5.7
11.4
14.1
22.8
57.0
Result
DCM reported
negative in
B. subtilis and
positive for
both strains in
S. typhimurium
TA100 TA98
+S9 -S9 +S9
152+19 129+12 21+4
329+37 248+32 54+5
515+76 407+4-7 74+4
757+82 582+56 93+9
865+82 653+89 123+10
1201+191 740+94 149+42
-S9
19+5
44+8
56+10
66+12
96+11
110+42
Comments
1. Results summarized
in abstract form.
2. Positive results
in Ames test supports
reports by other authors
using same system.
1. Testing conducted
in gastight perspex
boxes.
2. Only highest dose
exhibited less than
83% survival.
3. Purity of DCM not
reported.
4. Positive response.
5. Results from 3
experiments, 5
plates per dose.
(continued on the following pege)
-------
TABLE 4-3. (continued)
Reference Test system Strain
Snow et al., Salmonella/S9 TA98
1979 vapor exposure TA100
Activation
system Dose
DCM-induced Syrian (ul /chamber)
golden hamster
liver S9 microsome 0
mix 100
300
500
1000
Result
+S9
66
177
463
642
972
TA100
-S9
63
142
274
468
632
.
+S9
3S
47
69
92
39
TA98
-S9
19
31
46
61
72
Comments
1. Purity of DCM
not reported.
2. No information
about variability
of results.
3. Positive
response.
4. Mean calculated
from 3 plates per
dose.
(continued on the following page)
i
U)
-------
TABLE 4-3. (continued)
Reference Test system
McGregor, Salmonella/59
1979 vapor exposure
Nestmann Salmonella/59
et al., vapor exposure
1980
Activation
Strain system
TA1535 None Atmospheric
Theoretical
0
0.5
1.0
2.0
4.0
10
TA1535 Arocl or- induced
TA1537 rat liver S9
TA1538
TA98
TA100
Concentration/Result
concentration
Actual
ND
0.14
0.33
0.67
1.60
ND
% plate concentration
(ug)
ND
245
600,595,530
1400
2425
ND
Revertants
' 15
20
25
50
75
80
Comments
1. Purity of DCM
not reported.
2. Positive
response.
1. Data not
presented.
2. Negative
response in
standard test.
3. Positive
response in gas-
tight chamber.
Doubling in
revertant counts
for TA1535;
six-fold increase
for TA100.
(continued on the following pageT
-------
TABLE 4-3. (continued)
Reference Test system Strain
Green, 1980 Salmonella/S9 TA1535
vapor exposure TA100
Activation
system
Rat liver
fractions
Dose
Result
Concentration TA 100
(% in air) +S9 -S9
0
1.4
2.8
5.5
8.3
69+3
283+10 267+20
506+27 462+28
825+34 872+27
1050+88 997+88
Comments
1. Preliminary results pre-
sented in abstract form.
2. Metabolic studies con-
ducted in rat tissue and
TA100. Similar metabolism
in both systems. Radiolabel
reported to bind to bacterial
00
DNA but not to rat liver DNA.
3. Purity of DCM not reported.
4. Positive response. Green
thinks that this is due to
close proximity of cytoplasmic
enzymes and intermediates
to DNA in bacteria and that
negative responses would be
obtained in higher organisms.
Positive responses in other
tests argue against this. See
discussion in section 4.3.1.1.
(continued on the following page)
-------
TABLE 4-3. (continued)
Vf)
Reference Test system ppm Vapor umol/plate
Barber et Salmonella/S9 0 0
al ., 1980 vapor
exposure
3,600 38
7,200 76
9,100 96
St
,;;s 10,900 115
X!_,
Revertants/Plate
TA1535 TA98 TA100
-S9 +S9 -S9 +S9 -S9 +S9 Comments
23 28 23 39 254 264 1. Tested redistilled
sample of DCM > 99.9%
pure.
40 36 259 288 752 1152 2. Revertants/nmol at
highest dose for TA1535,
TA98, and TA100 were
0.0006, 0.006, and
0.03, respectively.
59 51 441 297 1440 960 3. Data shown for
testing in gastight
chamber.
78 78 459 322 2640 1096 4. Negative response
in standard test;
64 50 741 479 3060 3240 positive response in
gastight chamber.
5. DCM concentra-
tion measured by
GC/MC in aqueous
phase of petri dish.
(continued on the following page)
-------
TABLE 4-3. (continued)
Reference Test system
Nestmann Salmonella/S9
et al., vapor exposure
1981
Strain
TA1535
TA1537
TA1538
TA98
TA100
Material
Type
Paint
remover
Mix
(90:5:5
v/v/v DCM/
methanol/
ethanol)
Activation
system
Concentration/Result
Aroclor 1254-
induced rat
liver S9
his+ Revertants/Plate3
Weight (mg)
Added
0
203
370
790
1435
0
0.1
0.2
0.4
0.8
Vaporized
—
144
241
469
903
(ml)
—
0.1
0.2
0.4
0.8
TA1535
16
22
14
23
31
13
15
24
25
34
TA100
144
310
433
563
785
154
268
401
789
1084
TA98
25
31
42
76
60
32
43
73
138
164
Exposure
Comments
1. Levels of DCM in exposure
chambers related directly to
the mutational dose-effect
curves of three paint removers.
2. Data shown for one paint
remover only. Other two gave
similar response.
3. Purity of DCM not reported,
4. Positive response for paint
. removers likely due to DCM.
level (mg/L)
DCM
Time
Averaged0
—
12.7
21.9
40.1
80.2
—
12.2
26.9
50.6
94.1
Max 6h
Methanol
Ethanol0
Calculated0 Measured
—
15.5
25.5
49.9
95.4
—
12.8
25.3
50.8
101 .0
—
13.0
23.0
45.0
86.0
—
11.5
27.5
50.0
94.5
—
<0.5
<0.5
0.7
1.9
—
<0.5
<0.5
0.9
2.6
—
—
—
—
—
—
<0.9
0.7
1.1
2.7
(continued on the following page)
aAverage values from triplicate plates.
"Maximum measured.
cDetermined from an area under curve for concentration against time.
^Calculated from amount vaporized assuming only DCM vaporized in 9 L chamber.
-------
TABLE 4-3. (continued)
Reference Test system
Green, Salmonella/59
1981 vapor exposure
Gocke et Salmonella/59
al . , 1981 vapor exposure
Jongen et Salmonella/59
al., 1982 vapor exposure
Activation
Strain system
TA100 Aroclor-1254
induced rat li
59, microsomes
and cytosol
TA1535 Aroclor-1254
TA1537 rat liver S9
TA1538
TA98
TA100
TA100 Aroclor-1254
rat liver S9,
microsomes,
and cytosol
Dose Result
Revertants
ver % Vapor +59 -S9
0 100 100
2.8 458 386
5.0 700 720
8.4 950 900
Revertants
ul/desiccator +S9 -59
0 30+0 40+0
125 54+7 85+7
250 68+32 110+14
500 105+7 195+21
750 203+32 295+7
% DCM
Activation 0 0.35 0.7
150 210 350
59 150 240 410
Cytosol 150 220 420
Microsomes 150 215 380
Comments
1. Bacterial and mammalian
metabolism similar.
2. Cytosol and glutathione
catalyze DCM to formaldehyde
and C02. S-chloromethyl gluta-
thione is a putative intermediate
3. DCM converted to carbon
monoxide in the presence of
microsomes. Formyl chloride is a
putative intermediate.
4". Purity of DCM not given.
5. Positive response. See table
entry for Green, 1980.
1. Spontaneous revertants for
TA100 too low.
2. No information presented
for toxicity.
3. Purity of DCM not given.
4. Positive response.
1.4 1. Purity of DCM not given.
550 2. Positive response.
730
810
610
-------
formaldehyde, a metabolite of DCM, could form nonenzymatically by hydrolysis
of DCM in the aqueous solutions used for biological testing (March, 1977).
Because of the consistency of positive responses with several different samples,
however, it is highly likely that the DCM itself is mutagenic. For instance,
in their tests of DCM, Barber et al. (1980) used a redistilled sample of DCM
estimated to be >99.9% pure, containing only traces of 1,1- and 1,2-dichloroethane,
chloroform, chloromethane, and an unidentified CgH^Q aliphatic material
(Dr. E. Barber, Eastman Kodak, personal communication). Nestmann et al. (1981)
reported that their sample was "gas chromatographically pure," and Gocke et
al. (1981) checked their sample for the "correct melting point (sic) and elementary
analysis." The consistency of the positive responses indicate that the mutagenicity
1n Salmonella can be attributed to DCM. Barber et al. (1980) conducted their
tests in a chemically inert, closed incubation system and analyzed the concentrations
of DCM in the vapor-phase head space and in the aqueous phase of a test plate
by gas-liquid chromatography (Barber et al., 1981). The mutagenic responses
at the highest dose (i.e., 115 umol/plate) for TA1535, TA98, and TA100 were
0.0006, 0,006, and 0.03 revertants per umol, respectively, indicating that DCM
is a weak mutagen for Salmonella under the conditions of the test.
Although the results clearly show that DCM is mutagenic in Salmonella, questions
have been raised about their applicability to predicting mutagenicity in other
species, especially mammals. DCM is metabolized, apparently via mutagenic
intermediates, to CO and C02 in both rodents and humans. CO is produced by
oxidative dechlorination of DCM by the microsomal P-450 mixed-function
oxidase system. Formyl chloride is believed to be an intermediate in this
pathway. A second cytosolic glutathione transferase system dehalogenates DCM
to produce formaldehyde, which is further oxidized to C02. This pathway 1s
4-42
-------
thought to proceed via an S-chloromethyl glutathione intermediate (Ahmed and
Anders, 1978; Kubic and Anders, 1975). Formyl chloride and S-chloromethyl
glutathione are highly reactive alkylating agents. Salmonella also metabolizes
DCM to C02 and CO, apparently by reaction pathways similar to those occurring
in mammals (Green, 1980, 1981, and 1983).
Because of the reactivity of formaldehyde, formyl chloride, and S-chloromethyl
glutathione and the proximity of bacterial DNA to bacterial cytoplasmic enzymes,
it has been hypothesized that these chemical substances are more effective as
mutagens when they are formed by bacterial metabolism than when they are formed
outside the bacterial cell by rat liver fractions (Green, 1980, 1981» and
1983). The basis for this hypothesis is that rat liver fractions used for
metabolic activation have little effect on the mutagenicity of DCM in the Ames
test. The implication is made that as organismic complexity is increased,
there is less likelihood of DCM causing mutations. It is argued that
compartmentalization of DNA into the nucleus protects the genetic material
from exposure to the mutagenic metabolites of DCM (i.e., they would react with
other cellular constituents first) so there is little or no mutagenic
risk. Mhile the intraeellular compartmentalization of eucaryotes may reduce
the frequency with which short-lived, highly reactive metabolites interact
with DNA, the positive results discussed in the following paragraphs show that
genetic damage occurs in eucaryotes exposed to DCM as well.
4.3,1.2. Yeast--Callen et al. (1980) studied the ability of DCM obtained
from Fisher Scientific Company (purity not reported) and six other halogenated
hydrocarbons to cause gene conversion, mitotic recombination, and reverse
mutations in S. cerevisiae (Table 4-4). Strain 07 log phase cells were
incubated for 1 hour in culture medium containing 0, 104, 157, and 209 mM DCM.
The percent survival for these doses was 100, 77, 42, and <0.1, respectively.
4-43
-------
TABLE 4-4. GENE MUTATIONS AND MITOTIC RECOMBINATION IN YEAST
Response/10^ survivors
Reference Test system Strain
Dose
(mM)
Survival
trp-5
Conversion
Mitotic
crossing
over
ilv-1
Total genetic
alterations Revertants
Comments
Callen
et al.,
1980
Saccharomyces
cerevisiae
D7
0
104
157
209
100
77
42
18
28
107
310
190
4490
3,300 2.7 1. Positive
3,900 4.4 response.
14,000 5.8 2. Active meta-
— — bolites produced by
this system are made
intracellularly
rather than by an
exogenous activation
system.
Simmon
et al . ,
1977
Saccharomyces D3
cerevisiae
suspension
test
1. Data not pro-
vided, but reported
negative for mi tot i<
recombination.
2. Strain dif-
ferences and
differences in
treatment conditions
(i .e., time and
temperature) may
be the cause of
differences between
this study and that
of Callen et al.
(1980).
3. Cytochrome P-450
concentration not
known. Callen et
al. (1980) report
different yeast
strains have
different levels.
-------
Due to the toxicity of the compound, the genetic endpoints were not measured at
the highest dose. The responses for the other doses (0, 104, and 15? mM) expressed
per 106 survivors were: gene conversion at the trp-5 locus {18, 28, and 107);
mitotlc recombination for ade-2 (310, 190, and 4,490); total genetic alterations
for ade-2 {3,300, 3,900, and 14,000); and reverse mutations for ilv-1 (2.7,
4.4, and 5.8). A greater than twofold dose-related increase over negative
controls was observed for each endpoint measured. The magnitude of the
recombinogenic response at the ade locus may have been overestimated in this
study because the treatment regime used for estimating the recombinants overlaps
that used for estimating the number of trp-5_+ convertants. No exogenous
metabolic activation was used in these experiments, which indicates that yeast
cells metabolize DCM intracellularly to a mutagenic intermediate(s) that
reaches nuclear DNA. Simmon et al. (1977) reported that DCM (source and purity
not given, but stated to be the highest available purity) did not induce mitotic
recombination in strain D3 of Sj, cerevisiae when cells {1 x 108) in suspension
culture were exposed for 4 hours at 30°C (Table 4-4). The doses and the experimental
values obtained for mitotic recombination were not reported. The discrepancies
between the work by Callen et al. (1980) and Simmon et al. (1977) may be due
to a number of factors including the different strains used (D3 vs. 07), exposure
time differences (4 hours vs. 1 hour), or differences in the incubation temperature
(30°C vs. 37°C). Callen et al. (1980) reported that increasing the treatment
time from 1 hour to 4 hours significantly reduced the genetic activity detected in
strain D7. Other variables, such as a lower level of cytochrome P-450 enzymes
in strain D3, could also account for the discrepancy in the results.
On the basis of the results in strain D7, DCM is considered to be a mutagen
and recombinogen in yeast.
4-45
-------
4.3.1.3. Drosophi1a--Two reports are available concerning the ability of DCM
to induce sex-linked recessive lethal mutations in D. melanogaster (Table
4-5). Abrahamson and Valencia (1980) reported negative results, whereas a
positive response was reported by Gocke et al. (1981). Abrahamson and Valencia
(1980) conducted their sex-linked recessive lethal tests using two routes of
administration, adult feeding and injection. Due to the low solubility of DCM in
aqueous solutions, high concentrations of the test substance were not used in
these experiments, which may account for the negative response observed.
In the feeding study, Canton-S males were placed in culture vials containing
glass microfiber paper soaked with a saturated solution of 1.9% DCM (224 mM)
in a sugar solution for 3 days. The feeding solution was added twice daily
to compensate for evaporation of the compound. At this dose, there was no
evidence of toxicity. After mating, chromosomes from the 14,682 offspring
of treated parents and chromosomes from the 12,450 offspring of concurrent
control parents were assessed for recessive lethal mutations. No evidence of
mutagenicity was observed. DCM gave a level of 0.204% lethal mutations compared
with 0.215% in controls. Because of the volatility and insolubility of DCM,
the actual dosages may have been less than expected.
In the injection study, 0.3 ul of an isotonic solution containing 0.2%
DCM was administered to male flies. This exposure level resulted in 30% post-
injection mortality. The post-injection mortality observed for the controls was
not reported. This is a critical omission because the mortality in injection
studies is due not only to the test chemical, but also to the damage caused -by
injection, and concurrent negative controls are necessary to reach conclusions
about the effects of the treatment. After mating, 8,262 chromosomes from the
offspring of treated parents and 8,723 chromosomes from the offspring of control
4-46
-------
TABLE 4-5. GENE MUTATIONS IN MULT ICE LLULAR EUCARYOTES IN VIVO
Numbers of
Chromosomes
Reference Test system
Abrahamson Drosophila
and Valencia, sex-linked
1980 recessive
lethal test
SL.
S;
NJ
Strain Chemical Route
FM6 EMS AFa
females, Tris-BP AF
Canton Neg. controls AF or AIb
S males DCM AF
AI
tested
773
2,442
94,491
14,682
8,262
Numbers
of
Lethal s
44
35
230
34
18
Corrected
(%)lethals
5.69
1.43
0.233
0.204
0.157
Comments
1. No exposure
chambers designed
to prevent evap-
oration of the
compound in feeding
experiments.
2. No concurrent
negative controls
reported for the
injection experi-
ment.
3. Negative response
at dose tested (224
mM).
Gocke et Drosophila Base
al., 1981 sex-linked females, DCM (mM)
recessive Berlin- 0
lethal test K males
125
620
Lethal s/B rood
1
19/7130
(0.27)
16/3632
(0.44)
8/1213
(0.66)
2
8/5525
(0.14)
2/2579
(0.08)
3/735
(0.41)
3
13/3416
(0.38)
6/1310
(0.46)
5/1005
(0.50)
Total
treated
40/16071 (0.25)
24/7521 (0.32)
16/2953 (0.54)
X^ = 3.17
P < 0.05
1. Positive response
for Brood 1 indicating
DCM is mutagenic in
sperm of Drosophila.
2. Higher dose used
than in test by
Abrahamson and
Valencia (1980).
aAdult feeding
t>Adult injection
(continued on the following page)
-------
TABLE 4-5. (continued)
Reference Test system
Concentration
(mol/L)
Mutation frequency
(Lethal mutations/lf)5 loci)
Toxicity
(Survival re!
to controls)
Comments
i
4>
00
Samoiloff Panagrelus
et al . , redivivus
1980 sex-linked
recessive
DCM
0
10-8
10-6
10-4
12
Juveniles
2.2
6.0 1.02
10.1 1.02
9.8 1.00
L2-L3
Molt
0.99
1.00
1.00
L3-L4
Molt
0.97
0.86
0.88
L4
Adult
Molt
0.46
0.15
0.17
1. Equivocal
response
2. No dose-
related effects.
3. Some positive
controls gave
negative (e.g.,
EMS) or only
marginally posi-
tive (e.g., 3-
methylchol-
anthrene) response
4. Test system
not validated.
-------
parents were assessed for recessive lethal mutations. No evidence of mutagenicity
was observed by this route of administration. Flies injected with 0.2% DCM
had 0.157% lethals compared to 0.206% for controls.
Gocke et al. (1981) also tested DCM (Merck, Darmstadt, FRG, purity not
given) for its ability to induce sex-linked recessive lethal mutations in
Drosophila. Two solutions, 125 mM and 625 mM in 2% DMSO and 5% saccharose,
were fed to wild-type Berlin-K male flies for an unreported period of time.
The highest dose (620 mM) is reportedly close to the LDsg. The males were
then mated to Base females and, three broods were scored (i.e., offspring from
virgin females mated to treated males on days 1 through 3, 4 through 6, and
7 through 10 after exposure). There was a dose-related increase in lethals.
Results shown in Table 4-5 indicate that DCM is not only positive but appears
to induce a dose-related increase (see data from brood I), providing strong
evidence that the compound is mutagenic in germ cells of an in vivo multicellular
eucaryote test system. The discrepancy between the results of Abrahamson and
Valencia (1980) and Gocke et al. (1981) are likely due to the fact that larger
doses of DCM were employed by Gocke et al. (1981).
4.3.1.4. Nematodes--In another sex-linked recessive lethal test, Samoiloff et
al. (1980) tested DCM for its ability to induce mutations in the nematode
Panagrellus redivivus (Table 4-5). Individual females homozygous for the
X-linked mutation b? (coiled phenotype in liquid medium) were grown for 120 h
in the presence of several concentrations of DCM ranging from 10~8 to 10"^^.
They were then washed and mated to S-15 males that carry an X-chromosome inversion
extending at least 15 recombination units to either side of b7. One hundred
female progeny were collected and mated to wild-type (C-15) males, and their
progeny were scored for the presence of the b7 phenotype. The absence of b7
4-49
-------
male progeny indicates lethality of the X-chrornosome marked with 57 derived
from a female grown on DCM, Three replicate experiments were performed. A
non-dose-related increase in the level of lethals was observed in the progeny
of DCM-treated worms compared with the negative controls. For worms treated
with 10-8, 10-6, and 10-4M 0CM, the corresponding lethal mutations/105 loci
were 6,0, 10.1, and 9.8, respectively, compared with an estimated spontaneous
mutation frequency of 2.2 x 10~6 mutations per locus. Some of the positive
controls tested concurrently, such as proflavine, yielded a positive response
(12.5, 10.0, and 28.6 lethals/105 loci at lO"8, ID"6, and 10~4M, respectively);
but others, such as aflatoxin B and ethyl methanesulfonate (EMS), did not
cause an increase in lethal mutations. The investigators suggest that DCM is
mutagenic in nematodes, but firm conclusions cannot be made because the assay
is not validated and because negative responses were obtained with some of the
positive controls, especially EMS.
4.3.1.5. Mamma1ian eel 1s in cu1 _ture-~Jongen et al. (1981) tested DCM for its
mutagenic potential in several tests in cultured mammalian cells. Tests for
the induction of forward mutations at the HGPRT locus are described here
(Table 4-6), whereas tests for the ability of DCM to cause sister chromatid
exchange (SCE), unscheduled DNA synthesis (UDS), and inhibition of DNA synthesis
(IDS) are discussed in section 4.3.3. on other studies indicative of DNA damage.
To determine whether DCM induces forward mutations in cultured Chinese
hamster cells, Jongen et al. (1981) incubated log phase CHO up to 5% DCM and
V79 cells up to 4% DCM at 37°C for 1 hour in a closed glass container without
S9 mix. Analytical grade DCM was obtained from Merck. The cells were exposed
to gaseous DCM and then DCM in solution for 15-minutes intervals each by alternately
tilting the plates and then placing them horizontally. After growth to allow
4-50
-------
TABLE 4-6. GENE MUTATIONS IN MAMMALIAN CELLS IN CULTURE
V79 CHO
Reference
Jongen et
al., 1981
Test system
6-Thioguanine
resistance in
V79 and CHO
cells
Concentration
DCM 0
1
2
3
4
5
Mutants/10D
survivors
2
1.8
2
1.7
1.6
Survival
(I)
100
98
95
85
80
Mutants/100
survivors
1.9
1.8
1.2
0.9
2.1
2.5
Survival
100
90
85
- 80
73
76
Comments
1. Equivocal negative
reponse.
2. Highest dose resulted
in 20-25% decrease in
survival. Higher doses
should be tested.
-------
for an 8-day (CHO cells) or 6-day (V79 cells) expression period, mutant cells
were selected in thioguanine-containing medium. DCM failed to increase the
mutation frequency of either cell line at any dose. The positive control, EMS,
yielded a dose-dependent Increase in mutation induction in V79 cells, but was
not tested in CHO cells. DCM was not very cytotoxic to either cell line; at
the highest dose, survival decreased by only 20-25%. It would be appropriate
to repeat the experiment using higher doses of DCM.
Based on the positive responses in bacteria, yeast, and Drosophila,
and the suggested positive in the nematode Panagrellus, DCM is capable of
inducing gene mutations. Metabolic activation to highly reactive
mutagenic metabolites apparently accounts for this response; and although
these are thought to be short-lived unstable intermediates, they appear
capable of interacting with the genetic material of both procaryotes and
eucaryotes.
4.3,2. Cytogenetic Studies
Three studies on the ability of DCM to cause chromosomal aberrations were
evaluated. Burek et al. (1984) subjected four groups of 10 Sprague-Dawley
albino rats (Spartan substrain, SPF-derived, 5 males and 5 females) to 0, 500,
1,500, or 3,500 ppm DCM by inhalation 6 hours per day, 5 days/week, for 6 months.
The animals were then sacrificed, bone marrow cells collected, chromosome
preparations made, and slides coded and analyzed. Two hundred metaphases per
animal were scored and aberrations were tabulated (Table 4-7). No increase in
the total frequency of abnormal cells or in the frequency of any specific type
of aberration was reported in the treated compared with the control animals.
There were 1.1 _+ 1.3, 0.6 +_ 0.7, 0.8 ± 1.2, and 1.1 +_ 0.9% cells with chromosome
aberrations in animals treated with 0, 500, 1,500, and 3,500 ppm DCM, respectively.
4-52
-------
TABLE 4-7. TESTS FOR CHROMOSOMAL ABERRATIONS
Reference
Burek et
al., 1984
Reference
T hi lager
and
Kumaroo,
1983
aRCG = Rel
Reference
Gocke et
al., 1981
Strain/tissue
Male and female
Sprague-Dawley
rat/bone marrow
test system
Cultured CHO DCM
cells
ative cell growth.
Strain/tissue
Male and female
NMRI mice/bone
marrow
Route of Dose Breaks
exposure (ppm) Chromatid Chromosome Dicentrics Rings Exchanges
Inhalation 0 0.9 + 0.99 0.2 + 0.42
500 0.5 + 0.71 0.2 + 0.42
1500 0.5 + 0.97 0.1 + 0.32
3500 0.7 +_ 0.48 0.2 ^ 0.42
Breaks
Dose RCGa Chromatid Isochromatid Exchange
(ul/ml)
0 100 2 0 0
2 98.4 4 02
5 75.3 8 14 8
10 66.7 12 34 10
Route of Dose
exposure (ppm)
i.p. No. injection x mg/kg Micronuleated
Injection
0
2 x 425
2 x 850
2 x 1700
000
000
0 0 0.1 -(• 0.32
0 0.2^0.42 0
Number of % Aberrant
aberrations/cell cells
0.02 2
0.06 6
0.34 26
0.56 38
polychromatic erythrocytes (%)
0.19
0.19
0.35
0.28
Comments
1. 5 animals/
sex/dose.
2. 200 cells/
animal .
3. Dose to
bone marrow
cells may have
been low.
4. Negative
response.
Comments
1. Positive
response.
2. Four
experiments
yielded
similar
response.
Comments
1. Suggestive
of positive re-
sponse; authors
interpret as
negative res-
sponse.
2. Dose to
bone marrow
cells may have
been low.
-------
Thilagar and Kumaroo (1983) treated CHO cells grown in either plastic or
glass culture flasks with 0, 2, 5, 10, and in one experiment 15 ill/ml (i.e., 0,
31, 78, 156, and 234 mM) DCM for 2 hours with or 12 hours without S9 mix derived
from Aroclor-induced rat livers. DCM was obtained from Fisher Scientific
{certified A.C.S., lot no. 713580). After the exposure period, the cells were
washed, placed in fresh media and allowed to grow before being arrested at
metaphase with colcemid and harvested for chromosome preparation. Slides were
coded and read "blind"; 100 cells were scored for each dose level (50 cells per
duplicate flask). DCM induced a dose-related increase in chromosome aberrations
(Table 4-7) ranging from 0.02 aberrations per cell in the negative controls to
1.44 aberrations/cell at 15 ul/ml (234 mM). The response was not dependent on
the presence of the exogenous metabolic activation system.
Gocke et al. (1981) assessed the ability of DCM (Merck, Darmstadt; purity
not given) to cause micronuclei in polychromatic erythrocytes (PCE). Two male
and two female NMRI mice were used for each of three dose levels (425, 850, and
1,700 mg/kg per intraperitoneal injection). The highest dose approximated the 1059
for mice. Intraperitoneal injections of each dose were given at 0 and 24 hours,
the animals were sacrificed at 30 hours, bone marrow smears were made, and
1,000 PCEs per animal were scored for the presence of micronuclei. An increase
in PCEs with micronuclei was observed at the two highest doses, but the response
was not dose-related and was not double the control value. There were 0.19%
micronuclei in the untreated controls compared with 0.35% micronuclei in the
animals receiving two Injections of 850 mg/kg, and 0.28% micronuclei at the
highest dose. The results are therefore suggestive of a positive response,
but are not conclusive.
Based on the positive response reported by Thilagar and Kumaroo (1983), DCM
4-54
-------
is tentatively judged to be capable of causing chromosomal aberrations. The
negative responses reported by Burek et al. (1984) and Gocke et al. (1981) are
not inconsistent with these results. Thilagar and Kumaroo (1983) exposed
mammalian cells in culture to DCM. The studies by Burek et al. (1984) and
Gocke et al. (1981) where exposure occurred in vivo are not comparable to the
in vitro studies.
4.3.3. Other Studies Indicative of DNA Damage
4.3.3.1. Sister ChromatidExchange (SCE)—Two papers and one abstract have
been published on the ability of DCM to induce SCEs (Table 4-8). Jongen et
al. (1981) tested the ability of 0.5, 1.0, 2.0, 3.0, and 4% DCM (i.e., 58,
118, 235, 353, and 471 mM) to induce SCEs in V79 cells. Log phase cells
were incubated at 37°C for 1 hour in a closed glass container. The cells were
exposed to DCM in the gaseous phase and in the medium by tilting the plates
for 15 min and then placing them horizontally. The experiment was conducted
7 times and each yielded a dose-related increase in SCEs per cell, which approached
but did not exceed a twofold increase above the control level. An analysis of
variance of effects of different doses within experiments showed the increases in
frequency of SCEs to be statistically significant (P < 0.001). Increasing the
exposure time to 1 hours or 4 hours or using S9 from rat liver did not alter
the shape of the dose-response curve, which reached a plateau at 1% DCM.
Jongen et al. suggest that this phenomenon is due to a saturation of the
metabolic activation system of V79 cells.
Thilagar and Kumaroo (1983) exposed CHO cells to 0, 2, 5, 10, and in one
experiment 15 ul DCM/ml of medium (0, 31, 78, 156, and 234 mM DCM) for 2 hours
with and 24 hours without metabolic activation. The cells were grown for 24
hours in BrdUrd followed by a mitotic shake-off, fixing, and staining by a
4-55
-------
TABLE 4-8. TESTS FOR SISTER-CHROMATID EXCHANGE
Reference Test system Dose
Jongen et V79 cells %
al.t 1981 In culture DCM
0
0.5
1.0
2.0
3.0
4.0
Results
SCE/cell
experiment #
1 7
0.26 +_ 0.02
0.40 ^0.02
0.46 +_ 0.02
0.45 +_ 0.03
—
0.51 +_ 0.03
0.03 +_ 0.03
—
0.47 ±0.02
0.51 +_ 0.03
0.58 +_ 0.03
0.61 +_ 0.03
Comments
1. Exposure time 1 h.
2. Positive response.
Significant increases in
SCEs (P < 0.001).
3. Same type of dose
response observed in
. experiments 2-6 (data not
shown).
4. DMSO did not increase
the frequency of SCE.
PI . II »,!-. ••.- — .— ,1-i • |1 .1 1. . 1 _"'•"'•. '" •.--'• ~ •— -•
(continued on the following page)
-------
TABLE 4-8. (continued)
Reference Test system
Thilagar CHO
and cells
Kumaroo,
1983
SCE/Cell
Dose (X +_ SD) Range of SCEs MI MI + M2 Comments
DCM (ul/ml)
0 10.28+ 3.17 5-17 0 3 97 1. Marginal, but not
significant increases
2 11. 36 Jh 3. 09 3-19 0 16 84 in SCEs.
2. Three other experi-
5 12.56 + 2.95 7-18 6 54 40 ments yielded similar
responses.
10 12.36 + 3.35 7-21 4 56 40 3. Results not incon-
sistent with test by
Jongen et al. (1981)
where highest dose was
three times greater
(i.e., 471 mM vs.
156 mM).
-------
fluorescence-pius-Siemsa technique. The coded slides were scored "blind."
Slight dose-related elevations in SCE values were noted (Table 4-8), but they
never exceeded a 50% increase at the highest dose. Thilagar and Kumaroo
judged their test to be negative.
McCarroll et al. (1983) reported 1n an abstract that consistent and dose-
related increases were observed in SCEs in CHO cells following 24-hour exposures
to 1, 3,6, 5.4, and 1% atmospheres of DCM. A 7% atmosphere was required to
elicit a statistically significant increase. Based on the reports of Jongen et
al. (1981), Thilagar and Kumaroo (1983), and McCarroll et al. (1983), DCM is
capable of causing DNA damage that results in SCE.
4.3.3.2. DNA repair assays—Jongen et al. (1981) measured DOS and inhibition
of DNA synthesis (IDS) in V79 cells and primary human fibroblasts (AH cells).
These experiments were conducted by exposing 10^ cells attached to glass
covers!ips (UDS assay) or to glass petri plates (IDS assay) to 0.5, 1.0, 2.0,
3.0, and 5.0% DCM (58, 118, 235, 353, and 471 mM, respectively) without metabolic
activation. UDS experiments were done in duplicate, and at least 25 nuclei of
non-S phase cells were scored for the number of silver grains per nucleus at
each dose level, DCM had no detectable effect on UDS in either cell line. In
the IDS assays, the relative rate of DNA synthesis was determined radioisotopically
immediately after DCM exposure and 0.5, 1.5, and 3.5 hours later. The average of
duplicate samples revealed that DCM inhibited DNA synthesis in V79 and AH cells
at all dose levels compared with controls but that synthesis recovered with
time after exposure in all cases. This is unlike the persistent inhibition of
DNA synthesis by the positive control 4-nitroquinoline-l-oxide. The investigators
conclude that DCM was not inducing genetic damage in cells but was inhibiting
DNA synthesis by an effect on cell metabolism.
4-58
-------
Perocco and Prodi (1981) also performed a UDS assay using DCM. Blood
samples were collected from healthy individuals, the lymphocytes were separated,
and cultured 5 times 10& cells in 0.2-ml medium for 4 hours at 37°C in the
presence or absence of DCM (Carlo Erba, Milan, Italy or Merck-Schuchardt,
Darmstadt, FRG, 97 to 99% pure). The tests were conducted with and without
PCB-induced rat liver S9 mix. No difference was noted between the treated and
control groups of cells with respect to scheduled DNA synthesis measured as dpm
of [3H] deoxythymidylic acid (TdR) after 4 hours of culture (2,661 +_ 57 dpm in
untreated cells compared with 2,356 +_ 111 dpm in cells treated with 5 ul/ml [78 mM]
DCM). Subsequently, 2.5, 5, and 10 ul/ml (39, 78, and 156 mM) DCM was added
to cells cultured in 10 mM hydroxyurea to suppress scheduled DNA synthesis. The
amount of unscheduled DNA synthesis was estimated by measuring dpm from incorporated
[3H]TdR 4 hours later. At 10 ul/ml DCM, 532 _+ 31 and 537 _+ 39 dpm were counted
without and with exogenous metabolic activation, respectively. Both values
were lower than the corresponding negative control values of 715 _+ 24 and 612
_+_ 26 dpm, respectively. No positive controls were run to ensure that the
system was working properly, although tests of chloromethyl methyl ether (CMME)
with activation resulted in a doubling of dpms over the corresponding negative
control values (1,320 ^ 57 at 5 ul/ml CMME vs. 612 +_ 26 untreated). The
investigators calculated an effective DNA repair value (r) for each chemical
based on the control and experimental values with and without metabolic activation.
Perocco and Prodi (1981) evaluated DCM as negative in the test, but they did
not state their criteria for classifying a chemical as positive. None of the
experimental values from cells treated with DCM had higher dpm values than the
controls.
Based on these experiments there is no evidence that DCM specifically
4-59
-------
inhibits DNA synthesis or causes UDS, Certain kinds of DNA alterations that
lead to mutation either do not stimulate repair processes at all or do so to
such a small extent that detection is not practical (Larsen et al., 1982),
4.3.4. Summary and Conclusions
Dichloromethane has been tested for its ability to cause gene mutations
(in Salmonella, yeast, Drosophila, Panagrellus, and cultured mammalian cells),
chromosomal aberrations (in rats, mice, and cultured mammalian cells), and
other indicators of DNA damage in cultured cells (sister chromatid exchange,
unscheduled DNA synthesis, and inhibition of DNA synthesis).
Commercially available samples of DCM have been shown to be mutagenic in a
wide range of organisms, including bacteria (Salmonella), fungi (Saccharomyces),
and insects (Drosophila). The responses were weak under the treatment
conditions used and were obtained without the addition of exogenous metabolic
activation systems (e.g., S9 mix). The data suggest that DCM is metabolized
in vivo in various organisms to form mutagenic metabolite(s). Some negative
results have been reported in tests for mitotic recombination in fungi
(Saccharomyces) and gene mutations in cultured mammalian cells, but
these may be false negatives because of the treatment conditions used. DCM
has also been reported to induce chromosomal aberrations in cultured mammalian
cells but not in bone marrow cells of animals exposed in vivo, perhaps because
a sufficient dose of DCM did not reach the bone marrow. No tests have been
conducted to assess the ability of DCM to cause chromosome nondisjunction,
DCM causes a weak increase in SCEs, but has not been shown to cause UDS or to
inhibit DNA synthesis.
Mutagenicity tests of DCM have given positive responses in four different
organisms based on the weight of available evidence. DCM is judged to be a
4-60
-------
mutagen with the potential of inducing gene mutations in exposed human cells.
A positive response in cultured mammalian cells indicates that it also causes
chromosomal aberrations, but additional testing in another in vivo or in vitro
chromosomal aberration assay is needed to confirm the available data. If such
tests are conducted, care should be taken to ensure that the test cells are
exposed to sufficiently high doses of DCM, otherwise false negative responses
may be obtained. The magnitude of the mutagenic responses obtained with DCM
is much less than those obtained for similar endpoints after treatment with
ethylene dibromide (EDB).
4-61
-------
4,4. ETHYLENE DICHLORIDE (1,2-DICHLOROETHANE)
Ethylene dichloride (EDC) has been tested for mutagenic activity in bac-
teria, plants, Drosophila, mammalian cells in vitro, and intact rodents. These
studies are discussed below and are summarized in Tables 4-9 to 4-16. The
reader may also refer to published reviews of the mutagenic potential of EDC
(e.g., Fishbein, 1976, 1979; Fabricant and Chalmers, 1980; Rannug, 1980;
Simmon, 1980).
4.4.1. Gene Mutation Studjes
4.4.1.1. Bacteria and Fungi —Many investigators have studied the ability of
EDC to produce gene mutations in bacteria (Table 4-9). Most of them reported -
marginal positive responses without metabolic activation and stronger positive
responses with exogenous hepatic metabolic activation, indicating that EDC is
weakly mutagenic but that metabolites, such as S-(2-chloroethyl)-L-cysteine,
are more potent mutagens.
Ethylene dichloride has been reported positive in four Salmonella/microsome
plate incorporation assays (McCann et al.» 1975; Rannug, 1976; Rannug and Ramel,
1977; Rannug et al., 1978), in assays testing the mutagenicity of bile
obtained from EDC-perfused rat livers or livers from EDC-treated mice (Rannug
and Beije, 1979), and in two Salmonella spot tests. In one of the Salmonella spot
tests (Brem et al., 1974), duplicate experiments carried out at different
times revealed a reproducible twofold increase in revertant counts for strains
TA1530 and TA1535 (mean values of 50 and 54 revertants on treated plates vs. 23
and 26 in control plates for TA1530 and TA1535, respectively). No difference
in revertant counts was noted in strain TA1538. This response is consistent
with that expected for an alkylating agent. Brem et al. stated that plate
incorporation tests could not be performed because of the volatility of the
4-62
-------
TABLE 4-9. SUMMARY OF MUTAGENICITY TESTING OF EDC: GENE MUTATIONS IN BACTERIA AND FUNGI
Test Activation
Reference system Strain system
Brem et al., Salmonella TA1530 None
1974 typhimurium TA1535
(spot test) TA1538
Chemical
information
10 umol on filter
disk
Source: Not given
Purity: Not given
Results Comments
Weak positive 1. Could not perform
plate incorporation
tests because of
volatility.
Salmonella revertants
TA1530
EDC 50
Water 23
Chloramphenicol 20
TA1535 TA1538
54 19
26 19
31 14
(continued on the following page)"
-------
TABLE 4-9. (continued)
Test
Reference system Strains
Principe et Salmonella/
al., 1981 microsome
assay (spot TA1535
test) TA1537
TA1538
TA98
TA100
Streptomyces
coelicolor
forward mutation
assay to Strr
Aspergiellus
nidulans
forward mutation
to 8-AGf
Dose ul/pl
-S9
0 100
39 46
17 8
19 12
82 83
188 188
Dose ul/plate
0
2
10
20
100
0
250
500
ate
+S9
0
33
10
21
77
171
Survival %
100
100
100
100
100
100
100
42
Comments
100 1. Positive controls indi-
103d cated system working properly
8 2. Positive results in
27 Salmonella in spot test with
84 - TA1535.
169 3. No precautions taken to
prevent excessive evaporation
of EDC and ensure adequate
exposure.
4. Toxicity results indicate
exposure was minimal.
Strr/p1ate
2.5 + 0.6
0.2 + 0.2
0.7 + 0.4
0.7 + 0.4
1.0 +_ 0.8
8-AG/plate
2.5 + 0.9
2.0 + 1.1
1.0^0.7
ap < 0.01.
(continued on the following page)
-------
TABLE 4-9. (continued)
Test
Reference system
Activation
Strain system
Chemical
information Results
Comments
McCann Salmonella/ TA100
et al., microsome
1975 assay (plate
test)
PCB-induced
rat liver/59
mix
I
Ov
Ul
Concentration tested:
1.3 X 104 ug/plate
(13 umol)
Source:
Purity:
Aldrich
Chemical Co.
Not given,
but stated
to be high-
est purity
Negative or at
best marginal
positive response.
Induced 25 colonies/
plate above back-
ground. (0.19
revertants/umol
in TA100)
1. Nonmutagenic or
extremely weak mutagen in
this study. Reproducible
dose-response curves not
obtained.
2. Metabolic activation
did not increase positive
response.
3. Chloroethanol and
chloroacetaldehyde (two
putative intermediates in
the metabolism of EDC in
mammals) were positive
(0.06 and 746
revertants/umol,
respectively).
(continuea on the following page)
-------
TABLE 4-9. (continued)
Reference
Test
system
Strain
Activation
system
Chemical
information
Results
Comments
Rannug,
1976
Salmonella/
microsome
assay
(plate test)
TA1535 Liver fractions
from Sprague-
Dawley or R strain
Wistar rats in-
duced with pheno-
barbital with and
without NADPH-
generating system
and with and with-
out glutathione S-
transferases A, B,
and C
Concentration tested:
Up to 60 umol/plate
Source:
Purity:
BDH Chem-
icals, Ltd.
Not given
but reported
to be checked
by glass cap-
illary column
chromatogra-
phy using a
flame
ionization
detector
Marginally
positive without
activation (two-
fold increases);
positive response
with activation
(ten-fold
increases). Spon-
"taneous background
8-14 revertants/
plate.
1. EDC activated by
the liver cytosol
fraction; mixed-func-
tion oxygenases not
involved.
2. NADPH-independent
GSH S-transferase de-
pendent activation.
3. Strain differences
noted in ability to
metabolize EDC.
4. Thought that muta-
genicity of EDC after
activation caused by
formation of highly
reactive half sulfur
mustard, S-(2-chloro-
ethyl)-L-cysteine.
(continued on the following page)
-------
TABLE 4-9. (continued)
Test Activation Chemical
Reference system Strain system information Results
Rannug and Salmonella/ TA1535 S9 mix from Concentration tested: Positive response
Ramel , 1977 microsome livers of un- Up to 45 umol/plate (two-fold increase)
assay (plate induced male R
test) strain Wistar Source:
rats plus NADPH-
generating system
Purity:
without activation;
BDH Chemicals, nearly ten-fold
Ltd. increase with
activation.
Not given Negative controls
yielded roughly 15
revertants/plate.
Comments
1. Compared
mutagenicity of
EDC tar with
EDC. The level
of EDC present
at the highest
dose tested for
EDC tar would
only exert a
weak mutagenic
effect, yet a
strong response
was observed.
2. Activation
of EDC tar de-
pendent on
NADPH. EDC
activation in-
dependent cf
NADPH.
(continued on the following page)
-------
TABLE 4-9. (continued)
Reference
Rannug and
Ramel, 1977
f
;*%
*%.
Test
system Strain
Salmonella/ TA1535
microsome
assay (plate
test)
Activation
system
S9 mix from
livers of un-
induced male R
strain Wistar
rats plus NADPH-
generating system
Chemical
information
Concentration tested:
Up to 45 umol/plate
Source: BDH Chemicals,
Ltd.
Purity: Not given
Results
Positive response
(twofold increase)
without activation;
nearly ten-fold
increase with
activation.
Negative controls
yielded roughly 15
revertants/plate.
Comments
1. Compared
mutagenicity of
EDC tar with
EDC. The level
of EDC present
at the highest
dose tested for
EDC tar would
only exert a
weak mutagenic
effect, yet a
strong response
was observed.
2. Activation
of EDC tar de-
pendent on
NADPH. EDC
activation in-
dependent of
NADPH.
(continued on the following page)
-------
TABLE 4-9. (continued)
Reference
Rannug and
Beije, 1979
k
~X>
Test
system Strains
Salmonella/ TA1530
Body fluid TA1535
analysis
(isolated
perfused
rat liver)
Salmonella/ TA1535
Body fluid
analysis
(bile)
Activation
system
Isolated per-
fused liver from
male R strain
Wistar rats
Bile from male
CBA mice
Chemical
information
Concentration tested:
0.1 ml (1.3 mM)
for up to 4 hours.
80 mg/kg EDC i.p.;
removal of liver and
collection of bile 30
and 60 minutes later.
Results
Positive. Highest
response 15-60
min after
addition of EDC
(45-60 revertants
compared to 7-10
in controls).
Positive. Greater
than two-fold
increases with bile
from liver removed
30 min after
addition of EDC
Comments
1. Positive
responses con-
sistent with
conjugation of
EDC with
glutathione.
(28.8 + 2.7 revertants
compared to 11.3 _+ 1
.1).
(continued on the following page)
-------
TABLE 4-9. (continued)
Reference
Test
system
Activation
Strains system
Chemical
information
Results Comments
King et al.,
1979
i
-~j
o
Salmonella/
microsome
assay (plate
test)
IE. coli K 12
strain 343/113
(suspension
test and intra-
sanguineous
host-mediated
assay)
TA1535
TA100
TA1537
TA1538
TA98
PCB-induced
rat liver S9
Concentration tested: Negative
36 umol/plate
10 mM (suspension assay) Negative
2 mM/kg i.p. injection Negative
female NMRI mice
Source: Merck Co.
Darmstadt, FR6
Purity: Not given.
Stated that
samples had
correct melting
point and ele-
mental analysis
1. Standard plate
incorporation test
was conducted. No
precautions were
taken to prevent
evaporation of
EDC.
(continued on the following page)
-------
TABLE 4-9. (continued)
Reference
Nestmann et
al., 1980
V.
~*F
•i?
Test
system
Salmonella/
microsome
assay (plate
test and
desiccator
exposure)
Strains
TA1535
TA100
TA1537
TA1538
TA98
Activation
system
PCB-induced
rat liver S9
mix
Chemical
information
Concentration tested:
up to 9 mg/plate (91
umol) in desiccators.
10 mg/plate in plate
tests.
Source: Chem Service
Purity: Not given
Results
Negative in
standard test.
Positive in
desiccator test
in strain
TA1535.
Comments
1. Stated that
maximum yield with
TA100 is 20 rever-
tants above back-
ground (i.e., nega-
tive). For TA1535
a doubling of
mutant colonies per
plate observed. No
other data
presented.
Solvent: DMSO
2. Cannot ade-
quately evaluate
results.
(continued on the following page)
-------
TABLE 4-9. (continued)
Reference
Stolzenberg
and Hine,
1980
Test
system Strains
Salmonella/ TA100
microsome
assay (plate
test)
Activation
system
PCB-induced
rat liver 59
mix (2 mg
protein/0.5
ml)
Chemical
information Results
Concentration tested: Negative
Up to 10 umol/plate
Source: Aldrich
Chemical Co.
Purity: 99% pure
Comments
1. All compounds
tested in triplicate
with and without
S9 mix.
2. No precautions
taken to prevent
evaporation of
test material .
2. Experimental
values minus back-
ground revertants.
umol/plate
Revertants
•S9 +S9
10-1
1
10
0
0
15
0
0
No growth
(continued on the ToTlowing page)
-------
TABLE 4-9. (continued)
Reference
Barber et
al., 1981
Test
system
Salmonel la/
microsome
assay (vapor
exposure)
Strains
TA1535
TA100
TA1538
TA98
Activation
system
PCB-induced
rat liver S9
mix
Chemical
information
Concentration tested:
Up to 231.8 umol/plate
as determined by 6LC
analysis of distilled
water samples.
Source: Eastman Kodak
Purity: 99.98%
Results
Negative in
standard plate
test in all
strains. Posi-
tive in desicca-
tor test in
strains TA1535
and TA100.
Comments
1. Bacteria exposed
in gastight ex-
posure chambers.
2. Plastic plates
found to absorb
dibromomethane in
parallel experi-
ments. Thus, glass
plates used for all
other testing.
.£=>
CO
3. Weak positive
result. 0.002
revertants/nmol in
T/U.535 with or with-
out activation.
0.001 revertants/
nmol TA100 with or
without activation.
4. Revertants sel-
ected from each ex-
periment and tested
to ensure that they
were actually his+.
-------
test agent. Positive and negative control tests were conducted for these
experiments and indicated that the systems were working properly. Principe
et al. (1981) conducted a spot test using Salmonella strains TA1535, TA1537,
TA1538, TA98, and TA100. A positive response was observed for TA1535 when a
triangular-shaped paper disc soaked with 100 ul EDC was placed on the agar in
the presence of S9 from Aroclor 1254-induced rat liver (i.e., 103 revertants
on the treated plate vs. 33 revertants for the negative control). Negative
responses were obtained with all the other strains, A negative response was
also obtained in plate incorporation tests conducted with strains TA100 and
TA1535 at doses up to 100 ul/plate, but these tests involved no precautions to
prevent excessive evaporation of the EDC. Similarly in forward mutation tests
conducted with St reptomyces coeli col or and Aspergillus nidulans, negative respon-
ses were obtained at doses up to 100 and 500 ul/plate in plate incorporation
and spot tests. There was a 100% survival in the test conducted with S^
coel icol or and a 60% reduction in cell survival at the highest dose with A.
nidulans. These tests provide less than adequate conditions for assessing the
mutagerticity of EDC, because exposure to the test organisms may have been
insufficient.
McCann et al. (1975) exposed Salmonella strains TA1535, TA10Q, and TA98 to
EDC (Aldrich, stated to be highest purity available) concentrations as high as
13 mg/plate (131 umol/plate). A weak positive response was observed in TA100
(0.19 revertants/umol), but reproducible dose-response curves were not obtained.
The presence of an exogenous S9 metabolic activation system (S9 mix from rat
livers induced with either phenobarbital or Aroclor 1254) did not increase the
weak positive response. Chloroacetic acid, which is a known mammalian metabolite
of EDC, and the putative intermediates chloroethanol and chloroacetaldehyde
4-74
-------
were also tested for their mutagenic potential in strain TA100, Chloroacetic
acid was negative, chloroethanol yielded a weak positive result, and
chloroacetaldehyde a strong positive result (0.06 and 746 revertants/umol,
respectively). McCann et al. (1975) The authors speculated that the weak
mutagenic response of EDC may have been due to inefficient conversion of
chloroethanol to chloroacetaldehyde.
Two reports (Rannug, 1976; Rannug and Ramel, 1977) compared the muta-
genicity of EDC with that of EDC tar, a complex mixture formed during the
manufacture of vinyl chloride from acetylene, ethylene, or a mixture of the
two. The mixture is called EDC tar because EOC is a component, comprising
about 301 of the mass. The sample of EDC tested in these studies was from
British Drug House (BDH) Chemicals, Ltd, (purity not given). Salmonella tester
strain TA1535 was treated with concentrations up to 45 umol/plate in the presence
or absence of an exogenous metabolic activation system from livers of male
strain R Wistar rats with and without NADP. Weak positive (almost twofold)
increases in revertant frequencies over background (13.0 +_ 1,76) were observed
in the plates receiving the highest dose (24.8 +_ 3,06) without metabolic activation,
With activation, however, a stronger positive response was observed. The revertant
count was elevated ninefold over the spontaneous level (132.8 +_ 8.35 vs. 15.5 +_
1,21), The response to EDC was independent of the presence of NADP; in contrast,
the mutagenicity of EDC tars was increased with metabolic activation only in
the presence of NADP. The mutagenicity of EDC tar cannot be ascribed primarily
to EDC, because EDC and EDC tar have different requirements for metabolic
activation. In addition, the concentration of EDC present in EDC tar yields
only a weak positive response at the highest dose if tested alone, and a
strong response was observed for the EDC tar. In subsequent studies, Rannug
4-75
-------
et al. (1978) tested Salmonella strain TA1535 with EDO (BDH Chemicals Ltd.)
at doses up to 60 umol/plate. Although the purity of EDC was not reported,
the material was checked by glass capillary chromatography using a flame ionization
detector. Tests were conducted with and without an exogenous rat liver metabolic
activation system. The components of the activation system were varied to
study the mechanism of activation of EDC. Phenobarbital-induced liver fractions
from Sprague-Dawley or R strain Wistar rats were prepared with and without an
NADPH-generating system and with and without glutathione transferases A, B, and
C. Rannug et al. (1978) observed that EDC is activated by the cytosol fraction of
liver homogenates; mixed-function oxygenases are not involved. The activation was
NADPH-independent but required glutathione (GSH) A or C S-transferase enzyme
activity. The extent of activation was dependent on the rat strain used (liver
fractions from R strain rats were more effective than those from Sprague-Dawley
rats) and the handling of the extract (e.g., storage on ice or freezing reduced
the effectiveness of the activation system). The report by McCann et al.
(1975) that EDC was not metabolized to a mutagenic intermediate may be due to
differences in the exogenous activation system used (S9 mix rather than cytosol
fraction). Rannug et al. (1978) hypothesized that the mutagenicity of EDC
after metabolic activation was caused by the formation of a highly reactive
half sulfur mustard, S-(2-chloroethyl)-L-cysteine. This compound (>B9% purity)
was found to be more strongly mutagenic in TA1535 than EDC. At 0.2 umol/plate,
12.8 +_ 1.5 and 176.8 _+ 11.1 revertants were observed per plate after treatment
with EDC and S(2-chloroethyl)-L-cysteine, respectively. At 5.0 umol/plate, the
observed numbers of revertants per plate were 13.0 +_ 1.1 and 1945 (only one
plate tested), respectively. Thus, S-(2-chloroethyl)-L-cysteine gives a strong
direct mutagenic effect in strain TA1535 at low doses at which no effect is
seen for EDC.
4-76
-------
In intact mammals most GSH conjugates are normally excreted in bile.
Rannug and Bieje (1979) reasoned that if EDC were metabolically activated by
conjugation with glutathione to form a half sulfur mustard, mutagenic products
would be found in the bile of EDC-treaied mammals or perfused livers. To
test this hypothesis, an EDC concentration of 0.1 ml (1.3 mmol) was perfused
through R strain Wistar rat livers for up to 4 hours. Bile was collected
immediately after addition of EDC and 0.25, 0.5, 1, 1.5, 1.75, 2, 3, and 4 hours
later. Bile was tested directly or diluted 5 to 10 times in sterile water and
added to top agar containing Salmonella strain TA1535 for plate incorporation
tests. Positive responses were obtained. The greatest response (45-60 revertants
per plate, compared with 7-10 in negative controls) was reached between 15 minutes
and 1 hour after addition of EDC. In a second experiment, 80 mg/kg EDC was given
to male CBA mice intraperitoneally. The animals were sacrificed, their livers
removed, and bile collected 30 and 60 minutes later for mutagenicity testing with
strain TA1535 in plate incorporation tests. An increase in revertants (greater
than twofold) was observed for bile collected 30 minutes after treatment compared
with bile from negative control animals (28.8 +_ 2.7 revertants and 11.3 +_ 1.1
revertants, respectively). The positive responses obtained with bile from the
perfused rat livers and intact mouse livers are consistent with the hypothesis
that EDC is activated by conjugation with glutathione.
Four studies have been reported in which EDC was found to be negative in
the standard Salmonella/microsome assay plate incorporation test (King et al.,
1979; Nestmann et al., 1980; Stolzenberg and Hine, 1980; Barber et al., 1981).
The maximum doses employed in the first three studies were 36 umol/plate, 91
umol/plate, and 10 umol/plate, respectively. The doses in these studies are
in the range in which positive responses have been reported in other studies.
4-77
-------
The fourth study did not report the doses used. Each test was conducted with
and without PCB-induced rat liver S9 mix. Except for the report by Stolzenberg
and Hine (1980), the negative Salmonella plate incorporation studies were
conducted with appropriate positive controls. The positive controls that
require activation, however, were not structurally similar to EDC. These
positive controls may not be able to determine the effectiveness of the components
in the S9 mix necessary for EDC activation. King et al. (1979) also reported
negative responses when E_. coli K12 strain 343/113 was tested in either a
liquid suspension test or an intrasanguineous host-mediated assay.
Two of the studies (Nestmann et al., 1980; Barber et al., 1981) reported
that EDC did not induce mutations in standard plate incorporation tests, but did
cause positive responses when the studies were conducted in airtight exposure
chambers. Nestmann et al. (1980) exposed Salmonella strains TA1535 and TA100
to doses from 3 to 9 mg/plate (30 to 91 umol/plate) in desiccators. It was
reported that this treatment yielded positive results, at least for TA1535, in
which there was a doubling in the number of mutant colonies over the control.
It was stated that concentrations were tested up to levels where cell killing
was observed, but no data are given and insufficient detail is provided to
allow the results to be independently evaluated.
Barber et al. (1981) exposed Salmonella tester strains TA1535, TA98, and
TA100 to four levels of EDC vapors (Eastman Kodak Co., 99.98% pure) in a 3.4 L
airtight exposure chamber. These exposures resulted in estimated plate
concentrations ranging from 31.8 to 231.8 umol/piate as determined by gas
liquid chromatography analysis of distilled water samples placed in the exposure
chamber. Linear, dose-related increases in revertant counts were observed for
TA1535 and TA100. The mutagenicity of EDC in these two strains was 0.002 and
4-78
-------
0.001 revertants/nmol, respectively. No difference in revertant counts or
potency of EDC was noted in comparisons of tests done with and without metabolic
activation. The positive results were obtained by Nestmann et al. (1980) and
Barber et al. (1981) only when tests were conducted in airtight exposure
chambers. This suggests that standard mutagenicity testing of EDC (bp 83° - 84°C)
may not provide an adequate assessment of its mutagenic potential due to
excessive evaporation.
In summary, positive responses obtained without metabolic activation
indicate that EDC is a weak direct-acting mutagen in bacteria. Positive
responses obtained with metabolic activation indicate that one or more of its
metabolites are more potent mutagens. The negative findings reported in some
bacterial tests are not considered to contradict the reported positive results,
because the negative results may have been due to evaporation of EDC or to
inadequacy of the S9 activation system.
4.4.1.2. Higher Plants—Two reports on the ability of EDC to induce mutations
in higher plants are summarized in Table 4-10. Ehrenberg et al. (1974) treated
barley seeds (variety Bonus) with 30.3 mmol EDC (Merck, purity not given) for
24 hours and scored for sterile spikelets at maturity or chlorophyll mutations
in about 600 spike progenies in the subsequent generation. The dose corresponds
to the 1050. EDC-treated kernels had an increased incidence of chlorophyll
mutations (6.8%) compared with untreated controls (0.06%).
Kirichek (1974) exposed eight varieties of pea seeds (100 each) to gaseous
EDC (purity and concentration not given) for 4 hours. Germination of treated
seeds differed with the variety tested but was reduced (15-50% germination)
compared with negative control seeds exposed to water vapor for 4 hours (100%
germination). From 5.4 to 28.13% of the seeds were reported to be mutated.
4-79
-------
TABLE 4-10. SUMMARY OF MUTAGENICITY TESTING OF EDC: HIGHER PLANTS
Reference
Test
system
Chemical
information
Results
Comments
Ehrenberg et
1974
al
Segregating
chlorophyll
(gene) mutations
in barley
I
00
o
Kirichek, 1974
Morphological
mutations in peas,
8 varieties
Concentration tested:
200 seeds treated
with 30.3 mmol/24
hours (LD50) in a
closed vessel.
Concentration tested:
EDC (concentration not
reported) or water
(negative controls)
vapors for 4 hours.
Source: Not reported
Purity: Not reported
Positive response.
(6-8% mutants from
treated progeny vs.
0.06% mutants from
control progeny).
Source:
Merck Co.
Darmstadt,
FRG
Purity: Not given
Reported positive.
5.42-28.13% of
seeds reported
to be mutated.
1. About 600 spike progeny
were tested.
1. English translation of
Russian article.
2. Germination of the
treated seeds varied with the
variety tested from 15% to
58% compared to 100% germ-
ination of the control seeds.
3. Control mutation freqency
not given.
4. Putative mutations not
characterized.
5. Not possible to evaluate
results adequately.
-------
Kirlchek (1974} reported that two times as many morphological mutants were
induced as chlorophyll mutations and considered this a positive response, but
the mutation frequency for negative controls was not given. This limitation
of the report plus a lack of information concerning the characterization of
the putative mutations precludes an adequate evaluation of the results.
4.4.1.3. Insects—Four studies on the ability of EDC to cause mutations in
Drosophila me1 a nogaster a re summarized in Table 4-11. Three studies demonstrated
the ability of EDC to cause sex-linked recessive lethal mutations (Shakarnis,
1969; Shakarnis, 1970; King et al., 1979). The fourth study demonstrated the
induction of somatic cell mutations by EDC (Nylander et al., 1979).
Shakarnis (1969, 1970) performed two experiments to assess the ability of
EDC (purity not given) to cause sex-linked recessive lethal mutations in
Drosophila. In the first study (Shakarnis, 1969), adult females from a
radiosensitive strain (Canton-S) were exposed to 0.07% EDC gas for 4 or 8 hours
at 24° - 25°C, Immediately after treatment, the females were mated to Base
males. The fertility of treated females was reduced 47% by the 4 hours treatment
and 91% by the 8-hour treatment. The $2 progeny were scored for lethality
(measured as the absence of Base males). A statistically significant (P < 0.05)
time-related increase in the incidence of sex-linked recessive lethals was
observed. The frequencies of lethals were 0.3% (negative controls), 3.2% (4-
hour treatment) and 5.9% (8 h treatment). In his second study, Shakarnis (1970)
exposed females from a radiostable strain (D-32) of JK melanogaster to 0.074
EDC vapors (source and purity not reported) for 4 or 6 hours. Immediately
after treatment they were mated to Base 5 males and the F£ progeny scored for
sex-linked recessive lethals. The 4-hour treatment did not significantly reduce
fertility, but the 6-hour treatment reduced it by 50%. As in the first study, a
4-81
-------
TABLE 4-11. SUMMARY OF MUTAGENICITY TESTING OF EDC: GENE MUTATION TESTS IN INSECTS
Test Chemical
Reference system information
Shakarnis, 1969 Drosophila Concentration tested:
melanogaster virgin 3-day-old Canton
sex-linked S females exposed to
recessive lethal 0.07% EDC gas for 4 or
test 8 h at 24-25°C.
Source: Not given
Purity: Not given
Duration
^ treatment
* (h) with
J 0.07% EDC
Control
4
8
Results Comments
Positive response 1. Base males.
2. Fertility of treated females
reduced significantly (47% reduc-
tion after 4-h treatment and 91%
after 8-h treatment).
of
Number of Lethal Mutations
Chromosomes Scored No. %
4910 16 0.32
2081 67 3.22
4750 281 5.91
P < 0.05
(continued on the following page)
-------
TABLE 4-11. (continued)
I
00
u>
Test Chemical
Reference system information
Shakarnis, 1970 Drosophila Concentration tested:
melanogaster virgin 3-day-old
sex-linked D-32 strain females
recessive lethal exposed to 0.07% EOC
test gas for 4 or 6 h
at 24-25°C.
Source: Not given
Purity: Not given
Duration
treatment
(h) with
0.07% EDC
Control
4
6
Results Comments
Positive response 1. Base males.
2. Experiment conducted twice
for 4-h exposure and three
times for 6-h exposure.
3. 4-h treatment did not
significantly reduce fertility
but 6-h treatment reduced it
by 50%.
of
Number of Lethal mutations
chromosomes scored No. %
1904 1 0.05
2205 46 2.00
2362 78 3.30
(P < 0.05)
(continued on the following page)
-------
TABLE 4-11. (continued)
Reference
King et al.,
1979
Test
system
Drosophila
melanogaster
Chemical
information
Concentration tested:
50 mM solution of EDC
Results
Positive response
Comments
1. Base females.
sex-linked
recessive lethal
test
5% sucrose fed to
1- to 2-day-old Berlin-K
males for 3 days (near
LD50).
Source: Merck Co.
Darmstadt, FRG
Purity: Not given but
stated to have
correct melting
point and elemental
analysis.
2. Lethal frequency increased
for all broods. Greatest effect
in brood II, which corresponds to
the spermatid stage.
i
00
-Er-
Cone.
(mM)
0
50
Brood
I-III
I
II
III
I-III
Days after
treatment
0-9
0-3
4-6
7-9
0-9
Number of
chromosomes scored
22,048
1,185
1,179
156
2,520
Lethal
No.
47
6
41
2
49
mutations
%
0.21
0.51
3.48
1.28
1.94
P value
<0.01
<0.01
-------
TABLE 4-11. (continued)
Reference
Test
system
Chemical
information Results
Comments
Nylander et al.,
1979
Drosophila
mel anogaster
somatic cell
mutations ^c
z w+ and z
stocks.
Concentration tested:
0.01% and 0.5% EDC in
food at 25°C and 75%
humidity during larval
development
Source:
Fisher
Scientific
Co.
Purity: Not given
Positive response
1. Mutations at z^ locus scored
in flies of stable and unstable
genotype. Both have same pheno-
type. Instability thought to be
caused by transposable genetic
element.
2. Survival of treated flies
reduced significantly in both
genetically stable (34% re-
duction for 0.1% EDC and 77% re-
duction for 0.5% EDC) and
genetically unstable stocks
(86% reduction for 0.5% EDC).
Treatment
Control
Stable
Unstable
0.01%
Stable
Unstable
0.05%
Stable
Unstable
Number
males
scored
4441
5363
6260
2889
610
201
Number
with
sectors
2
4
263
274
44
50
%
0.045
0.075
4.20
9.28
7.21
24.88
t value differences
within genotypes
—
18.743 p < o.OOl
24.31a p < 0.001
11.463 p < 0.001
13.123 p < 0.001
t value differences
between genotypes
0.60
9.16a p < 0
5.66a p < 0
.001
.001
-------
statistically significant (P < 0.05) time-related increase in sex-linked recessive
lethals was observed. A frequency of 0.05% lethals was observed in the negative
controls, and frequencies of 2 and 3.30% lethals were observed in offspring
from the 4-hour and 6-hour treatments, respectively.
King et al. (1979) also studied the ability of EDC to induce sex-linked
recessive lethals in Drosophila. One- to two-day-old Berlin-K males were fed a
50 mM solution of EDC (Merck and Co., purity not given but correct melting
point [sic] and elemental analysis reported) in 5% sucrose for 3 days. This
dose approximated the LD5Q. The males were mated to virgin Base females
every 3 days for a total of 9 days to determine if EDC preferentially damaged
particular stages in spermatogenesis. Progeny of the ¥2 generation were
scored for lethality. The lethal frequency was found to be elevated for all
three broods (0.51%, 3.48%, and 1.28% for broods, I, II, and III, respectively).
compared with the negative controls (0.21%). The highest lethal frequency
(3.48%) was found in brood II (P < 0.01), which corresponds primarily to the
spermatid stage of spermatogenesis.
Nylander et al. (1978, 1979) raised flies on food containing 0.1 and 0.5%
EDC (Fisher Scientific Co., purity not given) during larval development and scored
Fj progeny for the induction of somatic cell sex-linked mutations in the eye.
A genetically unstable stock (_sc.z w+) and a genetically stable stock (2 Dp
w+61el9) both having the same phenotype were used in these studies and mated to
attached X females. Mutations result in the expression of normal (dark red)
eye pigment sectors In emerging adult treated males. The genetic Instability
of S£.z w+ is caused by the insertion of a transposable genetic element.
The survival of flies raised on the EDC-treated food was reduced compared with
the negative control values (e.g., 77% reduction for the stable stock and 86%
4-86
-------
reduction of the unstable stock at 0.5% EDC). Statistically significant increases
in somatic cell mutations were observed in both stocks at both the low and
high doses (P < 0.001).
The above positive responses in Drosophila indicate that EDC is capable of
causing both somatic cell and heritable germinal mutations in a multicellular
eucaryote.
4.4.1.4. Mamma1i an Cells in Cu 11ure--Tan and Hsie (1981) found that EDC (Matheson,
Coleman and Bell, purity not given) causes a dose-related increase in HGPRT
mutations in cultured Chinese hamster ovary (CHO) cells (Table 4-12). CHO-Ki~BH4
cells were exposed in suspension culture for 5 hours to EDC concentrations
ranging up to 3 mM (30% survival) in tests with exogenous rat liver S9 mix and
concentrations ranging up to 50 mM (50% survival) in tests conducted without
metabolic activation. Weak positive responses were observed both in tests
with and without metabolic activation. EDC was detected as a direct-acting
mutagen only at high doses, but the induced mutation frequency was increased
about tenfold over control values. About a fourfold additional increase in
mutagenicity was noted with metabolic activation, but toxicity precluded testing
at concentrations greater than 3 mM. The metabolic activation system was only
effective in the presence of NADP; this result differs from that of Rannug
(1976) who found metabolic activation of EDC to be NADP-independent.
4.4.1.5. Whole Mammals--Gocke et al, (1983) studied the ability of EDC to
cause somatic cell mutations in the mouse spot test (Table 4-13). This test
was a follow-up to an earlier study (King et al., 1979) of EDC in the
Salmonella/microsome assay, Drosophila sex-linked recessive lethal test, and
mouse micronucleus test. One intraperitoneal injection of EDC (from Merck,
Darmstadt, FR6; purity not reported) at 300 mg/kg was administered in olive
4-87
-------
TABLE 4-12. SUMMARY OF MUTAGENICITY TESTING OF EDC: MAMMALIAN CELLS IN CULTURE
Reference
Test
system
Chemical
information Results
Comments
Tan and Hsie,
1981
Chinese hamster
ovary cell H6PRT
gene mutation
assay
Concentration tested:
Up to 3 mM in tests
with rat liver S9 mix
and up to 50 mM in
tests without metabolic
activation. LD^g is 1
mM with activation and
6 mM without activation.
Positive response:
60 vs. 3 mutants/106
clonable cells for
50 and 0 mM EX
without activation;
28 vs. 3 mutants/106
clonable cells for
1.5 and 0 mM EDC
with activation.
1. Mutagenic activity of
EDC without and with metabolic
activation calculated to be
1 and 5 mutants/106 cells/mM
respectively.
2. S9 mix increases muta-
genicity by about fourfold
and cytotoxicity 5- to
25-fold.
3. NADP required in S9 mix
for metabolic activation.
00
00
-------
oil to 525 C57B1 females on the 10th day of pregnancy after mating with T-stock
males. There were 1,104 progeny examined; 49% of the females gave birth and
the average litter size at birth was 4,9 and at weaning was 4.3. Seven of the
offspring had spots indicative of somatic cell mutagenicity (0.6%) compared
with 2/812 in the olive oil controls (0.3%) and 3/2,161 for all the controls
(0.1%). Analyses of these data by the Fisher exact test showed a signficant
(P = 0.03) effect against the cumulative control, but no significance (P =
0,18) against the olive oil control. The data suggest a possible weak mutagenic
effect of EDC in the mouse. With the control values reported in this study,
testing more than 10,000 animals would be necessary to secure a doubling of
the spontaneous relevant spot frequency.
The consistency of positive results obtained in higher plants, Drosophila,
and cultured mammalian cells and the suggestive evidence for mutagenicity in
the mouse spot test is sufficient to demonstrate that EDC causes gene mutations
in eucaryotes,
4.4.2. Cytogenetic Studies
Five studies were evaluated on the ability of EDC to cause chromosomal
damage (Table 4-14). One was an abstract on chromosome breakage and C-mitosis
in Allium root tips and cultured human lymphocytes (Kristoffersson, 1974).
Two were D. melanogaster sex chromosome nondisjunction tests (Shakarnis, 1969,
1970), and two were micronucleus tests (King et al., 1979; Jenssen and Ramsel,
1980).
In the abstract by Kristoffersson (1974) EDC (source, purity, and concentration
not given) was reported to produce C-mitosis in Allium root tip cells but not
chromosome breaks in Allium root tip cells or human lymphocytes or to induce
prophage from E. coli K 39 (?•). Insufficient information, however, was available
4-89
-------
TABLE 4-13. SUMMARY OF MUTAGENICITY TESTING OF EDC: MOUSE SPOT TEST
Reference
Test
system
Chemical
information Results
Comments
Gocke et al.,
1983
C57B1 female X
T-stock male
One i.p. injection
of 300 mg/kg EDC in
olive oil 10 days
after conception.
Source:
Merck Co.
Darmstadt,
FRG
Purity: Not given
7/1,104 offspring
had spots compared
with 2/812 in olive
oil controls and 3/2,
161 for 211 the cumula-
tive controls.
Data suggestive of a positive
response.
i
\o
o
-------
TABLE 4-14. SUMMARY OF MUTAGENICITY TESTING OF EDC: "CHROMOSOMAL DAMAGE TESTS"
I
VO
Test Chemical
Reference system information
Shakarnis, 1969 Drosophila Concentration tested:
melanogaster virgin Canton-S females
X chromosome exposed to 0.7% EDC in
nondisjunction 1.5 liter desiccator
for 4 or 8 h at
24-25°C.
Source: Not given
Purity: Not given
0.07% EDC
duration of
treatment
(h)
Control
4
8
Results
Comments
Statistically significant
(P < 0.05) increases in
exceptional female
progeny for 4 h
exposure and male and
female progeny for 8 h
exposure.
Normal progeny
No. females No. males
5,848 5,727
24,125 21,297
8,437 7,773
Exceptional Progeny
females males
No. % No. %
0 Qa 0 Ob
8 0.03 3 0.01
(p«0.05)
15 0.18 7 0.09
(p«0.05) (p«0.05
aBased on female progeny only.
on male progeny only.
(continued on the following page)
-------
lABLb 4-14. (continued)
Test Chemical
Reference system information
Shakarnis, 1970 Drosophlla Concentration tested:
melanogaster 3-day-old females
X chromosome from radiostable D-32
nondisjunction strain exposed to 0.07%
EDC in a 1.5 L
desiccator for 4 or 6
h at 24-25°C.
Source: Not given
Purity: Not given
0.07% EDC
duration of
treatment
(h)
Control
4
6
Results
Incidence of non-
disjunction greater
in treated group than
1n control. Increase
not statistically
significant.
Normal .progeny
No. females No. ma
2472 2205
3584 3401
4043 4090
Comments
1. Not as many Individuals
scored as 1n 1969 study.
Sample size may not have been
sufficiently large.
2. Longest exposure times 2 h
shorter than in previous
experiment; may have been too
short.
Exceptional Progeny
females males
les No. % No. %
0 0 1 0.03
1 0.03 1 0.03
4 0.10 3 0.07
(continued on the following page)
-------
TABLE 4-14. (continued)
Reference
Test
system
Chemical
information Results
Comments
King et al.,
1979
Micronucleus test:
NMRI mice
I
\o
U)
Jenssen and
Ramel,
1980
Micronucleus test:
CBA mice
Concentration tested:
2 i.p. injections of
4 mmol/kg (400 mg/kg)
given 24 h apart;
4 animals/dose sacrificed
after second injection.
Source: Merck Co.
Darmstadt, FRG
Purity: Not specified,
but melting
point and ele-
mental analysis
were performed
Concentration tested:
single i.p. injection
at 100 mg/kg.
Source: BOH Chemicals,
Ltd.
Purity: Not given
Negative 1. 1,000 polychromatic erythrocytes
analyzed/animal.
2. Frequency of micronuclei not
given.
Negative.
(0.15 ±0.14)
polychromatic
erythrocytes
with micronuclei
in controls vs.
0.17 +_ 0.10 in
treated animals).
-------
to substantiate the reported results.
In studies by Shakarnis (1969, 1970} using D. melanogaster, females were
exposed to 0.07% EDC vapors (source and purity not given). In the first study
(1969) a statistically significant (P < 0.05) increase in exceptional F^
progeny, indicative of meiotic nondisjunction, was seen after a 4 h treatment
(0.03% exceptional females) and an 8-hour treatment (0.18% exceptional females
and 0.09% exceptional males) compared with untreated controls (0%; 0/11,575
progeny). However, not only was the longest treatment time some 25% shorter
than in the first study (6 hours vs. 8 hours) but a sample size at least two-
to-threefold as large was required to confirm the data as statistically significant
for the observed differences in rates between the control and treated series.
Accordingly, the second study is of considerably less value in the assessment
of the mutagenic potential of the compound.
Micronucleus tests were performed by King et al. (1979) and Jenssen and
Ramel (1980). Both studies reported negative results. King et al. (1979)
gave NMRI mice two intraperitoneal injections of 4 mmoles/kg (400
mg/kg) EDC (Merck Co., purity not specified but melting point [sic] and chemical
analysis reported to be correct). King et al. (1979) state this treatment
corresponds to an "approximate lethal dose" (the LDjQ for intraperitoneal
injections of EDC in mice is 250 mg/kg). The injections were given 24 hours
apart, and the animals were killed 6 hours after the second injection. Bone marrow
smears were made; one thousand polychromatic erythrocytes (PCEs) were analyzed
per animal. Frequencies of micronuclei were not given, but the results were
regarded by King et al. as negative.
Jenssen and Ramel (1980) also reported negative results in a micronucleus
test. CBA mice were given a single intraperitoneal injection of EDC (BDH
4-94
-------
Chemicals Ltd., England, purity not given) of 100 mg/kg. The animals were
sacrificed the next day and PCEs (number not given) were scored for micronuclei.
The frequencies of PCEs with micronuclei were 0.15 _+ 0.14 in the controls and
0.17 +_ 0.10 in treated animals.
The positive response in the X-chromosome loss test in Drosophila (Shakarnis,
1969) suggests EDC is capable of causing meiotic nondisjunction resulting in
numerical chromosomal abnormalities. The negative responses obtained in the
micronucleus tests may indicate that EDC does not cause chromosomal damage in
mice. However, because EDC has not been adequately tested for its ability to
cause structural chromosomal aberrations, it would be appropriate to perform
mammalian in vitro and in vivo cytogenetic tests. Such testing is required
before a judgment can be made on the ability of EDC to cause chromosomal aberrations,
4.4.3. Other Studies Indicative of DNA Damage
Three other tests have been conducted on the genotoxicity of EDC. These
tests do not measure mutagenic events per se in that they do not demonstrate
the induction of heritable (i.e., somatic or germinal) genetic alterations, but
positive results in these test systems are taken as evidence that DNA has been
damaged. Such test systems provide supporting evidence that is useful for
assessing genetic risk.
4.4.3,1. Bacteria—EDC is reported to be positive in the gplA assay, which
measures toxicity associated with unrepaired damage in DNA (Table 4-15). Brem
et al. (1974) soaked sterile filter disks with 10 ul (80 umol) EDC. The disks
were centered on the agar surface of petri dishes of bacteria. One set of plates
contained a polA* strain, while the other contained a jpglA" strain. After
incubation the plates were scored for differential inhibition of growth.
An 8-mm zone of growth inhibition was observed in the polA* strain compared
4-95
-------
TABLE 4-15. SUMMARY OF MUTAGENICITY TESTING OF EDC: polA ASSAY
Reference
Test
system
Strains
Activation
system
Chemical
information
Results
Comments
Brem et al., polA
1974 differential
cell killing
assay
E_. coli
polA"
po!A+
None Concentration tested:
10 ul on filter disk
Questionable
positive
1. All assays carried out
in duplicate on at least
three different occasions.
2. Only a small difference
in diameter (i.e., 1 mm)
was noted between the zones
of killing in the two
strains. Thus, the A+/A-
ratio may be misleading.
polA zone of inhib.
polA* polA" polAVpolA"
mm mm
EDC
MMS
Chloramphenicol
8
45
28
9
54
28
1.26
1.44
1.00
-------
with a 9-mm zone of Inhibition In the polA" strain. These responses were reported
to be reproducible. The ratio between the zones of inhibition (pj)lA+/polA")
was 1.26, which is interpreted by Brem et al. as a positive response. Since 1
ram is not a big difference, however, the result could also be regarded
as equivocal.
4.4.3.2. Eucaryotes—One test has been performed to assess the ability of
EDC to cause UDS (Perocco and Prodi, 1981). Perocco and Prodi (1981) collected
blood samples from healthy humans, separated the lymphocytes, and cultured 5 x
1Q5 in 0.2 ml medium for 4 hours at 37°C in the presence or absence of EDC
(Carlo Erban, Milan, Italy or Merck-Schuchardt, Darmstadt, FRG, 97-99% pure).
The tests were conducted both in the presence and in the absence of PCB-induced
rat liver 59 mix. A comparison was made between treated and untreated cells
for scheduled DNA synthesis (i.e., DNA semi-conservative replication) and UDS.
No difference was noted between the groups with respect to scheduled DNA synthesis
measured as dpm of [^H] deoxythymidylic acfd (TdR) after 4 hours of culture
(2,661 +_ 57 dpm in untreated cells compared to 2,287 +_ 60 dpm in cells treated
with 5 ul/ml [0,06 umol/ml] EDC). Subsequently 2.5, 5, and 10 ul/ml (0.03,
0.06, and 0.1 umol/ml) EDC was added to cells followed by 10 mM hydroxyurea
to suppress scheduled DNA synthesis. The amount of unscheduled DNA synthesis
was estimated by measuring dpm from incorporated [3|-i]TdR 4 hours later. At
10 ul/ml EDC, 483 +_ 37 and 532 _+ 21 dpm were counted without and with exogenous
metabolic activation, respectively. Both values were lower than corresponding
negative controls of 715 +_ 24 and 612 +_ 26 dpm, respectively. No positive
controls were run to ensure that the system was working properly, although testing
of chloromethyl methyl ether (CMME) with activation resulted in a doubling of dpms
over the corresponding negative control values (1,320 _+ 57 at 5 ul/ml CMME vs.
4-97
-------
612 +_ 26 untreated). Perocco and Prodi (1981) calculated an effective DNA
repair value (r) for each chemical based on the control and experimental
values with and without metabolic activation. Ethylene dichloride was evaluated
by the authors as positive in the test, but they did not state their criteria for
a positive. None of the experimental values from cells treated with HOC without
metabolic activation had higher dpra values than the controls. Although two
out of three experimental values were greater than the controls with metabolic
activation (673 +_ 45 at 5 ul/rol and 630 +_ 34 at 2.5 ul/ml compared with control
value of 612 +_ 26), the increases were not statistically significant. The
results reported in this work are inconclusive,
4.4.4. DNA Alkylation Studies
Reitz et al. (1982) compared the pharmacokinetics of HOC administered to
Osborne-Mendel rats by inhalation and by gavage. DNA alkylation was also
measured in bacteria at EDC cytosol concentrations corresponding to those used
in the DNA binding study (Table 4-16), Two gram aliquots of TA1535 were incubated
with 7.06 umol C^C] EDC/ml (sp. act, = 3.2 mCi/mmol) and varying amounts of
cytosol. DNA alkylation values at cytosol concentrations of 2.2, 7.8, 27, and
71% were 8.65, 27, 107, and 137 dpm/mg purified DNA, respectively. This corresponds
to 4, 12.5, 49, and 64 alkylations x 10-6 Q^A nucleotides. The corresponding
reversion frequencies were 4.6 +_ 0.82, 23.5 _+ 3.0, 80.2 +^ 9.6, and 111 _+ 2.6,
respectively (n = 3 in each case). A direct correlation between the degree of
alkylation and an increase in mutation frequency was indicated by linear regression
analysis (r = 0.9976). In the DNA alkylation studies with rats [14c] EDC
(sp. act. = 0.32 fflCI/mmol) was administered to groups of three animals by gavage
(150 mg/kg) or inhalation (150 ppm, 6 hours). The animals were sacrificed and
DNA was extracted from the liver, spleen, kidney, and stomach for measurement
4-98
-------
TABLE 4-16. SUMMARY OF MUTAGENICITY TESTING OF EDC: DNA BINDING STUDIES
Test Activation
Reference system Strains system
Reitz et DNA alkylation TA1535 Phenobarbital-
al., 1982 and mutagenesis induced rat % Cytosol
in Salmonella liver cytosol 2.2
7.8
27
71
DNA alkylation Osborne- NA
in rats Mendel rat Route of
Exposure
Gavage
150 mg/kg
Inhalation
150 ppm,
6 hours
Chemical information
Revertants
4.6 + 0.82
23.5 + 3.0
80.2 + 9.6
111 +_ 2.6
Tissue
Liver
Spleen
Kidney
Stomach
Liver
Spleen
Kidney
Stomach
Alkylation x 10~6
Nucleotides
4
12.5
49
64
Alkylation x 10-6
Nucleotides (means
from 2 experiments)
21.3 ; 13.9
5.8 ; 2.5
17.4 ; 14.5
14.9 ; 6.7
8.2 ; 3.3
1.8 ; 1.8
5.2 ; 2.0
2.8 ; 1.9
Comments
1. Bacteria incu-
bated with 7.06 umol
EDC/ml .
2. Significant cor-
relation between
degree of alkylation
and increased re-
version frequency
(r = 0.9976).
3. Rats sacrificed
4 h post gavage
or immediately after
inhalation.
4. Specific acti-
vity 3.2 mCi/mmol
for bacteria; 0.32
mCi/mmol for rats
-------
of DNA .alkylation. Overall, three to five times more DNA alkylation was detected
after gavage than after inhalation. The values ranged from 5.8 +_ 0.7 to 23.1
+_ 7.4 alkylation/106 nucleotides for gavage vs. 1.8 +_ 0.3 to 8,2 _+ 3.3 alkylation/
10$ nucleotides for inhalation. Under the conditions of the test (Reitz et al.»
1982), EDC exposure resulted in a similar degree of addyct formation in rats
and bacteria. Because ONA is the genetic material in both bacteria and rats,
these data predict that mutations were induced in the rat at the exposures
used. This prediction is in keeping with the positive results in mutagenicity
tests in other eucaryotes including Drosophila and cultured CHO cells. DNA
alkylation was not measured in rat gonads, so it is not possible to estimate
the heritable genetic risk.
4.4.5. Summary and Conclusions
Ethylene dichloride (EDC) has been shown to induce gene mutations in
bacteria, plants, Drosophila, and cultured Chinese hamster ovary (CHO) cells.
Weak positive responses were observed in the bacterial and CHO tests in the
absence of an exogenous metabolic activation system. Stronger positive responses
were found when hepatic metabolic activation systems were incorporated. Based
on these positive findings in different test systems representing a wide range
of organisms, EDC is capable of causing gene mutations,
EDC has been reported to cause meiotic chromosomal nondisjunction in
Drosophila. The induction of meiotic nondisjunction is a significant genotoxic
effect; however, a positive response in another test system is needed to permit
a judgment on the generality of this effect. With respect to its ability to
cause structural chromosomal aberrations, sufficient testing has not been per-
formed. There is only one abstract published on the ability of EDC to cause
structural chromosomal aberrations (i.e., in Allium root tip cells and human
4-100
-------
lymphocytes). Because no data were presented, an Independent assessment of
the author's conclusions is not possible. Even though negative results
are reported from micronucleus tests, additional information is needed to draw
conclusions on the ability of EDC to cause chromosomal aberrations. For example,
it would be appropriate to test EDC in in vitro and in vivo mammalian cytogenetic
assays including a sister chromatid exchange assay.
There are no available data on the ability of EDC to damage DNA in mammalian
germ cells. Studies on the ability of EDC to reach germinal tissue would
be appropriate to determine whether EDC has the potential to cause heritable
mutations which may contribute to genetic disease. The finding that EDC causes
heritable mutations in Drosophila and alkylates DNA in several somatic tissues
in the rat reinforces the need for germ-cell studies in mammals.
Based on the weight of evidence, EDC is a weak direct-acting mutagen.
Several of its putative metabolites, thought to be formed in mammals, are more
potent mutagens (e.g., S-[2-chloroethyl]-L-cysteine or chloroacetaldehyde)
than EDC. Such metabolites have the potential of causing adverse effects in
humans. The array of damage produced by EDC is similar to that of EDB. In
keeping with the relative electrophilicities of the chlorine and bromine atoms,
EDB is a better alkylating agent and more potent mutagen.
4-101
-------
4.5. METHYL BROMIDE {BROMOMETHANE)
Methyl bromide is a gaseous compound that has been evaluated for mutagenic
activity in bacteria and in eucaryotes (Drosophila, mammalian cells in culture,
intact rodents). The ability of methyl bromide to alky!ate ONA has been determined
in liver and spleen tissue from the mouse. The available studies are
discussed below and summarized in Table 4-17.
4.5.1. Gene Mutation Studies
4.5.1.1. Bacteria—Methyl bromide has been tested for mutagenicity 1n the
Salmonella/microsome test for reverse mutation and in E. coli and Kj_ebsje_11a_
pneumoniae for forward mutation. Because methyl bromide is a gaseous compound
(the boiling point is 3.56°C, vapor pressure at 20°C is 1420 mm Hg), studies
were generally conducted in sealed containers or desiccators. Some liquid
suspension studies were also performed. Methyl bromide was detected as mutagenic
in all bacterial tests. Metabolic activation was not required. In those
cases where S9 activation was used, the mutagenic responses were not enhanced
(or decreased).
In Salmonella tests, methyl bromide was active in base-pair substitution
strains while ineffective in frameshift-sensitive strains. Most studies, however,
used tester strain TA100, which responds both to base-pair substitution mutagens
and frameshift mutagens.
Moriya et al. (1983) evaluated 228 pesticides, including methyl bromide
(purity not reported)*, in Salmonella tester strains TA98, TA100, TA1535, TA1538,
and TA1537 and in £._ coli WP2 her. Methyl bromide was injected into a sealed
container from a syringe and incubated at 37°C for 2 days with constant stirring
*Most pesticide samples were obtained from the Agricultural Chemicals
Inspection Station of the Ministry of Agriculture, Forestry and Fisheries in
Japan (Kodaira).
4-102
-------
TABLE 4-17. SUMMMARY OF MUTAGENICITY TESTING OF METHYL BROMIDE
Test system
Reported
results
Comment
Reference
A. Gene Mutation
Bacteria:
Salmonella/microsome assay
o
Co
Escherichia coli WP2
reverse mutation assay
and Sd-4 forward mutation assay
Klebsiella pneumom'ae
Eucaryotes:
L5178Y mouse lymphoma cell
forward mutation assay
Drosophila sex-linked recessive
lethal test
dose-response; active in
base-pair substitution
strains (TA100, TA1535);
no enhanced response with
S9 activation, tested in
gaseous phase or liquid
suspension
dose-related response
without S9
dose-related response
without S9
dose-related response
without S9
1.25% total lethals is
highest frequency observed
Moriya et al., 1983
Simmon, 1981; Kramers
et al., unpublished;
Voogd et al., 1982
(Abstract)
Moriya et al., 1983
Djalali-Behzad, 1981
Kramers et al.,
unpublished; Voogd et
al., 1982; (Abstract)
Kramers et al.,
unpublished; Voogd et
al., 1982, (Abstract)
Kramers et al.,
unpublished; Kramers
and Bissumbhar, 1983
(Abstract); Voogd et
al., 1982, (Abstract)
nontoxic doses tested,
small sample size, no
negative control data,
results considered
inconclusive
McGregor, 1981
(continued on the following page)
-------
TABLE 4-17. (continued)
Test system
Reported
results
Comment
Reference
B. Chromosome Aberrations
Rat bone marrow assay
Rat dominant lethal assay
dosage levels may have
been too low to preclude
weak activity, gaps
observed in males only
low dosage levels
McGregor, 1981
McGregor, 1981
o
-C-
C. Other Indicators of DNA Damaging Potential
Unscheduled DNA synthesis (UDS) in
primary rat liver cells
UDS in human diploid fibroblasts
results considered in-
conclusive because of
lack of control data and
experimental data
large variation among
different samples
Kramers et al.,
unpublished; Voogd et
al., 1982 (Abstract)
McGregor, 1981
DNA and hemoglobin alkylation
in the mouse
guanine-N-7 adducts
measured in liver and
spleen
Djalali-Behzad et al.,
1981
-------
of the inside air by a small electric fan. Petri plates without Ifds were
placed 1n the container. Methyl bromide was tested at 5 concentrations with
S9 mix and 6 concentrations without S9 mix; the type of inducer and species
were not specified. A dose response was observed in strain TA1QQ in the concentration
range of 0.5 g/m3 (0.005 mmol/1) to 5 g/tn3 (0.05 mmol/1) in the absence of
S9. At 5 g/m3, an approximately fivefold increase was observed in numbers
of revertants per plate over the background control value. The spontaneous
mutant frequency for TA100 was within the normal range. At concentrations
higher than 5 g/m3 the response dropped quickly to zero revertants as toxldty
most likely became a factor in the test. Mutagenic activity was similar in
the presence and absence of S9 mix. A positive effect was also reported for
the base-pair substitution strain TA1535 and E. coli MP2 her, whereas negative
findings were for the frameshift-sensitive strains TA98, TA1538, and TA1537
(data were not reported). Although methyl bromide causes clear mutagenic
responses, its activity cannot be compared with the other test agents because
its exposure was in the gaseous phase, while the other test agents were added
directly to the top agar (i.e., plate incorporation assay).
Methyl bromide (99% purity) and other alkyl halides were evaluated by
Simmon (1978, 1981) for mutagenicity in S. typhimurium TA100 without S9 activation.
A desiccator procedure was used. Uncovered petri plates inoculated with Salmonella
were placed in a gastight chamber. Methyl bromide was Injected into the desiccator,
and the vapors were dispersed by a fan. Assays were conducted at 37°G for 21
hours. Methyl bromide caused a dose-related positive response at four concentrations
ranging from 0.01% to 0.11 in the atmosphere of the desiccators: there were
approximately 600 revertants per plate at 0.1%, compared with about 90 revertants
per plate in the control. Dichloromethane was mutagenic, whereas carbon tetrachloride
4-105
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was found to be negative in this study by Simmon.
Voogd et al. (1982) and Kramers et al. (1984, in press)*, found methyl
bromide (99% purity) to be mutagenic in the bacteria S. typhimurium and K»_
pneumoniae. Methyl bromide exhibited dose-related mutagenicity in a fluctuation
assay for streptomycin resistance using Klebsiella (ur~, pro'); the bacteria
were incubated for 20 hours at 37°C at three concentrations of methyl bromide
in the air of 4.75 g/m3 (0.05 mmol/1) to 19 g/m3. Salmonella strains TA98 and
TA1QQ were incubated in closed 1,5-1 jars at 37°C for 5 days at 9 concentrations
(0 to 50 g/m3). Dose-related mutagenicity was reported for TA100 at concentrations
of 1.9 g/m3 {0.02 mmol/1) to 19 g/m3 in the absence of S9 mix (Aroclor 1254-induced
rat liver). The maximum response was approximately fourfold over the background
counts, which were within the normal range for TA100. At concentrations greater
than 19 g/m3, the mutagenic response dropped because of toxicity. The addition
of S9 mix had no effect on the mutagenicity of methyl bromide. However, no postive
control requiring S9 activation was reported to insure the 59 was functional.
A preincubation assay with Salmonella TA100 showed mutagenicity at 285 mg/1 (3
mmol/1) for 6 hours and at 9.50 mg/1 for 4 hours. There was no increase in the
spontaneous frequency at lower concentrations at these treatment times. A
treatment time of 2 hours resulted in no mutagenic effect when tested up to
1000 mg/1. Tester strain TA98 was not reverted by methyl bromide in these studies.
Djalali-Behzad et al. (1981) evaluated methyl bromide (purity not reported,
source as a gift) in a forward mutation assay in £. coli Sd-4 (a streptomycin
dependent strain). Bacteria were treated in tightly closed tubes for 1 hour at
*The report by Voogd et al. 1982 is an abstract. A written request was made to
obtain the original data. In response to this request, Dr. Voogd sent an
unpublished report by Kramer et al. (1984), which has been accepted for publication
in Mutation Research (memo to Dr. V. Vaughan-Dellarco).
4-106
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37°C at 5 concentrations ranging from 0 to approximately 5mM. About a fivefold
increase in the mutation frequency was found at 5mM after a 1-hour treatment.
Other bacterial tests in Ju_ typhimunum and E_. coli her have been reported
as positive by Shirasu et al. (1982), Original data were not presented in
this article, and the results appeared to be based on those described by Moriya
et al. (1983).
4.5.1.2. Drosophna--Kramers et al. (1984, in press; Kramers and Bissumbhar,
1983; Voogd et al., 1982) examined the ability of methyl bromide to cause
sex-linked recessive lethals in D_. me1_anogaster. Berlin-K males were treated
by inhalation to methyl bromide for 6 hours at 150-750 mg/m^; 6 hours per day
for 5 days at 150-600 mg/m^; and 6 hours per day, 5 days per week, for 3 weeks
at 70-600 mg/m^. Methyl bromide was lethal at exposures of 600 mg/m^ for 30
hours or 90 hours. Five broods were examined; males were mated to virgin
females every 2 or 3 days for a total period of up to 12 days. Dose-related
responses were observed (Table 4-18). A clear, germ-cell stage-specific
response was not obtained. The highest lethal frequency, however, was after
the 6-hour exposure and was generally observed in brood I, which represents
mature sperm. Table 4-18 presents data from the experiment following a 6 hour
exposure per day for 5 days at 375 and 487 mg/m^ methyl bromide. As can be
seen from this table* the induced mutation frequency was approximately eightfold
and fourteenfold greater than the control. This experiment provides strong
evidence that methyl bromide is a mutagen in Drosophila.
McGregor (1981) evaluated methyl bromide (source, 8DH Limited, Poole,
Dorset, England) in the Drosophila sex-linked lethal test and reported negative
results at concentrations of 20 and 70 ppm in air for 5 hours (100 and 350
ppm»h). There were increased responses but they were not dose-response or
4-107
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TABLE 4-18. Methyl bromide: Drosophila Sex-Linked Resessive Lethal Test
Exposure Time
5d x 6h
Exposure
Concentration
0
375
487
Number of Lethal s/
Number of Tests
2/2320
17/2339
16/1282
%
Lethal s
0.09
0.73
1.25
SOURCE: Kramers et al., 1984, -in press
reproducible. The concentrations evaluated 1n this study were nontoxic and
the sample sizes were small (approximately 1000 chromosomes per dose). Furthermore,
negative control data were not given in the study by McGregor. The results of
this study are inconclusive.
4.5.1.3. Mammalian Cells 1n_Culture—Kramers et al. (1984, in press) and
Voogd et al. (1982) reported methyl bromide to produce a dose-related positive
response in gene mutation assays (TK and HGPRT loci) in mouse lymphoma L5178Y
cells. Cells were treated in medium for 24 hours at 37°C at five concentrations
ranging from 0.3 mg/1 (0.003 mmol/1) to 3 mg/1, The mutant frequency in the
control culture (treated with 1% ethanol) was 4.6 x 10-5 for TK-deficiency and
1 x 10~5 for HGPRT-deficiency. At the TK locus, the induced mutation frequency
was dose-related at five concentrations of methyl bromide. The highest induced
mutant frequency, which was observed at 1 mg/1 (survival was approximately 60%
at this concentration), was approximately a twofold increase In the control
frequency. At the next concentration (3 mg/1), the induced mutant frequency
dropped slightly as the toxicity increased. At the HGPRT locus, a dose-related
response was observed at five concentrations of the chemical. At the highest
concentration (3 mg/1), a 14-fold increase was observed over background (survival
was approximately 40%).
4-108
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4.5.2. Chromosomal Aberration Studies
McGregor (1981) examined bone marrow cells from male and female CD rats for
chromosomal aberrations after methyl bromide exposure. Animals were exposed to 20
and 70 ppm methyl bromide by inhalation 7 hours per day for 5 consecutive days
or for a single exposure of 20 and 70 ppm for 7 hours. Treatment was followed
by sampling after 6, 24, and 48 hours. No increases in aberrations were seen
after the acute exposures. In the subacute experiments, negative results were
obtained except at the 70 ppm for 7 hours per day for 5 days dose for males, where
an increase in the frequency of gaps was found. Because gaps are not considered
true chromosomal aberrations, their presence alone is insufficient to conclude
clastogenic activity. On the other hand, the data do not support a clear
negative conclusion because the criteria used to select dosage levels are not
stated. It is likely that a maximum tolerated dose was not reached. A toxic
dose was apparently not evaluated because the highest exposure (70 ppm) did
not affect the rats body weights. The only evidence of adverse effects during
or after exposure was traces of blood around the nostrils.
McGregor (1981) conducted a dominant lethal test in male rats exposed to
20 or 70 ppm methyl bromide 7 hours per day for 5 consecutive days. No increase
in dominant lethal effects was found. Higher dosage levels should have been
examined, especially since the dominant lethal test is not regarded as a sensitive
assay (as discussed in section 4.1.2).
4.5.3. Other Studies Indicative of D_NA Damage
The potential DNA-damaging activity of methyl bromide was examined in an
unscheduled DNA synthesis (DOS) assay referred to as the hepatocyte primary
culture DNA repair test (Kramers et a!., 1984, in press; Voogd et a!., 1982).
Hepatocytes were isolated from livers of 6-week-old S.P.F. male Wistar rats.
4-109
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Cell cultures were incubated with concentrations of methyl bromide {in ethanol}
that ranged from 10 mg/1 (0.1 mM) to 30 mg/1 (0.3 mM) and [3H]thymidine (ZO uCi)
for 24 hours at 37°C. UDS was measured by autoradiographic determination of the
amount of [3H]thymidine that is incorporated into nuclear DNA. For each
concentration, nuclear counts are reported after the highest cytoplasmic grain
count of three nuclear-sized areas adjacent to the nucleus is subtracted from
the nuclear counts (see Williams and long, 1980, for details of method).
Negative results were reported but no experimental data were presented. The
authors' conclusions, therefore, can not be evaluated independently. Also,
subtraction of the highest cytoplasmic grain count reduces the possibility
of false positives but increases the chance of missing a weak UDS inducer.
McGregor (1981) tested the ability of methyl bromide to induce UDS in
human embryonic intestinal cells after exposures of up to 70% in air for 3 hours.
The incorporation of [3H]thymidine (10 uCi/ml) was determined by autoradiography.
No increase in UDS was detected in the presence or absence of S9 mix (Aroclor
1254-induced rat liver). The positive control, vinyl chloride, induced UDS in
the absence of S9 mix; an enhanced response was observed in the presence of S9
mix. High standard deviations, however, were reported among samples; the large
variation may be due to the presence of heterogeneous cell populations with
different abilities to incorporate [3H]thymidine.
4.5.4. DNA Alkylation Studies
Methyl bromide alkylates DNA in intact mammals (Djalali-Behzad, 1981).
DNA alkylation (N-7 guanine) was measured in mouse (male, CBA) liver and spleen
after exposure to ^-labeled methyl bromide (4.9 and 5mCi/mmol) by inhalation
for 4 hours. The amount of alkylation was also determined in vitro. Hemoglobin
alkylation was measured to determine whether it could be used for an estimation
4-110
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of DNA alkylation in vivo. It was found that the extent of alkylation of DNA
in vivo (3.4 x 10~9 alkylations/nucleotide at 144 ppm h) was considerably
less than the alkylation of hemoglobin in vivo and of DNA and hemoglobin in
vitro (Table 4-19).
In a study on the presence of methylated purines in humans after suspected
dimethylnitrosamine (DMN) poisoning, DNA samples from the liver and kidney
were also examined from one case of methyl bromide poisoning (Herron and Shank,
1980). None of the samples from the methyl bromide poisoning contained
7-methylguanine or Q^-methylguanine, whereas these alkylation products were
found in people exposed to DMN, Conclusions cannot be reached from the negative
findings on methyl bromide because only one victim was examined, the magnitude
of the exposure was not given, and information was not reported on how the
tissues were prepared and frozen.
4.5.5. Gonadal Effects
McGregor (1981) examined mice (86C3F1) for altered sperm morphology after
treated exposure to methyl bromide at 20 and 70 ppm by inhalation 7 hours per day
for 5 consecutive days. One mouse in 10 died 5 days after exposure. Mice
were killed 5 weeks after the last day of treatment. Animals were not sacrificed
at different time intervals to examine the effects on various germ-cell stages.
Ethyl methanesulfonate (EMS) administered by i.p. injection was the positive
control; a gaseous compound given by inhalation would have been more appropriate.
There was no evidence of morphological sperm abnormalities induced by methyl
bromide. However, these negative findings alone are insufficient to conclude
that methyl bromide does not reach the germinal tissues of whole mammals.
4.5.6. Summary and Conclusions
Methyl bromide is mutagenic in bacterial tests using S. typhimuriuin, E. coli,
4-111
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TABLE 4-19. THE ALKYLATIQN OF CYSTEINE IN HEMOGLOBIN AND LIVER PROTEINS
AND OF GUANINE N-7 IN DNA FROM LIVER AND SPLEEN AFTER EXPOSURE OF MICE TO
RADIOLABELED METHYL BROMIDE
Administered
amount of
methyl bromide
Degree of alkylation
of cysteine-s
Degree of alkylation
of guanine-N-7 in DNA
(uCi/kg b.w.)
{uC1/g protein)
found (expected)3 in
Hb
Li ver
(uCi/gDNA)
found (expected)8 in
Liver
Spleen
340
(inhalation)
174
(inhalation)
21.6
(i.p. injection)
2.2 x 10-2
1.6 x 10-2
4.2 x 10-3
1 x 10-3
(>2.2 x 10-2)
5 x ID'5 5 x 10-4
(1 x 10-2) (1 x 10-2)
aExpected from the reactivities in vitro of cysteine-S in hemoglobin and guanine-N-7
in DNA, respectively, assuming that the dose of methyl bromide is equal to the dose
obtained in red blood cells.
SOURCE: Djalali-Behzad et a!., 1981.
4-112
-------
and K. pneumoniae. Clear dose-dependent mutagenlc responses have been reported.
The mutagenieity of methyl bromide is consistent with its alkylating properties
in that it is a direct-acting mutagen and induces primarily base-pair substitution
mutations. The mutagenic responses were not enhanced by an 59 metabolic
activation system.
In addition to its being a bacterial mutagen, methyl bromide is reported
to be mutagenic in eucaryotic organisms. It has been shown to induce gene
mutations in Drosophila and at two different loci in mammalian cells in culture,
The positive findings in bacteria, cultured mammalian cells, and Drosophila,
coupled with the ability of methyl bromide to alkylate DNA in intact mammals 1s
sufficient evidence to presume that methyl bromide has the potential to be
mutagenic in whole mammals. Methyl bromide does not appear to be as potent a
mutagen as ethylene dibromide in that more chemical is needed to induce a response.
Data regarding its ability to cause chromosomal aberrations have been primarily
negative. However, these results appear to have been generated using inadequate
exposure levels. Furthermore, chromosome tests are generally not as sensitive
as gene mutation tests (Voogd et al., 1977). Although, methyl bromide does
not appear to elicit UDS, the testing has been limited and inadequate. Moreover,
certain kinds of DNA lesions that lead to mutations may not stimulate the
repair process or do so to such a small extent that UDS would not be detected.
Methyl bromide's potential to reach the germinal tissue of intact mammals has
not been sufficiently investigated; one test for altered sperm morphology and
a test for dominant lethal effects have been reported as negative. In view of
the fact that methyl bromide induces heritable effects in the germ cells of
Drosophila, it should be further evaluated for such effects in mammalian germ
cells.
4-113
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TECHNICAL REPORT DATA
ff lease read Instructions on the reverse before completing)
1, REPORT NO.
EPA/600/6-85/001
3. RECIPIENT'S ACCESSION*NO.
4.TITLE AND SUBTITLE Assessment of the Mutagenic
Potential of Carbon Bisulfide, Carbon Tetra-
cloride, Dvchloromethane, Ethylene Bichloride
and Methyl Bromide: A Comparative Analysis in
8. REPORT DATE
August 1985
6, PERFORMING ORGANIZATION CODE
7.AUTHORJS) Relation to Ethylene Dibromide
Vicki L. Vaughan-Dellarco,
John R. Fowle III. SheilaRosenthal.
I. PERFORMING ORGANIZATION REPORT NO,
S. PERFORMING ORGANIZATION NAME AND ADDRESS
U.S. Environmental Protection Agency
Office of Health and Environmental Assessment
Reproductive Effects Assessment Group (RD-689)
401 M St., SW., Washington, DC 20460
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
12, SPONSORING AGENCY NAME AND ADDRESS
U.S. Environmental Protect ion,. A,ge.ncy , „*
Office of Health and Environmental Assessment
Reproductive Effects Assessment Group (RE>-689)
401 M St., SW, Washington, DC 20460
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA/600/21
15. SUPPLEMENTARY NOTES
ie. ABSTRACTThig document provides an evaluaton of the rautagenic potential of five
Iternative fumigants to ethylene dibroroide(EDB). These include carbon disulfide(CS2>,
arbon tetrachloride(CCl4), dichloromethane(DCM), ethylene dichloride(EDO), and methyl
romide(MB). Of the five proposed alternatives, DCM, EDC, and MB caused gene mutations
n lower and higher eucarvotes. These three agents have not been well studied for
heir potential to cause chromosome damage. There are no available data on the ability
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