"Current Awareness"
Program
vol.
March 1983
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LINEAR HALOALKANES AND HALOALKENES
CARCINOGENICITY AND STRUCTURE-ACTIVITY
RELATIONSHIPS. OTHER BIOLOGICAL PROPERTIES.
METABOLISM. ENVIRONMENTAL SIGNIFICANCE.
Yin-tak Woo, Ph.D.
Joseph C. Arcos, D.Sc., and
Mary F. Argus, Ph.D. '
Preparation for the Chemical Hazard
Identification Branch "Current
Awareness" Program
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TABLE OF CONTENTS
5.2.2.1 Halogenated Linear Alkanes and Alkenes
5.2.2.1.1 Introduction
5.2.2.1.2 Physicochemical Properties and Biological Effects
5.2.2.1.2.1 Physical and Chemical Properties
5.2.2.1.2.2 Biological Effects Other Than Carcinogenicity
5.2.2.1.3 Carcinogenicity and Structure-Activity Relationships
5.2.2.1.3.1 Overview
5.2.2.1.3.2 Halomethanes
5.2.2.1.3.2.1 Carbon Tetrachloride
5.2.2.1.3.2.2 Chloroform
5.2.2.1.3.2.3 Halomethanes Other Than Carbon Tetrachloride and
Chloroform
5.2.2.1.3.3 Haloethanes
5.2.2.1.3.4 Halopropanes and Higher Haloalkanes
5.2.2.1.3.5 Haloethenes
5.2.2.1.3.5.1 Vinyl Chloride
5.2.2.1.3.5.2 Haloethenes Other than Vinyl Chloride
5.2.2.1.3.6 Halopropenes
5.2.2.1.3.7 Halobutenes, Halobutadienes, and Arylalkyl Halides
5.2.2.1.3.8 Modification of Carcinogenesis
5.2.2.1.4 Metabolism and Mechanism of Action
5.2.2.1.4.1 Metabolism and Mechanism of Action of Haloalkanes
5.2.2.1.4.1.1 Halomethanes
5.2.2.1.4.1.2 Haloethanes
5.2.2.1.4.1.3 Halopropanes
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TABLE OF CONTENTS (cont'd)
5.2.2.1.4.2 Metabolism and Mechanism of Action of Haloalkenes
5.2.2.1.4.2.1 Haloethenes (Haloethylenes)
5.2.2.1.4.2.2 Halopropenes, Halobutenes, and Halobutadienes
5.2.2.1.5 Environmental Significance
5.2,2.1.5.1 Epidemiologic Evidence
5.2.2.1.5.2 Environmental Sources, Occurrences and Exposures
5.2.2.1.5.2.1 Haloalkanes and Haloalkenes in the Air
5.2.2.1.5.2.2 Haloalkanes and Haloalkenes in the Water
5.2.2.1.5.2.3 Haloalkanes and Haloalkenes in Foodstuffs
References to Section 5.2.2.1
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5.2.2.1 Halogenated Linear Alkanes and Alkenes.
5.2.2.1.1 Introduction.
Halogenated hydrocarbons, also known as halocarbons or organohalogen
compounds, represent one of the most important classes of synthetic chemi-
cals. First synthesized in the 1820's, a great number of halogenated hydro-
carbons are now known. Many of these compounds are produced in enormous
quantities and occupy an indispensable role in modern technology. This sec-
tion focuses on the halogenated aliphatic hydrocarbons, which include the
saturated haloalkanes (alkyl halides) and the unsaturated haloalkenes (alkenyl
halides).
Haloalkanes and haloalkenes are widely used in chemical manufacturing; as
industrial degreasers; in automotive, aircraft, textile, food processing, and
various other industries; in anesthesiology; in agriculture; and in innumer-
able other industrial and commercial applications. Table I summarizes the
production data, the major uses and applications of 14 haloalkanes and halo-
alkenes that have annual production in excess of one million pounds in the
United States alone and are known or suspected carcinogens. Other haloalkanes
and haloalkenes with limited information on carcinogenic!ty and with estimated
production volumes exceeding one million pounds include (in decreasing order
of volume): ethyl chloride, dichlorodifluoromethane (Freon 12), methyl
chloride, trichlorofluoromethane (Freon 11), chlorodifluoromethane (Freon 22),
1,2-dichloropropane, trichlorotrifluoroethane, methyl bromide, 1,2-difluoro-
1
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1,1,2,2-tetrachloroethane (Freon 112), and tetrafluoroethylene (40). In
addition, halothane (1,1,l-trifluoro-2-bromo-2-chloroethane) is a well known
anesthetic agent. A survey of the industrial, commercial, and medical appli-
cations of a variety of haloalkenes has been published (41).
As may be expected from the high production volumes and extensive uses, a
large number of workers are occupationally exposed to these compounds. The
U.S. National Institute for Occupational Safety and Health has published a
series of criteria documents on occupational exposure to various haloalkanes
and haloalkenes (1, 4, 8, 11, 12, 17, 20, 21, 31, 34, 37, 42-45). Human
exposure to halogenated compounds is not confined to occupational setting;
emission from industrial production and uses result in the release of large
amounts into the environment. Secondary sources such as water chlorination,
burning of gasoline, plastics, tobacco, and plant materials also contribute to
environmental contamination. Halogenated hydrocarbons have been detected in
ambient and indoor atmospheres, in drinking water, in foodstuffs, and in
various consumer products (see Section 5.2.2.1.5.2).
The potential health hazard of human exposure to haloalkanes and halo-
alkenes has been the subject of intensive investigations in recent years.
Historically, prior to adequate testing, many halogenated compounds were
erroneously assumed to be safe. Chloroform was first introduced as an "ideal"
anesthetic agent by Fluorens and Simpson in 1847 and was used for this purpose
until the mid 20th century. In the 1920's, carbon tetrachloride was hailed as
the drug of choice for the treatment of hookworm. Lambert (46) reported
remarkable success in treating 50,000 hookworm patients on Fiji with 3-4 ml of
carbon tetrachloride and ascribed several ensuing deaths to impurities. The
carcinogenic potential of haloalkanes was first discovered in the 1940's. In
1941, Edwards (47) first reported the induction of liver tumors in the mouse
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Table I
Production Volumes, Major Uses, and Recent Health Effects Reviews of Some
Important Haloalkanes and Haloalkenes
page 1 of 3
Compound, Synonyms, CAS No. Production Volume0
Major Uses and Applications
Reviews
Dichloromethane*, methylene
chloride, 75-09-2
Chloroform*, trichloro-
methane, 67-66-3
Carbon tetrachloride*; tetra-
chloromethane, 56-23-5
1,2-Dichloroethane*, ethylene
dichloride, 107-06-02
497 (1975)
262 (1975)
907 (1975)
7977 (1975)
1,2-Dibromoethane*, ethylene 275 (1975)
dibromide, 106-93-4
Paint remover; degreasing solvent; aerosol propel-
lant; solvent applications in food, pharmaceutical,
synthetic fiber and photography industries
Production of chlorodifluoromethane (used as
refrigreant, propellant); solvent applications in
pharmaceutical (e.g., antibotics), dyes, plastics,
dry cleaning Industries; component of toothpaste,
cough medicine, liniments, salves
Production of difluorodlchloromethane and tri-
chloromethane (used as refrigerant, propellant);
solvent; grain fumigant; pesticide; fuel additive
Production of vinyl chloride and other chlorinated
ethenes; lead scavenger in antiknock fuel additive;
fumigants
Lead scavenger in antiknock fuel additive; fumigant;
production of vinyl bromide
NIOSH (1); IARC
(2); USEPA (3)
NIOSH (4);
Winslow & Gerstner
(5); Reuber (6);
IARC (2); USEPA
(3)
NIOSH (8), IARC
(2); Louria and
Bogden (9); USEPA
(10)
NIOSH (11, 12);
Drury & Hammonds
(13); IARC (2);
Ames et al. (14);
Rannug (15)
Kover (16); NIOSH
(17); IARC (2,
18); Rannug (15)
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Table I (continued)
page 2 or 3
Compound, Synonyms, CAS No. Production Volume0
Major Uses and Applications'
Reviews
1,1,1-Trichloroethane*, methyl 459 (1976)
chloroform, 71-55-6
1,1,2,2-Te trachloroe thane*,
acetylene tetrachloride, sym-
tetrachloroethane, 79-34-5
l,2-Dibromo-3-chloropropane,
96-12-8
Vinyl chloride*, chloroethene
75-01-4
Trichloroethylene, 79-01-6
37 (1974)
25 (1975)
5736 (1976)
Vinylidene chloride*, 1,1-di- 170 (1975)
chloroethylene, 75-35-4
303 (1976)
Cold cleaning and vapor degreasing of metals and
other materials; production of vinylidene chloride;
aerosol propellant; lubricant carrier; coolant in
metal cutting oil
Production of trichloroethylene; industrial solvent
Soil fumigant (for protection of field crops,
vegetables, fruits, nuts, ornamental plants)
Production of PVC resins (used in plastic pipes,
floor tiles, consumer goods, electrical appliances);
limited use as aerosol propellant (now banned)
Production of copolymers (used mainly for food
packaging films and coatings) and modacrylic fibers
Vapor degreasing of fabricated metal parts; solvent
in textile industry, for adhesives and lubricants,
and in commercial cleaning solutions; anesthetic
agent
Aviado et al.
(19); IARC (2)
NIOSH (20); IARC
(2)
NIOSH (21); IARC
(2, 18)
IARC (22, 23);
USEPA (24, 25);
Bins (26); Wagoner
et al. (27);
Hopkins (28, 29)
IARC (23); USEPA
(30)
NIOSH (31); IARC
(2, 32); Aviado et
al. (19); USEPA
(33)
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Table I (continued)
page 3 of 3
Compound, Synonyms, CAS No. Production Volume
Major Uses and Applications
Reviews
Tetrachloroethylene*, per-
chloroethylene, 127-18-4
657 (1976)
2-Chloro-l,3-bu tadiene*,
chloroprene, 126-99-8
349 (1975)
Hexachloro-l,3-butadiene*, per- 7-14 (1972)
chlorobutadiene, 87-68-3
Drycleaning and textile industries; industrial metal
cleaning; production of fluorocarbons; anti-
helminthic agent
Production of polychloroprene (neoprene) elastomers
(used in automobile rubber goods, wire, cable,
construction and adhesive applications)
Recovery of "snift" (chlorine-containing) gas in
chlorine plants; production of lubricants, rubber
compounds; gyroscope fluid
NIOSH (34);
Utzinger &
Schlatter (35);
IARC (2); USEPA
(36)
NIOSH (37); Haley
(38); IARC (23)
IARC (2); USEPA
(39)
aMajor sources of information: IARC Monographs Volumes 19 and 20 (1979); SRI International "A Study of Industrial Data on
Candidate Chemicals for Testing," EPA Publ. 560/5-77-006, U.S. Environmental Protection Agency, Washington, D.C., 1977; L.
Fishbein [Sci. Total Environ., 11, 111 and 163 (1979)]; NAS, "Nonfluorinated Halomethanes in the Environment," National
Academy of Sciences, Washington, D.C., 1978.
Only the most commonly used synonyms are listed. Names with an asterisk are those used in this review. CAS numbers are
the Chemical Abstract Services Registry Numbers.
cProduction volumes in the United States in millions of pounds in the year indicated in parenthesis.
Produced as byproducts or waste products of tetrachloroethylene, trichloroethylene, carbon tetrachloride, and other
chlorinated compounds.
Major uses in United States in decreasing order of the volumes of usages.
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by carbon tetrachloride. Shortly afterwards, Eschenbrenner and Miller (48)
found that chloroform was also hepatocarcinogenic in the mouse. However, very
few findings had greater impact on occupational health than the discovery of
the carcinogenicity of vinyl chloride. Viola (49) reported first at the 10th
International Cancer Congress in 1970 the carcinogenicity of the compound in
rats exposed by inhalation. An extensive series of experiments was subse-
quently undertaken by Maitoni and associates, who not only confirmed the
carcinogenicity of vinyl chloride but also showed its high potency and multi-
target effect. In 1974, Creech arid Johnson (50) reported the development of
liver angiosarcoma, a rare form of liver cancer, in 4 vinyl chloride-exposed
workers in a vinyl chloride polymerization plant in the United States. A wave
of similar findings ensued throughout • the world. By October 1977, at least 64
confirmed cases were reported in 12 countries. The cause-effect relationship
was established beyond doubt (see Section 5.2.2.1.5.1). The discovery of the
carcinogenicity of vinyl chloride stimulated an-explosive growth in investiga-
tions of the health effects of related compounds. The rapid growth is
reflected by a plethora of recent reviews and symposia on haloalkanes and
haloalkenes in general (2, 14, 18, 22, 23, 27, 32, 51-60) and reports on
individual compounds (see Table I). Some 40-50 haloalkanes and haloalkenes
have been tested in various carcinogenesis bioassays and the list is
continually growing (see Section 5.2.2.1.3). This section reviews the
comparative carcinogenicity of various sub-classes of haloalkanes and halo-
alkenes, with emphasis on structure-activity relationship, as well as their
mutagenicity, metabolism, mechanism of action, and their environmental
significance.
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5.2.2.1.2 Physicochemical Properties and Biological Effects.
5.2,2.1.2.1 PHYSICAL AND CHEMICAL PROPERTIES.
The physical and chemical properties of haloalkanes and haloalkenes
depend largely on the nature of the halogen substituent. The electronega-
tivity of halogens- is greater than that of hydrogen and decreases in the
order: F > Cl > Br > I (see Section 3.1.2.3, Vol. I). With the exception of
iodine, all halogens are more electronegative than carbon, so that the C-X
bonds are expected to be partially polarized to yield electron-deficient
(partially positively charged) carbon atoms and electron-rich halogen atoms.
Yet, despite the partial polarization of the C-F bond, fluorinated compounds
are relatively inert as alkylating agents because the strength of the C-F bond
is actually higher than that of the C-H bond. As the size of halogen atom
increases (in the order F < Cl < Br < I) the bond length increases and the
bond energy decreases thus weakening the C-X bond and facilitating the leaving
of halogen atom in nucleophilic reactions. The leaving of the halide ion of
larger size (e.g., iodide) in an aqueous system may be further facilitated by
the lower energy of solvation than that of an ion of smaller size (e.g.,
fluoride).
The physical and chemical properties of haloalkanes and haloalkenes have
been extensively discussed in many reviews (2, 23, 56-58, 61, 62) and standard
textbooks. Some important physical properties of several haloalkanes and
haloalkenes are summarized in Tables II and III. In general, the volatility
of the halogenated compounds decreases with an increase in the number of
halogen subs tituents. With the same degree of halogenation, the volatility
decreases in the order: F > Cl > Br > I. For liquid haloalkanes and halo-
alkenes, solubility in water also tends to decrease with an increase in
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halogenation. Comparable chlorinated compounds are generally more soluble
than brominated compounds, which are in turn more soluble than iodinated
compounds. The partition coefficient of haloalkanes and haloalkenes corre-
lates usually inversely with their water solubility. The partition
coefficient of the many haloalkanes and haloalkenes may be calculated, with
reasonable agreement with experimental values, by a "fragment method"
developed by Hansch and Leo (63). In this method, a given molecule may be
"dissected" into small fragments with known assigned constants; the log of the
partition coefficient (log P) is then calculated by simply adding up the
constants, of each fragment. Among comparable halogenated compounds, branching
r
tends tc jlower the partition coefficient. Multiple halogenation on the same
carbon (jgjeminal substitution) or adjacent carbons (vicinal substitution)
results in a higher partition coefficient than simple additivity predicts.
Fluorinated alkanes are so stable that they are of ten .referred to as
"inert"; nonetheless, fluorocarbons readily react with highly reactive
materials such as alkali metals (59). The chemical reactivity of haloalkanes
increases in the order: Cl < Br < I. The estimated half-lives of hydrolysis
of monohalomethanes in aqueous media are of the order of 10,000, 480, and 8
hours for-chloro-, bromo-, and iodomethane, respectively (58). The reactivity
of chlorinated methanes decreases with an increase in the degree of chlorina-
tion as evidenced by the increase in the half-lives (58). Based on theoreti-
cal calculations, 1,2-dihaloalkanes (vicinally substituted) are expected to be
more reactive than their 1,1- (geminally substituted) counterpart; this pre-
diction is supported by thermochemical data, microwave spectroscopic data, and
quantum mechanical calculations (64). Most haloalkanes are quite resistant to
oxidation; some polyhalogenated alkanes are in fact used as flame retar-
dants. Haloalkanes are susceptible to photolysis by high energy u.v. light
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Table II
Physical Properties of Some Haloalkanes3
Compound
B.P. (°C)
Vapor Pressure
(mm Hg)
Solubility in Water
(gm/100 ml)
Partition Coefficient
(log P)
Chloromethane (Methyl chloride) -23.7
lodoraethane (Methyl iodide) 42.4
Dichloromethane (Methylene chloride) 40.1
Chloroform 61.2
lodoform 218b
Difluorodichloromethane (Freon-12) -29.8
Fluorotrlchloromethane (Freon-11) 23.8
Carbon tetrachloride 76.7
1,1-Dichloroethane 57.3
1,2-Dichloroethane 83.4
1,2-Dib romoe thane 131.6
1,1,1-Trichloroethane 74.1
1,1,2-Trichloroethane 113.5
1,1,2,2-Tetrachloroethane 146.3
Halo thane 50.2
Hexachloroethane 188^
1,2-Dibromo-3-chloropropane 196
3,756 at 20°C
400 at 20°C
400 at 24°C
200 at 26°C
4,306 at 20°C
667 at 20°C
91 at 20°C
230 at 25°C
85 at 25°C
11 at 25°C
103 at 20°C
16 at 20°C
5 at 21°C
243 at 20°C
1 at 32.7°C
0.8 at 21°C
0.74 at 25°C
1.4 at 20°C
2.0 at 20°C
0.82 at 20°C
0.01 at 25°C
0.028 at 25°C
0.11 at 20°C
0.078 at 20°C
0.5 (unspecified)
0.87 at 20°C
0.43 at 30°C
0.03 at 25°C
0.45 at 20°C
0.29 at 25°C
0.35 (unspecified)
0.005 at 22°C
0.1 (unspecified)
0.91 (octanol)
1.69 (octanol)
1.25 (octanol); 1.32 (oil)
1.97 (octanol); 2.06 (oil)
2.16 (octanol)
2.53 (octanol)
2.83 (octanol)
1.79 (octanol); 1.84 (oil)
1.48 (octanol); 1.60 (oil)
2.49 (octanol); 2.58 (oil)
2.12 (oil)
2.57 (oil)
2.30 (octanol)
2.49 (octanol)
a Summarized from data compiled by International Agency for Research on Cancer [lARC Monogr. No. 15 (1977), No. 19
(1979), and No. 20 (1979)]; G. McConnell, D.M. Ferguson, and G.R. Pearson [Endeavor 34, 13 (1975)]; National Academy
of Sciences, "Nonfluorinated Halomethanes in the Environment," National Academy of Science, 1978; A. Sato and T.
Nakajima [Arch. Environ. Health 34, 69 (1979)]; C. Hansch and A. Leo, "Substituent Constants for Correlation Analysis
in Chemistry and Biology," Wiley, New York, 1979.
Sublimes.
Octanol-water or olive oil-water partition coefficient as indicated.
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Table III
Physical Properties of Some Haloalkenes*
Compound
B.P. (°C)
Vapor Pressure
(mm Hg)
Solubility in Water
(gm/100 ml)
Partition Coefficient
(log P)
Vinyl chloride -13.4
1,1-Dichloroethylene
(Vinylidene chloride) 32.0
cis-1,2-Dichloroe thylene 60.6
trans-1,2-Dichloroe thylene 47.7
Trichloroethylene 87
Tetrachloroethylene 121
3-Chloropropene (Allyl chloride) 45.1
1,3-Dichloropropene (racemic mixture) 104
2-Chloro-l,3-butadiene (Chloroprene) 59.4
Hexachloro-l,3-butadiene 210-220
2,530 at 20°C
400 at 14.8°C
208 at 25°C
324 at 25°C
77 at 25°C
20 at 26.3°C
368 at 25°C
215 at 25°C
22 at 100°C
0.11 at 25°C
0.04 at 20°C
0.35 at 20°C
0.63 at 20°C
0.1 at 20°C
0.015 at 25°C
0.1 (unspecified)
0.1 at 20°C
slightly soluble
insoluble
1.38 (octanol)c
2.18 (octanol)
1.53 (octanol)c; 1.97 (oil)
1.53 (octanol)c; 1.96 (oil)
2.29 (octanol); 2.74 (oil)
2.60 (octanol); 3.65 (oil)
Summarized from data compiled by International Agency for Research on Cancer [IARC Monogr. No. 15 (1977), No. 19
(1979), and No. 20 (1979)]; C.R. Worthing (ed.), "The Pesticide Manual," 6th ed., The British Crop Protection Council,
1979; D.D. Irish, In; "Patty's Industrial Hygiene and Toxicology," 2nd. ed., Vol. LI, 1963; A. Sato and T. Nakijima
[Arch. Environ. Health 34, 69 (1979)]; C. Hansch and A. Leo, " Sub stitu tent Constants for Correlation Analysis in
Chemistry and Biology," Wiley, New York, 1979.
'Octanol-water or olive oil-water partition coefficient as indicated.
'Calculated values.
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and form free radicals (except fluorinated compounds); susceptibility to
photolysis follows the order: I > Br > Cl > F.
The ability of haloalkanes to alkylate nucleophiles is of great bio-
logical importance. There are two possible mechanisms — the S^J. and the S-^2
J1-
(for a brief discussion of the SN! and SN2 reactions, see Section 3.2.4 in
Vol. I). In the SN! type of reaction, electrical effects (hyperconjugation,
inductive) on the alkyl group play a determining role in the reactivity of
haloalkanes. The relative reactivity follows the order: tert » sec > pri >
CHo; for example, the relative SN1 solvolysis rate of tert-butyl bromide is
100,000 times greater than that of sec-propyl bromide, which in turn is 12
times greater than that of ethyl or methyl bromide (65). In a S^2 type reac-
tion, steric hindrance around the carbon which is in transition state is of
greater importance. Under ordinary conditions, haloalkanes with a primary or
secondary alkyl group predominantly react by S*,2 mechanism. Reactivity
follows the order: CH-, > pri > sec » (tert) and the relative SN2 displace-
ment rates are of the order of 30, 1, 0.4, 0.02 and near zero, for methyl,
ethyl, primary alkyl, sec-propyl, tert-butyl halides, respectively (65). The
strength of the carbon-halogen bond plays a crucial role in determining the
leaving tendency of the halide ion. The bond energy of the C-X bond decreases
in the order: F > Cl > Br > I. Moreover, halides with lower solvation energy
in aqueous media are also expected to have a higher leaving tendency. The
reaction rates of monohalomethanes have in fact been shown to follow the
order: I > Br > Cl > F (66). -
The alkylating activity of several haloalkanes has been measured by the
NBP (4-p-nitrobenzyl pyridine) color reaction of Preussmann et _al_. (67).
Methyl iodide has been consistently found to be quite active (68, 69); among
the several haloalkanes tested, methyl iodide was the most active alkylating '
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agent. The relative alkylation rate (expressed as change in absorbance at 560
nm in 60 minutes) is 110, 55, 3, and 0 for methyl iodide, ethyl iodide, 1,2-
dibromoethane, and 1,2-dichloroethane, respectively (69). This relative order
has also been demonstrated by the use of a biological nucleophile, deoxyguano-
sine (69). Two primary alkyl chlorides, 1-chloropropane and 1-chlorobutane,
have marginal activity and are considered inactive in the NBP reaction (70).
The chemical reactivity of haloalkenes is dependent on the nature, the
number, and the position of halogen substitutents, as well as the number and
position of the double bond(s). In the ethylene (ethene) series, introduction
of the electronegative halogen atoms decreases .the electron density in the
double bond and exerts a stabilizing effect. The reactivity of ethylene
decreases dramatically with the increase in the degree of halogenation. The
relative rates of reaction with ozone for ethylene, vinyl chloride (mono-
chloroethylene), trans-1,2-dichloroethylene, cis-1,2-dichloroethylene, 1,1-di-
chloroethylene, trichloroethylene, and tetrachloroethylene are 25000, 1180,
591, 35.7, 22.1, 3.6, and 1, respectively (71). The C-X bond in vinyl halides
is expected to be stronger than that in haloalkanes, because of the possi-
bility of resonance as represented below (56):
[TEXT-FIGURE lj
Theoretical calculations predict that cis-1,2-dihaloethylenes are more reac-
tive than trans-1,2-dihaloethylenes, which in turn are more reactive than
1,1-dihaloethylenes (64). In the propene series, allyl halides (3-halopro-
penes) are expected to be much more susceptible to nucleophilic substitution
-------
X
CH2-(
H
H
Text-Figure 1
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of the halogen atom than the corresponding haloalkanes, in both the S^l and
SN2 reactions. The increase in SvJ reactivity can be readily explained by
resonance stabilization of the carbonium ion,
© <§> V® fc®
CH2 - CH - CH2 <—* CH2 - CH = CH2 «—> CH2 .- CH - CH2
The higher S«2 reactivity may be due to stabilization of the transition state
by delocalization of the -H -electrons, lower C-X bond strength, or other
factors. In addition, a modified S»r2 reaction (Sv,2 ), involving nucleophilic
(Nu:) attack on the unsaturated Y-carbon with subsequent shift of double bond
[TEXT-FIGURE 2]
and expulsion of halide ion, may proceed concurrently with the S-^2 reaction.
The chemical reactivity of halobutenes is also expected to be dependent on the
position of the double bond and the halogen atom.
The alkylating activity of a variety of haloalkenes in the NBP reaction
has recently been extensively studied by investigators in Germany (70, 72) and
in the laboratories of the International Agency for Research on Cancer (73,
74). Table IV summarizes the available data. The Table illustrates the fact
that, as expected, vinyl halides are poor alkylators, and metabolic activation
(S-9) is needed to bring to fore the alkylating activity. On the other hand,
allyl halides are good alkylating agents. The alkylating activity of allyl
halide increases in the order: Cl < Br < I (70). In contrast to 3-chloropro-
pene (allyl chloride), the two nonallylic isomers, 1-chloro- and 2-chloropro-
penes, are completely inactive in the NBP reaction. Substitution of a second
chloride atom at the 1-position of allyl chloride (yielding 1,3-dichloro-
8
-------
ICI -CH=CH-CH2-CI
Text-Figure 2
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page 1 of 2
Table IV
Relative Alkylating Activity of Haloalkenes (NBP-Reaction)*
Compound
Wurzburg Study
IARC Study
A) Haloethenes (Haloethylenes)
Vinyl chloride
Vinyl bromide
1,1-Difluoroethylene
1,1-Dichloroe thylene
(Vinylidene chloride)
Trichloroe thylene
0 (0.03 with S9)b»c
0 (0.05 with S9)b'c
0 (with S9)c
0 (with S9)c
0 (with S9)c
B) Halopropenes
1-Chloropropene
2-Chloropropene
3-Chloropropene
(Allyl chloride)^
3-Bromopropene
(Allyl bromide)^
3-Iodopropene
(Allyl iodide)S
3-Chloro-2-me thylpropene^
cis-1,3-DichloropropeneS
trans-1,3-Dichloropropene^
2 > 3-DichloropropeneS
Od
Od
0.285d'e
1.2086
1.828s
0.570d
2.240d
1.933d
0.248d
C) Halobutenes and Halobutadienes
3-Chloro-l-buteneS
4-Chloro-l-butene
3-Chloro-2-methyl-1-buteneS
3,4-Dichloro-l-buteneg
1-Chioro-2-b u teneS
n.a.
d,f
d,f
n.a.
1.035d
0.03C
-------
page 2 of 2
Table IV (continued)
Compound Wurzburg Study IARC Study
2-Chloro-2-butene Od
l-chloro-2-methyl-2-buteneg 2.057d
l,4-Dichloro-2-buteneg 13C
l-Chloro-l,3-butadiene 4.3C
2-Chloro-l,3-butadieneg 0.2 (0.08 with S9)c
aThe alkylating activity of the compound with 4-(p-nitrobenzyl)-pyridine (NBP)
was expressed asAE560nm in the Wurzburg study and AE at^max in the IARC
study. In some cases (as stated), liver 9,000 x g postmitochondrial fraction
(S9) was included in the incubation mixture.
From A. Barbin, H. Bresil, A. Croley, P. Jacquignon, C. Malaveille, R.
Montesano, and H. Bartsch [Biochem. Biophys. Res. Commun. 67, 596 (1975)].
cFrom H. Bartsch, C. Mallaveille, A. Barbin, and G. Planche [Arch. Toxicol.
.41_, 249 (1979)].
From T. Neudecker, D. Lutz, E. Eder, and D. Hsnschler [Biochem. Pharmacol.
_29_, 2611 (1980)].
eFrom E. Eder, T. Nuedecker, D. Lutz, and D. Henschler [Biochem. Pharmacol.
J29_, 993 (1980)].
NBP reaction not applicable due to interference (see text).
gContains allylic structure.
-------
propene) substantially increases the alkylating activity of the compound.
Neudecker e_t _al_. (72) attributed the increase in activity to the chlorine-
induced positive mesomeric (+ M) effect, which exceeds its negative inductive
(-1) effect and weakens the allyl C-C1 bond.
[TEXT-FIGURE 3]
The resulting carbonium ion may be stabilized by resonance. The cis-1,3-
dichloropropene is more active than its trans isomer due to a possible neigh-
boring group effect that is absent in the trans isomer.
[TEXT-FIGURE A]
In contrast to 1,3-dichloropropene, 2,3-dichloropropene is slightly less
reactive than allyl chloride. The positive mesomeric effect is absent when
the chlorine substituent is at the central carbon of the allylic structure.
In this compound, only the negative inductive effect is operative, which
stabilizes the allylic C-C1 bond. Halobutenes with allylic structure (e.g.,
l-chloro-2^-butene) are active in the NBP reaction while those with nonallylic
structure (e.g., 2-chloro-2-butene, 4-chloro-l-butene) are not. Substitution
in the allylic structure by a methyl group increases the alkylating activity
of the haloalkene probably by a combination of positive inductive and hyper-
-------
Nu-CH2-CH =CH2
Text-Figure 3
-------
H
y
(£'
H
e ,.CH2
tr
cr
c H
xc=cx
/ \
H CH2
C!
Text-Figure 4
-------
conjugation effects of the methyl group. • Activation is substantial if methyl
substitution is on carbon-1 or carbon-3 of the allylic structure (e.g.,
compare l-chloro-2-butene with allyl chloride), but relatively smali if methyl
substitution is on the central carbon (e.g., 3-chloro-2-methyl-propene).
3-Chloro-l-butene and 3-chloro-2-methyl-l-butene, which have allylic struc-
tures (Table IV), are expected to be active in the NBP reaction but cannot be
tested because of the reagent-induced dehalo^enation of the compound (72).
Two chlorinated 1,3-butadienes have been tested in the NBP reaction and the
results indicate that alkylating activity is not dependent on the allylic
structure. The vinylic 1-chloro-l,3-butadiene is considerably more reactive
than 2-chloro-l,3-butadiene (chloroprene), which may be considered to have
both an allylic and a vinylic structure.
The chemical properties of benzyl chloride, an arylalkyl halide, resemble
more closely those of haloalkanes and haloalkenes than those of haloaro-
matics. Benzyl chloride is more reactive than allyl chloride in both S^l and
S*j2 type °f reactions (65). Resonance stabilization of the benzyl cation
[TEXT-FIGURE 5}
greatly enhances the Sxrl reactivity of the compound. Benzyl chloride has been
shown to be highly reactive in the NBP reaction (68, 72).
5.2.2.1.2.2 BIOLOGICAL EFFECTS OTHER THAN CARCINOGENICITY.
Toxic Effects. The toxicology of haloalkanes and haloalkenes has-been
studied for decades. There are many reviews and monographs (e.g., 9, 19, 57,
10
-------
©
CH2
=CH2
=CH2
Text-Figure 5
-------
58, 61, 62, 75-77) on this subject. Only a brief discussion emphasizing
comparative toxicity is presented in this section. Some representative acute
toxicity data of haloalkanes and haloalkenes that have been tested for
carcinogenic!ty are summarized in Table V. In general, fluoroalkanes have
very low toxicity unless their bio transformation leads to fluoroacetic acid.
Fluoroalkenes have not been thoroughly studied; however, the available data
suggest that fluoroalkenes are more toxic than fluoroalkanes and should be
more carefully investigated (reviewed in 109). Chlorinated alkanes and
alkenes are substantially more toxic than their fluorinated counterparts. The
..' .v
principal toxic effects in humans and in mammalian animal species are central
nervous system depression (e.g, narcosis), liivet and kidney pathology (je^.g.,
l\ /!' , 'If" '
fatty changes, necrosis, degeneration), and ;wy<&cardial sensitization 'tif> »
UH ' 1?i
endogenous epinephrine (resulting in symptoms s(aoh as ventricular fibrilla-
y/.yl"."
tion). Inhalation exposure to dichloromethane may also result in hypoxia due
to metabolic formation of carbon monoxide which binds to hemoglobin. The
position of chlorine substitution on the alkane may play a significant tole in
determining the toxicity. 1,1,2-Trichloroethane, for example, is considerably
more toxic than its 1,1,1-isoraer.
The toxicity of haloalkanes and haloalkenes may be modified by'a variety
of exogenous and endogenous factors. The toxicity of carbon tetrachloride,
the most extensively studied haloalkane, has been shown to be potentiated by a
long list of chemical and biological factors. Based on their possible
mechanism of action, these factors may be loosely classified as (a) inducers
of mixed-function oxidases (MFO), such as phenobarbital, 3-methylcholanthrene,
polychlorinated biphenyls, polybrominated biphenyls (110-113), (b) conditions
known to deplete tissue glutathione (GSH) level, such as fasting, diurnal
11
-------
Table V
Acute Toxicity of Haloalkanes and Haloalkenes
page 1 of 3
Compound
Species & Route
LD50 or LC
50
Reference
A) Haloalkanes
Chlorome thane
(Methyl chloride)
lodome thane
Dichloromethane
Mouse, inhalation
Rat, inhalation
Mouse, inhalation
Mouse, i.p.
Rat, inhalation
Rat, i.p.
Rat, s.c.
Mouse, inhalation
(Methylene chloride) Mouse, i.p.
Rat, oral
Dog, i.p.
Chloroform
Dichlorobromomethane
Tribromome thane
Triiodome thane
Carbon tetrachloride
1,2-Dichloroe thane
Mouse, inhalation
Mouse, i.p.
Mouse, oral
Mouse, s.c.
Rat, oral
Dog, i.p.
Mouse, oral
Rat, oral
Chlorodibromomethane Mouse, oral
Rat, oral
Mouse, oral
Mouse, s.c.
Rat, oral
Mouse, s.c.
Mouse, inhalation
Mouse, i.p.
Rat, oral
Dog, i.p.
Rat, inhalation
Rat, oral
3,146 ppm for 7 h (61)
152 mg/liter for 30 min (78)
5 mg/liter for 57 min (79)
173 mg/kg (80)
1.3 mg/liter for 4 h (80)
101 mg/kg (80)
110 mg/kg (68)
13,500 ppm for 9-12 h (81)
1.5 ml/kg (82)
2.3 ml/kg (83)
0.95 ml/kg (84)
4,500 ppm for 9-12 h (81)
1.2 ml/kg (82)
1,120 mg/kg (M); (85)
1,400 mg/kg (F)
3,283 mg/kg (78)
908 mg/kg (M); (86)
1,117 mg/kg (F)
1.0 ml/kg (84)
450 mg/kg (M); (85)
900 mg/kg (F)
916 mg/kg (M); (86)
969 mg/kg (F)
800 mg/kg (M); (85)
1,200 mg/kg (F)
1,186 mg/kg (M); (86)
848 mg/kg (F)
1,400 mg/kg (M); (85)
1,550 mg/kg (F)
1,820 mg/kg (78)
1,388 mg/kg (M); (86)
1,147 mg/kg (F)
629 mg/kg (78)
8,500 ppm for 9-12 h (81)
2.7 ml/kg (84)
1.77 ml/kg (87)
1.5 ml/kg (84)
1,000 ppm for 7.2 h (88)
680 mg/kg (89)
-------
Table V (continued)
page 2 of 3
Compound
1 , 2-Dibromoe thane
1,1 ,1-Trichloroe thane
1 ,1 , 2-Trichloroe thane
1,1,2, 2-Te trachloro-
e thane
Halo thane
Hexachloroe thane
1 , 2-Dibromo-3-chloro-
propane
Species & Route
Mouse , oral
Rat, oral
Mouse, inhalation
Mouse, i.p.
Rat, oral
Dog, i.p.
Mouse, inhalation
Mouse, i.p.
Rat, oral
Dog, i.p.
Mouse, i.p.
Rat, oral
Mouse, inhalation
Mouse, i.p.
Rat, oral
Mouse, oral
Rat, inhalation
Rat, oral
LD5Q or LC50a
420 mg/kg
146 mg/kg (M) ;
117 mg/kg (F)
13,500 ppm for 9-12 h
3.8 ml/kg
12,300 mg/kg (M);
10,300 mg/kg (F)
3.1 ml /kg
3,750 ppm for 9-12 h
0.35 ml /kg
0.58 ml/kg
0.45 ml/kg
820 mg/kg
250 mg/kg
22,000 ppra for 10 min
4,500 mg/kg
6,000 mg/kg
257 mg/kg
103 ppm for 8 h
150-370 mg/kg (M) ;
260-620 mg/kg (F)
Reference
(90)
(90)
(81)
(82)
(91)
(84)
(81)
(82)
(92)
(84)
(93)
(94)
(78)
(78)
(78)
(78)
(78)
(95)
B) Haloalkenes
Vinyl chloride
Vinylidene chlorideb
(1,1-Dlchloro-
ethylene)
trans-1,2-Dichloro-
ethylene
Trichloroe thylene
Mouse, inhalation
Rat, inhalation
Mouse, inhalation
Mouse, oral
Rat, inhalation
Rat, oral
Mouse, i.p.
Rat, oral
Rat, i.p.
Mouse, inhalation
Mouse, i.p.
Rat, oral
Dog, i.p.
113,000 ppm for 2 h
150,000 ppm for 2 h
98-105 ppm for 23 h
194-217 mg/kg
10,000-15,000 ppra
for 4 h (fed)
500-2,500 ppm
for 4 h (fasted)
1,550 mg/kg
3.2 ml/kg
1.0 ml/kg
6.0 ml/kg
5,500 ppm for 9-12 h
2.2 mlk
..... ml/kg
4.92 ml/kg
1.9 ml/kg
(96)
(96)
(97)
(98)
(99)
(100)
(lOla)
(lOla)
(lOla)
(81)
(82)
(92)
(84)
-------
page 3 of 3
Table V (continued)
Compound
Te trachloroe thylene
1-Chloropropene
Allyl chloride
( 3-Chloropropene)
1 , 3-Dichloropropene
(cis/ trans mixture)
1 , A-Dichloro-2-bu tene
2-Chloro-l , 3-buta-
diene (Chloroprene)
Hexachloro-1 ,3-buta-
diene
Species & Route
Mouse, inhalation
Mouse, i.p.
Rat, oral
Dog, i.p.
Rat, oral
Rat, oral
Rat, oral
Rabbit, topical
Rat, inhalation
Rat, oral
Rabbit, topical
Rat, inhalation
Rat, oral
Mouse, i.p.
Rat, oral
Rat, i.p.
LD5Q or LC5Qa
3,700 ppm for 9-12 h
2.9 ml/kg
8.0 ml/kg
2.1 ml/kg
1,950 mg/kg
700 mg/kg
713 mg/kg (M);
470 mg/kg (F)
504 mg/kg
86 ppm for 4 h
89 mg/kg
0.62 ml/kg
8.2 mg/liter
(2,280 ppm) for 4 h
670 mg/kg
76 mg/kg
580 mg/kg (M) ;
200-400 mg/kg (F)
190 mg/kg
Reference
(81)
(84)
(lOlb)
(84)
(78)
(102)
(103)
(103)
(78)
(104)
(104)
(105)
(106)
(107)
(108)
(107)
•Median lethal concentration (via inhalation) or median lethal dose (via all
other routes); (M) • male, (F) = female.
^Substantial variation dependent on the condition of animals (see text).
-------
variation (lower GSH at night), diethyl maleate administration (113, 114), (c)
ketones such as acetone, Kepone, methyl _n-butyl ketone, and 2,5-hexanedione
(115 and refs. therein), (d) ketogenic compounds and conditions such as iso-
propanol, 1,3-butanediol, _n-hexane and neonatal or uncontrolled diabetes (115-
118), and (e) agents with unclear mechanisms such as ethanol (115, 116, 119
and refs. therein). On the other hand, free radical scavengers (e.g., di-
ethyldithiocarbamate, propyl gallate, GSH, cystamine), antioxidants (e.g.,
tinoridine), inhibitors of MFO (e.g., SFK 525A), zinc, and cyclohexamide all
exhibit protective effect against carbon tetrachloride toxicity (113, 120-
122). The toxicity of a number of other haloalkanes and haloalkenes such as
chloroform (115, 123-126), vinylidene chloride (97, 127, 128), vinyl chloride
(129-131) is also subject to modifiers in an essentially similar manner as
carbon tetrachloride. However, there is some evidence that the toxicity of
methylene chloride, 1,1,1-trichloroethane, trichloroethylene, and tetrachloro-
ethylene is not potentiated in certain target organs by some inducers of MFO
(110, 112).
Mutagenic Effects. The mutagenicity of haloalkanes and haloalkenes has
been extensively studied in a variety of test organisms including bacteria
(e.g., 132-136; see also Ames Salmonella test described below), yeasts (137-
144), Neurospora (145, 146), higher plants (133, 147, 148), Drosophila (15,
136, 149-152), cultured mammalian cells (133, 137, 153-156), and experimental
animals (136, 157-161). A number of studies have been undertaken to evaluate
the potential mutagenic effects of vinyl chloride and other haloalkanes and
haloalkenes in humans (reviewed in 2, 23, 29, 162). The following discussion
focuses only on mutagenicity studies using the Ames Salmonella test, which has
been widely employed for the pre-screening of chemical carcinogens.
12
-------
Close to 80 haloalkanes and haloalkenes have been tested in the Ames
Salmonella test. The results of these studies are summarized in Tables VI and
VII. With few exceptions, mutagenic haloalkanes and haloalkenes induce muta-
tion predominantly in base-pair substitution mutants (TA100, TA1535). Only
dichloromethane (163), trichloroethylene (164), 1,1-difluoro-2-bromo-2-chloro-
ethylene (165), 1,3-dichloropropene (166), and 1,2,3,3-tetrachloropropene
(167) display some activity in the frame-shift mutants (TA98, TA1537, TA1538,
or TA1978); in all these cases, the activity toward frame-shift mutants is
substantially lower than that toward base-pair substitution mutants. Por this
reason, many of the Ames tests of haloalkanes and haloalkenes are carried out
using TA100 and TA1535 only.
It should be emphasized that a number of factors must be considered in
the Interpretation of mutagenicity data to avoid erroneous conclusions or
false negatives: (a) Most low-molecular-weight haloalkanes and haloalkenes
are highly volatile and therefore may be lost through evaporation in the
standard Ames test. These compounds must be assayed in tightly closed
containers to ensure that the bacteria are actually exposed to the level of
the chemical added, (b) Some compounds (e.g., allyl chloride) display greater
mutagenic activity in suspension assays, in which the chemical is preincubated
with the bacteria before plating. The suspension assay should be employed if
results from standard assays and assays in closed containers are inconclu-
sive, (c) Some dihalomethanes (e.g., dibromomethane, diiodomethane) and
1,2-dihaloethanes (e.g., 1,2-dichloroethane, 1,2-dibromoethane) are metabolic-
ally activated by cytosolic enzymes (S-100 or S-115 fractions) as well as by
the microsomal (S-9) fraction (see also Section 5.2.2.1.4). For the 1,2-di-
haloe thanes, cytosol is actually the better source of activating enzymes.
Suspension assays with the inclusion of cytosol should be used for compounds
13
-------
page 1 of 3
Table VI
Comparative Mutagenicity of Haloalkanes in the Ames Salmonella Test
Mutagenicity
Compound
System3 Without Activation0
With Activationd
A) Hal oine thanes
Chloromethane (methyl chloride)
Broraomethane (methyl bromide)
lodomethane (methyl iodide)
Fluorochlorome thane
Dichloromethane (methylene
chloride)
Bromochlorome thane
Dibromomethane
Diidome thane
Difluorochlorome thane
Trichloromethane (chloroform)
Bromodichlorome thane
Dibromochlorome thane
Tribromomethane (bromoform)
Difluorodichlorome thane
Fluoro trichlorome thane
Tetrachloromethane (carbon
tetrachloride)
Enclosed
Enclosed
Standard
Enclosed
Unspecified
Standard
Enclosed
Enclosed
Enclosed
Suspension
Enclosed
Standard
Enclosed
Suspension
Standard
Enclosed
Standard
Enclosed
Standard
Enclosed
Enclosed
Suspension
Standard
Enclosed
Suspension
+ (168-170)
+ (168, 169)
+ (171)
+ (168, 169)
+ (172)
- (167)
+ (163, 167-169)
+ (168, 169)
+ (168, 169)
-I- (173)
Suspension + (173)
+ (174)
- (167, 168)
- (168, 169)
- (168, 175)
- (168)
+ (168, 169)
- (168)
+ (168, 169)
- (168)
+ (168, 169)
- (174)
- (175)
- (167, 168, 171)
- (168, 169)
- (175)
+ (168+, 169, 170)
n. t.
n. t.
n.t.
-1- (17 2+)
- (167)
n. t.
n. t.
+ (173"*") (S9
or S100)
+ (173"1") (S9
or S100)
+ (174)
- (167)
- (169)
- (175)
n. t.
n. t.
n. t.
n. t.
n. t.
n.t.
- (174)
- (175)
- (167, 171)
- (169)
- (175)
-------
page 2 of 3
Table VI (continued)
Mutagenicity
Compound
B) Hal oe thanes
1,1-Dichloroe thane
1,1-Dibromoe thane
1,2-Dichloroe thane (ethylene
dichloride)
l-Bromo-2-chloroe thane
1,2-Dibromoe thane (ethylene
dibromide)
1,1,1-Trichloroe thane
(methyl chloroform)
1,1, 2-Trichloroe thane
1,1,1, 2-Te trachloroe thane
1,1,2, 2-Te trachloroe thane
l,l,l-Trifluoro-2-bromo-2-
chloroe thane (halo thane)
Trif luoro trichloroe thane
Hexachloroe thane
C) Halopropanes
1-Chloropropane
2-Chloropropane
1 , 2-Dichloropropane
1 , 2-Dibromopropane
Sys tern3
Standard
Standard
Standard
Enclosed
Suspension
Standard
Standard
Suspension
Standard
Enclosed
Standard
Standard
Standard
Enclosed
Enclosed
Suspension
Standard
Standard
Suspension
Enclosed
Standard
Standard
Without Activation0
- (168)
wf (132)
- (136, 167, 176)
wf (132, 177, 178)
wf (167)
- (179)
+ (132)
+ (132, 171, 177,
178, .180)
+ (181)
- (167)
wf (167, 168)
- (168, 178)
- (168)
- (167)
wf (132)
- (167)
- (182)
- (175, 182)
- (168)
- (cited in ref.
144)
- (70)
+ (168)
- (176); + (166)
+ (176)
With Activationd
n. t.
n. t.
- (136, 167, 176)
wf (177)
wf (167)
- (179) (M);
+ (179+) (S100)
n. t.
+ (177+, 178+, 180)
+ (181") (M);
+ (181 ) (S100)
- (167)
wf (168)
- (178)
n. t.
- (167)
n. t.
- (182)
- (175, 182)
n.t.
- (cited in ref.
144)
- (70)
+ (168+)
- (176); + (166)
+ (176+)
-------
page 3 of 3
Table VI (continued)
Mutagenicity
Compound
1 , 3-Dichloropropane
1 , 3-Dib romopropane
1,2, 3-Trichloropropane
1 , 2-Dibromo-3-chloropropane
(DBCP)
1 , 2, 3-Trib romopropane
D) Higher Haloalkanes
1-Chlorobutane (n-butyl chloride)
1-Bromobutane (n-butyl bromide)
l-Bromo-2-methyl propane
(i-butyl bromide)
2-Broraobutane (s -butyl bromide)
l,2-Dibromo-2-methylpropane
1 , 5-Dib r omopen tane
System3 Without Activation0
, Standard
Standard
Standard
Standard
Standard
Suspension
Enclosed
Enclosed
Enclosed
Standard
Standard
+ (176)
± (176)e
± (176)
- (176, 183);
+ (180)
+ (176, 180)
- (70)
+ (168)
- (168)
+ (168)
+ (132)
+ (132)
With Activationd
+ (176)e
+ (176)e
+ (176+)
+ (176+, 180+, 183+)
+ (176+, 180)
- (70)
n. t.
n. t.
n. t.
n. t.
n. t.
a"Standard" refers to standard Ames Salmonella tests (plate incorporation or spot test);
"Enclosed" refers to Salmonella tests carried out in desiccators or closed glass
containers; "Suspension" refers to modified Salmonella tests in which bacteria and test
chemical were pre-incubated in liquid suspension before plating.
Mutagenicity in base-pair substitution mutants (TA100, TA1535): "+" = positive;
"w+" = weakly positive; "_+" = marginal; "-" = negative; n.t. = not tested.
-cWithout added mammalian activation system.
Unless specified, the activation system was liver postmitochondrial fraction (S9) plus
cofactors. "S100" or "S115" refers to cytosolic fraction plus glutathione. "M" refers
microsomal fraction plus cofactors. A superscript "+" or "-" sign over the reference
number denotes an increase or a decrease in mutagenicity by the inclusion of the
activation system, respectively.
eResults obscured by the cyto toxic effects of the chemical.
-------
;a^
page 1 of 3
Table VII
Comparative Mutagenicity of Haloalkenes in the Ames Salmonella Test
Mutagenicity
Compound
A) Haloethenes (Haloethylenes)
Vinyl chloride
Vinyl bromide
1 ,1-Difluoroe thylene
1 ,1-Dichloroe thylene
(Vinylidene chloride)
cis-1 , 2-Dichloroe thylene
trans-1 , 2-Dichloroe thylene
Trichloroe thylene
Te trachloroe thylene
1 ,1-Dif luoro-2-bromo-2-
chloroe thylene
B) Halopropenes
l-Chloropropenee
l-Bromopropenee
2-Chloropropenee
2-Bromopropenee
Sys tern3
Enclosed
Enclosed
Enclosed
Enclosed
Standard
Standard
Standard
Enclosed
Suspension
Standard
Enclosed
i
Suspension
Enclosed
Suspension
Standard
Suspension
Standard
Without Activation0
+ or w+ (74, 168,
170, 174, 177,
184-187)
+ (74)
- (74)
- (74, 168, 188)
+ (182)
- (164, 168)
- (164, 168)
+ (164)
w+ (168)
- (74, 182)
- (182)
+ (164)
- (74)
+ (165)
+ (168, 169)
- (72)
+ (176)
- (72)
+ (176)
With Activationd
+ (74+, 168+, 170+,
174+, 177+, 184-186+
187)
+ (74+)
w+ (74+)
+ (74+, 168+, 182+,
188+)
n. t.
n. t.
n. t.
+ (168+); xrt- (74+) ;
- (182)
- (182)
n. t.
- (74)
+ (165)
+ (168-)
- (72)
+ (176~)
- (72)
± (176")
-------
page 2 of 3
Table VII (continued)
Mutagenicityb
Compound
3-Chloropropene (Allyl chloride )f
3-Bromopropene (Allyl bromide)
3-Iodopropene (Allyl iodide)
3-Chloro-2-me thylpropene
1 ,3-Dichloropropenee ' )g
cis-isomer
trans-isomer
unspecified
2 , 3-Dichloropropenee ' f
2 , 3-Dib romopr opene6 '
1,2, 3-Tr ichloropropene6 » f
1 , 2 j 3 , 3-Te trachloropropene6 » f
1,1,2,3, 3-Pen tachloropropene6 » f
C) Halobutenes and Halobutadienes
3-Chloro-l-butene
4-Chloro-l-butene
3-Chloro-2-methyl-l-butenef
3 , 4-Dichloro-l-bu tenef
Sys tern3
Standard
Enclosed
Suspension
Suspension
Suspension
Suspension
Standard
Suspension
Standard
Suspension
Standard
Standard
Suspension
Standard
Standard
Standard
Standard
Suspension
Suspension
Suspension
Enclosed
Without Activation0
- (140, 166);
+ (189)
+ (169)
+ (70, 72, 140)
+ (70)
+ (70)
+ (72)
+ (166)
+ (72)
+ (166)
+ (72)
+ (176)
+ (166, 176)
+ (72)
+ (176)
+ (176)
+ (167)
+ (167)
+ (72)
- (72)
+ (72)
+ (74)
With Activationd
- (140, 166);
+ f T Q Q^^ \
1 A. O -^ )
n.t.
- (70", 72~);
+ (140)
- (70")
- (70")
+ (72-)
+ (166)
+ (72")
+ (166)
+ (72")
+ (176~)
+ (166, 176)
+ (72+)
+ (176~)
+ (176+)
» \ JL D / /
i \ J. D / )
+ (72")
- (72)
n. t.
+ (74+)
-------
«6^>i^
' page 3 of 3
Table VII (continued)
Mutagenicity
Compound
l-Chloro-2-bu tenef
2-Chloro-2-butenee
l-Chloro-2-methyl-2-butenef
l,3-Dichloro-2-butenee>f
l,4-Dichloro-2-butenef
1-Chloro-l ,3-butadienee
2-Chloro-l , 3-bu tadiene6 ' f
Hexachloro-1 , 3-bu tadiene6 ' ^
Sys tema
Suspension
Suspension
Suspension
Standard
Standard
Enclosed
Enclosed
Suspension
Without Activation0
+ (72)
- (72)
+ (72)
- (166)
+ (74)
+ (74)
+ (74)
+ (169)
With Activationd
+ (72")
+ (72+)
+ (72")
- (166)
+ (74+)
+ (74+)
+ (74+)
+ (169)
a"Standard" refers to standard Ames Salmonella tests (plate incorporation or spot test);
"Enclosed" refers to Salmonella tests carried out in desiccators or closed glass
"containers; "Suspension" refers to modified Salmonella tests in which bacteria and test
chemical were pre-incubated in liquid suspension before plating.
bMutagenicity in base-pair substitution mutants (TA100* TA1535): "+" = positive;
"w+" = weakly positive; "+" = marginal; "-" = negative; n.t. = not tested.
cWithout added mammalian activation system.
In most of these studies, the activation system was liver postmitochondrial fraction (S9)
plus cofactors. A superscript "+" or "-" sign over the reference number denotes an
increase or a decrease in mutagenicity by the inclusion of the activation system,
respectively.
eContains a vinylic structure.
Contains an allylic structure.
SA purified sample of this compound was reported to be nonmutagenic [R.E. Talcott, The
Toxicologist 1, 41 (1981)].
-------
with similar structure, (d) Many haloalkenes are actually more mutagenic in
the absence of S-9 fraction that in its presence. The mutagenicity of allyl
halides (3-halopropenes) in suspension assay, for example, is completely
abolished if S-9 fraction is included, (e) The results of some mutagenicity
assays may be obscured by the cyto toxic effect of the chemical (e.g., 1,3-di-
chloropropane, 1,3-dibromopropane). These considerations suggest that some of
the negative findings summarized in Tables VI and VII should be further inves-
tigated before firm conclusions can be made.
The data available lead to some interesting structure-activity relation-
ships for mutagenicity of haloalkanes and haloalkenes. Comparison of the
relative mutagenic potency of various compounds must be limited to the same
laboratories because of inter-laboratory variations and differences in experi-
mental procedures. Among the mutagenic haloalkanes (Table VI), the compounds
are either active as such or after metabolic activation. In the fluorochloro-
methane series both tetrahalomethanes (CF2Cl2> CFC1.,) are inactive whereas the
trihalomethane (CHF^Cl) and dihalomethane (CH-FCl) show some mutagenic
activity. In the chloromethane series, there is some evidence that mutagenic-
ity declines with an increase in chlorination. Despite extensive studies (see
Table VI), carbon tetrachloride and chloroform (which are carcinogenic) have
been consistently found inactive in the Ames test. On the other hand, all
three monohalomethanes (CH^Cl, CHoBr, CHoI) are active in the Ames test. The
mutagenicity of chloromethane is enhanced by metabolic activation by S-9
fraction. All five dihalomethanes are mutagenic. Of the 4 dihalomethanes
tested, inclusion of S-9 fraction increases the mutagenicity of the
compounds. Consistent with their relative chemical reactivity and metabolic
rate, the relative mutagenic potency of bromo- and chloro- compounds in the
study of Simmon (169) follows the order: CIUBr, > CH-BrCl > CH2C12. Dibromo-
14
-------
and diiodomethanes are activated by microsomal as well as by cytosolic
enzymes. Cytosolic glutathione S-transferase is believed to be the activating
enzyme for the dihalomethanes. In contrast to the higher chemical reactivity
and metabolic rate, diiodomethane appears to be less mutagenic than dibromo-
methane. Van Bladeren _e_t _al_. (173) suggested that the reactive intermediate
from diiodomethane may be too reactive to reach target macromolecules (i.e.,
it may be scavenged by water or genetically unimportant nucleophiles). In the
trihalomethane group, chloroform has been consistently found to be nonmuta-
genic in a variety of test conditions. Substitution of either one or two
chlorine atoms by either bromine or fluorine yields mutagenic compounds
(detected by testing in the closed system). In the tetrahalomethane group,
none of the three compounds tested demonstrated any mutagenic activity.
Carbon tetrachloride, in particular, has been tested in various systems.
Twelve haloethanes have been tested in the Ames test. With the exception
of 1,2-dihaloethanes, haloethanes are either nonmutagenic or weakly muta-
genic. 1,2-Dibromoethane has been found to be the most potent mutagen of the
group either in the presence or the absence of mammalian activation system.
The mutagenicity of 1,2-dibromoethane may be substantially reduced by substi-
tution with chlorine; thus, the mutagenicity of dihaloethane decreases in the
order: 1,2-dibromo > l-bromo-2-chloro > 1,2-dichloro (132). This is consis-
tent with the higher reactivity of bromo compounds as compared to chloro
compounds. The position of the halogen may also affect the mutagenicity:
1,2-dibromoethane is considerably more potent than its 1,1-isomer (132).
1,2-Dihaloethanes appear to be more effectively activated by cytosolic than by
microsomal enzymes. Guengerich ^_t _al_. (179) showed that cytosol does activate
1,2-dichloroethane in suspension assay, while microsomes do not. Van Bladeren
^_t_al_. (173) demonstrated that the inclusion of microsomes actually decreases
15
-------
the mutagenicity of 1,2-dibromoethane in suspension assay; on the other hand,
cytosol increases the mutagenicity of the compound.
A comparative study of the relative mutagenicity of seven halopropanes .
has been carried out by Stolzenberg and Hine (176). The activities follow the
order: 1,2,3-tribromo > 1,2,3-trichloro > l,2-dibromo-3-chloro > 1,3-dichloro
> 1,2-dibromo > inactive 1,2-dichloro. 1,3-Dibromopropane is also mutagenic
but its mutagenicity is masked by its cytotoxicity. Blum and Ames (180) have
shown that 1,2,3-tribromopropane is more mutagenic than 1,2-dibromo-3-chloro-
propane. Only limited data are available to compare the mutagenicity of
higher haloalkanes. In the study by Brem £_t__al_. (132), 1,5-dibromopentane was
as potent as 1,2-dibromoethane which, in turn, is more potent than 1,2-di-
bromo-2-methylpropane.
The mutagenicity of haloalkenes (Table VII) is determined by the posi-
tion^) and the number, as well as the nature, of halogen subs titutent. In
general, there is a good correlation between the alkylating activity (see
Table IV) and the mutagenicity of haloalkenes, although exceptions have been
noted. Haloalkenes with vinylic structure are either inactive or weak
mutagens without activation; inclusion of an S-9 activation system enhances
substantially the mutagenicity of most of these compounds. In contrast, most
haloalkenes with allylic structure are mutagenic as such and the inclusion of
S-9 tends to decrease rather than increase the mutagenicity.
In the haloethene series, vinyl chloride is the most extensively studied
compound. Vinyl chloride is a relatively weak mutagen, but its mutagenicity
is substantially enhanced by the inclusion of an S-9 activation system.
Bartsch _e_£_al^. (74) have undertaken an extensive study of the mutagenicity of
10 haloalkenes; the relative potency of 6 haloethenes (with metabolic activa-
16
-------
tion) follows the order: vinyl bromide > 1,1-dichloroethylene > vinyl
chloride > trichloroethylene and 1,1-difluoroethylene (which are weakly or
marginally active) > tetrachloroethylene (inactive). Compared to the 4 halo-
butenes and halobutadienes tested in the same study, the haloethenes are
significantly less mutagenic (74). Using Escherichia coli as the test
organism for mutagenicity, Henschler and associates (135, 190-192) postulated
a rule which predicts that chlorinated ethenes with urisymmetric substitution
(vinyl chloride, 1,1-dichloroethylene, trichloroethylene) are mutagenic,
whereas those with symmetric substitution (1,2-dichloroethylene, tetrachloro-
ethylene) are not (see also Section 5.2.2.1.4.2.1). This rule is partially
supported by results obtained using the Ames test (see Table VII). However,
exceptions to this rule, such as the lack of mutagenicity of trichloroethylene
(182) and the positive result with tetrachloroethylene (164), have been
observed by some investigators. In addition, the mutagenicity of cis- and
trans-1,2-dichloroethylene has not been adequately tested. 1,1-Difluoro-2-
bromo-2-chloroethene, a presumed metabolite of halo thane, appears to be an
unusual haloethene; it is mutagenic without metabolic activation (165).
In the halopropene series, a very good correlation between alkylating and
mutagenic activities of at least 9 compounds has been noted by Eder et al.
(70) and by Neudecker e_t_ al. (72). In agreement with the expected order of
relative chemical reactivity, the mutagenic potency (with and without meta-
bolic activation) of allyl halides follows the order: iodide > bromide »
chloride (70). Similarly, the relative order of potencies (without metabolic
activation) —cis-1,3-dichloro- > trans-1,3-dichloro- » 3-chloro-2-methyl- >
2,3-dichloro- > 3-chloro- > inactive 2-chloro- and 1-chloropropenes — is
consistent with their relative alkylating activity (72). All the mutagenic
halopropenes listed above have allylic structure and are mutagenic as such.
17
-------
The inclusion of an S-9 system reduces the mutagenicity of all the halopro-
penes with the exception of 2,3rdichloropropene, which contains a vinylic as
well as an allylic structure and whose mutagenicity may be increased by more
than 30-fold by metabolic activation (72). The mutagenicity of brominated
halopropenes appears to be less predictable from the chemical structure. The
data of Stolzenberg and Hine (176) suggest the relative order of potencies
(without metabolic activation): 2,3-dibromo- > 1-bromo- > 1,3-dichloro- >
2,3-dichloro- 2. 2-bromo- > 1,2,3-trichloropropene. Mammalian S-9 enhances the
mutagenicity of 1,2,3-trichloropropene, has little effect on 2,3-dichloro-
propene, and greatly reduces the mutagenicity of the other compounds. The
potent direct-actiiig mutagenicity of 1-bromo- and 2-bromopropenes is somewhat
,'lj
unexpected from their vinylic structure.
' ,1
In the halobutene group, the correlation between direct-acting mutageni-
city and allylic structure is also quite evident. In the study of Neudecker
et al. (72), the mutagenic potency of 6 halobutenes follows the order (in the
absence of metabolic activation): l-chloro-2-butene > 3-chloro-l-butene >
2-methyl-3-chloro-l-butene _>. l-chloro-2-methyl-2-butene; 2-chloro-2-butene and
4-chloro-l-butene are inactive. The four mutagenic compounds have allylic
structures. The vinylic type compound, 2-chloro-2-butene, may be metabolic-
ally activated whereas 4-chloro-l-butene is inactive even in the presence of
the S-9 system. No good correlation between mutagenicity and alkylating acti-
vity of the four halobutenes and halobutadienes was observed by Bartsch et al.
(74). The relative potencies follow the order: 3,4-dichloro-l-butene >
l,4-dichloro-2-butene > 1-chlorobutadiene > 2-chlorobutadiene (without meta-
bolic activation). 3,4-Dichloro-l-butene has a very weak alkylating activity
in NBP reaction whereas 1,4-dichloro-2-butene and 1-chlorobutadiene are very
strong alkylating agents. The mutagenicity of the four compounds is enhanced
v
18
-------
by the S-9 fraction, irrespective of whether the structure is allylic or
vinylic. With metabolic activation, 1,4-dichloro-2-butene becomes the most
potent mutagen of the group.
The mutagenicity of benzyl chloride has been tested by Neudecker et al.
(72). Consistent with its potent alkylating activity, the compound is a
strong, direct-acting mutagen. Neudecker _e_t_al_. (72) have pointed out that
benzyl chloride may be considered to have an allylic structure.
Teratogenic Effects. Compared to mutagenicity and carcinogenicity, the
teratogenicity of haloalkanes and haloalkenes has not been extensively inves-
tigated. A number of these compounds are being actively studied at the time
of this writing.
Among halomethanes, dichloromethane (methylene chloride), chloroform, and
carbon tetrachloride have been tested. Dichloromethane has been assayed in
Sprague-Dawley rats and Swiss Webster mice by Schwetz _e_t _al_. (193) and in
Long-Evans rats by Hardin and Manson (194). In the former investigation
rodents were exposed to an atmosphere containing 1,250 ppm dichloromethane for
7 h/day on days 6-15 of gestation, while in the latter study, rats were
exposed to 4,500 ppm 6 h/day before and during gestation. Neither study
revealed any statistically significant teratogenic effects. However, a
critical review of these studies by U.S. Environmental Protection Agency (3)
revealed methodological shortcomings which could cast doubt on the conclu-
sion. There is evidence that inhalational exposure of rats to dichloromehane
may induce behavioral changes in the offspring, as exhibited by the altered
rates of behavioral habituation to novel environments (195).
Chloroform has been tested by inhalational and oral routes. Schwetz et
al. (196) exposed Sprague-Dawley rats to air containing 30, 100, or 300 ppm
19
-------
chloroform for 7 h/day on days 6-15 of gestation. The compound was found to
be fetotoxic and teratogenic; the highest dose caused a significant increase
in fetal resorption. and the conception rate was only 15% (compared to 88% in
the control group). Exposure to 100 ppm chloroform led to significant
increases in the incidence of malformations including acaudia, imperforate
anus, and missing ribs, while exposure to the lowest dose elicited delayed
skull ossification and wavy ribs. Thompson j_t _al_. (197) administered daily
oral doses of 20, 50, or 126 mg/kg to rats or 20, 35, or 50 mg/kg to rabbits
on days 6-15 or 6-18 of gestation, respectively; no significant teratogenic or
embryocidal effects were observed.
Carbon tetrachloride has no teratogenic effects in Sprague-Dawley rats
exposed to 300 or 1,000 ppm of the compound for 7 h/day on days 6-15 of gesta-
tion (198). The compound was, however, slightly embryotoxic, inducing some
degree of retarded fetal development, such as delayed ossification of
s ternebrae.
A number of haloethahes have been tested for teratogenicity in mammalian
species. 1,1-Dichloroethane is not teratogenic in Sprague-Dawley rats after
inhalational exposure to 3,800 or 6,000 ppm for 7 h/day on days 6-15 of gesta-
tion (198). 1,2-Dichloroethane is currently being tested in ICR Swiss mice by
administration in drinking water in a multi-generation study (199). 1,2-Di-
bromoethane was tested in Charles River CD rats and CD-I-mice by inhalational
exposure at concentrations of 20, 38, and 80 ppm for 23 h/day on days 6-15 of
gestation. The compound had little primary effect on fetal development and
was not considered teratogenic by the authors (200).
The teratogenicity of 1,1,1-trichloroethane has been tested by several
groups of investigators. In the study of Schwetz et al. (193), the incidences
20
-------
of fetal anomalies in Sprague-Dawley rats and Swiss Webster mice exposed to an
atmosphere containing 875 ppm of the compound (7 h/day on day 8-15 of gesta-
tion) were not sigificantly higher than the control values. However, the
study was considered inconclusive by the U.S. Environmental Protection Agency
(201) owing to methodological shortcomings. In a preliminary communication,
York jet_£l_. (202) reported that 1,1,1-trichloroethane did not induce signifi-
cant teratogenic effects (neither structural nor behavioral) in Long-Evans
rats continuously exposed by inhalation of 2,100 ppm of the haloethane before
or during gestation. The teratogenic potential of 1,1,1-trichloroethane is
also being investigated in ICR Swiss mice by administration in the drinking
water in a multi-generation study (199).
The teratogenicity of halothane (1,1,1-trifluoro-2-bromo-2-chloroethane)
has been extensively studied. Basford and Fink (203) exposed Sprague-Dawley
rats to an anesthetic concentration (0.8%) of halothane for a prolonged period
of time (12 h) on various days of gestation. Significant increases in the
incidence of skeletal malformations were observed following exposure on days 8
and 9 of gestation. Even short periods (3 h) of anesthesia with halothane (1
or 1.5%) during organogenesis period were teratogenic in C-57B1 mouse,
inducing cleft palate, limb hematomas, and ossification defects (204). How-
ever, in Charles River albino rats and New Zealand albino rabbits, brief
exposures (1 h/day for 5 days) to anesthetic concentrations (1.35-1.43% for
rats; 2.16-2.30% for rabbits) of halothane on days 1-5, 6-10, or 11-15 of
gestation did not bring about significant teratogenic effects (205). Neither
did prolonged exposures of Sprague-Dawley rats on days 8-12 of gestation to
subanesthetic concentrations (50 to 3,200 ppm) of halothane produce no terato-
genic effects (206). However, ultrastruetural study of the neonatal brain
tissues of rats continuously (40 h/week) exposed to 10 ppm halothane through-
21
-------
out gestation revealed central nervous system damage including focal
weakening, vacuoles, myelin figures, and occasional neuronal necrosis. The
authors (207, 208) suggested that these anomalies could contribute to
behavioral changes and poorer learning ability in the offspring.
Hexachloroethane has been tested in Sprague-Dawley rats by inhalation
(15, 48, or 260 ppm) and gavage (50, 100, or 500 mg/kg) from day 6 through day
16 of gestation. The highest dose by each route caused slight maternal
toxicity as shown by slight to moderate tremors. There were no significant
skeletal or soft tissue anomalies in fetuses indicating a lack of terato-
genicity of the compound. Doses that were maternally toxic elicited slight
retardation of fetal development (14A).
Relatively little information is available on the teratogenicity of
haloalkenes. Vinyl chloride has no significant teratogenic effects in CFY
rats exposed to air containing 4,000 mg/m of the compound between the 8th and
14th days of gestation (209, 210). Its lack of teratogenicity was also
reported by John _et_ £l_. (211), who exposed CF-1 mice, Sprague-Dawley rats, and
New Zealand white rabbits to an atmosphere containing 500 ppm vinyl chloride
for 7 h/day during the period of organogenesis. Vinylidene chloride has been
tested in Sprague-Dawley rats and New Zealand rabbits by inhalation (20, 80,
or 160 ppm) or by ingestion (200 ppm in drinking water) in the study of Murray
_e_t_al_. (212). At doses which were not maternally toxic, no teratogenic
effects were noted. Trichloroethylene and tetrachloroethylene were tested in
Sprague-Dawley rats and Swiss Webster mice by Schwetz et _al_. (193). Exposure
of the rodents to 300 ppm of either compound for 7 h/day during the period of
organogenesis caused no maternal toxicity, fetotoxicity, or teratogenic
effects. This study alone is probably inadequate to fully assess the terato-
genic potential of trichloro- and tetrachloroethylene. Soviet researchers
22
-------
have extensively studied the various health effects .of 2-chloro-l ,3-butadiene
t
(chloroprene). The compound was found embryotoxic and teratogenic in rats by
gastric intubation at 0.5 mg/kg or by inhalation at 1.0 ppm (213). However,
this finding was not confirmed by Culik e_t al_. (214), who failed to observe
significant teratogenic effects in rats exposed to up to 25 ppm of the
compound.
5.2.2.1.3 Carcinogenicity and Structure-Activity Relationships.
5.2.2.1.3.1 OVERVIEW.
Since the discovery of the carcinogenicity of carbon tetrachloride and
chloroform in the early 1940's, some 40-50 haloalkanes and haloalkenes have
been tested for carcinogenic activity. Actually, many of these studies were
conducted in recent years in response to the concern which arose from the
potent carcinogenicity of vinyl chloride in humans.
Although at least 24 haloalkanes have been investigated in long-term
carcinogenicity studies, there is no firm evidence for or against carcinogenic
activity. Among the halomethanes, only carbon tetrachloride and chloroform
are carcinogenic in rodents by oral administration. lodomethane, an active
alkylating agent, is a locally active carcinogen. There is only a preliminary
evidence for the carciongenicity of chlorofluoromethane. If confirmed, the
generally held assumption of inactivity of fluorinated alkanes may have to be
reassessed. Seven other halomethanes have been tested and found to have no
convincing evidence for carcinogenicity; however, with the exception of tri-
idomethane (iodoform), most of these studies are either equivocal or incom-
pletely reported.
The most significant finding among studies of haloethanes is the potent
carcinogenicity of 1,2-dibromoethane (ethylene dibromide). This compound is
23
-------
carcinogenic by topical, oral, or inhalational route. The closely related
1,2-dichloroethane is also carcinogenic (although less potent) by topical or
oral administration, but appears to be inactive via inhalation. Besides
1,2-dichloroethane, 6 other chlorinated ethanes have been investigated. The
majority of these compounds have been shown to be carcinogenic in B6C3F. mice
but inactive in Osborne-Mendel .or F344 rats. All the carcinogenic chlorinated
ethanes appear to have a structural similarity — they are all 1,2-disubsti-
tuted (i.e., they have at least one halogen substitution on both carbons).
The evidence for noncarcinogenicity of the geminally substituted (1,1-di-, and
1,1,1-tri-) chlorinated ethanes cannot be considered conclusive because of the
high incidence of early mortality. .,•
/I'll
1,2-Dibrorao-3-chloropropane (DBCP) is probably thjei flnost potent carcino-
Hi
genie haloalkane thus far discovered. The compound is! cuijrcinogenic by
topical, oral, or inhalational route, inducing malignant tumors with a high
incidence. Significant carcinogenic effects were observed in rodents exposed
to as little as 0.6 ppm DBCP in the air or 3 mg/kg/day in the diet. The
potent carcinogenic!ty of DBCP is consistent with the structural requirement
of 1,2-dihalogenation observed in haloethanes.
Eleven haloalkenes have thus far been tested in long-term studies. The
well-known carcinogenic!ty of vinyl chloride has been established in at least
16 different studies (mostly by inhalation). The compound induces a variety
of tumors in different strains of mice and rats, in the hamster, and in the
rabbit. Inhalation appears to be the most effective route. Atmospheric
concentration of 5 ppm of vinyl chloride is significantly carcinogenic in the
rat. The closely related vinyl bromide is also active. Among the other
chlorinated ethenes, vinylidene chloride (1,1-dichloethylene) was found to be
a relatively weak carcinogen in early studies. However, more recent invest!-
s
24
-------
gations do not provide convincing evidence for its carcinogenicity.
Similarly, trichloroethylene has been shown to be carcinogenic in two studies
but inactive in 7 other studies; the role of possible impurities present in
the samples of trichloroethylene used in these studies has not been thoroughly
investigated. Technical-grade tetrachloroethylene was reported to induce
hepatocellular carcinomas in mice after oral administration but is tentatively
considered inactive in the rat. Considerably less information is available on
higher haloalkanes. A preliminary report suggests the carcinogenicity of
trans-1,4-dichloro-2-butene toward the nasal cavity after inhalation.
2-Chloro-l,3-butadiene (chloroprene) appears to be noncarconigenic whereas
hexachloro-1,3-butadiene is carcinogenic at high doses.
To provide a bird's eye view of the relative carcinogenic potency of
various haloalkanes and haloalkenes, the mouse bioassay data of U.S. National
Cancer Instiute, and the skin carcinogenesis data of Van Duuren and coworkers
(215-217) are summarized in Tables VIII and IX, resepctively. It can be
assessed from the data in Table VIII that, on a molar basis, 1,2-dibromoethane
and l,2-dibromo-3-chloropropane are the most potent carcinogens, followed by
1,1,2,2-tetrachloroethane, chlorofrom, 1,1,2-trichloroe thane, and carbon
tetrachloride. Among chloroethanes, 1,1,2,2-tetrachloroethane is most potent;
the potency tends to diminish with either an increase or a decreae in chlori-
nation. When tested by the topical route (Table IX), all four 1,2-disubs ti-
tuted haloalkanes are carcinogenic, inducing "distant" tumors in the lung or
forestomach; only 1,2-dibromoe thane is locally active. The induction of
"distant" tumors may be ascribed to skin absorption or ingestion, through
animal grooming, of the haloalkanes. In two-stage skin carcinogenesis
studies, only l,2-dibromo-3-chloropropane, vinylidene chloride and allyl
chloride are active as tumor-initiators. Among compounds tested by subcu-
25
-------
i or j
Table VIII
Comparative Carcinogenic!ties of Haloalkanes and Haloalkenes in B6C3FJ Mice by Oral Administration0
Incidence (%)
Compound
A) Haloalkanes
Chloroformb
lodoform
Carbon tetrachloride
Trichlorof luorome thane
1 ,1-Dichloroe thane
1 ,2-Dichloroethaneb
N
1 , 2-Dibromoe thane
Sex
M
F
M or F
M
F
M or F
M
F
M
F
M
F
Dose (mg/kg)
138 or 227
238 or 447
47 or 93
1,250 or 2,500
1,250 or 2,500
1,962 or 3,925
1,442 or 2,885
1,665 or 3,331
97 or 195
149 or 229
62 or 107
62 or 107
Significant Neoplasm
Hepatocellular carcinoma
Hepatocellular carcinoma
None
Hepatocellular carcinoma
Hepatocellular carcinoma
Nonec
Nonec
Nonec
Alveolar /bronchiolar adenoma
Alveolar /bronchiolar adenoma
Mammary adenocarcinoraa
Endome trial stromal polyp or
sarcoma
Fores toraach squaraous cell
carcinoma
Alveolar/bronchiolar adenoma
Fores tomach squamous cell
Control
6
1
—
5
0
—
—
0
5
0
0
0
0
0
Low-dose
36
80
—
100
100
—
• —
2
14
18
10
90
9
94
High-dose
98
95
—
98
96
—
—
31
31
15
11
59
21
56
carcinoma
Alveolar/bronchiolar adenoma
0
23
13
-------
Table VIII (continued)
page /
Incidence (%)
Compound
1,1,1-Trichloroe thane M
1 ,1 ,2-Trichloroe thane
1,1,2, 2-Te trachloroe thane
Pen tachloroe thane
Hexachloroe thane
1 ,2-Dibromo-3-chloropropane
B) Haloalkenes
Vinylidene chloride
(1,1-Dichloroethylene)
Trichloroe thylene
Sex
or F
M
F
M
F
M
F
M
F
11
F
^
M
F
M
F
Dose (mg/kg)
2,870 or 5,615
195 or 390
195 or 390
142 or 282
142 or 282
250 or 500
250 or 500
590 or 1,190
590 or 1,190
114 or 219
114 or 219
2 or 10
2 or 10
1,169 or 2,339
869 or 1739
Significant Neoplasm
Nonec
Hepatocellular carcinoma
Adrenal pheochromocytoma
Hepatocellular carcinoma
Adrenal pheochromocytoma
Hepatocellular carcinoma
Hepatocellular carcinoma
Hepatocellular carcinoma
Hepatocellular carcinoma
Hepatocellular carcinoma
Hepatocellular carcinoma
Fores toraach squamous cell
carcinoma
Fores tomach squamous cell
carcinoma
None
None
Hepatocellular carcinoma
Hepatocellular carcinoma
Control
—
10
0
0
0
6
0
8
0
15
10
0
0
—
5
0
Low-dose
—
37
0
33
0
26
63
55
60
30
40
93
100
—
52
8
High-dose
—
76
17
89
28
90
91
22
31
63
31
98
98
—
65
23
-------
Table VIII (continued)
Incidence (%)
Compound
Te trachloroe thylene
Allyl chloride
Sex
M
F
M
F
Dose (mg/kg)
536 or 1,072
386 or 772
172 or 119
129 or 258
Significant Neoplasm Control
Hepatocellular carcinoma 10
Hepatocellular carcinoma 0
None6
None6
Low-dose
65
40
—
High-dose
56
40
— —
aSummarized from National Cancer Institute/National Toxicology Program Carcinogenesis Technical Reports No. 2, 3,
13, 27, 28, 55, 66, 73, 74, 86, 106, 110, and 228.
Also carcinogenic in the rat.
Considered inconclusive because of insufficiently high dose (below maximal tolerated dose) or because inadequate
number of mice survived long enough to be at risk from late-developing tumors.
Preliminary data.
Suggestive of positive association with neoplastic lesions of the forestomach.
-------
Table IX
Carcinogeniclty of Haloalkanes and Haloalkenes by Topical Application or Subcutaneous Injection
Compound
As Initiator
Repeated Topical Applications
Local Tumor '. Distant Tumor
Local Sarcoma
After s.c. Injection
A) Haloalkanes
1,2-Dichloroethane
1,2-Dibromoethane
1,1,2,2-Te trab romoe thane
1,2-Dibromo-3-chloropropane
Lung
Lung
Fores tomach
Lung, fores tomach
n. t.1
n. t.
n. t.
n. t.
B) Haloalkenes
Vinyl bromide - -
Vinylidene chloride +
Trichloroe thylene
Tetrachloroethylene - -
1-Chloropropene - -
Ally! chloride +
cis-1 ,3-Dichloropropene - -
trans-1 ,4-Dichloro-2-butene
Hexachlorobutadiene
Summarized from B.L. Van Duuren, B.M. Godlschmidt, and
Duuren [Environ. Hlth. Persp. 21, 17 (1977)]; B.L. Van
Smith, S. Melchionne, I. Seidman, and D. Roth [J. Natl
None
None -
None -c
None n.t.
None -d
None -
None +
None +e
None n.t.
I. Seidman [Cancer Res. 35, 2553 (1975)]; B.L. Van
Duuren, B.M. Goldschmidt, G. Loewengant, A.C.
. Cancer Inst. 63, 1433 (1979)].
D Not tested.
Q
Also inactive by oral administration.
Induced stomach tumors after oral administration.
Q
Inactive by i.p. administration.
-------
taneous administration, only cis-1,3-dichloropropene and _trans_-l ,4-dichloro-2-
butene display local carcinogenic activity, inducing local sarcoma at the site
of injection.
A number of haloalkanes and haloalkenes have been tested in three short-
term assays: pulmonary adenoma (218), in vitro cell transformation (219), and
preneoplastic hepatocellular foci (220-222). Of the twenty-two haloalkanes
tested in the pulmonary assay (Table X), eleven (mostly iodoalkanes and butyl
halides) are active while two (dichloromethane and 1,1,2,2-tetrachloroethane)
display marginal activity. With the exception of _t-butyl iodide (tested at a
very low dose) and iodoethane, all iodoalkanes tested are active. The nega-
tive finding of iodoethane is surprising in view of the fact that its lower
and higher homologs are all active. Among the butyl halides tested, jt- and
£-butyl chlorides are both active while their ji-isomer is not. This is
consistent with the expected higher alkylating activity (via SjJ. mechanism) of
tertiary and secondary than of primary alkyl halides. The same relationship
may also hold for butyl bromides, although the negative finding of _n-butyl
bromide is not as convincing because of the low dose administered. In the
cell transformation assay of Price _e_t_al_. (219), dichlorome thane, 1,1,1-tri-
chloroethane, trichloroethylene and tetrachloroethylene have been shown to
induce phenotypic transformation of F1706 rat embryo cells. Some of the
transformed cells grew as undifferentiated fibrosarcomas after innoculation
into newborn rats. The transformation activity of dichloromethane is not
substantiated by the study of Sivak (cited in ref. 223) using a purified
sample of the compound. Bolt and associates (220-222) have tested the ability
of seven haloethanes to induce ATPase-deficient preneoplastic hepatocellular
foci in newborn rats. The relative activity follows the order: vinyl
chloride > vinyl fluoride > vinyl bromide > vinylidene fluoride > vinylidene
26
-------
page 1 of 3
Table X
Relative Carcinogenic Potency of Haloalkanes and Haloalkenes
(Pulmonary Adenoma Bioassay)3
Compound
Total Dose
No. Lung Tumors/Mouse
"A/HeA/Si
Negative controls
lodomethane (methyl iodide)
Dichloromethane
(methylene chloride)
Trichlorome thane
(chloroform)
Bromodichlorome thane
Tribromome thane
(bromoform)
Bromoethane (ethyl bromide)
lodoethane (ethyl iodide)
1,2-Dichloroe thane
(ethylene dichloride)
1,1,2,2-Te trachloroe thane
1-Iodopropane
(n-propyl iodide)
2-Iodopropane
(i-propyl iodide)
0.31
0.15
151.0
32.0
40.2
16.2
14.6
9.5
4.4
55.0
38.4
24.3
38.1
17.6
7.1
35.2
17.6
7.0
0.21-0.36
0.55*
0.30
0.19-0.39
0.35
0.15
0.70*
0.22
0.58*
0.44
0.53*
0.50
0.94°
0.00
0.35
0.85
0.67
1.13*
0.75
1.00C
-------
Table X (continued)
page 2 of 3
Compound
Total Dose
No* Lung Tumors/Mouse
"A/He A/lTt
1-Chlorobutane
(jn-butyl chloride)
2-Chlorobutane
(s-butyl chloride)
2-Chloro-2-me thylpropane
(t-butyl chloride)
1-Bromobutane
(n-butyl bromide)
l-Bromo-2-rae thylpropane
(i-butyl bromide)
2-Bromobutane
(s-butyl bromide)
2-Bromo-2-me thylpropane
(t-butyl bromide)
1-Iodobutane
(n-butyl iodide)
2-Iodobutane
(s-butyl iodide)
65.0
35.0
17.5
65.0
32.4
12.9
1.2
43.7
21.8
8.7
43.7
21.8
8.7
43.7
21.8
8.7
13.1
6.6
2.6
32.6
16.3
0.31
1.20*
0.67
1.00*
0.73*
0.64
0.14
0.75*
0.64*
0.42
1.15*
1.00*
0.35
0.78*
0.73*
0.53
0.63*
0.60*
0.63*
0.63*
0.33
-------
Table X (continued)
page 3 of 3
Compound
Total Dose
No. Lung Tumors/Mouse
A/HeA/Si
2-Iodo-2-methylpropane
(_t-butyl iodide)
1-Chlorooctane
2.7
0.42
(n-octyl chloride)
Benzyl chloride
1-chloromethyl naphthalene
Te trachloroe thene
( te trachloroe thylene)
Hexachloro-1 ,3-butadiene
Positive control (ure thane)
aSummarized from L. A. Poirier,
64.4
15.8
7.0
57.9
0.37
—
G. D. S toner and M.
0.15
0.25
0.22
0.50
0.36
8.1-17.8* 19.6*
B. Shimkin, Cancer Res.
35, 1411 (1975); J. C. Theiss, G. D. Stoner, M. B. Shimkin and E. K.
Weisburger, Cancer Res. 37. 2717 (1977).
Total dose over the 24-week period (mmoles/kg). For negative results, only
the highest dose was listed.
cNot significant but with p-value close to 0.05.
*Significantly higher than negative controls with p < 0.05.
-------
chloride > trichloroethylene and tetrachloroethylene (which are inactive).
The difference in activity is believed to be related to the rate of metabolic
activation to the presumed epoxide intermediates and the selectivity of the
epoxides to react with DNA in target organs.
5.2.2.1.3.2 HALOMETHANES.
5.2.2.1.3.2.1 Carbon Tetrachloride. Investigations on the carcinogenicity of
carbon tetrachloride (CCl,) have been the subject of several recent reviews
(2, 9, 10); the major findings are summarized in Table XI. Carbon tetra-
chloride has also been frequently used in syncarcinogenesis and modification
studies discussed in Section 5.2.2.1.3.8. Carbon tetrachloride has been shown
to be a liver carcinogen in 3 mammalian species and in the rainbow trout.
The hepatocarcinogenicity of CCl, was first reported in 1941 by Edwards
(47). Subsequent experiments by Edwards and coworkers (224, 225) showed that
CCl, was carcinogenic in five strains of mice. These mice received oral
administration (gavage) of 0.1 ml of a 40% CCl, solution in olive oil 2-3
times per week for various periods of time and were sacrificed around 1 year
of age. A slight degree of strain difference in susceptibility of the mice to
the hepatocarcinogenic effect of CCl, was observed. The respective incidences
of liver tumor were: C3H, 88%; A, 98%; Y, 60%; C, 83%; L (male), 47-54%; L
(female), 27-38%. The spontaneous liver tumor incidence at the age of around
1 year was below 4% for all five strains. The liver tumors usually emerged
following acute necrosis and subsequent cirrhosis.
Eschenbrenner and Miller (226, 227) confirmed the hepatocarcinogenicity
of CCl ^ using strain A mice. In addition, they investigated the effects of
size and spacing of multiple doses on the incidence of CCl/-induced hepatomas
and the relationship between liver necrosis and tumor induction. Mice were
27
-------
Table XI
Carcinogenic!ty of Carbon Tetrachloride
Species & Strain
Mouse, A, C3H,
C, L or Y
Mouse, A
Mouse, C3H
Mouse BUB
Mouse, C3H
Mouse, C3H
Mouse, XVII/G
Mouse, B6C3F,
Rat, albino
Rat, Buffalo or
Wis tar
Rat, Japanese or
Osborne-Mendel
Rat, —
Rat, Osborne-Mendel
Rou te
oral
oral
oral
oral
oral
rectal instillation
oral
oral
inhalation
subcutaneous
subcutaneous
subcutaneous
oral
Principal
Organs
Affected
Liver
Liver
Liver
Liver
Liver
Liver (nodules)
Liver
Liver
Liver
Liver
Liver, thyroid gland
Mammary gland
Liver
References
(47, 224,
225)
(226, 227)
(228, 229)
(230)
(231)
(232)
(233)
(234)
(235)
(236-238)
(237)
(239)
(234)
Hamster, Syrian golden
oral
Liver
(240)
-------
given 30 graded doses (0.1, 0.2, 0.4, 0.8, and 1.6 ml/kg of body weight) of
CC1/; the interval between consecutive doses ranged from 1 to 5 days. They
observed that the tumor incidence and the size of hepatomas progressively
increased with increase in dose, as well as with the increase of the interval
between the consecutive doses (226). To investigate whether liver necrosis is
a prerequisite for tumor induction, Eschenbrenner and Miller (227) divided
carcinogenic "necrotizing" doses into smaller "nonnecrotizing doses" and
administered the smaller doses more frequently to provide the same total
doses. Hepatoma induction was observed in mice with no signs of liver
necrosis. The authors concluded that: "While it was found that a correlation
exists between the degree of liver necrosis and the incidence of hepatomas in
relation to dose, the use of a graded series of necrotizing and nonnecrotizing
doses indicated that repeated liver necrosis and its associated chronic
regenerative state are probably not necessary for the induction of tumors with
carbon tetrachloride" (227).
The hepatocarcinogencity of CC1, in the mouse has also been confirmed in
several other studies using C3H, BuB, XVII/G, and B6C3F, strains (see Table
XI). In a NCI study (234), nearly all the mice (see Table VIII) developed
hepatocellular carcinomas by the end of the bioassay. A study by Confer and
Stenger (232) showed that CCl^ was also hepatocarcinogenic by intrarectal
administration. Thirteen of the 25 male C3H mice that received biweekly doses
of 0.1 ml of a 40% solution of CCl^ in olive oil for 20-26 weeks developed
liver tumors described as nodular hyperplasia. No such tumors occurred in 10
vehicle-treated control mice.
The carcinogenicity of CC1/ in the rat has been tested by inhalational,
subcutaneous, and oral routes. Costa et al. (235) exposed a group of albino
rats to an atmosphere containing CCl^ for 7 months. Among the 30 survivors,
28
-------
10 had liver nodules diagnosed histologically as early or established liver
carcinomas. The details of the study and the type of control were not
given. Reuber and Glover (236-238) investigated the carcinogenicity of CCl^
in'6 different strains (Buffalo, Japanese, Osberne-Mendel, Wistar, Black,
Sprague-Dawley) of rats following biweekly subcutaneous injections of 1.3
ml/kg of a 50% solution of CCl^ in corn oil. A low incidence of small '
hepatomas was observed in Buffalo strain rats of both sexes treated at the age
of 24 or 52 weeks (236). The hepatocarcinogenicity of CCl^ in Buffalo strain
rats was enhanced by simultaneous administration of 3-methylcholanthrene
(238). Japanese, Osborne-Mendel, and Wistar rats appeared to be considerably
more susceptible to the hepatocarcinogenic action of CC1,; their respective
incidences of hepatocellular carcinomas were 80% (12/15), 62% (8/13), and 33%
(4/12). Black and Sprague-Dawley rats died shortly after treatment. This
strain difference was found to be due to the hepatotoxicity of CC1,. As shown
in Table XII, Black and Sprague-Dawley rats developed severe liver cirrhosis
and survived an average of 11 and 13 weeks, respectively. The susceptible
strains had milder cirrhosis and survived long enough for the development of
tumors (237). In addition to liver tumors, there were carcinomas of the
thyroid gland in three Osborne-Mendel and three Japanese rats (237). The
mammary gland appeared to be the principal target organ of an unspecified
strain of white female rats in the study of Alpert _e_t _al_. (239). Of the
thirty rats that received biweekly subcutaneous injections of 1 ml/kg CC1, for
2 years, eight developed mammary adenocarcinomas, one had a mammary fibro-
adenoma. No such tumors were observed in fifteen untreated control rats. In
sharp contrast to the potent hepatocarcinogenicity in mice, CC1, appears to be
inactive or at most marginally active in the Osborne-Mendel rat by oral
«
administration (234).
29
-------
Table XII
Strain-dependent Induction of Liver Cirrhosis, Nodules and Carcinomas in Male Rats
by Carbon Tetrachloride3
No. of rats No. of rats
Strain at start Mild
Japanese
Osborne-
Mendel
Wis tar
Black
Sprague-
Dawley
a
Summarized
15
13
12
17
16
from M.
9
2
0
0
0
D. Reuber
with cirrhosis
Moderate Severe
5
7
6
4
0
and E.
1
4
6
13
16
L. Glover
Total
15
13
12
17
16
[J. Nat.
No. of rats
with No. of
Hyperplastic rats with
Nodules Carcinoma
3
4
7
7
2
Cancer Inst.
12 (80%)
8 (62%)
4 (33%)
0 ( 0%)
0 ( 0%)
44, 419 (1970).]
Average
Survival (weeks)
47
44
33
11
13
All rats were 12
weeks old at the start of the experiment. They were given subcutaneous injections of a 50% solution of
in corn oil twice a week and were sacrificed when they became moribund.
-------
In addition to rats and mice, CCl^ has been shown to be hepatocarcino-
genic in the hamster and the rainbow trout. Delia Porta et al. (240)
administered 30 weekly oral doses of 6.25-12.5yxl CCl^ in 5% corn oil solution
to 20 (10 of each sex) 12-week-old Syrian golden hamsters. All 10 (5 of each
sex) animals that survived 10 or more weeks beyond the end of treatment
developed liver-cell carcionmas. Halver (241) fed rainbow trout diets con-
taining 3.2 and 12.8 ppm CCl^. After 20 months, 4/44 low dose group and 3/34
high dose group developed hepatomas; none were found in controls.
5.2.2.1.3.2.2 Chloroform. The carcinogenicity of chloroform (CHCl^) has been
tested in the mouse, the rat, and the dog. These bioassay studies have been
reviewed at some preliminary stages by Winslow and Gerstner (5), Reuber (6),
and IARC (2); the final reports on some of the data have been published (242-
245). Table XIII summarizes these findings. Chloroform was first reported to
be hepatocarcinogenic in the mouse by Eschenbrenner and Miller (48) in 1945.
Groups of 5 strain A mice were given 30 oral doses of 0.1, 0.2, 0.4, 0.8, or
1.6 ml/kg (body weight) CHC1, in olive oil. The high doses were toxic and
killed many mice early in the experiment. A high incidence of hepatomas and
liver cirrhosis was observed among the survivors. No hepatomas were found in
the low dose groups and the control group. Rudali (233) administered by •
gavage 0.1 ml of a 40% solution of CHC1, in oil twice a week for 6 months to
24 NLC mice. Of the five animals that survived 297 days, three had
hepatomas. No hepatomas were observed in several other groups of mice given a
number of tetrahalomethanes other than CC1, (see Section 5.2.2.1.3.2.3). The
hepatocarcinogenicity of CHC1-, in the mouse has been confirmed in a NCI study
(242). Groups of 50 E6C3F^ mice of each sex were given (by gavage) a 2-5%
solution of CHCl-j in corn oil 5 times/week for 78 weeks. The average doses
were 138 and 277 mg/kg for males and 238 and 477 mg/kg for females. The mice
30
-------
Table XIII
Carcinogenic!ty of Chloroform
Species & Strain
Route
Principal
Organs Affected
References
Mouse, A
Mouse, NLC
Mouse, B6A2FJ
Mouse, BSCSFj
Mouse, ICI-Swiss
Mouse, C57BL, CBA
or CF-1
Rat, Osborne-Mendel
Rat, Sprague-Dawley
oral
oral
subcutaneous
oral
oral
oral
oral
oral
Liver
Liver
No significant effect
Liver
Kidney (males only)
None
Kidney (males only)
No significant effect
(48)
(233)
(246)
(242)
(243)
(243)
(242)
(244)
Dog, beagle
oral
No significant effect
(245)
-------
were sacrificed atf 92-93 weeks. Significant hepatocarcinogenic effect of
CHC13 were observed (see Table VIII); nearly all the mice in the high dose
group had hepatocellular carcinomas. In contrast to these studies, Roe e t al .
(243) observed no significant carcinogenic effects of.CHCl-j in 3 strains
(C57BL, CBA, or CF-1) of male mice that had received CHC1., by gavage at doses
up to 60 mg/kg/day, 6 days/week for 80 weeks. In a fourth strain (ICI-Swiss),
males (but not females) in 60 mg/kg/day group had significantly higher inci-
dence of epithelial tumors of the kidney. No significant effects were noted
in mice exposed to a lower dose (17 mg/kg/day) of CHC1,. Besides oral admini-
stration, Roe e_t__al_« (246) al'sp tested CHC1- injected subcutaneously as a
i '
siifigl/2 dose of 0.2 mg in aracjhjis oil, or 8 daily doses of 0.2 mg to newborn
II ''I' jj
(CjJ7/i DBA2)F, mice in the indrascapular region. After 77-80 weeks, no evi-
! 1
defl.ce.jof significant carcinogenic effects of CHC1., was found.. The signifi-
V;';'. yJV! J
cance of this study may be questionable because of the low dose.
The carcinogenici ty of CHC1-, in the rat has also been evaluated. In an
NCI study (242), Osborne-Mendel rats were given 90 and 180 mg/kg (males) or
100 and 200 mg/kg (females) CHCl^ in corn oil 5 days/week for 78 weeks and
were then sacrificed after 111 weeks. The most significant finding was the
induction of kidney epithelial tumors in male rats with incidences of 0, 8, .
and 24% in the control, low dose, and high dose groups, respectively. A
•
statistically significant increase in thyroid tumors in treated female rats
was also observed; however, this finding was not considered to be "bio-
logically significant" (242). Reuber (6) has recently re-examined the NCI
data; combining the data on cholangiof ibromas, hyperplastic nodules, and
carcinomas of the liver, he concluded that CHC1., was hepatocarcinogenic in the
rat with the females being more susceptible than the males. In another study
in the rat, Palmer _e_t _al_. (244) found no significant carcinogenic effects of
-------
CHCU in Sprague-Dawley rats of both sexes which received oral doses of 60
ing/kg/day CHCl-^ 6 days/week for 80 weeks and then observed for up to a total
of 95 weeks. The tumor incidence was 39% in CHCl-j-treated rats and 38% in
vehicle-treated controls.
The potential carcinogenicity of CHCl^ has also been evaluated using
beagle dogs. Heywood _e_t _al_. (245) gave beagle dogs CHC1, equivalent to 15 or
30 mg/kg/day, 6 days/week for 7.5 years and the animals were observed for an
additional 20-24 weeks. A number of macroscopic and microscopic tumors
(mostly tes ticular and mammary) were found in both the CHClo-treated and the
control groups. No tumors were seen in the liver and kidney. Despite rela-
tively high incidences of testicular and mammary tumors in the low dose group
the authors (245) did not attribute tumor induction to CHClo treatment because
of the long duration of the study and the lack of dose-dependence. Heywood et
al. (245) concluded that exposure of beagle dogs to CHClo had no effect on the
incidence of tumors in beagle dogs.
5.2.2.1.3.2.3 Halomethanes Other than Carbon Tetrachloride and Chloroform.
Besides carbon tetrachloride and chloroform, 12 other halomethanes have been
tested for carcinogenicity and the results of these studies are summarized in
Table XIV. Only one monohalomethane has- thus far been studied. Druckrey et
al. (68) reported that iodomethane (methyl iodide) induces local sarcomas at
the site'of subcutaneous injection. Groups of BD rats were given either a
single dose of 50 mg/kg or weekly injections of 10 or 20 rag/kg iodomethane in
vegetable oil for one year; the incidences of local sarcomas after lifetime
observation were 4/14, 9/12, and 6/6, respectively. The average latent period
was 580-610 days. No local sarcomas were found in the vehicle-treated control
rats. Iodomethane appears to be also carcinogenic by intraperitoneal injec-
tions. Moreover, in the pulmonary adenoma bioassay study by Poirier et al.
32
-------
Table XIV
Carcinogenic!ty of Halomethanes Other Than Chloroform and Carbon Tetrachloride
Compound
lodome thane (Methyl iodide)
Chlorofluorome thane
Dichlorome thane
(Methylene chloride)
Bromodichlorome thane
Dibromochlorome thane
Tribromome thane (Bromoform)
Triiodome thane (lodoform)
Trichlorof luorome thane
(Freon 11)
Trichlorobromome thane
Dibromodichlorome thane
Te tr ab roraome thane
Species & Strain
Mouse, A/ He
Rat, BD
Rat, —
Rat, Sprague-Dawley
Hams ter , —
Mouse, XVII/G, NLC or RHI/f
Mouse, XVII/G, NLC or RHI/f
Mouse, A/St
Mouse, B6C3Fj
Rat, Osborne-Mendel
Mouse, Swiss
Mouse, B63F,
Rat, Osborne-Mendel
Mouse, XVII/G, NLC or RHI/f
Mouse, XVII/G, NLC or RHI/f
Mouse, XVII/G, NLC or RHI/f
Route
i.p.
s.c.
unspecified
inhalation
inhalation
oral
oral
i.p.
oral
oral
s.c.
oral
oral
oral
oral
oral
Principal
Organs Affected
Lung
Local sarcoma
unspecified
None0
None
Noned
Noned
Lung
None
None
None
None
None (inconclusive)6
None
Noned
Noned
Reference
(247)
(68)
(172)
(248-250)
(248-250)
(233)
(233)
(251)
(252)
(252)
(253)
(254)
(254)
(233)
(233)
(233)
aln addition to the studies listed above, dichloromethane, and bromodichloromethane have been tested in pulmonary
adenoma assay and found to have no significant effect (see Table X).
Lung adenomas (see Table X).
CA possible increase in the incidence of benign mammary tumors was noted (cited in ref. 3).
It is not certain whether the duration of the experiment was sufficiently long (see text).
f* *
Inadequate number of rats survived long enough to be at risk from late-developing tumors.
-------
(247), iodomethane caused a significant increase in the number of tumors/mouse
(see Table X).
Very little information is available on the carcinogenicity of dihalo-
methanes. In an industry-sponsored study published in the trade literature
(248-250), it was reported that dichloromethane (methylene chloride) is not
carcinogenic in rats and hamsters of both sexes. In these studies, approxi-
mately 2,000 animals were exposed via inhalation to 0, 500, 1,500, and 3,500
ppm dichloromethane for 6 hr/day, 5 days/week for 2 years; details of the
methodology have not been given. With the exception of benign mammary tumors
in rats of both sexes, there was no increase in the incidence of malignancies
in exposed animals. The increase in benign mammary tumor was attributed to
spontaneous incidence in this strain (Sprague-Dawley) of rats. However, the
U.S. Interagency Regulatory Liaison Group suggested that the observation may
be an indication of the oncogenic potential of the compound (3). Dichloro-
me thane slighly increased the number of lung tumors/mouse (see Table X) in the
pulmonary adenoma assay by Theiss _e_t _al_. (251); the increase had marginal
statistical significance (p = 0.054). In a recent abstract, Green (172) has
claimed that chlorofluoromethane is carcinogenic in the rat. Chlorofluoro-
methane was found to be mutagenic in the Ames Salmonella test.
Bromodichloromethane and dibromochloromethane were reported to be noncar-
cinogenic in a brief report by Rudali (233). In this study, groups of mice
were given oral doses of 0.1 ml of a 40% solution of the trihalomethanes in
oil. Although the duration of the treatment and the length of the observa-
tional period were not reported, in the same study chloroform was hepatocar-
cinogenic after 297 days. In the pulmonary adenoma assay by Theiss et al.
(251), bromodichloromethane was inactive while tribromomethane (bromoform) had
a significant effect (see Table X). Triiodomethane (iodoform) has been tested
33
-------
in Osborne-Mendel rats and BeCSFj^ mice of both sexes in a NCI bioassay (252);
no significant carcinogenic effects were observed.
Besides carbon tetrachloride, four tetrahalomethanes have been bioassayed
for carcinogenicity. Trichlorofluoromethane (Freon 11) has no significant
carcinogenic effects one year after repeated subcutaneous injections into
neonatal Swiss ICR/Ha mice (253). An NCI study (254) confirmed the lack of
carcinogenicity of trichlorofluoromethane in the mouse; B6C3F, mice that
received average oral doses of 1,962 and 3,925 mg/kg/day, 5 days/week for 78
weeks did not develop tumors attributable to the treatment. Osborne-Mendel
rats were also used in the study. However, the doses administered (488 and
977 mg/kg/day for males; 538 and 1,077 mg/kg/day for females) caused a high
rate of early deaths so that an insufficient number of rats survived long
enough to exclude the possibility of late-developing tumors. Three tetra-
halomethanes (trichlorobromomethane, dibromodichloromethane, and tetrabromo-
methane) were tested by Rudali (233). Groups of mice received 0.1 ml of
either a 10% solution of trichlorobromomethane or dichlorodibromomethane or a
40% solution of tetrabromomethane for an unspecified period of time. None of
these compounds was carcinogenic. The two chlorinated compounds (CCl-Br and
CCljBr^) were hepato toxic, while tetrabromomehane was not. In the same study,
oral administration of a 40% and a 25% solution of carbon tetrachloride (used
as positive control) led to hepatoma incidences of 91% and 5%, respectively.
5.2.2.1.3.3 HALOETHANES.
Over a dozen haloethanes have been tested for carcinogenicity. The
relative carcinogenic potency of various haloethanes tested orally in B6C3Fi
mice is given in Table VIII (Section 5.2.2.1.3.1). More detailed information
on the conditions of carcinogenicity testing of haloethanes is tabulated in
Table XV.
34
-------
Table XV
Carcinogenic!ty of Haloethanes8
page 1 of 3
Compound
Species & Strain
Route
Principal
Organs Affected
Reference
A) Dihaloe thanes
1,1-Dichloroethane
1,2-Dichloroe thane
(Ethylene dichloride)
1,2-Dib romoethane
Mouse, B6C3F.
Rat, Osborne-Mendel
Mouse, B6C3F.
Mouse, Swiss ICR/Ha
Mouse, Swiss
Rat, Osborne-Mendel
Rat, Sprague-Dawley
Mouse, B6C3Fj
Mouse, Swiss ICR/Ha
Rat, Osborne-Mendel
Rat, F344
Rat, Sprague-Dawley
oral None (inconclusive)
oral None (inconclusive)
oral Lung, mammary gland,
uterus
topical Lung
inhalation None
oral Forestomach, mammary
gland, circulatory
system, subcutaneous
tissues
inhalation None
oral Forestomach, lung
inhalation Lung, subcutaneous
tissue, nasal cavity,
mammary gland
topical Skin, lung
oral Forestomach, circula-
tory system, liver
inhalation Nasal cavity, circula-
tory system, pituitary
gland, genital tract,
lung, mammary gland
inhalation Spleen (preliminary)0
(255)
(255)
(256, 257)
(216)
(258)
(256, 257)
(258)
(259,
(261)
(216)
(259,
(261)
(262)
260)
260)
-------
Table XV (continued)
page 2 or 3
Compound
Species & Strain
Route
Principal
Organs Affected
Reference
B) Tribalomethanes
1,1,1-Trichloroe thane
(Methyl chloroform)
1,1,2-Trichloroe thane
C) Te trahaloe thanes
1,1,2,2-Te trachloroe thane
1,1,2,2-Te trab romoe thane
Mouse, B6C3FJ
Rat, Osborne-Mendel
Rat, Sprague-Dawley
Mouse, B6C3FJ
Rat, Osborne-Mendel
Mouse, B6C3FJ
Rat, Osborne-Mendel
Mouse, Swiss ICR/Ha
oral
oral
inhalation
oral
oral
oral
oral
topical
None (inconclusive)
None (inconclusive)
None (1-year study
only)
Liver, adrenal gland
None
Liver
None (inconclusive)
Fores tomach
(263)
(263)
(Quast et al 1978,
cited in ref. 201)
(264)
(264)
(265)
(265)
(216)
D) Pentahaloethane
Pen tachloroe thane
Mouse, B6C3FJ
Rat, F344
oral
oral
Liver (preliminary)
None (preliminary)
1,1,l-Trifluoro-2-bromo- Mouse, Swiss ICR inhalation None
2-chloroethane (Halothane) (perinatal exposure)
J. Mennear, NCI/NTP,
personal communication
J. Mennear, NCI/NTP,
personal communication
(266)
-------
page 3 of 3
Table XV (continued)
Compound
Species & Strain
Route
Principal
Organs Affected
Reference
E) Hexahaloe thanes
l,l,2-Trlchloro-l,2,2-tri-
fluoroethane (Freon-113)
1,1,2,2-Te trachloro-1,2-
difluoroethane (Freon-112)
Hexachloroe thane
House, Swiss
Mouse, Swiss
Mouse, B6C3FJ
Rat, Osborne-Mendel
s.c,
s.c,
oral
oral
None
None
Liver
None
(253)
(253, 267)
(268)
(268)
In addition to the studies listed above, bromoethane, iodoethane, 1,2-dichloroethane, and 1,1,2,2-tetrachloroethane
have been tested in pulmonary adenoma assay and found to have no significant effect (see Table X).
Considered inconclusive either because of insufficiently high doses or because of early mortality.
cAs a part of a synergism study with Disulfiram (see Section 5.2.2.1.3).
-------
With the exception of the inactivity on bromoethane (ethyl bromide) and
iodoethane (ethyl iodide) in the pulmonary adenoma assay by Poirier et al.
(247) (see Table X), there is no information on monohaloethanes. The inac-
tivity of iodoethane is surprising in view of the fact that iodomethane,
iodopropanes, and 1- and 2-iodobutanes were all tumorigenic in the same
study. Iodoethane is about 50% as active as iodomethane as an alkylating
agent in the NBP test (69).
Of the three dihaloethanes tested, the results on technical grade 1,1-di-
chloroethane (255) were inconclusive due to poor survival rate. The survival
rate was 32-80% for the mice and only 4-40% for the rats; pneumonia occurred
in 80% of the rats. There was suggestive evidence of an increase in the
incidence of mammary adenocarcinomas and in hemarigiosarcomas among female rats
and of endometrial stromal polyps among female mice. A re testing of the
compound is needed before conclusions can be made.
Technical grade 1,2-dichloroethane (ethylene dichloride) is carcinogenic
by oral administration. Significant increase in the incidence of alveolar/
bronchiolar adenoma in mice of both sexes, and of mammary adenocarcinoma and
endometrial stromal polyp or sarcoma in female mice was observed (see Table
VIII). In Osborne-Mendel rats (dosed 47 and 95 mg/kg/day, 5 days/week for 78
weeks), 1,2-dichloroe thane brought about significant increase in the incidence
of squamous cell carcinomas of the forestomach (control 0%, low dose 6%, high
dose 18%), hemangiosarcoma, and subcutaneous fibromas in males and of mammary
adenocarcinoma (control 0%, low dose 2%, high dose 36%) in females (256,
257). Also, by topical route, 1,2-dichloroethane (126 mg/application, 3
times/week, 440-594 days) was carcinogenic, inducing lung tumors in 26/30
female Swiss ICR/Ha mice; however, no local tumors were observed (216). In
contrast to oral and topical administration, Maltoni et '_a_l_. (258) observed no
35
-------
significant carcinogenic effects in Sprague-Dawley rats and Swiss mice after
exposing the animals to atmospheres containing 5, 10, 50, or 150-200 ppm of a
relatively pure (> 99.8%) sample of 1,2-dichloroethane. The nature for the
discrepancy is not clear. In an assessment of the above data, Hooper et al.
(269) suggested that the apparent discrepancy may be due to differences in the
route, the strain of the animal, or to the statistical consideration of the
effect of "intercurrent mortality."
Whereas the carcinogenicity of 1,2-dichloroethane may be debatable or is
dependent on the route of administration, there is little doubt that its bromo
analog, 1,2-dibromoethane (ethylene dibromide) is a potent carcinogen. At
least six studies concur on the carcinogenicity of the compound. By oral
administration, 1,2-dibromoethane (technical grade) induced squamous cell
carcinoma of the forestomach in over 90% of the mice in the low dose group
(see Table VIII) with some tumors appearing as early as the 24th week of
treatment. Alveolar/bronchiolar adenomas were also noted. No such tumors
were observed in the control mice. In Osborne-Mendel rats of both sexes,
which received 37-41 mg/kg/day, 5 days/week for 78 weeks, the incidences of
forestomach squamous cell carcinomas were 58-90% with the first tumor
appearing as early as the 12th week of treatment. Increases in hemangiosar-
comas and hepatocellular carcinomas were also noted in male and female rats,
respectively. By topical route, 1,2-dibromoethane (25 or 50 mg/application, 3
s
times/week for 440-594 days) induced lung tumors in 50 and papillomas (5
progressing to squamous cell carcinomas) in 10 of 60 Swiss ICR/Ha mice
(216). By inhalational route, 1,2-dibromoe thane is a multi-potential car-
cinogen in B6C3F, mice, F344 rats, and possibly in Sprague-Dawley rats. In a
NCI inhalation study (261), groups of 50 B6C3FJ mice and F344 rats of each sex
were exposed to atmospheres containing 10 or 40 ppm of L,2-dibromoethane
36
-------
(> 99.3% pure) for 78-103 weeks. In B6C3F1 mice, significant increases in the
incidence observed were: alveolar/bronchiolar carcinomas or adenomas in males
(control 0%, low dose 6%, high dose 50%) and females (8%, 22%, 82%); and
hemangiosarcomas of the circulatory system (0%, 22%, 46%), fibromas of the
subcutaneous tissue (0%, 8%, 22%), tumors of the nasal cavity (0%, 0%, 24%),
and adenocarcinomas of the mammary gland (4%, 28%, 16%) in females. In F344
rats, 1,2-dibromoethane caused significant increases in the incidence of:
tumors (many malignant) of the nasal cavity (males, 0%, 78%, 82%; females, 2%,
68%, 86%), hemangiosarcomas of the circulatory system (males, 0%, 2%(, 30%;
females 0%, 0%, 10%), adenomas of the pituitary gland (males, 0%, 15%, 4%;
females, 2%, 37%, 9%) in both sexes; and mesotheliomas in the tun/Ics* vaginalis
(0%, 14%, 50%) in males; and alveolar/ bronchiolar carcionmas or Qjdtenomas (0%,
I 1 I
0%, 11%), and fibroadenomas of the mammary gland (8%, 58%, 48%) ilvi, whales.
In the Sprague-Dawley rat, preliminary results (262) indicate increase in the
incidence of hemangiosarcomas in the spleen of rats exposed to 20 ppm 1,2-
dibromoe thane for 18 months. Simultaneous administration of 1,2-dibromoethane
and the apparently innocuous disulfiram led to dramatic increases in inci-
dences of tumors in the liver, spleen, kidney, and omen turn (see Section
5.2.2.1.3.8).
1,1,1-Trichloroethane (methyl chloroform) was found in two studies to be
not carcinogenic in rodents (Table XV); however, neither bioassay can be
considered adequate because of insufficient duration of the experiment. In
the NCI oral study (263), groups of 50 B6C3F1 mice and Osborne-Mendel rats of
each sex were given technical grade 1,1,1-trichloroethane in corn oil 5
days/week for 78 weeks. A large number of animals had short life spans due to
the toxicity of the compound or pneumonia (only 31% of the mice and 3% of the
rats survived to the end of the experiment). At the time of this writing, the
37
-------
compound is being re tested by the U.S. National Toxicology Program. In an
inhalation study (Quas t et_ al_., 1979, cited in ref. 201), in which groups of
92-94 Sprague-Dawley rats of each sex were exposed to atmosphere containing
875 or 1,750 ppm of the compound 6 hr/day, 5 days/week for 52 weeks, no signi-
ficant effects were noted. The animals survived an average of 628-677 days.
The U.S. Environmental Protection Agency (201) considered the study inadequate
because of insufficient duration of exposure. However, it should be noted
that the study was specifically designed to simulate the proportion of the
-total life span to which an average human would be occupationally exposed.
In contrast to the 1,1,1-isomer, 1,1,2-trichloroethane was found to be
carcinogenic in B6C3F, mice (264). As seen in Table VIII, the compound caused
significant increase in the incidence of hepatocellular carcinomas in mice of
both sexes; the high dose was also associated with the induction of
pheochromocytoma of the adrenal gland. However, in Osborne-Mendel rats of
either sex, no tumors were observed following oral doses of 46 or 92
mg/kg/day, 5 days/week for 78 weeks (Table XV).
Two isomers of tetrachloroethane have been tested by the NCI by oral
administration. The data on the 1,1,1,2-isomer are under final review at the
time of this writing. The 1,1,2,2-isoraer (technical grade) was hepatocar-
cinogenic in B6C3F. mice, but noncarcinogenic in Osborne-Mendel rats (265).
The incidence of hepatocellular carcinomas was over 90% in the mice of the
high dose group (see Table VIII). The doses that the rats received were 62
and 108 mg/kg/day for males and 43 and 76 mg/kg/day for females. The carcino-
genicity of 1,1,2,2-tetrabromoethane was also reported (216). Topical appli-
cation of 15 mg of the compound to the dorsal skin of female Swiss ICR/Ha mice
3 times/week for 440-594 days led to the induction of stomach tumors in 4/30
animals; no local carcinogenic effects were observed, however (Table XV).
38
-------
Pentachloroe thane (270) appears to have the same carcinogenic effects as
1,1,2,2-tetrachloroethane. As shown in Table VIII, pentachloroethane causes a
significant increase in the incidence of hepatocellular carcinomas in B6C3F1
mice. The compound is also noncarcinogenic in the rat (Table XV). The strain
used in this study was F344; the dosages were 75 and 150 mg/kg/day. Halothane
(l,l,l-trifluoro-2-bromo-2-chloroethane), an anesthetic agent, has been
suspected for some time to be carcinogenic. This compound has recently been
tested by Eger _e£_al/ (266). Perinatal exposures of Swiss ICR mice to 1/32,
1/8, and 1/2 maximum allowable concentrations (MAC) of halothane 2 hr/day on
days 11, 13, 15, and 17 of gestation and 2 hr/day, 3 days/week for 8 weeks
post-partum elicited no significant carcinogenic effects. The investigators
emphasized that the doses administered (up to 1/2 MAC), at an age of rapid
growth (a period of high susceptibility to carciriogenesis), were sufficient to
have revealed a potent carcinogen.
Three fully halogenated ethanes (hexahaloethanes) have been tested for
carcinogenicity. Both 1,1,2-trichloro-l,2,2-trifluoroethane (Freon 113) and
1,1,2,2-tetrachloro-1,2-difluoroethane (Freon 112) were found to have no
significant carcinogenic effects by subcutaneous injections into the neck of
neonatal Swiss ICR/Ha mice (253, 267). In these studies, doses of 0.1 ml of
10% solution of either Freon in tricaprylin were injected into 1- and 7-day-
old mice, and 0.2 ml into 14- and 21-day-old mice. The animals were allowed
to survive until the experiments ended after one year. It is interesting to
note that while the Freons tested in this study were noncarcinogenic, simulta-
neous administration of either Freon and piperonyl butoxide led to induction
of liver tumors (see Section 5.2.2.1.3.8). Hexachloroethane was tested by the
NCI (268) in rodents by oral administration. Like its lower homologs
(1,1,2,2-tetrachloro- and pentachloroethanes), hexachloroethane was hepatocar-
39
-------
cinogenic in the B6C3F1 mouse (see Table VIII). Osborne-Mendel rats given
doses of 212 and 423 mg/kg/day did not develop any tumors attributable to the
treatment (Table XV).
5.2.2.1.3.4 HALOPROPANES AND HIGHER HALOALKANES.
There is a paucity of data regarding the carcinogenicity of halopropanes
although one compound in this group, l,2-dibromo-3-chloropropane, may prove to
be the most potent carcinogenic haloalkane. The information available on
halopropanes is summarized in Table XVI. Two iodopropanes have been tested in
the pulmonary adenoma assay by Poirier _et__al_« (247). Both compounds led to
significant increases in tumor incidence in the lung adenoma assay (see
Table X).
1,2-Dibromo-3-chloropropane (DBCP), a soil fumigant, was first reported
to be a potent carcinogen in a preliminary communication of NCI data by Olson
et al. (259). A final report of the study was subsequently published (271).
Virtually all the B6C3F, mice that received oral doses of DBCP developed
squamous cell carcinomas of the forestomach ('see Tables VIII and XVI).
Osborne-Mendel rats which received oral doses of 15 and 29 mg/kg/day, 5
days/week for 64-78 weeks also developed the same tumor with high incidence
(94% for both low and high dose males; 76 and 59% for low and high dose
females, respectively). Some of these carcinomas were accompanied by pulmo-
nary metastases. In addition, female rats had significantly increased inci-
dences of adenocarcinomas of the mammary gland (control 10%, low dose 48%,
high dose 62%). Palpable mammary tumors were noted already after 14 weeks of
treatment. The carcinogenicity of DBCP has been confirmed by a chronic
feeding study conducted for the Dow Chemical Company (272). Groups of 50
Charles River albino rats and HAM/ICR Swiss mice of each sex were fed diets
40
-------
Table XVI
Carcinogenic!ty of Halopropanes
Compound
Species & Strain
Route
Principal
Organs Affected
Reference
1-lodopropane
2-Iodopropane
1,2-Dibromo-3-chloro-
propane
Mouse, A/He i.p.
Mouse, A/He i.p.
Mouse, B6C3Fj oral
Mouse, Swiss ICR/Ha topical
Mouse, Swiss HAM/ICR oral
Mouse, B6C3Fj inhalation
Rat, Osborne-Mendel oral
Rat, Charles River albino oral
Rat, F344 inhalation
Lung
Lung3
Fores tomach
Lung, fores tomach
Fores tomach
Nasal cavity, lung
Forestomach, mammary
gland
Forestomach, liver,
kidney
Nasal cavity, tongue
pharynyx, adrenal
gland
(247)
(247)
(259, 271)
(216)
(272)
(273-275)
(259, 271)
(272)
(275)
aLung adenomas, see Table X0
'Also active as an initiator.
-------
containing DBCP (equivalent to 0.3, 1.0, and 3.0 mg/kg/day) for 104 or 78
weeks, respectively. The statistically significant increases in tumors
include the following histological types: squamous cell carcinomas and papil-
lomas of the forestomach in rats and mice of either sex, renal tubular
adenomas and carcinomas in rats of either sex, and hepatocellular carcinomas
in male rats. Dibromochloropropane was also carcinogenic by topical applica-
tions to female Swiss ICR/Ha mice. Van Duuren _e_t _al_. (216) showed that
repeated applications (3 times/week for 440-594 days) of 11.7 or 35 mg DBCP
led to the induction of lung tumors in 52/60 mice and of tumors of the fore-
stomach in 35/60 mice, including 15 squamous cell carcinomas. Interestingly,
no skin tumors were observed, although the compound was active in an initia-
tion-promotion study. Considering the possibility of inhalational exposure,
the NCI (275) has recently re tested DBCP by this route. Groups of 50 B6C3F,
mice and F344 rats of each sex were exposed to an atmosphere containing 0.6 or
3.0 ppm DBCP 6 hr/day, 5 days/week for 103 weeks. Significant increases in
the incidences of tumors of the nasal cavity (control 0%, low dose 2%, high
dose 44% in males; 0%, 22%, 76% in females) and alveolar/bronchiolar
carcinomas or adenomas (0%, 8%, 16% for males; 8%, 10%, 28% for females) were
observed in mice of both sexes. Increased incidence of tumors of the nasal
cavity (0%, 80%, 88% in males; 2%, 54%, 84% in females) were also noted in the
rat. In addition, rats of both sexes had higher incidences of squamous cell
carcinomas or adenomas of the tongue (0%, 2%, 22% in males; 0%, 8%, 18% in
females) and females developed squamous cell papillomas or carcinomas of the
pharynx (0%, 0%, 12%) and cortical adenomas of the adrenal gland (0%, 14%,
10%).
In addition to halopropanes, a number of higher haloalkanes have been
tested in the pulmonary adenoma assay by Poirier et al. (247) and Theiss et
41
-------
al. (251). The results of these studies are shown in Table X in Section
5.2.2.1.3.1. As previously discussed, the pulmonary adenoma assay should be
considered as a limited test for carcinogenicity; a positive result is
strongly indicative of potential carcinogenicity whereas a negative result is
of little predictive value. The haloalkanes found positive in the assay
include _s_-, _t-butyl chloride, JL_-, _s_-, and _t-butyl bromide, and _n_- and _s_-butyl
iodide.
5.2.2.1.3.5 HALOETHENES.
5.2.2.1.3.5.1 Vinyl Chloride. Vinyl chloride (VC) has attracted a great deal
of attention since the discovery of its carcinogenic action in humans. The
s
extensive carcinogenicity bioassays of VC have been reviewed in a number of
publications (22, 23, 25, 276, 277). Only a brief account of these studies
will be presented in this section; the major findings are summarized in Table
XVII. Vinyl.chloride has been found to be carcinogenic in at least 4 animal
species and in humans (see Section 5.2.2.1.5.1). It is a highly potent multi-
target carcinogen in rodents by inhalation. The histopathological types of
tumors are: liver angiosarcoma, carcinoma of the Zymbal glands (in ear duct),
nephroblastoma, neuroblastoma, mammary adenocarcinoma, forestomach papilloma,
lung tumor, vascular tumor, and epithelial tumor of the skin.
The carcinogenicity of VC was first discovered by Viola _e_t _al_. (285) in
1970. Male Ar/IRE rats exposed to an atmosphere containing 30,000 ppm of VC,
4 hr/day, 5 days/week for 52 weeks, developed tumors (reported to be skin
tumors) of the submaxillary parotid region, and tumors of the lung and
bones, Maltoni and Lefemine (286) examined the slides from this experiment
and concluded that the cutaneous tumors actually arose from Zymbal glands and
that the pulmonary tumors were most likely metastases from Zymbal gland car-
42
-------
Table XVII
Carcinogenicity of Vinyl Chloride
Species & Strain
Mouse, Swiss
Mouse, GDI Swiss /ChR
Mouse, NMRI
Mouse , CD-I
Rat, Ar/ IRE Wistar
Rat, Sprague-Dawley
Rat, Wistar
Rat, CD
Hamster, Golden
Rabbit, —
Route
inhalation
inhalation
inhalation
inhalation
inhalation
inhalation
oral
s.c.
i.p.
inhalationb
inhalation
inhalation
inhalation
inhalation
inhalation
Principal Organs Affected
Lung, mammary gland,
liver, vascular
system, skin
Lung, liver, mammary
gland
Lung, various sites
Lung, mammary gland,
liver
Ear duct (Zymbal gland)3
Ear duct, liver, kidney,
brain, mammary gland,
fores tomach
Liver
Kidney? (preliminary)
Kidney?, subcutaneous
tissue? (preliminary)
Ear duct, subcutaneous
tissue
Liverc (preliminary)
Liver, kidney, brain,
ear duct
Liver, lung
Fores tomach , skin
Skin, lung
Reference
(278, 279)
(280)
(281)
(282-284)
(285, data re-
examined in
ref. 286)
(278, 279,
287, 288)
(279, 287)
(279)
(279)
(279)
(289)
(279)
(282, 283)
(279)
(290)
Originally reported as skin tumors of the submaxillary parotid region, and
tumors of the lungs and bones (see text).
Prenatal exposure from 12th to 18th day of gestation.
cMarkedly potentiated by simultaneous administration of ethanol.
-------
cinemas.. Beginning in 1971, an extensive series of experiments were under-
taken by Maltoni and associates (278, 279, 286-288) to investigate the effects
of dose, length of treatment, route of administration, and species, strain,
sex, and age of animals on VC-induced carcinogenesis. The most pertinent and
interesting findings of these studies are summarized below.
A clear-cut dose-response relationship has been observed in the induction
of liver angiosarcomas in Sprague-Dawley rats (287). Exposure (4 hr/day, 5
days/week for 52 weeks) of rats to air containing 1, 5, 10, 25, 50, 100, 150,
200, 250, 500, 2,500, 6,000, 10,000, or 30,000 ppm VC led to tumor incidences
of 0, 0, 0.8, 4.2, 4.8 or 1.7 (2 groups exposed to 50 ppm), 0.8, 5.0, 10.0,
5.1, 10.0, 21.7, 22.0, 11.7, and 30.0%, respectively. The average latent
period progressively decreased from 79-135 weeks for low doses (below 50 ppra)
•to less than 54 weeks for the highest dose. By.oral administration (5
days/week for 52 weeks), doses of 0.03, 0.30, 1.0, 3.33, 16.65, or 50.0
mg/kg/day produced incidences of 0, 0.7, 2.0, 0, 12.0, and 21.0%, respec-
tively. The lowest doses capable of inducing statistically significant inci-
dences in various types of tumors were: Zymbal gland carcinoma, 10,000 ppm;
liver angiosarcoma, 200 ppm or 50 mg/kg for males, and 50 ppm or 16.65 mg/kg
for females; nephroblastoma, 100 ppm for males and 250 ppm for females; neuro-
blastoma, 10,000 ppm for females; mammary gland adenocarcionma, 5 ppm for
females; forestomach papilloma, 30,000 ppm (287).
A reduction in the length of treatment may markedly decrease the carcino-
genicity of VC. Maltoni (288) showed that Sprague-Dawley rats exposed to
6,000 ppm VC for 52, 17, or 5 weeks had liver angiosarcoma incidences of 22,
0.6, and 0%, respectively.
43
-------
The route of administration may significantly affect the carcinogenicity
of VC. In contrast to the multi-target carcinogenicity of VC by inhalation
*if
exposure, the induction of liver angiosarcoma seemed to be the only signifi-
cant carcinogenic effect of VC by oral administration (287). The carcino-
genicity of VC by intraperitoneal or subuctaneous injection may be doubtful.
Maltoni (279) reported preliminary data showing that one nephroblastoma and
one subcutaneous angiosarcoma were found among 240 rats which received 1-4
intraperitoneal injections of 4.25 mg VC. One nephroblastoma was observed
among 75 rats that received a single subcutaneous injection of 4.25 mg VC.
,' \
Significant! species- and strain-differences in. VC carcinogenesis have
been reported-by Maltoni (279, 288). Swiss mice are quite susceptible to the
n '' >! j
M / i I
carcinogenic ac kion of VC. Exposure tc'i Jair containing 50-10,000 ppm for 30
111 ''!
weeks caused hitjh incidences of lung tumors, mammary carcinomas, vascular
V!':\/fc"
tumors, and liver angiosarcomas. Epithelial tumors of the skin were also
occasionally observed. Marked increases in the incidence of mammary and
vascular tumors were evident even at the lowest dose (279). Golden hamsters
appeared to be considerably less susceptible than Swiss mice. Exposure to 50-
10,000 ppm VC in air for 30 weeks led to the induction of forestomach epi-
thelial tumors, skin trichoepitheliomas, and occasional liver angiosarcomas
and melanomas (279). Wistar rats had a similar carcinogenic response to VC as
Sprague-Dawley rats; however, the relative response of different organs or
tissues differed. The most notable difference was the considerably higher
incidence of Zymbal gland carcinomas in Sprague-Dawley than Wistar rats (279,
288).
The influence of age on VC carcinogenesis is striking. Newborn Sprague-
Dawley rats seem to be extremely susceptible to the hepatocarcinogenic action
of VC. Exposure of 1-day-old rats (a group of 43 rats to 6,000 ppm, another
44
-------
group of 46 rats to 10,000 ppm) to VC in air, 4 hr/day, 5 days/week for only 5
weeks resulted in the induction of 20 liver angiosarcomas and 28 hepatomas
after 104 weeks. Only one hepatoma was found among 240 rats similarly terated
starting at the age of 13 weeks (279, 288). Vinyl chloride is also an active
transplacental carcinogen. Exposure of pregnant Sprague-Dawley rats to 6,000
or 10,000 ppm VC in air, 4 hr/day from 12th to 18th day of gestation, was
sufficient to induce tumors in a number of offspring (279).
The influence of sex on VC carcinogenesis depends on the target organ
involved. The data of Maltoni _e_t _al_. (287) indicate that the lowest effective
carcinogenic .dose for the induction of liver angiosarcoma is lower in females
that in male Sprague-Dawley rats. On the other hand, male rats may be more
susceptible to the induction of nephroblastoma by VC. Excess mammary gland
adenocarcinomas were observed in female rats exposed to as low as 5 ppm VC in
air.
In addition to the studies summarized above, the carcinogenic!ty of VC
has also been demonstrated in several other strains of mice and rats and in
rabbits by the inhalational route. In agreement with Maltoni's results on
Swiss mice, the most affected organ in GDI Swiss/ChR (280), NMRI (281), and
CD-I (282-284) was the lung. Angiosarcomas of the liver and various other
sites, and mammary gland adenocarcinomas were also found in most of these
studies. Preliminary data of Radike et al. (289) confirmed the hepatocar-
cinogenic action of VC in Sprague-Dawley rats; in addition, there is some
evidence that ethanol potentiates the carcinogenic!ty of VC (see Section
5.2.2.1.3.8). Lee e_t al_. (282, 283) exposed groups of 36 CD strain rats of
each sex to 50, 250,- or 1,000 ppm VC in air, 6 hr/day, 5 days/week for 12
months. Exposure of rats to 250 or 1,000 ppm VC induced hemangiosarcomas of
the liver (12/58 in 250 ppm group; 22/51 in 1,000 ppm group) and the lung
45
-------
(3/58; 13/51). Caputo e_t _al_. (290) exposed a group of 40 rabbits to 10,000
ppm VC in air, 4 hr/day, 5 days/week for 12 months. After 15 months of obser-
vation, 12 skin acanthomas and 6 lung adenocarcinomas were seen; no such
tumors occurred in 20 controls.
5.2.2.1.3.5.2 Haloethenes Other Than Vinyl Chloride. The discovery of the
carcinogenic!ty of VC has spurred great interest and concern about the poten-
tial health hazards of related compounds. A number of haloethenes have been
tested for carcinogenicity; the major findings of these studies are summarized
in Table XVIII.
Vinyl Bromide. The data on vinyl bromide are sparse or incomplete at the
time of this writing. Van Duuren (217) reported that vinyl bromide was
completely inactive as an initiator (15 rag as initiating dose plus phorbol
myristate acetate as promo tor) or complete carcinogen (tested by repeated
applications of 15 mg vinyl bromide, 3 times/week for 60 weeks) on mouse
skin. Weekly subcutaneous injections-of 25 mg vinyl bromide for 60 weeks also
failed to elicit any tumor. In a short-term assay described in Section
5.2.2.1.3.1, Bol t e_t_ _al_. (220, 222) found that vinyl bromide induced preneo-
plastic lesions in newborn rats; consistent with their relative rates of
metabolism, the potency of vinyl bromide was lower than that of vinyl
chloride. Preliminary or unpublished results cited by Bahlman _et__al_. (291)
and Infante and Marlow (60) indicated that inhalation exposure of Sprague-
Dawley rats to vinyl bromide induced tumors in the liver, ear duct, and
possibly also in the lung, lymphatic system, and mammary gland. The details
of the study were not given.
Vinylidene Chloride (1,1-Dichloroethylene). The question of the carci-
nogenicity of vinylidene chloride (VDC) was first raised by Viola at the llth
46
-------
Table XVIII
Carcinogeniclty of Haloethenes Other Than Vinyl Chloride
page 1 ot I
Compound
Species & Strain
Route
Principal
Organs Affected
Reference
Vinyl bromide'
Mouse, Swiss ICR/Ha
Rat, Sprague-Dawley
Vinylidene chloride
(1,1-Dichloro-
ethylene)
Trichloroe thylene
Mouse, Swiss
Mouse, CD-I
Mouse, Swiss ICR/Ha
Mouse, B6C3Fj
Rat, Sprague-Dawley
Rat, CD
Rat, BD IV
Rat, F344
Hamster, Chinese
Mouse, NLC
Mouse, B6C3Fj
Mouse, Swiss ICR/Ha
topical None
s.c. None
inhalation Liver, ear duct, lung,
lymphatic system,
mammary gland,
(preliminary)
inhalation Kidney
inhalation No significant effect?5
topical None0
s.c. None
oral None
inhalation Mammary gland
inhalation None (preliminary)
oral None (preliminary)
inhalation None (preliminary)
inhalation No significant effect
oral6 No significant effect
oral None
inhalation None (preliminary)
oral
oral
topical
s.c.
oral
None**
Liver
None
None
None
(217)
(217)
(Huntingdon Res. Ctr.,
cited in ref. 291;
W. Busey, cited in
ref. 60)
(288,
(282,
(216)
(216)
(293)
(292)
(294)
(294)
(295)
(282,
(296)
(293)
(288)
(233)
(234)
(216)
(216)
(216)
292)
283)
283)
-------
Table XVIII (continued)
page 2 ot 2
Compound
Trlchloroe thylene
(cont'd)
Te trachloroe thylene
( Perchloroe thylene)
Species & Strain
Mouse, NMRI
Rat, Osborne-Mendel
Rat, WIST
Hams ter , Syrian
Mouse, Swiss ICR/Ha
Mouse, B6C3F.
Rat, Osborne-Mendel
Route
inhalation
oral
Inhalation
inhalation
topical
oral
oral
Principal
Organs Affected
Lympha tic sys tern
None
None
None
None •
Liver
None (inconclusive)
Reference
(297)
(234)
(297)
(297)
(216)
(298)
(298)
Vinyl bromide has also been shown to induce preneoplastic lesions in newborn rats [H.M. Bolt, Arbeitsmed.
Sozialmed. Praventivmed. 15, 49 (1980)].
Three mice developed hemangiosarcoma of the liver and two mice developed skin keratoacanthomas after exposure to
55 ppm of vinylidene chloride.
cActive as an initiator.
Two rats developed hemangiosarcomas in the mesenteric lymph node or subcutaneous tissue.
Q
Prenatal and lifetime exposure.
There was a significant increase in the incidence of liver hyperplastic nodules.
^It is not certain whether the duration of the experiment was sufficiently long.
Tetrachloroethylene has been tested in pulmonary adenoma assay and found to have no significant effect (see
Table X).
Due to high incidence of early death among treated animals.
-------
International Cancer Congress in 1974. The compound has since been tested in
at least 13-14 studies and was found to be a relatively weak carcinogen in
some studies, but inactive in others. Maltoni and coworkers (288, 292)
exposed Swiss mice to 25 ppm (maximum tolerable dose), Sprague-Dawley rats to
10-200 ppm, and Chinese hamsters to 25 ppm VDC in air, 4 hr/day, 4-5 days/week
for 52 weeks. Several additional groups of rats were given oral doses of 0.5
mg/kg/day VDC in water or 5, 10, or 20 mg/kg/day VDC in olive oil at the same
schedule. Preliminary data after 98 weeks indicated that the most significant
carcinogenic effect was the induction of kidney adenocarcinonias in mice.
Males (16% incidence) were considerably more susceptible than females (0.7% .
incidence) (288). In rats exposed by inhalation, an increase in the incidence
of mammary tumors was noted; however, no dose-response relationship was
observed. One Zymbal gland carcinoma was found in a rat in the 100 ppm
group. No increase in mammary tumors was observed among rats exposed by
ingestion. One Zymbal gland carcinoma occurred in a rat in the 10 mg/kg group
(292). In the hamsters, no tumors were found after 74 weeks (292).
A low incidence of tumors in rodents following VDC exposure was also
noted by Lee _e_t _al_. (282, 283) in a 1-year inhalation study. Among 70 CD-I
mice that survived exposure to 55 ppm VDC for 1-3, 4-6, 7-9, or 10-12 months,
3 developed hemangiosarcomas of the liver and 2 had skin keratoacanthomas. In
CD rats, 2/36 male rats exposed to 55 ppm VDC in air for 12 months developed
angiosarcomas — one in the mesenteric lymph node and one in subcutaneous
tissue. There were no tumors in exposed females.
Vinylidene chloride has also been tested by other routes of administra-
tion (see Table XVIII); none of these studies gave evidence of significant
carcinogenicity. Van Duuren £££!_• (216) did not find any carcinogenic effect
after repeated topical applications of 40 or 121 mg VDC'(3 times/week for 440-
47
-------
594 days) or subcutaneous injections of 2.0 mg VDC (weekly for 518-694 days)
to female Swiss ICR/Ha mice. The compound (125 mg) was, however, active as an
initiator in a two-stage skin carcinogenesis bioassay, inducing 1 squamous
cell carcinoma and 9 papillomas in 8/30 mice. Rampy £t_ _al_. (294) exposed
Sprague-Dawley rats to VDC orally (68-220 ppm in drinking water) or by inhala-
tion (10-75 ppm in air) for 18 months. Preliminary data indicated that the
tumor incidence in VDC-exposed rats was not greater than in the controls. The
same conclusion was reached by Viola and Caputo (295) from their preliminary
data of an inhalation study in which Sprague-Dawley rats were exposed to 75 or
100 ppm VDC.
Additional bioassay studies support the lack of carcinogenicity of VDC.
In the study of Ponomarkov and Tomatis' (296), pregnant female BD IV rats were
given an oral dose of 150 mg/kg body weight of VDC on day 17 of gestation and
their offspring were administered orally 50 mg/kg/week VDC from weaning to the
end of the life span. There was an increase, although not statistically
significant, of liver and meningeal tumors. However, the increase in the
incidence of liver hyperplastic nodules was significant. In a U.S. National
Toxicology Program carcinogenesis bioassay (293), groups of 50 B6C3F, mice and
50 F344 rats of each sex were given VDC orally for 104 weeks. The doses were
2 or 10 mg/kg for mice and 1 or 5 mg/kg for rats. No significant increase in
tumor incidence was observed in mice and rats of either sex.
Trichloroethylene. Information on the carcinogenicity of trichloro-
ethylene (TCE) is summarized in Table XVIII. Trichloroethylene was found
noncarcinogenic in a limited study by Rudali (233). Twenty-eight NLC mice
were given orally 0.1 ml of a 40% TCE solution, twice a weekly for 6 months.
No tumors were found at the termination of the experiment of unspecified dura-
tion. The carcinogenicity of TCE (industrial grade, epoxide-stabilized,
48
-------
> 99%) was first detected in B6C3F1 mice (234; see also Table VIII). Signifi-
cant increase in the incidence of hepatocellular carcinoma was observed in
mice of both sexes, the males being more susceptible. In male mice of the
high dose group, the first tumor was seen at the 27th week, 9 other tumors
were found by the 78th week. In contrast to BSCSF^ mice, Osborne-Mendel rats
were not susceptible to TCE. The tumor incidence in rats given 549 or 1,097
mg/kg/day of TCE, 5 times/week for 78 weeks was not significantly different
from those in control rats.
The carcinogenicity of TCE has also been tested in female Swiss ICR/Ha
mice by Van Duuren et al. (216) by skin painting, subcutaneous injection, or
by oral administration. None of these treatments brought about any signifi-
cant increase in tumor incidence.
Henschler et al. (297) have investigated more recently the carcino-
genicity of TCE in 3 animal species via inhalation exposure. Groups of NMRI
mice, WIST rats, and Syrian hams te'rs of each sex were exposed to 100 or 500
ppm purified TCE, 6 hr/day, 5 days/week for 18 months. The only significant
effect observed was an increase in the incidence of malignant lymphomas in
NMRI mice: 9/29 in controls, 17/30 in 100 ppm group, 18/28 in 500 ppm
group. Since this strain of mice is known to have a relatively high spon-
taneous incidence of malignant lymphoma, the authors did not consider the
effect highly significant. In. rats and hamsters, no carcinogenic effects were
observed. The authors concluded from their findings that purified TCE is not
carcinogenic. However, they stressed that their conclusions may apply only to
pure TCE stabilized by an amine base instead of epoxide-stabilizers used in
industrial grade TCE.
49
-------
Te trachloroethylene. The carcinogenic!ty of tetrachloroethylene (USP
grade) has been tested by oral administration (298; see also Table VIII).
Significant increases in the incidence of hepatocellular carcinomas were
observed in B6C3F, mice of both sexes. The first tumor was detected in a male
animal (in the low dose group) that died during the 27th week. In the same
study, no significant increase in the tumor incidence was observed in Osborne-
Mendel rats receiving 471 and 941 mg/kg/day (for males) and 474 and 949
mg/kg/day (for females), respectively. However, since a high incidence of
early death occurred among treated rats due to toxic nephropathy, the negative
finding in rats was not considered conclusive. Tetrachloroethylene has also
been tested by Van Duuren et al. (216) by skin painting; repeated applications
(3 times 18 or 54 mg/week for 426-576 days) did not elicit any significant
increase of local or distant tumors.
5.2.2.1.3.6 HALOPROPENES.
Only three halopropenes have -thus far been tested for carcinogenicity
(see Table XIX). Van Duuren et _al_. (216) showed that repeated skin painting
(3 times/week for at least 342 days) of 1-chloropropene (2.5 mg/application),
allyl chloride (31 or 94 mg/application), or cis-1,3-dichloropropene (41 or
122 mg/application) to female Swiss ICR/Ha mice did not induce any significant
carcinogenic effects. Only allyl chloride (99 mg) was weakly active as an
initiator in the 2-stage skin carcinogenesis assay, inducing 10 papillomas in
7/30 mice. By subcutaneous injection (once a week for over 538 days), both
1-chloropropene (1.0 mg/injection) and allyl chloride (1.5 mg/application)
were inactive, while cis-1,3-dichloropropene (3.0 mg/injection) was signifi-
cantly carcinogenic, inducing local sarcomas in 6/30 mice. Only 1-chloropro-
pene was tested by oral administration; weekly intragastric administration of
1.0 mg of the compound led to significant increase in the incidence of fore-
50
-------
Table XIX
Carcinogenic!ty of Halopropenes
Compound Species & Strain
1-Chloropropene Mouse, Swiss ICR/Ha
Allyl chloride Mouse, B6C3FJ
(3-Chloropropene) Mouse, Swiss ICR/Ha
Rat, Osborne-Mendel
cis-l,3-Dichloro- Mouse, Swiss ICR/Ha
propene
Route
topical
s.c.
oral
oral
topical
s.c.
oral
topical
s.c.
Principal
Organs
Affected Reference
None
None
Fores tomach
None3
Noneh
None
None
None
Local sarcoma
(216)
(216)
(216)
(299)
(216)
(216)
(299)
(216)
(216)
There was some suggestive evidence of positive association with neoplastic
lesions of the forestomach.
'Active as an initiator.
-------
stomach tumors (13/30, including 3 squamous cell carcinomas) in female mice.
Four out of 30 male mice also developed forestomach tumors but the increase in
the incidence was not statistically significant.
In addition to the above study, a bioassay for possible carcinogenicity
of technical-grade allyl chloride was conducted by NCI (299) using BSCSF^ mice
and Osborne-Mendel rats. The time-weighted average doses were: 172 and 199
mg/kg/day for male mice; 129 and 258 mg/kg/day for female mice; 57 and 77
mg/kg/day for male rats; and 55 and 83 mg/kg/day for female rats. The animals
were dosed orally 5 times/week for 78 weeks and observed for an additional 14
weeks for mice and 30-33 weeks for rats. The survival rate of high dose male
mice and high dose rats of both sexes was extremely poor. Based on observa-
tions on the surviving animals, some "suggestive" evidence of a relatively
weak carcinogenicity of the compound was noted in mice of both sexes. Low
incidence of squamous-cell carcinomas (2/46 low dose males; 2/47 low dose
females) or papillomas (1/47 low dose males; 3/45 low dose females) of the
forestomach were observed. In addition, proliferative nonneoplastic lesions
(e.g., acanthosis and hyperkeratosis) occurred in the stomach of many treated
mice but not in controls. No convincing evidence for the carcinogenicity of
allyl chloride was found in Osborne-Mendel rats of both sexes.
5.2.2.1.3.7 HALOBUTENES, HALOBUTADIENES AND ARYLALKYL HALIDES.
One halobutene and two halobutadienes have been bioassayed for carcino-
genicity; the results of these studies are summarized in Table XX. Van Duuren
££^i.' ^215) tested trans-1,4-dichloro-2-butene by topical, subcutaneous, or
intraperitoneal route in female Swiss ICR/Ha mice. By subcutaneous injection
(1 dose of 0.05 mg/week for 537 days), the compound was weakly carcinogenic,
inducing local sarcomas in 3/30 mice. By intraperitoneal administrations
51
-------
Table XX
Carcinogenic!ty of Halobutenes and Halobutadienes
Compound
trans-1 , 4-Dichloro-
2-butene
2-Chloro-l , 3-buta-
diene (Chloro-
prene)
Hexachlpro-1 ,3-buta-
dieneb
Species & Strain
Mouse, Swiss ICR/Ha
Rat, —
Mouse, random-bred
Rat, random-bred
Rat, BD IV
Mouse, Swiss ICR/Ha
Rat, Sprague-Dawley
Route
topical
s.c.
i.p.
inhalation
topical
oral
s.c.
intratracheal
oral3
topical
oral
Principal
Organs
Affected
None
Local sarcoma
No significant
effect
Nasal cavity
(preliminary)
None
None
None
None
None
None
Kidney
Reference
(215)
(215)
(215)
(B.C.
cited
(300,
(300,
(300,
(300,
(296)
(216)
(108,
McKusick
in ref. 51)
301)
301)
301)
301)
302)
Prenatal and lifetime exposure.
'inactive in pulmonary adenoma assay (see Table X).
-------
(1 x 0.05 mg/week for 537 days), the compound induced local sarcomas in 2/30
mice; however, the increase in the, tumor incidence was not statistically
significant. By topical route, the compound was inactive both as a complete
carcinogen (3 x 1.0 mg/week for 537 days) or as an initiator (one initiating
dose of 1.0 mg followed by promotion with phorbol myristate acetate). The
compound was subsequently tested by inhalation and a significant incidence of
malignant nasal tumors in rats exposed to 5 ppm was reported (McKusick, cited
in ref. 51).
2-Chloro-l,3-butadiene (chloroprene) has been tested in randombred albino
mice and rats by Zil'fyan et_ _al_. (300, 301) by several routes of adminis-
tration. Their data have been reviewed by an IARC Study Group (23). The
compound was noncarcinogenic in the mouse tested by repeated skin applications
(twice-weekly applications of a 50% solution of chloroprene in benzene for 25
weeks). In the rat, the compound did not induce tumors after oral (2 x 200
mg/kg/week for 25 weeks), intratracheal (5 x 200 mg/kg at 20-day intervals),
or subcutaneous (10 x 400 mg/kg) administration. However, the IARC Study
Group (23) pointed out the incomplete reporting of the experimental details.
A long-term ingestion study of chloroprene has recently been described by
Ponomarkov and Tomatis (296). Pregnant female BD IV rats were given an oral
dose of 100 mg/kg body weight of chloroprene on day 17 of gestation. Their
offspring were given by stomach tube 50 mg/kg/week of the compound from the
time of weaning to the end of the lifespan. The test yielded no evidence for
the carcinogenicity of the compound.
Kociba _e_t _al_. (108, 302) tested hexachloro-1,3-butadiene (HCBD) in
Sprague-Dawley rats. The rats were fed, for up to 2 years, diets containing
sufficient HCBD to maintain daily intakes of 0.2, 2.0, or 20 mg/kg body
weight. Ingestion of the highest dose produced a signficant increase in the
52
-------
incidence of renal tubular adenomas and adenocarcinomas. Males (18% inci-
dence) were more susceptible than females (7.5% incidence). No renal tumors
were observed in rats fed lower doses of HCBD. In the pulmonary adenoma assay
of Theiss e_t__al_. (251), HCBD was inactive (see Table X). Van Duuren et al.
(216) have tested HCBD by skin application in female Swiss ICR/Ha mice. The
compound was inactive in tests as a complete carcinogen (3 x 2.0 or 6.0
mg/week for over 440 days) or as an initiator (a single dose of 15 mg followed
by phorbol myristate acetate).
Benzyl chloride (C^HrCH^Cl), an arylalkyl halide, has been included for
. i'
discussion in this section because its chemical properties (see Section
!i ' '
5.2.2..U.2.1) resemble those of allyl halides more closely than those of aryl
']'
halidefs1. Druckrey et_ _al_. (68) reported that benzyl chloride induces local
i' •
sarcomas at the site of subcutaneous injections into BD strain rats. The
tumor incidences were 3/14 and 6/8 in two groups of rats that received weekly
injections of 40 and 80 mg/kg benzyl chloride, respectively. The mean latent
period was 500 days. The carcinogenic potency of benzyl chloride was compar-
able to that of iodomethane. In the pulmonary adenoma assay, Poirier et al.
(247), found benzyl chloride inactive (see Section 5.2.2.1.3.1).
5.2.2.1.3.8 MODIFICATION OF CARCINOGENESIS.
Considering the importance of this class of compounds, surprisingly
little is known on the potential role of environmental factors in modifying
the carcinogenicity of haloalkanes and haloalkenes. However, carbon tetra-
chloride has been extensively used in syncarcinogenesis studies and in studies
on the modification of the carcinogenicity of other compounds. Carbon tetra-
chloride acts synergistically with a variety of synthetic or naturally
occurring carcinogens, such as 2-N-fluorenylacetamide (303), 3-methylchol-
53
-------
anthrene (238), 2,7-bis(acetamide)fluorene (304), aflatoxin B^ (305), and
flower stalk of a type of Japanese coltsfoot, Petasites japonica (306) in the
induction of liver tumors in rodents. In addition, combined treatment of
/3-naphthylamine and carbon tetrachloride induces tumors in both the liver and
the urinary bladder in the dog (307). Carbon tetrachloride has also been
shown to potentiate the carcinogenic action of N-nitroso compounds (such as
dimethyl-, diethyl-, and methylethylnitrosamine, N-butyl-N-nitrosourea, and
dimethylnitrosamine precursors) (308-312; see also Section 5.2.1.2.3.7),
/
polycyclic aromatic hydrocarbons such as benzo[a]pyrene (313), azo dyes such
as 3'-methyl-4-dimethylaminoazobenzene (314), and aromatic amines such as
2-N-fluorenylacetamide (315, 316). In most of these studies, hepatonecrotic
doses of carbon tetrachloride were required and for this reason the potentia-
tion is generally considered to be due to the promoting, co-carcinogenic or
"chemical traumatic" (a form of "chemical hepatectomy") effects of carbon
tetrachloride. With the N-nitroso compounds, maximum potentiation was
observed when carbon tetrachloride was administered shortly (1 day) before the
N-nitroso compounds (309, 311). However, carbon tetrachloride also poten-
tiates the carcinogenicity of diethylnitrosamine when given repeatedly af ter
the administration. There is some evidence that the two potentiating effects
(i.e., before and after diethylnitrosamine) are additive and independent of
each other (310). Not all carcinogens are potentiated by carbon tetra-
chloride, however. For example, administration of carbon tetrachloride before
urethan reduces the incidence of lung adenomas by 32-47% in CC57Br mice (317).
The carcinogenicity of vinyl chloride is modified by ethanol and disul-
firam. A preliminary report by Radike et al. (289) suggests potentiation of
carcinogenicity of vinyl chloride by ethanol. Male Sprague-Dawley rats
receiving the combined treatment (73%) developed more liver tumors than those
54
-------
receiving vinyl chloride (38%) or ethanol (0%) alone. Wins ton _et__al_. (318)
reported that disulfiram protects against the carcinogenic effects of vinyl
chloride in CD-I mice. Disulfiram delayed the induction of bronchiole-
alveolar adenomas and reduced the incidence of hepatic hamangiosarcomas and
mammary gland tumors.
In sharp contrast to the protective effect of disulfiram against vinyl
chloride and a number of other carcinogens (see Section 5.2.1.6.3.9), disul-
firam has been shown to enhance the carcinogenic!ty of 1,2-dibromoethane in
Sprague-Dawley rats. Rats receiving the combined long-term treatment had
significantly higher incidences of hepatic, splenic, and renal tumors than
those receiving 1,2-dibromoethane alone and developed moreover hemangiosar-
comas of the omentum (Midwest Research. Institute, 1979. cited in ref. 262).
The effect is believed to be related to the inhibition of alcohol dehydro-
genase by disulfiram (262; see also Section 5.2.2.1.4.1.2).
Another unusual case of synergism was noted by Epstein _e_t_ _al_. (253) in a
study on fluoroalkanes (Freon 112, Freon 113) and piperonyl butoxide. When
administered singly, neither fluoroalkane nor piperonyl butoxide was carcino-
genic in neonatal Swiss ICR/Ha mice; however, combined treatment of Freon 112
(l,l,2,2-tetrachloro-l,2-difluoroethane) and piperonyl butoxide led to the
induction of hepatomas in 31% of male mice. Three female mice developed
malignant lymphomas.. Combined treatment of Freon 113 (1,1,2-trichloro-l,2,2-
trifluoroethane) and piperonyl butoxide also enhanced the incidence of hepa-
tomas in male mice. The mechanism of the synergism is not known. It was
suggested (253) that piperonyl butoxide may modify a presumed in vivo dehalo-
genation of the fluoroalkanes.
55
-------
5.2.2.1.4 Metabolism and Mechanism of Action.
The metabolism and mechanisms of action of haloalkanes and haloalkenes
have been extensively studied. Depending on the chemical structure, halo-
alkanes and haloalkenes may either interact directly with cellular macromole-
cules or may first undergo metabolic activation to reactive intermediates to
initiate carcinogenesis and mutagenesis.
5.2.2.1.4.1 METABOLISM AND MECHANISM OF ACTION OF HALOALKANES.
5.2.2.1.4.1.1 Halomethanes. A comparative metabolic study of haloalkanes and
haloalkenes has been conducted by Nakajima and Sato (319) in Wistar rats.
Among chlorinated halomethanes, the in vivo relative rates of metabolism
follow the order: dichloromethane > chloroform » carbon tetrachloride. Male
rats display higher metabolic activity than female rats. Food deprivation
increases the metabolic rates in both sexes.
Monohalomethanes. Very little information is available on the metabolism
of monohalomethanes. The U.S. Environmental Protection Agency (320) reviewed,
in 1980, the available information on chloromethane. Several reports indicate
the presence of methanol, formaldehyde or formate in the blood or urine of
animals or humans exposed to chloromethane. However, these findings have not
been consistently confirmed. Monohalomethanes may be capable of reacting
directly with nucleophiles. lodomethane (methyl iodide) is a well known
methylating agent. Among the haloalkanes tested in the NBP reaction, methyl
iodide is the most active alkylator (see Section 5.2.2.1.2.1). lodomethane,
bromomethane and chloromethane are all mutagenic in the Ames test without
activation. There is some evidence that chloromethane may react with tissue
nucleophiles by an enzyme-catalyzed reaction. Redford-Ellis and Gowenlock
(321, 322) detected the presence of S-methyl-cysteine, S-methyl-glutathione
56
-------
and traces of methylated histidine and methionine after in vitro reaction
of ^C-chloromethane with human blood or tissue homogenates. Covalent binding
was substantially reduced when the blood or tissue homogenates were heated.
Dihalomethanes. The metabolism of dihalomethanes has. been extensively
investigated by Anders, Ahmed, Kubic and coworkers (reviewed in 323, 324). In
vitro studies indicate that the rates of metabolism of dihalomethanes ranks as
follows: CH2I2 > CH2Br2 > CH2BrCl > CH2C12 (325-328). The two major
metabolic pathways of dihalomethanes are shown in Fig. 1. In the first path-
way, dihalomethane is hydroxylated by mixed function oxidases to yield
hydroxydihalomethane, which spontaneously decomposes to give formyl halide and
carbon monoxide. This pathway is supported by the increase in carboxyhemo-
globin in humans and animals exposed to dihalomethanes. The production of
carbon monoxide and carboxy-hemoglobin from the dihalomethane was confirmed
1 O
using C-labeled dichloromethane (329). The involvement of mixed function
oxidases is indicated by the requirement of NADPH, and molecular oxygen.
Agents (e.g., phenobarbital, 3-methylcholanthrene, SKF-525A) that modify the
v
activity of cytochrome P-450 also bring about a corresponding change in the
metabolism of dihalomethanes to carbon monoxide (325). Metabolic studies
1 8
using deuterated dihalomethanes and °02 substantiate the view that the inser-
tion of oxygen (from OO in the C-H bond is the rate-limiting step (330). The
formyl halide intermediate is a potential acylating agent and has been postu-
lated to be a reactive intermediate responsible for covalent binding of
dichloromethane to tissue nucleophiles. The covalent binding of dichloro-
me thane to microsomal lipids and proteins has been demonstrated and shown to
have the same requirement for NADPH and oxygen, the same response to pheno-
barbital pretreatment and similar kinetic properties as its metabolism to
carbon monoxide (323).
57
-------
H-O-C-X
NE
-H+ X-'
+GSH
-HX
0
II
H-C—X
ME m
-H+X-*
CO
[
HCHO] + GSH
VFDH
GS-CHO -- GSH + HCOOH
Fig. 1. Proposed metabolic pathways of dihalomethanes. The abbrevia-
tions used are: MFO = mixed function oxidases; NE = nonenzymic process;
GSH Tr = glutathione transferase; FDH = formaldehyde dehydrogenase; SFGH =
S-formyl glutathione hydrolase. Compounds with;an asterisk are potential
reactive intermediates. [Adapted from A.E. Ahmed, V.L. Kubic, J.L.
Stevens, and M.W. Anders: Fed. Proc. 39, 3150 (1980)]
-------
In addition to the above pathway, dihalomethanes are also metabolized to
formaldehyde and inorganic halide. Ahmed and Anders (326, 331) have studied
this pathway in detail (see Fig. 1). The reaction is catalyzed by enzymes
present in hepatic cytosolic fraction and requires reduced glutathione (GSH)
,as a co-factor. The reaction may be inhibited by sulfhydryl reagents (e.g.,
p-chloromercuribenzoate, diethyl maleate) and by known substrates of gluta-
thione transferase (e.g., iodomethane, l-chloro-2,4-dinitrobenzene) indicating
the involvement of the enzyme in the metabolism of dihalomethane. Similar
metabolic rates have been observed with the use of dibromomethane, deuterated
dibromomethane and bromochioromethane suggesting that nucleophilic attack on
the carbon with subsequent displacement of halide is the initial, rate-
limiting step. S-Halomethyl glutathione conjugate has been postulated to be
the reaction intermediate, which is expected to undergo nonenzymic hydrolysis
to S-hydroxymethyl glutathione, which in turn may be converted to formaldehyde
and glutathione. The production of formaldehyde may be substantially
decreased in the presence of NAD ; instead, formic acid is produced. It has
been suggested (324) that cytosolic formaldehyde dehydrogenase may oxidize
S-hydroxymethyl glutathione to S-formyl glutathione, which is then hydrolyzed
by cytosolic S-formyl glutathione hydrolase to formic acid and glutathione.
The biological significance of this metabolic pathway is not clear. Although
the pathway appears to be detoxifying in nature, it should be pointed out that
the S-halomethyl glutathione intermediate is an o(-halomethyl thioether, which
may possess reactivity similar to that of the potent carcinogen, bis(chloro-
methyl)ether (see Section 5.2.1.1.2). As expected, the mutagenicity of
dibromo- and diiodomethane is also enhanced by the inclusion of microsomal or
cytosolic fraction (see Section 5.2.2.1.2.2).
58
-------
Trihalomethanes (haloforms). Comparative in vivo and in vitro studies by
Anders and associates (324) showed that the relative metabolic rate of tri-
halomethanes, follows the order: CHIj > CHBr3 > CHBr2Cl > CHBrCl2 = CHCLj.
Substantial species differences have been noted in the in vivo metabolism of
chloroform. Brown _e_£ _al_. (332) showed that after the administration of a
single oral dose (60 mg/kg) of ^C-labeled chloroform, mice, rats, and monkeys
excreted 6%, 20%, and 79% of the dose unchanged and 80%, 66%, and 18% of the
dose as C0? in expired air, respectively. A study by Fry et_ al_. (333) indi-
cated that in humans orally administered chloroform is largely expired
unchanged. VI
• ; i1 .,•_
The possible metabolic pathways of tJ-ihalomethanes are depicted! jin Fig.
2. Several investigations concur that'mi'fir^somal hydroxylation of ' i'hte C-H
Li 11 ''•'
bond is the rate-limiting initial step. Ttife hydroxytrihalomethane th'us formed
V'.yjv'
is highly unstable and may decompose to dihalocarbonyl intermediate, which may
(a) be hydrolyzed to carbon dioxide, (b) react with cysteine to form 2-oxothi-
azolidine-4-carboxylic acid (OTZ), (c) react with sulfhydryl compounds to
yield disulfide and carbon monoxide, or (d) covalently bind to tissue macro-
malecules. In the study of Mansuy ^_t_jil_. (334), aerobic incubation of chloro-
form in the presence of rat liver microsomes and NADPH yielded a reactive'
intermediate which reacted with cysteine to form OTZ. The intermediate was
deduced to be dichlorocarbonyl, more familiarly known as phosgene. A subse-
quent study by the same investigators (335) showed that phosgene was
responsible for the covalent binding of chloroform to microsomal macromole-
cules and that human microsomes also metabolize chloroform to phosgene.
In vitro and in vivo studies by Pohl and coworkers (336-340) support the
conclusion that phosgene is the reactive intermediate of chloroform. The
production of phosgene was significantly decreased when deuterated chloroform
59
-------
X
H-O-C-X
X
NE
-HX
•t-H^O
-HX
-HX/
ACntai
0 NH2 /COOH
X-C-S-CHgCHCOOH —» ^ JV-H
-HX
0
II
X—C-X
'fCystein
0
QIZ
R-S-C-X
R-S-S-R -I- CO
Covolent binding to tissue
mocromolecules
Fig. 2. Proposed metabolic pathways of trihalomethanes. The abbre-
viations used are: MFO = mixed function oxidases; NE = nonenzymic process;
RS~ = glutathione or other sulfhydryl compounds; OTZ = 2-oxothiazolidine-4-
carboxylic acid.
-------
was used, supporting the view that the breakage of the C-H bond is the rate-
limiting step (338, 340). Binding studies with the use of 14C-, 3H-, or 36C1-
labeled chloroform indicate that only the C-label becomes appreciably bound
by covalence to inicrosomal proteins. Covalent binding is proportionately
decreased when phosgene is trapped as OTZ by the addition of cysteine. These
results led Pohl _et>_al_. (339) to suggest that phosgene is the major, if not
the only, reactive intermediate formed from chloroform. Phosgene produced
from chloroform may also react with glutathione; diglutathionyl dithiocar-
bonate (GS-CO-SG) has been demonstrated to be a final metabolite of chloroform
(341).
Like dihalomethanes, trihalomethanes are also metabolized to carbon
monoxide; this pathway has been investigated in detail by Anders and
associates (323, 324, 342-345). In vitro production of carbon monoxide from
trihalomethanes requires the presence of active microsomes, NADPH and
molecular oxygen. Pretreatment of animals with phenobarbital or 3-methylchol-
anthrene increases whereas cobaltous chloride or SKF-525A decreases the.
activity. Addition of glutathione or sulfhydryl compounds greatly increases
the production of carbon monoxide, although glutathione alone was found inef-
fective without NADPH and molecular oxygen (343). The use of 13C-labeled
tribromomethane and molecular Oo showed that the oxygen of carbon monoxide
originated from molecular oxygen (343) whereas the carbon was from tribromo-
methane (342). The lower metabolism of deuterated tribromomethane supports
the view that breakage of C-H bond is the rate-limiting step (342, 345). The
presence of dibromocarbonyl, the bromo analog of phosgene, as a reactive
intermediate of tribromomethane has also been demonstrated by the trapping of
the intermediate by cysteine as OTZ (345). Anders1 group (324, 345) concluded
that tribromomethane is first hydroxylated by microsomal mixed function oxi-
60
-------
dase. The successive attacks by two molecules of sulfhydryl compounds on .
dibromocarbonyl yield a disulfide compound and carbon monoxide (gee Fig. 2).
The covalent binding of chloroform to microsomal proteins and lipids has
been demonstrated by various investigators (175, 335, 339, 340, 346-350).
There is ample evidence to suggest that the covalent binding may be related to
the toxic action of chloroform (e.g., 340, 349, 351). However, there is no
evidence of covalent binding of chloroform to RNA (175, 348, 350) or DNA
(350). Diaz Gomez and Castro (350) have recently demonstrated significant
covalent binding of chloroform to hepatic histones and to nonhistone proteins;
they suggested that the covalent binding to nuclear protein may be related to
chloroform-induced hepatocarcinogenesis. This epigenetic mechanism is consis-
tent with the lack of mutagenic action of chloroform in the Ames test (see
Section 5.2.2.1.2.2).
Tetrahalomethane. Carbon tetrachloride is the only tetrahalomethane that
has been extensively studied. The possible metabolic pathways of carbon
tetrachloride are depicted in Fig. 3. It is now generally accepted that the
first metabolic step involves reductive dehalogenation through interaction
with cytochrome P-450 with the formation of the extremely short-lived tri-
chloromethyl free radical (reviewed in 111, 113, 352, 353). Formation of the
free radical has recently been demonstrated in vitro (352, 354) and in vivo
(355) by electron spin resonance studies with the use of "spin-trapping"
compounds although the exact form of the free radical is uncertain. Under
anaerobic conditions, the trichloromethyl free radical may undergo a variety
of reactions including (a) dimerization to hexachloroethane (356, 357), (b)
addition of a proton and an electron to form chloroform (356-359), (c) binding
to microsomal proteins and lipids (175, 348, 357, 360, 361), and (d) further
reductive dehalogenation to carbon monoxide probably via a dichlorocarbene
61
-------
COOH
Macromolecules
Covalent
A NMembrane lipids—
Lipid peroxidation
Conjugation
Molonaldehyde production
CO,
CO + HCOOH
^ [C.3C-OH] -
+2GSH
Covalent binding GS-C-SG
to tissue macro-
molecules DGC
Fig. 3 Proposed metabolic pathways of carbon tetrachloride. The
abbreviations used are: P-450 = cytochrome P-450; GSH = glutathione; OTZ
2-oxothiazolidine-4-carboxylic acid; DGC = diglutathionyl carbonate.
-------
) intermediate (362). Under aerobic condition, however, it appears that
the trichloromethyl free radical is predominantly oxygenated, with the forma-
tion of carbon dioxide as the final product (353, 363, 364). Shah et al.
(353), and Kubic and Anders (365) have recently identified phosgene (dichloro-
carbonyl or carbonylchloride) as an intermediate in the aerobic metabolism of
carbon tetrachloride, by trapping the intermediate with cysteine to yield
2-oxothiazolidine-4-carboxylic acid. Further evidence of phosgene as the
intermediate has been provided by Pohl et al. (341), who isolated digluta-
«
thionyl carbonate (GS-CO-SG) as a final metabolite of carbon tetrachloride
indicating the interaction of phosgene with 2 molecules of glutathione. The
production of phosgene requires the presence of molecular oxygen and NADPH, is
not affected by glutathione, and may be inhibited by carbon monoxide or SKF-
525A, suggesting the involvement of cytochrome P-450 (365). Shah _e_t _al_. (353)
postulated the formation of hydroxytrichlorome'thane as the precursor of phos-
gene.
Despite extensive studies, the mechanism of carcinogenic action of carbon
tetrachloride remains obscure. At least two possible mechanisms have been
proposed. It is well known that carbon tetrachloride binds covalently to
microsomal proteins and lipids following metabolic activation. Using 3"C1-
labeled carbon tetrachloride, Reynold (360) showed the incorporation of ^°C1
into microsomal lipids and proteins; he regarded trichloromethyl free radical
CCClo) as the reactive intermediate. The evidence for covalent binding of
carbon tetrachloride to nucleic acids appears to be less convincing. A number
of investigators (175, 348, 356, 357, 360, 362) were unable to detect signifi-
cant levels of covalent binding of carbon tetrachloride to nucleic acids.
Only two groups of investigators reported positive findings. Ro.cchi et al.
(366) did not observe any covalent binding in untreated animals; however,
62
-------
after 3-methylcholanthrene pretreatment, in vivo covalent binding to mouse
liver DNA or rat liver RNA was detected. In in vitro studies, covalent
binding was observed only if hepatic microsomes from 3-methyclcholanthrene-
pretreated rodents were used along with "pH 5 enzyme"; significant level of
covalent binding to nuclear proteins also occurred (366). The covalent
binding of carbon tetrachloride to mouse hepatic DNA and nuclear proteins has
recently been confirmed by Diaz Gomez and Castro (367), who suggested that
both processes could be relevant to the hepatocarcinogenic action of the
compound. The recent identification of phosgene as a reactive intermediate of
carbon tetrachloride metabolism invites further investigations. Phosgene has
two highly reactive chlorines and may well act as a bifunctional alkylating
agent.
The peroxidation of microsomal lipids by free radicals originating from
carbon tetrachloride has been suggested to play a major, if not obligatory,
role in its toxic and possibly carcinogenic action (113, 368). Considering
the extremely short half-life (estimated to be around 1 microsecond) of
the "CClo radical, Slater (368) postulated a cascade type of events in
which 'CC1., first reacts with microsomal polyunsaturated fatty acids to form
fatty acid radicals and peroxy radicals, which then break down to yield dif-
fusible hydroperoxides and unsaturated hydroxy-aldehydes such as malonaldehyde
(which has been reported to be a carcinogen, see Section 5.2.1.7.1.) Alter-
^
natively, peroxidation of membrane lipids which is known to bring about a
spectrum of pathological changes leading to fatty liver, loss of protein
synthesizing capability, structural disorganization of the endoplasmic reti-
culum, and eventually cell necrosis (113). It is possible that some of these
pathological changes represent aspects of epigenetic mechanism for carcino-
genesis.
63
-------
Besides carbon tetrachloride, the metabolism of fluorotrichloromethane
(Freon 11), difluorodichloromethane (Freon 12), and bromotrichloromethane has
been studied. The presence of a fluorine atom increases while the presence of
the bromine atom decreases the stability against reductive dehalogenation.
Thus, Cox et al. (369) were unable to detect reductive dehalogenation of
fluorotrichloromethane by microsomes from phenobarbital-pretreated mice, rats,
guinea pigs or hamsters. No evidence of free radical or fluorodichloromethane
was found. An in vivo study by Blake and Mergner (370) provided no firm.
evidence of metabolism of fluorotrichloromethane in dogs. For difluorodi-
chlorome thane, at most, only about 1% of the compound appeared to be metabo-
lized in dogs. Nonetheless, in vitro binding study of fluorotrichloromethane
by Uehleke and his associates (175, 348) showed that the fluorocarbon does
bind covalently to microsomal proteins and lipids; however, the extent of
binding was substantially lower than that of carbon tetrachloride. Pohl et
al. (341) have shown subsequently that bromotrichloromethane is probably
metabolized in a similar manner as carbon tetrachloride but higher amounts of
dihalocarbonyl were produced from bromotrichloromethane.
5.2.2.1.4.1.2 Haloethanes. Several comparative metabolic studies have been
undertaken on haloethanes. In a series of reports, Yilner (371-375) studied
the metabolism of 5 chloroethanes; his major findings are summarized in Table
XXI. It is evident from the Table that both the metabolic rate and the meta-
bolic fate of chloroethanes are dependent on the number and the position(s) of
chlorine substituent(s). 1,1,2,2-Tetrachloroethane is metabolized much faster
than its 1,1,1,2-isomer. The major metabolite is S-carboxymethyl cysteine for
1,1-di- and 1,1,1-trichloroethane, whereas for 1,1,1,2-tetra- and pentachloro-
ethanes, trichloroethanol and trichloreacetic acid appear to be the only
urinary metabolites. Trace amounts of chlorinated ethylenes may be detected
64
-------
Table XXI
Comparative Metabolism of C-Labeled Chloroethanes in Mice3
Chloro-
e thane
1 , 2-Di-
1,1,2-Trl-
1,1,1,2-
Te tra-
1,1,2,2-
Te tra-
Penta-
o
Summarized
Dose
(g/kg) -
0.05-0.17
(i.p.)
0.1-0.2
(i.p.)
1.2-2.0
(s.c.)
0.21-0.32
(i.p.)
1.1-1.8
(s.c.)
Expired
Unchanged
(% of dose)
10-45
6-9
21-62
< 4
12-51
from the data of S. Yilner
Metabolites
(% of dose)
Expired Urinary
12-15 (as C02) 51-73
10-13 (as C02) 73-87
< 0.02 18-56
(as CHC1=CC12)
45-61 (as C02) 23-34
0.2-0.4
(as CHC1=CC17)
0.2-0.4
(as CC12=CC12)
2-16 (as CHC1=CC12) 25-50
3-9 (as CHC1=CC12)
[Acta Pharmacol. Toxicol. 30, 257
Major Identified
Urinary Metabolite
(% of urinary 14C activity)
S-Carboxyme thylcys teine
(44-46%, free)
(0.5-5%, conjugated)
Thiodiacetic acid (33-34%)
Chloroacetic acid (6-23%)
S-Carboxyme thylcys teine
(29-46%, free)
(3-10%, conjugated)
Thiodiacetic acid (38-42%)
Chloroacetic acid (6-31%)
Trichloroethanol (89-94%)
Trichloroacetic acid (6-12%)
Dichloroacetic acid (20-34%)
Trichloroacetic acid (2-8%)
Trichloroethanol (3-15%)
Oxalic acid (5-10%)
Glyoxylic acid (0.4-1.4%)
Unidentified (approx. 50%)
Trichloroethanol (64%)
Trichloroacetic acid (36%)
(1971); 30, 248 (1971); 29, 471
(1971); 29, 499 (1971); 29, 481 (1971).] The excretion of radioactivity was followed by 3 days."
-------
in the expired air of. mice given 1,1,1,2-tetra-, 1,1,2,2-tetra-, or penta-
chloroethanes. A comparative study of Nakajima and Sato (319) shows that the
relative in vivo metabolic rate of chloroethanes follows the order: 1,2-di- >
1,1,2-tri- > 1,1-dl- > 1,1,2,2-tetra- > 1,1,1,2-tetra- > 1,1,1-tri- in male
Wistar rats, and 1,1,2,2-tetra- > 1,2-di- > 1,1-di- > 1,1,2-tri- > 1,1,1,2-
tetra- > 1,1,1-tri- in female rats. The relative in vitro metabolic rate (as
measured by percent ^°Cl enzymatically removed from °Cl-labeled haloethane by
rat liver microsomes) was shown by Van Dyke and Wineman (376) to follow the
approximate order: 1,1-di- > 1,1,2-tri- > 1,1,2,2-tetra- > penta- > mono- =
1,2-di- = 1,1,1-tri-. Among the haloethanes, only 1,2-dihaloethane and
halo thane have been extensively studied; these are discussed below.
1,2-Dihaloethanes. Owing to their mutagenic and carcinogenic properties
and industrial uses, 1,2-dihaloethanes (ethylene dihalides) have attracted
much attention. Rannug (15) and Anders and Livesey (377) have reviewed, in
1980, the metabolic studies of these compounds. Several subsequent studies
(179, 181, 378) have since been reported. The known metabolites of 1,2-di-
chloro- and 1,2-dibromoethanes are: inorganic halides (379, 380), S-carboxy-
methylcysteine, thibdiacetic acid, chloroacetic acid (375), N-acetyl-S-(2-
hydroxyethyl)-cysteine and its S-oxide (381), S-(2-hydroxyethyl)-cysteine
(381, 382), S-(2-hydroxyethyl)-glutathione and its S-oxide, S,S'-ethylene-bis-
(glutathione) (380), bromoacetaldehyde (383), and ethylene (384). At least
two major routes of metabolism have been proposed (see Fig. 4). In the first
route, oxidative metabolism of 1,2-dihaloe thane (_i_) by microsomal cytochrome
P-450-dependent mixed-function oxidase yields the highly unstable hydroxy
intermediate (ii), which spontaneously decomposes to haloacetaldehyde (iii).
Haloacetaldehyde is highly reactive and may (a) covalently bind to nucleo-
philic macromolecules, (b) react with glutathione to form a conjugate which
65
-------
OH
1 /
Cowtent binding
?HZ
ix-CHzCH-x! -£• X-CHzCHO ^ GS-CHzCHO —• GS-CHzCOOH —- HOOC-CH-CHz-S-CHzCOOH—-tf
L (ii) J (iii)\ (lvl
X-CHzCOOH
(vi)
(viii)
GS-CHzCHzX /
(v'i) V , . /. ,
]t2!L.GS-CHzCHz-SG 0
(x) G-S-CHzCHzOH
\ NHj
-• " —»N-ocetytotion
Sulfoiidotion
(xiii)
Fig. 4. Proposed metabolic pathways of 1,2-dihaloethanes. The chemi-
cal names of the compounds are: i = 1,2-dihaloethane; ii = hydroxy-1,2-
dihaloethane; iii = haloacetaldehyde; iv = S-carboxymethyl-cysteine; v =
thiodiacetic acid; vi = haloacetic acid; vii = S-(2-halpethyl)-glutathione;
vii = ethylene; ix = episulfonium ion intermediate; x = S,S'-ethylene-bis-
(glutathione); xi = S-(2-hydroxyethyl)-glutathione; xii = S-(2-hydroxy-
ethyl)-glutathione sulfoxide; xiii = S-(2-hydroxyethyl)-cysteine. [Modi-
fied from M.W. Anders, and J.C. Livesey: _In_ "Ethylene Dichloride: A
Potential Health Risk?" (B. Ames, P. Infante, and R. Reitz, eds.), Cold
Spring Harbor Laboratory, New York, 1980, p. 331]
-------
gives rise to S-carboxylmethyl cysteine (iv), and thiodiacetic acid (v) after
further metabolism by dehydrogenase, peptidase and deaminase, or (c) be oxi-
*3,
dized to halocetic acid (vi). In the second route, nucleophilic attack of
1,2-dihaloethane by reduced glutathione catalysized by glutathione transferase
yields S-(2-haloethyl)-glutathione (vii), which may be attacked by a second
molecule of reduced glutathione to yield ethylene (viii). Alternatively,
since S-(2-haloethyl)-glutathione is actually a half-sulfur mustard, it may
cyclize to the highly reactive episulfonium ion (ix), which may (a) be
attacked by reduced glutathione to form S,S' -e thylene-bis-(glutathione) (_x_),
\
(b) hydrolyze to- S-(2-hydrOxyethyl)-glutathione (xi), or (c) possibly act as
' ' (
an alkylating agent. The S-(2-hydroxethyl)-glutathione (xi) may either be
i| j
oxidized to itsi/ ^ulfoxide or hydrolyzed by peptidase to yield S-(2-hydroxy-
'U
ethyl)-cysteine1 (xiii), which can be further N-acetylated to N-acetyl-S-(2-
hydroxyethyl)-cysteine and sulfoxidized to its sulfoxide. In addition to the
above routes, several other possibilities have been proposed (179, 385). The
formation of an extremely reactive l-chloroso-2-chloroethane (C1CH,?CH?C1=0)
intermediate by microsomal oxidation of 1,2-dichloroethane has been suggested
(179). This intermediate is impossible to detect directly due to its high
reactivity; it is expected to (a) rearrange to a hypochlorite (C1CH9CH,OC1),
^ L.
which may give rise to chloroacetaldehyde or 2-chloroethanol, or (b) react
with glutathione to form S-(2-chloroethyl)-glutathione. Theoretically,
1,2-dihaloethanes may also undergo reductive dehalogenation to yield chloro-
ethyl free radical or dehydrohalogenation to vinyl chloride; however, there
appears to be no sufficient experimental evidence to support these pathways
(179).
66
-------
The role of metabolism in the generation of mutagenic or carcinogenic
intermediates from 1,2-dihaloethanes has been extensively investigated but
still remains unresolved. Haloacetaldehyde and the episulfonium intermediate
have been regarded as the principal reactive intermediates. Hill £t_ _al_« (383)
identified bromoacetaldehyde as a microsomal metabolite of 1,2-dibromoethane,
demonstrated that bromoacetaldehyde was capable of binding directly (without
metabolic activation) to nucleophiles, and suggested the role of this inter-
mediate in the macromolecular binding of 1,2-dibromoethane. This finding was
confirmed by Banerjee, Van Duuren and coworkers (385, 386). The microsomal
mixed function oxidases-mediated covalent binding to macromolecules has also
been shown with 1,2-dichloroethane (378, 385). There is preliminary evidence
for a correlation between the microsome-mediated binding and species and organ
susceptibility to 1,2-dichloroethane-induced carcinogenesis (378, 385). The
observation that disulfiram enhances the carcinogenicity of 1,2-dibromoethane
(262) is consistent with the proposed role of haloacetaldehyde; indeed, the
inhibition of aldehyde dehydrogenase by disulfiram is expected to block
further oxidation of bromoacetaldehyde and this results in increased tissue
level of this intermediate. In contrast to the above findings, there is
sufficient evidence to indicate that the mutagenic activity of 1,2-dihalo-
ethanes is mainly due to cytosol-catalyzed activation via conjugation with
reduced glutathione (GSH). Studies by Rannug _e_t ai_. (178), Van Bladeren et
al. (181), and Guengerich et _al_. (177) all indicate that cytosol is the better
source of the activating enzyme (GSH transferase) for 1,2-dihaloethanes in the
Ames test. Metabolic activation of 1,2-dihaloethanes by the commonly used S-9
fraction (9000 x g supernatant which contains both cytosol and microsomes) is
most likely due mainly to cytosol component because the inclusion of micro-
somes alone decreases rather than increases the mutagenic activity of 1,2-di-
67
-------
haloethanes (in suspension assay, see Section 5.2.2.1.2.2). The episulfonium
ion (ix) has been suggested to be the most likely nutagenic intermediate.
Episulfonium ions are active electrophiles capable of readily reacting with
nucleophiles (reviewed in 387). The above observations may imply a possible
bifurcation of the metabolic activation of 1,2-dihaloethanes into pathways
leading to carcinogenic and to mutagenic intermediates. A major discrepancy
between the binding studies of Banerjee _e_t _al_. (378, 385) and Guengerich et
al. (179) has been noted. The former group showed that the covalent binding
of 1,2-dichloroethane to DNA was not catalyzed by cytosol and was inhibited by
glutathione whereas the latter group demonstrated that glutathione actually
enhanced the microsome-mediated binding to DNA arid that cytosol catalyzed the
covalent binding in the presence of glutathione.
Halo thane. The metabolism of halo thane has been extensively studied
because of the widespread use of the compound as an anesthetic agent. This
topic has been thoroughly reviewed in 1976 and 1977 (44, 59, 388) and will not
be further elaborated in this section because of the lack of evidence of
carcinogenic!ty or mutagenicity of the compound. It is important to point
out, however, that covalent binding of halothane metabolites to proteins and
lipids (but not RNA, DNA) does occur (e.g., 175, 389) and that 1,1-difluoro-2-
bromo-2-chloroethylene, a "presumed" metabolite of halothane has been shown to'
be mutagenic (165).
5.2.2.1.4.1.3 Halopropanes. Little information is available on halopropanes
and higher haloalkanes. Nakajima and Sato (319) reported that ,1-chloropropane
is metabolized in the rat at a rate higher than most halomethanes, halo-
ethanes, and haloethenes. Hutson ££_al_. (390) noted rapid metabolism of ^C-
labeled 1,2-dichloroprbpane in the rat; 80-90% of the radioactivity is
68
-------
excreted via the exhaled air and the urine within the first 24 hours. About
45% of the radioactivity in the exhaled air is in the form of carbon
dioxide. The identity of other exhaled and urinary metabolites has not been
investigated. The metabolism of the soil fumigant, 1,2-dibromo-3-chloro-
propane (DBCP) in the rat has recently been studied by Jones ja£.al/ (391).
Figure 5 depicts its possible metabolic pathways. The initial step is
presumed to be the dehalogenation of the central bromine atom of DBCP (_i_),
yielding the reactive carbonium ion (ii), which readily reacts with water to •
form l-bromo-3-chloro-propan-2-ol (iii). Dehydrohalogenation of the inter-
mediate (iii) readily occurs, especially under alkaline condition, with the
formation of either epibromohydrin (iv, X = Br) or epichlorohydrin (iv,
X = Cl). Glutathione conjugation of these epoxides, followed by hydrolysis by
peptidase, and then N-acetylation, produces the mercapturic acid intermediate
(v). Dehydrohalogenation of the intermediate (v) yields the epoxide (vi),
which may either be conjugated by a second molecule of glutathione to even-
tually yield 1,3-(bis-N-acetylcysteinyl)-propan-2-ol (vii) or be hydrolyzed to
S-(2,3-dihydroxypropyl)-mercapturic acid (viii). Hydrolysis of epihalohydrin
(iv) produces o(-halohydrin (ix), which may yield compound (viii) via epoxide
(x) or be oxidized to £>-halolactate (xi) and eventually to oxalic acid
(xii). Compounds (vii) and (viii) have actually been isolated from the urine
of rats given DBCP. The production of -bromolactate (xi, X
= Br), and the mercapturic acid conjugates (vii) and (viii) have been identi-
fied as the metabolites of 1,2,3-tribromopropane. For 1,2-dichloropropane,
69
-------
CHjCI
(i)
CHjBf
CH©
w
~ HX 1
(ii)
(iii)
>"
CHgSR
+6SH, -«l», glu
+N-octtyla(im
CH2SR
• rHHH
CHzSR
(vii)
(xii)
Fig. 5. Proposed metabolic pathways of l,2-dib;romo-3-chloropropane.
In the formulas, R = -CH2CHCOOH ; X =» Cl or Br. The chemical names of the
NHCOCH
compounds are: i = 1,2-dibromo-3-chloropropane; ii = carbonium ion inter-
mediate; iii = l-bromo-3-chloropropan-2-ol; iv = epihalohydrin; v = S-(2-
hydroxyl-3-halopropyl)-mercapturic acid; vi = S-(2,3~epoxypropyl)-mercap-
turic acid; vii = l,3-(bis-N-acetylcysteinyl)-propan-2-ol; viii = S-(2,3-
dihydroxypropyl)-mercapturic acid; ix = 3-halo-l,2-propanediol; x =
glycidol; xi = /:>-halo lac tic acid; xii = oxalic acid. [Modified from A.R.
Jones, G. Fakhouri, and P. Gadiel, Experientia 35, 1432 (1979)]
-------
the metabolites include S-(2-hydroxypropyl)-mercapturic acid (major
metabolite), [i-chlorolactate (xi, X = Cl), and S-(2,3-dihydroxypropyl)-
mercapturic acid (viii).
5.2.2.1.4.2 METABOLISM AND MECHANISM OF ACTION OF HALOALKENES.
5.2.2.1.4.2.1 Haloethenes (Haloethylenes). Haloethenes are the most exten-
sively studied haloalkenes because of their economic importance and because of
the potent carcinogenicity of vinyl chloride in this class. The role of
metabolism in the activation of chlorinated ethenes has been reviewed by
Henschler (135, 191) and Leibman and Ortiz (392). The general metabolic
scheme of these compounds is shown in Fig. 6. It is generally believed that
microsomal oxidation of chlorinated ethenes to their respective epoxide is the
first and obligatory step in the metabolic activation of the whole class. The
epoxide of chlorinated ethene is highly reactive and may undergo a variety of
reactions including (i) covalent binding to cellular macromolecules, (ii)
5
conjugation with soluble nucleophiles such as glutathione, (iii) enzymatic (by
epoxide hydrase) or nonenzymatic hydrolysis to chlorinated ethylene glycol,
and (iv) intramolecular rearrangement (Cl shift) to chlorinated acetaldehyde
(reaction a, X/ = H) or acetyl chloride (reaction b). The conjugation is
generally regarded to be a detoxification reaction. Chlorinated ethylene
glycol is unstable and is expected to undergo further decomposition (392).
The intramolecular rearrangement plays a predominant role in the metabolism.
Theoretical considerations and thermal rearrangement studies by Henschler and
coworkers (135, 190-192) suggest that, depending on the number and position(s)
of chlorine substituent(s), two types of products — acyl chlorides (tri-,
di-, or monochloroacetyl chlorides for tetra-, tri-, or 1,1-dichloroethylenes,
respectively) or aldehydes (di- or monochloroacetaldehyde for cis/trans
1,2-dichloroethylene and vinyl chloride, respectively) -- may arise. The
70
-------
Covalent binding to Conjugation with soluble
tissue macromolecules nucleophiles (eg., glutathione)
Intramolecular rear-
rangement (Cl shift)
, ,
X2-C—C—X4
II
OH OH
Fig. 6. General metabolic scheme of chlorinated ethenes. (In the
formula, Xt = Cl; X2> X3> X4 • Cl or H.)
-------
aldehydes may be further reduced or oxidized to alcohols or acids, respec-
tively, whereas the acyl chlorides may act as acylating agent or be hydrolyzed
to acids. These predictions have been supported by metabolic studies of
various chlorinated ethenes (see below) with trichloroethylene as the only
exception. Assuming the formation of ketocarbenium ion intermediate after C-0
heterolysis as the first step, trichloroethylene epoxide is expected to yield
[TEXT-FIGURE 6]
dichloroacetyl chloride [reaction (b)] because the ionized carbon is more
stable with one chlorine than with two chlorine substitutions. Metabolic
studies have, however, shown that reaction (a) with the formation of tri-
chloroacetaldehyde (or its hydrated form, chloral hydrate) is the preferred
route.
The epoxide of chlorinated ethene has been regarded as one of the prin-
cipal reactive intermediates responsible for the potential mutagenic or car-
cinogenic action of the parent compound. Henschler and his group (135, 190-
192) have postulated that epoxides of unsymmetrically substituted chlorinated
ethenes (vinyl chloride, 1,1-dichloroethylene, trichloroethylene) are less
stable and more electrophilic than those with symmetric chlorine substitutions
(cis or trans 1,2-dichloroethylene, tetrachloroethylene). Using a muta-
genicity test with Escherichia coli K12, a relationship between instability of
the epoxide and the mutagenicity of the parent compound has been noted: the
%
unsymmetric chlorinated ethenes are all mutagenic whereas the symmetric ones
are not. It is not known to what extent this rule may apply to other
71
-------
C,CH
Text-Figure 6
-------
systems.. This molecular rule is partially supported by the results pbtained
using the Ames test (see Table VII). In addition, tetrachloroethylene appears
to be at least as carcinogenic as trichloroethylene in B6C3F, mice. However,
a recent theoretical computational study by Politzer e_t _al_. (394) indicates
that there is no significant difference in the calculated stabilities of
epoxides of various symmetric and unsymmetric chlorinated ethenes.
A number of comparative metabolic studies of haloethenes have been
carried out. Using isolated perfused rat liver preparations Bonse et al.
(395) showed that, in general, an inverse relationship exists between the
number of chlorine substituents and the metabolic rate; (if chlorinated '
ethenes. This is supported by the in vivo study of tfak'aj ima and Sato (319), 1
M/|' : J
who showed that the metabolic rate in Wistar rats of! 'three chlorinated ethenes I
Ui I I
:hlo!ro. A more extensive il
study by Filser and Bolt (396) is also in agreement with this correlation
(with the exception of trichloroe thylene) . The estimated zero-order maximal
metabolic rates (¥„,„„) of six chlorinated ethenes follow the order: tri-
max
chloro > monochloro (i.e. , vinyl chloride) > 1,1-dichloro > cis-1 ,2-dichloro >
trans-1 , 2-dichloro > tetrachloro. Fluorinated alkenes are substantially less
susceptible than chlorinated alkenes to biotransf ormation. The zero-order
V__,, of 1,1-difluoroe thylene is nearly 100 times lower than that of 1,1-di-
chloroe thylene .(396). In the vinyl halide series, the zero-order V _„ follows
max
the order: vinyl chloride > vinyl bromide > vinyl fluoride (396). Although
Filser and Bolt (396) have cautioned that substantial differences in metabolic
rates may exist among different species and strains so that the above pharma-
cokinetic data may be valid only for the Wistar rats used, a pharmacokinetic
study by Monster (397) with trichloroe thylene and tetrachloroethylene in human
subjects shows that (in agreement with rat studies) trichloroe thylene is
72
-------
indeed rapidly metabolized (75% metabolized) whereas tetrachloroethylene is
very resistant to metabolism (2% metabolized). Many metabolic and mechanism
studies on the individual haloethenes have been conducted.
Vinyl Chloride. The metabolism of vinyl chloride has been thoroughly
reviewed by Plugge and Safe (398), IARC (23), and Fishbein (54). The major
metabolic pathways are depicted in Fig. 7. Vinyl chloride (_i_) is believed to
be oxidized to its epoxide (chloroethylene oxide, ii), which may undergo
intramolecular rearrangement (Cl shift) to generate chloroacetaldehyde
(iii). Oxidation of compound (iii) by aldehyde dehydrogenase yields chloro-
acetic acid (iv). Compounds (ii), (iii), and (iv) may be conjugated with
glutathione (GSH) to glutathione conjugates (v_ and _vi_), which give rise to
S-formylmethyl-cysteine (vii) and S-carboxymethyl-cysteine (viii) after hydro-
lysis by peptidase. Compound (viii) may be converted to thiodiglycolate
(thiodiacetate, ix) after deamination and decarboxylation, while compound
(vii) may be reduced to S-( 2-hydroxyethyl)-cys teine (x_) and then N-acetylated
to the mercapturic acid conjugate, N-acetyl-S-(2-hydroxyethyl)-cysteine
(xi). Compounds (iv), (viii), (ix), (x_), and (xi) have all been detected as
urinary metabolites (see ref. 398). The generation of ^C0« and a number of
minor metabolites in animals given ^C-labeled vinyl chloride was postulated
to occur via the tricarboxylic acid cycle or one-carbon or two-carbon pools
with chloracetic acid (iv) or chloroethylene glycol (xii) -as the starting
intermediates (398). In addition to above pathways, Green and Hathway (399)
detected S-(2-chloroethyl)cysteine and its N-acetylated derivative as urinary
metabolites and proposed a possible direct interaction between glutathione and
vinyl chloride per se. This pathway is, however, not supported by a 1979
study of Guengerich and Watanabe (400) using 36C1-labeled vinyl chloride; they
concluded that any chemical mechanism for activation and binding of vinyl
chloride involves release of the chlorine atoms as chloride ions.
73
-------
H-C-C-H
-^~ —»TCAC»ctyt,,C2pool
\ r/ •Q.
/C~\ NMPHOj
H H
(i)
Uii)
a « H
NH2
U)
HOOCCHCH2SCHJCH20H
NHOOCHj
aCHgCHO*
(iii)
\«»
GS-OfeCHO
W
-jlj.glu
HOOCCHCHzSO^CHO
(vii)
-*v
CICHzCOOH
(iv)
+€SH
GS-CHjCOOH
(vi)
-*».«!•
HOOCCHCHjSCHjCOOH
JNHZ
(viii)
r
HOOCCHjSCHjCOOH
Fig. 7. Major metabolic pathways of vinyl chloride. The chemical
names of the compounds are: i = vinyl chloride; ii = chloroethylene oxide;
iii = chloroacetaldehyde; iv = chloroacetic acid; v = S-formylmethyl gluta-
thione; vi = S-carboxymethyl glutathione; vii » S-formylmethyl-cysteine;
viii » S-carboxymethyl-cysteine; ix = thiodiacetic acid; x = S-(2-hydroxy-
ethyl)-cysteine; xi = S-(2-hydroxyethyl)-mercapturic acid; xii = chloro-
ethylene glycol. Compounds with an asterisk are potential reactive inter-
mediate. [Adapted from H. Plugge and S. Safe: Chemosphere 6, 309 (1977)]
-------
The pharmacokinetics of the metabolism of vinyl chloride has been exten-
sively investigated (396, 401-405). Since the metabolism of vinyl chloride
appears to be a saturable process, the incorporation of a pharmacokinetic
model in the risk assessment of low dose exposure to vinyl chloride has been
proposed (406, 407). It is interesting to point out that considerable species
differences in vinyl chloride metabolism have been observed. Buchter et al.
(408, 409) reported that the first order metabolic clearance rate (in
liter/h/kg body weight) for vinyl chloride in various animal species decreases
in the order: mouse (25.6) > gerbil (12.5) > rat (11.0 for Wistar strain) >
monkey (3.55) > rabbit (2.74) > man (2.02). They stressed that this species
difference should be taken into account in risk assessment.
The covalent binding of vinyl chloride metabolites to cellular macromole-
cules has been the subject of intensive investigations (400, 410-417) because
of its implication in the initiation of mutagenesis and/or carcinogenesis.
Both chloroethylene oxide (vinyl chloride epoxide) and chloroacetaldehyde have
been regarded as the possible "ultimate" mutagen or carcinogen of vinyl
chloride. Both compounds are highly reactive (with chloroethylene oxide being
much more so) (73, 412) and may react directly with adenosine to form
3- ft-ribofuranosyl-imidazo-[2,1-i] purine (1,N -etheno-adenosine) (73). Both
chloroacetaldehyde (154, 177, 418, 419) and chloroethylene oxide (154, 178,
418) are mutagenic in bacterial and V79 Chinese hamster cell test systems,
although the latter compound is much more potent and is also mutagenic in
yeast. A recent study by Zajdela _e£_al_. (412) shows, moreover, that chloro-
ethylene oxide is a potent local carcinogen by subcutaneous injection and an
active tumor-initiator by skin painting. Chloroacetaldehyde is inactive as a
tumor-initiator; however, its potential complete carcinogenic!ty cannot be
evaluated because of its potent necrotizing activity. Most investigators
74
-------
(410-412, 414) consider chloroethylene oxide as the principal reactive inter-
mediate, although some (400, 413) regard the less reactive chloroacetaldehyde
to be a more effective biological alkylating agent. The nature of covalent
binding between vinyl chloride metabolite and DNA (414, 415) or RNA (416, 417)
has been investigated. 9-(i -D-2'-Deoxyribofuranosyl-imidazo-[2,l-i] purine
(l,N^-etheno-deoxyadenosine), l-£ -D-2'-deoxyribofuranosyl-l,2-dihydro-2-oxo-
imidazo-[l,2-c] pyrimidine (3,N -etheno-deoxycytidine), 1,N -etheno-adenosine,
and 3,N -etheno-cytidine have been identified as reaction products.
[TEXT-FIGURE 1\
The introduction of such etheno groupings into DNA bases is expected to inter-
fere with the normal Watson-Crick type base pairing (412). Besides vinyl
chloride, vinyl bromide has also been shown to alkylate (after metabolic
activation) polyadenylic acid, pblycytidylic acid, or RNA to yield
l,N^-etheno-denosine and 3,N^-etheno-cytidine (421).
Vinylidene Chloride (1,1-Dichloroethylene). The metabolic fate of
vinylidene chloride has been investigated in several studies (98, 422-426).
The major metabolic pathways proposed are summarized in Fig. 8. Vinylidene
chloride (_i_) is expected to be oxidized to 1,1-dichloroethylene oxide (ii)
which rearranges to chloroacetyl chloride (iii) and is then oxidized to
chloroacetic acid (iv). The epoxide (ii) may also conjugate with glutathione
and eventually yield the N-acetyl-S-cysteinyl-acetyl derivative (_v_) as a final
metabolite (98, 425). Reichert et al. (426) detected methyl-thio-acetylamino-
ethanol (vi) as a major metabolite and postulated the interaction of chloro-
75
-------
Ado.
Etheno-Ado.
Cytd.
r\
0
Etheno-Cytd.
Text-Figure 7
-------
" JJ "
CI-C-C-H
1
QL
-
(xi
a H
a \
1
OH
«v 9°°"
— •— • — » C02 «— T
cor
3H
(xiv)
1
HOHzCCOOH
(xiii)
w
ax
NH
r t
^•CICHzCOCI* ^— CICHzCOOH
(iii) (iv)
(i) (ii) " |+pe
H
GS-CHjCOa ClCHzCONHCHzCHzOH GS-C
+6»
^COOH
NHCOCHj
(V)
+SSHor
CHjSCHzCONHCHzCHjOH HOOCCHCHzSCHzCOOH
NHj
(Vi) I (vii)
StCHzCOOHfe
(ix)
r
HSCHzCOOH
HOOCCHCHzSCHzCOOH
NHCOCHj
(viii)
Fig. 8. Proposed metabolic pathways of vinylidene chloride. The
abbreviations used are: MFO = mixed function oxidases; GSH = glutathione;
PE = phosphatidyl ethanolamine; P = phosphatidyi group. The chemical names
of the compounds are: i = vinylidene chloride; ii = 1,1-dichloroethylene
oxide; iii = chloroacetyl chloride; iv = chloroacetic acid; v = S-(N-
acetylcysteinyl)-acetyl derivative; vi = methylthioacetylaminoethanol;
vii = S-carboxymethyl-cys teine; vii = S-carboxymethyl-iaercapturic acid;
ix = thiodiglycolic acid; x =» thioglycolic acid; xi = dithioglycolic acid;
xii = 1,1-dichloroethylene glycol. Compounds with an asterisk are poten-
tial reactive intermediates.
-------
acetyl chloride (iii) with phosphatidyl ethanolamine in membrane lipid
followed by nucleophilic attack by a methylthio-containing compound (e.g.,
methionine) or glutathione, as the reaction mechanism. Thiodiglycolic acid
(ix) has been identified as one of the predominant metabolites (98, 423-426);
its formation may be accounted for by glutathione conjugation of chloroacetic
acid (iv), followed by hydrolysis of the glycine and glutamate moieties,
transamination, and decarboxylation. N-Acetyl-S-(carboxymethyl)cysteine
(viii), another major metabolite (426), may arise by N-acetylation of inter-
mediate (vii). Hydrolysis of thiodiglycolic acid by ft-thionase yields thio-
glycolic acid (x_) and dithioglycolic acid (xi) which have been detected as
minor metabolites (98, 422-425). In addition to the above metabolites, the
formation of N-acetyl-S-(2-hydroxyethyl)cysteine has been reported (423, 424);
no reaction mechanism has been proposed. The generation of carbon dioxide as
the major exhaled metabolite may be accounted for by the degradation of di-
chloroethylene glycol (xii) or chloroacetic acid (iv) via glycolic acid (xiii)
and oxalic acid (xiv) (98, 425). A recent study by Andersen ££_al_. (128)
suggests, however, that the epoxide hydratase pathway may be of minimal signi-
ficance in the metabolism of vinylidene chloride.
Both 1,1-dichloroethylene oxide and chloroacetyl chloride are regarded as
potential "ultimate" mutagenic or carcinogenic intermediates (98, 422, 425).
Reitz _e_t _al_. (427) investigated the potential of vinylidene chloride in
covalent binding to cellular macromolecules. Alkylation of DNA was observed
in the liver and kidney of both rats and mice exposed to 50 ppm ^C-labeled
vinylidene chloride. The level of binding was, however, quite low (about two
orders of magnitudes less than that reported for dimethylnitrosamine in
rats). Extensive tissue damage was associated with the administration of
carcinogenic doses of vinylidene chloride. The authors (427) are of the view
76
-------
that epigenetic mechanism(s) related to cytotoxicity may play a more important
role in the carcinogenic action of vinylidene chloride.
Trichloroethylene. The metabolism of trichloroethylene has been reviewed
by Kelley and Brown (428), Van Duuren (217), Leibman and Ortiz (392), Vaughan
_e_t_al/ (388), IARC (2), and Fishbein (54). Several studies (429-432) have
since then been published. The metabolic pathways of trichloroethylene are
depicted in Fig. 9. Like all chlorinated ethenes, trichloroethylene (_i_) is
expected to be metabolized to its epoxide (ii) by microsomal mixed function
oxidases. Intramolecular rearrangement of the epoxide yields' trichloroacet-
aldehyde (iii) which is readily hydrated to chloral hydrate (iv). Subsequent
reduction and oxidation of (iii) or (iv) give rise to the final major urinary
metabolites, trichloroethanol (y_) (and its glucuronide) and trichloroacetic
acid (vi), respectively. The formation of compounds (iii), (iv), (v_), and
(vi) is regarded to be detoxification. The enzymes involved in the reduction
and oxidation, and their subcellular distribution, have been described in a
recent study by Ikeda e t al. (432). Another route of detoxification is the
enzymatic (epoxide hydrase) or nonenzymatic hydrolysis of the epoxide (ii) to
trichloroethylene glycol (ix). The identification of a small amount of di-
chloroacetic acid (vii) as a "new" metabolite of trichloroethylene in the
mouse by Hathway (430) suggests a possible activating route. The interme-
diate, dichloroacetyl chloride (vii) may be expected to react with nucleotides
in DNA to form cyclized products the same way as chloroacetyl chloride
(422). Hathway (430) proposed that this minor pathway is significant only
when mice are given massive doses of trichloroethylene resulting in a buildup
of trichloroacetaldehyde and reversion to the epoxide.
Trichloroethylene epoxide is generally regarded to be the principal
reactive intermediate of the parent compound. Henschler _e_t _al_. (431) have
77
-------
.
(i)
t f '
CI-C—C-H
OH OH
| (ix)
i «
a ov xci
Y-V
Cl
H
Cl shift
J:.T.!L* ci3ccHo*
(ii)
ClgCHCOCI
(viii
CI3CCH2OH
(v)
glucuronide
CI3CCH(OH)2
(iv)
CI#COOH
(vi)
CfeCHCOOH
(viii)
Fig. 9. Proposed metabolic pathways of trichloroethylene. The chemi-
i
cal names of the compounds are: i = trichloroethylene; ii = trichloro-
I
ethylene oxide; iii = trichloroacetaldehyde; iv = chloral hydrate; v =•
trichloroethanol; vi = trichloroacetic acid; vii =• dichloroacetyl chloride;
viii = dichloroacetic acid; ix = trichloroethylene glycol. Compounds with
an asterisk are potential reactive intermediates.'
-------
synthesized the epoxide and studied its reactivity in aqueous systems. The
decomposition pattern of the epoxide appears to be quite different from that
derived from in vivo metabolism of trichloroethylene. 'Henschler _e_t _al_. (431)
suggested that, under normal in vivo conditions, the highly reactive epoxide
(produced from trichloroethylene) may be confined within the hydrophobic
milieu of microsomes and less likely to undergo reactions observed from the
synthetic epoxide in aqueous system. Nonetheless, covalent binding (although
relatively low) of trichloroethylene metabolite to cellular macromolecules has
been demonstrated in in vitro and in vivo studies (175, 429, 433, 434). The
covalent binding is modified correspondingly by inducers and inhibitors of
microsomal mixed function oxidases and is enhanced by 3,3,3-trichloropropene
oxide, a typical inhibitor of epoxide hydrase (429, 434). Trichloroethylene
metabolites appear to bind to more nucleophilic sites of proteins than do
vinyl chloride metabolites which mainly bind to sulfhydryl groups (433). A
substantial level of covalent binding of trichloroethylene to exogenously
added DNA in the presence of microsomes from male B6C3F, mice rather than from
^
female mice was observed (429). This observation is in good agreement with
the substantially lower carcinogenicity of the compound in female mice (234).
Tetrachloroethylene. The metabolism of tetrachloroethylene has been
reviewed by Leibman and Ortiz (392) and subsequent studies (435-437) have been
reported. Figure 10 depicts the major metabolic pathways. Like other chlori-
nated ethenes, tetrachloroethylene (_i) is believed to be metabolized to tetra-
chloroethylene epoxide (ii), which rearranges to trichloroacetyl chloride
(iii), which in turn is hydrolyzed to form trichloroacetic acid (iv). Tri-
chloroacetic acid has indeed been detected as the major urinary metabolite of
tetrachloroethylene by many investigators (392 and refs. therein, 436). The
epoxide (iii) may also be hydroxyzed to tetrachloroethylene glycol (v), which
78
-------
Cl Cl
Cl—C-C-CI
I !
OH OH
» «
(v)
-2HCI
fl fi
CI-C-C-CI
(VJ)
COOH
* COOH
(vii)
/ \ NADPH.02
Cl NCI
(i)
CICI
.
Cl8hift
*
(ii)
I CI3CCOCl
(iii)
CI3CCOOH
Fig. 10. Major metabolic pathways of tetrachloroethylene. The chemi-
cal names of the compounds are: i = tetrachloroethylene; ii = tetrachloro-
ethylene oxide; iii = trichloroacetyl chloride; iv = trichloroacetic acid;
v = tetrachloroethylene glycol; vi = acyl chloride!intermediate (oxalyl
chloride); vii = oxalic acid. Compounds with an asterisk are potential
reactive intermediates.
-------
may readily rearrange to yield trichloroacetyl chloride (iii) or decompose to
oxalic acid (vii) via the acyl chloride intermediate (vi). Pegg e_t _al_. (435)
recently identified oxalic acid as the major urinary metabolite and suggested
that the hydrolysis of the epoxide (ii) to the diol (v) may be a major pathway
in the metabolism of tetrachloroethylene. It is interesting to point out that
reaction with glutathione does not seem to be a significant route of
metabolism; the glutathione pool in rat liver is not depleted following tetra-
chloroethylene exposure (435).
Tetrachloroethylene epoxide and trichloroacetyl chloride are the presumed
reactive intermediates of (tetrachloroe.thylene. Bonse _e_t_ _al_. (395) detected
ythe in vitro covalent binding of some tetrachloroethylene metabolite in
p€ffused rat liver and po&tplated that trichloroacetyl chloride reacted with
K 'J
bell| cons tituents resulting in acylation. The acylation of hepatic microsomes
has also been demonstrated by Costa and Ivanetich (436). Schumann et al.
(437) reported the lack of evidence of covalent binding of tetrachloroethylene
to hepatic DNA of B6C3F, mice, a strain of mice that is susceptible to the
carcinogenic action of the compound. They have suggested that epigenetic
mechanisms involving cytotoxicity are probably involved in the tumor induction
by tetrachloroethylene.
5.2.2.1.4.2.2 ,Halopropenes, Halobutenes, and Halobutadienes, Very little
information is available on higher haloalkenes. As discussed in Section
5.2.2.1.2, haloalkenes with vinylic structure differ significantly from those
with allylic structure regarding metabolic activation. In general, vinylic
haloalkenes require metabolic activation (most likely, epoxidation) whereas
allylic haloalkenes may react directly with tissue nucleophiles. The muta-
genic activity of vinylic and allylic haloalkenes (Table VII) reflects this
difference. It is not known to what extent the direct-acting alkylating
', 79
.V
\
-------
activity of allylic haloalkenes may contribute to their potential carcinogenic
activity. Highly reactive compounds may react with the first available
nucleophile (including soluble tissue nucleophiles, non-essential proteins,
etc.) before they can reach the critical target site(s).
Allyl Halides (3-Halopropenes). The metabolism of allyl halides in the
rat was studied by Kaye £t_£l_. (438). S-Allyl-mercapturic acid and its sulf-
oxide are the major metabolites of allyl chloride, while S-allyl-cysteine has
been shown to be a metabolite of allyl bromide and iodide. These metabolites
can be accounted for by glutathione conjugation followed by hydrolysis by
peptidase (yielding cysteine derivatives) and N-acetylation (yielding mercap-
turic acid derivatives). S-(3-Hydroxypropyl)^mercapturic acid has also been
detected; it is not known whether hydroxylation occurs before or after gluta-
thione conjugation. The above metabolism appears to be mainly detoxifica-
tion. Allyl halides are direct-acting mutagens in the Ames test and their
mutagenicity is reduced by inclusion of the S-9 mix (see Table VII). Van
Duuren (217) hypothesized two possible activating metabolic pathways for allyl
halides. Allyl halides nay be converted to allyl alcohols and then oxidized
to acrolein (which is mutagenic; Section 5.2.1.7.1), and acrylic acid. Alter-
natively, allyl halides may be epoxidized to epihalohydrin (which is carcino-
genic; Section 5.2.1.1.5), and then converted to glycidol, glycidaldehyde
(carcinogenic; Section 5.2.1.1.5), and epoxyproprionic acid.
1,3-Dichloropropenes. Hutson _et__al_. (390) reported that 1,3-dichloro-
propenes are rapidly metabolized and excreted by the rat. Of the ^C-labeled
1,3-dichloropropene administered, 80-90% of the radioactivity was eliminated
in the urine, expired air, and feces within 24 hours. The trans isomer
yielded more ^C^ (23.6% of the dose) in the expired air while the cis isomer
yielded less ^C^ (3.9% of the dose) but correspondingly more radioactivity
80
-------
in the urine. Climie et al. (439) characterized the urinary metabolite of
cis-1,3-dichloropropene. Nearly all (92%) of the urinary radioactivity was
present as a mercapturic acid derivative, S-(cis-3-chloroprop-2-enyl)-N-
acetyl-cysteine. The same metabolite can be produced by in vitro incubation
with glutathione in the presence of rat liver cytosol. Thus, like allyl
halides, 1,3-dichloropropenes are probably detoxified by glutathione conjuga-
tion. 1,3-Dichloropropenes are direct-acting mutagens in the Ames test and
their mutagenic activity appears to be reduced by inclusion of the S-9 mix
(see Table VII).
1,4-Dichloro-2-fautene. The metabolic fate of 1,4-dichloro-2-butene has
not been investigated. Van Duuren et al. (215) hypothesized that the compound
may be bio transformed to its epoxide which could be its reactive interme-
diate. However, Bartsch _et_ _al_. (74) have synthesized this putative metabo-
lite, 1,4-dichloro-2,3-epoxybutane, and tested it for mutagenicity and
alkylating activity; at equimolar concentrations, the epoxide showed lower
mutagenicity in Ames test and lower alkylating activity in NBP test than the
parent compound, suggesting that other reactive intermediate(s) may be
involved. One such possibility is dechlorination as well as epoxidation of
the parent compound to a monochlorinated epoxide resembling in structure to
epichlorohydrin, a potent mutagen and carcinogen.
2-Chloro-l,3-butadiene (Chloroprene). The bio transformation of chloro-
prene has been postulated by Haley (38) to involve microsome-catalyzed epoxi-
dation and subsequent glutathione conjugation to form a mercapturic acid
derivative. This hypothesis is supported by a 1980 study by Summer and Greim
(440), who showed that administration of chloroprene to rats leads to the
depletion of hepatic glutathione and increased excretion of thioethers
(presumably glutathione conjugates and mercapturic acid derivatives) in the
81
-------
urine. In vitro study revealed that the glutathione conjugation of chloro-
prene appears to require the presence of microsomes, suggesting the invovle-
ment of an epoxide intermediate. Bartsch _e_t _al_. (74) showed that incubation
of chloroprene in the presence of mouse liver microsomes and cofactors yielded
a volatile alkylating intermediate (presumably an epoxide) that reacted with
4-(p-nitrobenzyl)pyridine to form an NBP adduct.
Hexachlorobutadiene. The disposition of hexachlorobutadiene in the rat
has been studied by Davis et_ al_. (441). Rats given a tracer dose (0.1 mg/kg)
of C-labeled hexachlorobutadiene excreted 40% of the dose in feces (indi-
cating biliary excretion) and 30% in urine within 48 hours. Rats given a
nephrotoxic dose (300 mg/kg) of the compound only excreted 7% in feces and 6%
in urine. All of the radioactivity in bile and 87% of that in urine was water
soluble indicating the bio transformation of hexachlorobutadiene (which is
lipophilic) to polar metabolites. The identity of the metabolites has not
been determined. There is some evidence that glutathione conjugation may be
involved, since the hepatic glutathione of the rat was depleted following
hexachlorobutadiene administration (442).
5.2.2.1.5 Environmental Significance.
As may be expected from the extensive production and widespread use of
haloalkanes and haloalkenes (see Section 5.2.2.1.1), human exposure to these
compounds is virtually inevitable. With the spread of halocarbons into the
environment and consumer products, the general population, especially those
living in the vicinity of emission sources, may be exposed to low levels of
halocarbons via the air, the drinking water, and the food. In the wake of the
discovery of human carcinogenicity of vinyl chloride, the potential insidious
health hazard of low level exposure to halocarbons has been the focus of great
82
-------
concern. This subsection discusses the epidemiologic evidence for or against
carcinogenic!ty of halocarbons (Section 5.2.2.1.5.1). The sources and occur-
rence of halocarbons in the ambient and indoor air, the drinking water, and in
foodstuffs are discussed (Section 5.2.2.1.5.2). Human exposure in the occupa-
tional environment (reviewed in 1, 4, 8, 11, 12, 17, 20, 21, 31, 34, 37, 42-
45) and environmental problems related to fluorocarbons in the stratosphere
are not touched upon.
.5.2.2.1.5.1 EPIDEMIOLOGIC EVIDENCE.
With the exception of vinyl chloride, there is insufficient epidemiologic
evidence to unequivocally establish or refute the human carcinogenic!ty of
haloalkanes and haloalkenes. The major problems encountered in epidemiologic
studies of these compounds include: (.a) insufficient latent period, (b) small
cohort size, (c) lack of accurate quantitative exposure data, and (d) presence
of confounding factors (such as other chemicals, cigarette smoking, alcohol
usage, etc.). A brief review of available epidemiologic evidence is presented
below.
Methylene Chloride (Pichloromethane). Only one epidemiological study
with long-term follow-up of exposed workers has thus far been published in the
open literature. Friedlander et al. (443) used several approaches (propor-
tionate mortality ratio, standardized mortality rate, and survivorship
analyses) to assess the health effects of chronic exposure of workers to
between 30 and 125 ppm of the solvent. There was no evidence of human carci-
nogenicity of the compound. A critique of the above study has been presented
by U.S. Environmental Protection Agency (3).
Chloroform and Other Trihalomethanes. Two epidemiologic studies of
occupational exposure to CHC1~ have been reported. In the first study, Bomski
83
-------
et al. (444) found no evidence of liver cancer among exposed workers. How-
ever, the study was considered "uninformative" by the International Agency for
Research on Cancer work group (2) with respect to CHCl, carcinogenicity
because of the small number of workers and short follow-up time since first
exposure. Since CHClo was used as an inhalation anesthetic during the latter
half of the 19th cenury and the early 20th century, anesthesiologists of that
era were likely to be occupationally exposed to the compound. A retrospective
epidemiologic study of this particular occupational group has recently been
conducted by Linde and Mesnick (445). The evidence of this s.tudy does not
suggest that CHC1-, is carcinogenic in humans. However, because of the small
population, the small number of cancer deaths involved, the different age
disributions, and the lack of quantitative data, this study cannot definitely
refute the human carcinogenicity of CHC1-.
Chloroform and a number of other trihalomethanes (THMs) have been
detected in the drinking water of many U.S. cities (see Section
5.2.2.1.5.2). A preliminary survey by U.S. Environmental Protection Agency
suggested positive correlation between THMs level in water supplies (measured
in 1975) and cancer mortality rates (recorded in 1969-1971). Various epi-
demiologic studies (e.g., 446, 447) have since been conducted using data with
indirect or direct evidence of the presence of THMs in water supplies. The
U.S. National Academy of Sciences (448) has reviewed these studies and
concluded that:
"The conclusions drawn in the second group of studies (i.e.,
studies with direct measurement of THMs), in which many cancer
sites were examined, suggest that higher concentrations of THMs in
drinking water may be associated with an increased frequency of
cancer of the bladder. The results do not established causality,
84
-------
and the quantitative estimates of increased or decreased risk are
extremely crude. The effects of certain potentially important
confounding factors, such as cigarette smoking, have not been
de termined."
Carbon Tetrachloride. There appears to be no epidemiologic studies
directly involving CC1,. However, at least three cases of liver cancer in
humans exposed to CCL, have been reported. In the first case (449), a woman
with previous history of periodic jaundice developed cirrhosis followed by
cancer of the liver shortly after several exposures. She died 3',;years after
the first exposure. A fireman with a long history of CC1, exposure from fire
K \
extinguishers developed cirrhosis and an "epithelioma" of thejti/Ver 4 years
1 ' i
after an acute intoxication by the haloalkane (450). A 59-yeiiy-i old man, who 7
ULi,
years earlier had had an episode of CC1,-induced acute renal f-aiU'ure and liver
damage, succumbed with the development of a hepatocellular carcinoma and
concomitant cirrhosis (451).
1,2-Dibromoethane (Ethylene Dibromide). An epidemiologic study of the
cancer mortality of 161 workers at two 1,2-dibromoethane manufacturing plants
has been conducted by 011 _e£ _al_. (452). Cancer mortality was significantly
higher in one plant (5 observed vs. 2.2 expected;) but lower (2 observed vs.
3.6 expected) in the other plant. The findings of this study neither estab-
lish nor rule out 1,2-dibromoethane as a human carcinogen.
Halo thane (1,1,l-trifluoro-2-bromo-2-chloroethane). Halo thane has been
extensively used in combination with other compounds as anesthetic agent in
contemporary medicine. Many epidemiologic studies of the potential carcino-
genicity of anesthetic gases have been carried out. These studies were
thoroughly reviewed by the National Institute for Occupational Safety and
85
-------
Health (44). It appears that no unequivocal conclusion can be drawn from
these studies.
Vinyl Chloride. The human carcinogenicity of vinyl chloride has been
well established; this subject has been extensively reviewed (23, 25, 27, 28,
277). In 1974, Creech and Johnson (50) were the first to report the develop-
ment of liver angiosarcoma, a rare form of liver cancer, in 4 vinyl chloride-
exposed workers. As of October, 1977,-a total of 64 cases of liver angio-
sarcoma were reported in 12 countries (453). A summary of these cases is
presented in Table XXII. The latent period for tumor induction ranged from 9
to 38 with a median of 21 years (453). Several studies (454, 455) indicated
that the tumor incidence was dependent on the intensity and duration of expo-
sure. Among the more heavily exposed workers, the incidence of liver cancer
may be as much as 4-5 (456) or 11 (457) times higher than that expected from
spontaneous incidence. In addition to the liver, vinyl chloride has been
found by some (but not all) investigators to increase significantly the inci-
dence of brain tumors (457, 458), lung tumors (457-459), and pancreatic tumors.
(456). Besides occupational exposure, there is also some evidence that indi-
viduals living near vinyl chloride polymerization plants may have a higher
cancer risk than the general population. Several cases of liver angiosarcoma
in individuals, whose only apparent exposure was that of living near PVC
plants, have been reported (460, 461).
Vinylidene Chloride (1,1-Dichloroethylene). One epidemiologic study of
the cancer mortality of 138 workers exposed to vinylidene chloride has been
reported (462). Only one death from respiratory cancer was noted in a worker
after a lapse of more than 15 years following the initial exposure; the
expected lung cancer rate for this group was 0.2. Since 40% of this small
cohort had less than 15-year lapse after first exposure, it would be premature
to draw any firm conclusion from this study.
86
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Table XXII
Summary of Case Reports of Liver Angiosarcoma
in Vinyl Chloride/PVC Workers3
Coun try
Belgiumb
Canada
Czechoslovakia
Fed. Rep. Germany
France
Great Britain
Italy
Japan
Norway
Sweden
U.S.A.
Yugoslavia
Total Reported
aSummarized from R.
No. of Cases
1
10
2
9
8
2
2
1
1
3
23
2
64
Spirtas and R.
Years of
Exposure
—
5-26
15, 16
10-22
10-29
4, 22
6, 21
22
21
18-31
4-28
18, 20
4-31
Kaminski [J. Occup.
Latency
(years)
—
11-28
15, 16
12-22
10-29
9, 28
15, 22
22
22
19-31
12-38
20, 23
9-38
Med. 20,
Age at
Diagnosis
—
41-61
40, 46
38-58
38-63
37, 71
43, 55
52
56
43-65
37-67
42, 59
37-71
427 (1978)].
Data not available.
-------
Trichloroethylene. Two epidemiologic studies on the possible cancer
hazard from trichloroethylene exposure have been conducted. In a Swedish
study, Axelson et al. (463) examined a cohort of 518 men and found no excess
cancer deaths (11 observed versus 14.5 expected). However, because of the
short follow-up period, a cancer hazard could not be ruled out. In fact, in
the subcohort with high exposure and > 10 years of latency time, there were 3
cancer deaths observed versus 1.8 expected. In a Finnish study, Tola e*t al.
(464) followed a cohort of 2117 workers and also observed no excess of cancer
deaths. However, the investigators cautioned that because of the short
follow-up (6-13 years), the carcinogenicity of trichloroethylene cannot be
excluded at this stage of the study.
Tetrachloroethylene and Other Chlorinated Solvents. A number of chlori-
nated solvents such as-carbon tetrachloride, trichloroethylene, and tetra-
chloroethylene (perchloroethylene) have been extensively used as dry cleaning '
fluids. Tetrachloroethylene, in particular, has been the predominant solvent
in use since the 1950s. An epidemiologic study of laundry and dry cleaning
workers is being carried out at the time of this writing by the U.S. National
Cancer Institute. A preliminary report (465) indicates slight excess of liver
cancer and leukemia among exposed workers and underscores the need for addi-
tional study of this occupational group.
2-Chloro-l,3-butadiene (Chloroprene). Two epidemiologic studies of
occupational exposure to 2-chloro-l,3-butadiene yielded contradictory
findings. In a Soviet study, Khachatryan (466, 467) reported excess of skin
and lung cancer among exposed workers. In a more recent U.S. study, Pell
(468) concluded that there was no significant excess of cancers associated
with 2-chloro-l,3-butadiene exposure. It has been pointed out, however, that
both studies have a number of methodological shortcomings (296, 469). The
87
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former study failed to distinguish prevalent from incident causes, did not
adjust the effect of sex and age, and did not consider the importance of
latency. The latter study had incomplete follow-up, small number of
persons/years of exposure, and inadequate consideration of potential con-
founding variables. There is one confirmed case of liver angiosarcoma in a
worker who had extensive exposure probably only to the finished polychloro-
prene product manufacured (Herbert, 1976, cited in ref. 469).
5.2.2.1.5.2 ENVIRONMENTAL SOURCES, OCCURRENCES AND EXPOSURE.
5.2.2.1.5.2.1 Haloalkanes and Haloalkenes in the Air.
Sources. There are three principal categories of emission sources of
haloalkanes and haloalkenes in the air: (a) environmental losses during
manufacturing, processing, distribution, use, and disposal of products, (b)
emission from secondary formation reactions or as incidental byproducts of
anthropogenic activities, and (c) production of natural origins.
(a) Environmental losses from product manufacture, distribution, and use
represent the most important source, of most haloalkanes and haloalkenes in the
ambient air. Owing to their high volatility and extensive uses (some of which
are dispersive, e.g., degreaser, solvent, fumigant, fuel additive), substan-
tial amounts of low-molecular-weight halogenated compounds are released into
the atmosphere. Since the passage of Clean Air Act, the U.S. Environmental
Protection Agency has been monitoring and assessing the environmental losses
of selected haloalkanes and haloalkenes into the ambient air. Table XXIII
lists the estimated annual U.S. emission of these compounds in recent years.
In addition to the compounds listed, substantial emission of 1,1,1-trichloro-
ethane (methyl chloroform), 1,2-dibromoethane (ethylene dibromide), and
fluorocarbons is expected. Lovelock (481) estimated that the worldwide annual
88
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Table XXIII
Estimated Annual U.S. Emissions of Some Nonfluorinated
Haloalkanes and Haloalkenes from Industrial Sources into the Atmosphere
Compound
Dichlorome thane
Emission
(million Ib/year)
43ga,b
Year
1973
Reference
(A.D. Little, Inc.
Chloroform
14C
1973
cited in ref. 58)
(A.D. Little, Inc,
cited in ref. 58)
Carbon tetrachloride
1 , 2-Dichloroe thane
Vinyl chloride
Vinylidene chloride
Trichloroe thylene
Te trachloroe thylene
Chloroprene
99
91
65
75
84
66
61
13
189
163
110
242
40
4
2.5
258d
553e
11
1973
1973
1973
1973
1974
1975
1976
1977
1973
1974
1977
prior to 1975
1977
1974
1975
1974
1974
1977
(470)
(471)
(472)
(58)
(58)
(58)
(58)
(473)
(474)
(475)
(476)
(24)
(473)
(477)
(478)
(479)
(480)
(473)
Equivalent to 84.2% total production
Uncorrected for emissions not reaching the atmosphere
Equivalent to 5.6% total production
Equivalent to 60% total production
Equivalent to 85% total domestic consumption
-------
emission of 1,1,1-trichloroethane, difluorodichloromethane (Freon 12), and
trichlorofluoromethane (Freon 11) from chemical industry was of the order or
0.5, 0.33, and 0.38 megatons, respectively. 1,2-Dibromoethane may be readily
released into the atmosphere through its dispersive use as a gasoline addi-
tive. The U.S. Environmental Protection Agency (cited in ref. 16) estimated
emission factors of 0.008 and 0.31 gm 1,2-dibromoethane/gm lead/gallon gaso-
line from automobile exhaust in the most likely and the worst cases, respec-
tively. Assuming lead content of 1.9 gm/gallon gasoline and annual consump-
tion of 100 billion gallons in the United States in 1973 (cited in ref. 16)
the corresponding crude estimates of annual emission into the atmosphere would
be 1.5 and 59 million kg. There is some evidence (see Table XXIII) that the
environmental emission of some haloalkanes and haloalkenes is diminishing as a
result of implementation of new control mechanisms, substitution of alterna-
tive processes or fuel, or leveling off of demand for production.
(b) A variey of anthropogenic activities may give rise to secondary
formation and subsequent emission of haloalkanes and haloalkenes into the
atmosphere, although most of these sources are difficult to quantify. Combus-
tion and chlorination are the two principal processes contributing to inci-
dental formation.
Incineration of plastic solid wastes is a potentially important source of
atmospheric halocarbons. Several investigators (e.g., 482-484) have found
evidence that atmospheric methyl chloride originates from the thermal decompo-
sition of polyvinyl chloride (PVC). The yield depends on the composition of
PVC and the type of combustion process and may range from 0.31 to 3.75 mg/gm
of PVC. Palmer (485) estimated that the U.S. annual emission of methyl
chloride originating from the combustion of PVC is 84 million kg. Thrune
(486) identified methyl chloride, methyl bromide, and methylene bromide as
89
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minor decomposition products of epoxy resins cured with methylene dianiline.
Boettner ej^_al_« (487) found that vinyl chloride is a combustion product in the
incineration of plastics. Ahling _e_t _al_. (488) showed that the amount of vinyl
chloride released from the combustion of PVC is higher than was expectable
from the residual monomer present in the plastic, suggesting pyrolytic forma-
tion. The emission factor (mg vinyl chloride/gm PVC) was 53.6 at combustion
temperature of 140°C, 69.9 at 420°C, but decreased "to 9.7 at 600°C and 1.7 at
790°C. The authors (488) concluded that incineration of PVC at high tempera-
ture is not a major emission source of vinyl chloride.
Automobile exhaust has been suggested to be a potential source of methyl
bromide and chloroform in the urban atmosphere. Harsch and Rasmussen (cited
in ref. 58) detected 18-55 ppb methyl bromide in the exhaust of automobiles
burning "leaded" gasoline; automobiles burning "unleaded" gasoline emitted
only 1-2 ppb in the exhaust. Thermal decomposition of the gasoline additive,
1,2-dibromoethane, is believed to be the source. Harsch _e_t _al_. (489) also
showed that the urban atmospheric concentration of chloroform was higher
during heavy traffic and adverse meterological conditions. The exhaust gases
from vehicles (burning "leaded" gasoline), in which pollution was not
controlled, contained 5.6-6.8 ppb chloroform while those from pollution-
controlled vehicles had substantially lower (0.066-0.091 ppb) levels of
chloroform.
Cigarette smoking may be one of the most important sources of halocarbons.
in the indoor atmosphere. The presence of methyl chloride in cigarette smoke
has been demonstrated by various authors (reviewed in 58). Chopra and Sherman
(490) detected methyl chloride in the smoke of various types of tobacco and
chloroform if the tobacco was previously fumigated with p,p'-DDT. The yield
of methyl chloride was 11.6 mg/pack smoked. On the basis of 1974 world
90
-------
tobacco production of 5.23 megaton/year and the data of Chopra and Sherman, it
was estimated (58) that the annual emission of methyl chloride to the atmos-
phere from cigarette smoking was in the order of 10.5 million kg. Besides
halomethanes, trace amounts (up to 30 ppb) of vinyl chloride were reported to
be present in tobacco smoke (491).
Chlorination of waste water and drinking water is another potential
secondary anthropogenic source of halomethanes in the atmosphere. Keith
(cited in ref. 58) found that the use of chlorine in the treatment of waste
water from paper mills results in very high concentrations of methylene
chloride and chloroform in the effluent. A portion.of these compounds may
conceivably escape into the atmosphere because of their volatility. It is now
generally accepted that chlorination of drinking water leads to the formation
of trihalomethanes (see Section 5.2.2.1.5.2.2). Barcelona (492) estimated
that a substantial amount (about 78.5 kg) of chloroform in the municipal water
supplies of the Los Angeles area may escape into the atmosphere every day.
•3
Such escape may boost the atmospheric level of chloroform by about 79 ng/m
air and may account in part for the difference in chloroform levels in urban
and rural atmosphere. Consistent with the above finding, Batjer _e_t _al_. (493)
found that the air in eight covered public swimming pools (with chlorinated
water) in Bremen, Germany contained significant amounts of chloroform.
(c) Natural production (either biosynthetic, pyrogenic, or photochemical)
is the most important source of a number of halomethanes in the environment.
Biosynthesis by marine algae is virtually the exclusive source of iodomethane
(methyl iodide) in the environment. Lovelock _e_t_al_. (494) estimated a world-
wide annual production of 40 megatons (36 billion kg) of iodomethane in the
ocean by this source. A fraction of the iodoraethane may escape into the
•atmosphere above the ocean. Methyl iodide may react with sodium chloride in
91
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the ocean to yield methyl chloride (495), which is considerably more volatile
than methyl iodide. The evaporation of methyl chloride from the ocean is
believed to be one of the two major sources (both*natural) of the compound in
the air (481). Singh _e_t _al_. (496) found si-gnificant concentration gradients
of methyl halides between marine and continental (non-urban) air masses
supporting the view that the methyl halides originate from the ocean. The
smoldering combusion of plant materials (e.g., forest, grass) is another
natural source of methyl chloride in the environment. Roughly 10% of the
chlorine content of smoldering vegetation is believed to be converted to
methyl chlori'de (481). Palmer (485)'estimated that the U.S. annual emission
of methyl chloride from forest fires; and agricultural burning to be around 126
.,
million kA /lLovelock (481) estimate'd
( -I
an annual worldwide emission rate of 10
megatons (4i / billion kg) as a resul't of marine algal biosynthesis and grass
111 Ll, >'
and fores t';' f\lr e s.
Atmospheric photochemical reaction has been suggested to be a possible
source of carbon tetrachloride (481, 497). Under laboratory conditions,
Lovelock (481, 497) was able to produce small but significant amounts of
carbon tetrachloride by irradiating methyl chloride with sunlight. Graedel
and Allara (498), however, considered the reaction too slow to be of any major
significance. Photochemical reactions may convert the relatively unstable
chlorinated ethylenes into the more stable halorae thanes. Singh _e_t al. (499)
simulated tropospheric irradiation of synthetic mixtures of tetrachloro-
ethylene in air and obtained carbon tetrachloride with an average yield of
about 8% by weight. Similarly, Appleby et al. (500) detected chloroform as a
solar-induced photochemical reaction product of trichloroethylene.
Occurrence in Ambient Air. As may be expected from the extensive emis-
sions discussed above, ambient air is contaminated by some haloalkanes and
\
92
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haloalkenes. The extent of contamination is dependent on the intensity of
emission, the distance from emission sources, the stability of the individual
halocarbons in the atmosphere, the geographic location, and the metereologic
conditions.
The U.S. Environmental Protection Agency has monitored the ambient air
for a number of halocarbons in the vicinity of selected industrial sites. In
1975, the Agency (24) reported the detection of vinyl chloride in the ambient
air of residential areas near VC/PVC plants. The concentration exceeded 1 ppm
in less than 10% of the time. The maximum concentration was 33 ppm in a grab
(instantaneous) sample collected at a distance of 0.5 km from the center of a
plant. For vinylidene chloride (501), the highest concentration was 14 ppb at
the property line downwind of a monomer production plant. Vinylidene chloride
was still detectable. at a station 1.5 miles away from the production
facilities. Environmental monitoring of trichloroethylene (502) and
1,1,1-trichloroethane (503) showed considerable variations with concentrations
ranging from 1 ppb (limit of detection) to 270 ppb and 0.03 ppb (limit of
detection) to 155 ppb, respectively. The atmospheric concentration of 1,2-di-
chloroethane near point sources was reported to be very low, although the
methodology used may not be appropriate (13). A high concentration of 75 ppb
1,2-dichloroethane was detected in the air near a vinyl chloride plant in the
Netherlands (504). The maximum atmospheric concentrations of 1,2-dibromo-
ethane at downwind locations near the property line of two major manufacturers
were reported (505) to be 90 and 115 IXg/m^ (0.011 and 0.014 ppb). The urban
air at locations near major streets and gasoline stations in three western
U.S. cities (Phoenix, Los Angeles, Seattle) contained 0.008, 0.014, and 0.010
ppb 1,2-dibromoethane (505). Close to 20 different brominated alkanes and
alkenes were identified in the ambient air surrounding bromine industrial
plants in the state of Arkansas (506).
93
-------
A number of halocarbons have been consistently detected in the ambient
air at various locations in the United States and around the world. Reviews
on the environmental data on trichloroethylene (507), 1,1,1-trichloroethane
(508), and several halomethanes (58) have been published. Information on
atmospheric occurrences of various haloalkanes and haloalkenes has also been
summarized in several IARC monographs (2, 18, 23) and in the reviews of
Fishbein (54, 55). Only a selection of representative studies is given in
Table XXIV to illustrate the general trend. All the numbers shown are either
typical or average values and considerably higher concentrations may be found
in ambient air near emission sources under adverse metereological condi-
tions. The most commonly detected compounds are chloroform, carbon tetra-
chloride, 1,1,1-trichloroethane, trichloroethylene and tetrachloroethylene.
In general, halocarbons with anthropogenic origins are found in substantially
higher concentration in urban areas than in rural or oceanic areas. For
compounds with natural origin (marine algal biosynthesis), only methyl
chloride showed distinctly higher concentrations in oceanic air than in either
urban or rural air.
Occurrence in the Air of Indoor Environments. Indoor or enclosed
environment represents a significant but often neglected source of human
exposure to air pollutants. Depending on the size of the enclosed space and
the ventilation, even a relatively minor emission may generate alarmingly high
atmospheric levels of pollutants. There is a paucity of monitoring data on
halocarbons in the nonindustrial indoor environment. The data available have
been reviewed by Bridbord £££!_• (515) and the U.S. National Academy of
Sciences (58). The most commonly detected halocarbons are difluorodichloro-
inethane (Freon 12), trichlorofluoromethane (Freon 11), methyl chloride*
methylene chloride, chloroform, carbon tetrachloride, and 1,1,1-trichloro-
94
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page 1 oi 2
Table XXIV
Representative Atmospheric Concentrations of Some Haloalkanes and Haloalkenes (ppt)'
Compound
Methyl chloride
Methyl bromide
Methyl iodide
Methylene chloride
Chloroform
Carbon tetrachloride
Rural Area Oceanic Area
713 ± 51 1260 ± 434
15 ±10 93 ± 100
9 ± 5 7 ± 7
<1 16
36 ± 11
35 ± 19
25 ± 8 40 ± 38
7
240 280
116 ±6 128 ± 16
180
—
Urban
834 ± 40
108 ± 138
24 ± 20
6
<20 - 144
—
102 ± 102
25
380
134 ± 20
220
1400
Area
(Los Angeles)
(Los Angeles)
(Los Angeles)
(New York)
(Los Angeles)
(Bremen, Germany)
(New York)
(Los Angeles)
(Los Angeles)
(Tokyo)
Reference
(496)
(496)
(496)
(509)
(Pierotti & Rasmussen,
cited in ref. 58)
(510)
(496)
(493)
(509)
(496)
(511)
(512)
-------
Table XXIV (continued)
page 2 oi" 2
Compound
1 , 1 , 1-Trichloroe thane
Trichloroe thylene
Te trachloroe thylene
Rural Area Oceanic Area
83 180
50
—
<20 180
2 1
—
9 73
<125
590
— —
Urban
280
370
800
110
156
1200
1200
1250
880
1200
Area
(New York)
(Los Angeles)
(Tokyo)
(New York)
(Liverpool)
(Tokyo)
(New York)
(Los Angeles)
(Munich)
(Tokyo)
Reference
(509)
(511)
(512)
(509)
(513)
(512)
(509)
(511)
(514)
(512)
aln parts per trillion (ppt) by volume. The numbers shown are either typical, average, or average ±
standard deviations. The conversion factors (1 ppt = x ng/m at 25°C, 760 mm Hg) are: CH^Cl, 2.07; CH-jBr,
3.88; CH3I, 5.80; CH2C12, 3.5; CHC13, 4.9; CCl^, 6.3; CH3CC13, 5.4; CHC1CC12, 5.45; CC12CCI2, 6.78.
-------
ethane. These compounds may escape into the atmosphere from their uses as
refrigerants, aerosol propellants or ingredients, or solvents. Methyl
chloride may be produced during the smoldering combustion of tobacco. Hester
_et_ al. (516) measured fluorocarbon levels in air samples taken from homes in
the Los Angeles area. Levels of Freon 11 and Freon 12 are generally higher
indoors than outdoors and, in a few cases, may exceed 12 ppb and 500 ppb,
respectively. Beauty shops, supermarkets, department stores, and drug stores
also have substantially higher levels of Freons than the outside air. The
effect of cigarette smoking on the indoor concentration of methyl chloride may
be quite dramatic; Harsch (cited in ref. 58) reported that the atmospheric
concentration of methyl chloride in an apartment increased from the usual
level of 0.5-0.7 ppb to 20 ppb after a cigarette had been smoked. A high
concentration of 23.4 ppb niethylene chloride in a beauty shop was attributed
to the use of hair spray aerosol (58). The extensive use of methylene
chloride as a paint remover in indoor spaces may also be a significant source
of exposure. The concentration of chloroform and carbon tetrachloride in the
indoor air in the study by Harsch was relatively low. However, Batjer et al.
(493) found that air collected 0.2-1 m away from the water surface in several
covered public swimming pools contained an average of 7.5-49 ppb chloroform
with a maximum of 78 ppb. The presence of chloroform was attributed to the
formation during chlorination. In the chemistry building at the University of
Montana, Taketomo and Grimsrud (517) detected high concentrations of chloro-
form (1.76 ppm) and carbon tetrachloride (14.3 ppb) and it is very likely that
similar levels may be found at university chemistry laboratories around the
world. A high concentration of 21 ppb 1,1,1-trichloroethane was also detected
by Taketomo and Grimsrud (517) in a grocery store. The air of some unventi-
lated new automobiles may contain measurable amounts of vinyl chloride emitted
95
-------
from the plastic interior. Hedley £t__al_. (518) reported that of the seven
different models of new 1975 automobiles tested, only 2 had vinyl chloride
ranging from 0.4-1.2 ppm. The other five were below the detection limits of
0.05 ppm.
5.2.2.1.5.2.2 Haloalkanes and Haloalkenes in the Water. The U.S. Environ-
mental Protection Agency completed, in 1980, a series of Ambient Water
Criteria Documents on carbon tetrachloride (10), chloroform (7), halqmethanes
(219), chlorinated ethanes (519), dichloropropanes and dichloropropenes (520),
vinyl chloride (25), dichloroethylenes (30), trichloroethylene (33), tetra-
chloroethylene (36), and hexachlorobutadiene (39). The readers are referred
to the documents for aquatic toxicity and risk assessment of health hazards on
these compounds. The following discussion focuses on sources and occurrence
of halocarbons in the drinking water.
Sources and occurrences in surface water. The sources of halocarbons
present in the water are essentially the same as those present in the atmos-
phere, with the exception that less volatile compounds have a greater chance
of entering the water than the atmosphere. Natural production probably plays
a negligible role in directly contributing halocarbons to surface waters that
are used as the principal source of drinking water.
Discharges of waste or byproducts represents one of the most important
sources of contamination of surface waters. Relatively few estimates of the
extent of such discharges are available. Neuf eld _e_t al. (478) estimated that
between 2,600 and 3,000 Ib of vinylidene chloride are discharged into waste-
water effluent every year. In a 1974 report, U.S. Environmental Protection
Agency (521) reckoned that two chemical plants in the Long Beach, California
area discharged.a total of 12.3 kg/day vinyl chloride into waste water. Mumma
96
-------
and Lawless (522) estimated that 7.3-14.5 million pounds of hexachlorobuta-
diene were produced in 1972 mainly as incidental waste product or byproduct of
tetrachloroethylene, trichloroethylene, and carbon tetrachloride. It is not
known what proportion of the waste product was discharged into the water. The
waste water effluents and surface water in the vicinity of a number of
industrial plants were monitored. The concentrations ranged from 0.05 ppm to
20 ppm (typically 2-3 ppm) for vinyl chloride (523); < 1 ppm to 550 ppb
(typically 200-400 ppb) for trichloroethylene (502); < 1 ppb to 16 ppm
(typically around 200 ppb) for 1,1,1-trichloroethane (503). A study by Keith
(cited in ref. 58) showed concentrations of 5 ppb to 132 ppm methylene-
chloride, 0.05-650 ppb chlorodibromomethane, 5 ppb to 22 ppm chloroform, and
10 ppb to 5 ppm carbon tetrachloride in waste water effluents. Hexachlorobu-
tadiene was found in water samples collected from inland sites bordering the
lower Mississippi River; a landfill pond near an industrial source was found
to contain 4.49 ppb of the compound (524). The surface waters from fourteen
heavily industrialized U.S. river basins contained 1-90 ppb of 1,2-dichloro-
ethane (525).
A number of large spills have been recorded by U.S. Coast Guard (cited in
ref. 58). These include a spill of 8,327 liters of methyl chloride into
Chesapeake Bay in June 1973, a spill of 1,892 liters of carbon tetrachloride
into the Kanawha River, West Virginia in April 1975, and a spill of 242,240
liters of chloroform into Lower Mississippi in September 1973. A second
massive spill into Mississippi of 1.75 million pounds of chloroform occurred
when tow barge tanks ruptured at Baton Rouge, Louisiana. Concentrations up to
300 ppb were found at two points 16.3 and 121 miles downstream, up to 7.5 days
after the spill (526).
97
-------
A variety of other factors suck as atmospheric fallout, agricultural and
roadway runoff may also contribute to surface water contamination by halo-
carbons. However, no quantitative data on these potential sources are avail-
able.
Sources and occurrence in finished drinking water. A variety of halo-
alkanes and haloalkenes have been detected in finished drinking water. The
majority of these compounds originate most likely from contaminated raw water
supply. Chlorination of water supplies contributes to the formation of tri-
halomethanes. Polyvinyl chloride pipe used in water distribution systems may
i
also be a source of low levels of vinyl chloride in drinking water. > I
The environmental impact and health effects of the chlorination of wsvte.r
i
,' 4
have attracted much attention in recent years and an annual conference ha
/
i
been devoted to the study of this problem (527-529). The production of \i \
tf; VV-"
chloroform and other trihalomethanes by chlorination of water containing
organic matters was first discovered in 1974 by Rook (530) and Bellar e_t al.
(531). The finding has since been confirmed by various investigators (532-
534). The formation of haloform is dependent on the presence of precursor
organic matter such as humic acid, phenols, aromatic amines, and simple ali-
phatic carbonyl compounds. The reaction mechanism involves a series of base-
catalyzed halogenation and hydrolysis reactions. The ratio of brominated
trihalomethanes to chloroform is related to the concentration of bromine in
the water supply. Chloroform and trihalomethanes have now been detected in
most municipal water supplies.
The use of polyvinyl chloride pipe in water distribution systems provides
a potential source of low levels of vinyl chloride in finished drinking
water. Dressman and McFarren (535) have studied five water distribution
98
-------
systems using PVC pipes of different age, length, and size. Three of these
*>
five systems contained water with measurable amounts of vinyl chloride. The
highest concentration (1.4 ppb) was found in water from the most recently
installed and the longest pipe system. Traces (0.03 and 0.06 ppb) of vinyl
chloride were still detected in the water from the two oldest systems about 9
years after installation. A voluntary standard of < 10 ppm residual monomer
in finished PVC pipe has been adopted.
The occurrence of halocarbons in finished drinking water has been closely
monitored by U.S. Environmental Protection Agency. Table XXV summarizes the
monitoring data in a 10-city study (536). At least 29 haloalkanes and halo-
alkenes have been identified. The compounds that were detected with high
frequencies are: all the trihalomethanes, methylene chloride, carbon tetra-
chloride, and tetrachloroethylene. The concentrations ranged from undetect-
able to the highest for chloroform at 301 ppb. A similar trend has been
observed in monitoring studies at other U.S. cities or regions (537-540).
Monitoring studies of German drinking water yielded qualitatively comparable
results (541). However, the concentrations of trihalomethanes in Germany were
considerably lowe,r than those in U.S. drinking water. The investigators (541)
attributed the quantitative differences to the use of different amounts of
chlorine in water treatment. Well water (539) and ground water (540) were
found to yield considerably less or no trihalomethanes after chlorination
probably because of lesser amounts of organic matters. However, wells are
susceptible to contamination by leaching. A well water supply on Long Island,
New York was reported (539) to contain 50 ppb vinyl chloride, 500 ppb tetra-
chloroethylene, and 65 ppb trichloroethylene due to leaching from a nearby air
base.
99
-------
Table XXV
Haloalkanes and Haloalkenes Detected in U.S. Drinking Water3
Compound Number
Chlorome thane
lodome thane
Dichlorome thane
Bromochlorome thane
Dibromome thane
Fluorodichlorome thane
Chloroform
Bromodichlorome thane
Dibromochlorome thane
Tribromome thane
Dichloroiodome thane
Fluoro trichlorome thane
Carbon tetrachloride
Chloroe thane
Bromoe thane
1 ,1-Dichloroe thane
1 , 2-Dichloroe thane
1 ,1 ,1-Trichloroe thane
1,1, 2-Trichloroe thane
Trichloro trif luoroe thane
Hexachloroe thane
2-Chloropropane
Vinyl chloride
Vinylidene chloride
cis-1 , 2-Dichloroe thylene
trans -1 , 2-Dichloroe thylene
Trichloroe thylene
Te trachloroe thylene
Hexachlorobu tadiene
of Citiesb
5
1
9
1
1
2
10
9
9
5
7
4
8
5
2
2
3
3
1
2
1
1
2
4
3
1
5
8
1
Concentration Range (jtg/liter)
*
n.q.c
n.q.
0.1-1.6
n.q.
n.q.
n.q.
0.08-301.0
0.1-73.0
0.01-32.0
0.2-3.0
n.q.
n.q.
0.1-0.13
n.q.
n.q.
n.q.
n.q.
n.q.
n.q.
n.q.
0.07-0.5
n.q.
0.27-5.6
0.1
0.1-16.0
1.0
0.1-0.5
0.01-0.46
<0.01
Summarized from USEPA: "Preliminary Assessment of Suspected Carcinogens in
Drinking Water. Report to Congress," U.S. Environmental Protection Agency,
Washington, D.C., 1975
"Number of cities with positive findings (in a 10-city survey)
cNot quantified
-------
5.2.2.1.5.2.3 Haloalkanes and Haloalkenes in Foodstuffs. In contrast to the
extensive monitoring of haloalkanes and haloalkenes in the air and the
drinking water, there is a paucity of data on their occurrence in food-
stuffs. An extensive study by McConnell ^_t__al_. (542) suggests that several
haloalkanes and haloalkenes occur ubiquitously in various foodstuffs in
England.
Sources and occurrence. A variety of sources may contribute to the
occurrence of halocarbons in foodstuffs. These include: (a) biosynthesis,
(b>) environmental contamination, (c) food fumigation, (d) food processing, and
• i
(e.)1 food packaging and storage.
(a) With the exception of lower marine plant organisms, biosynthesis of
halocarbons is virtually nonexistent. Nonetheless, human exposure to
r
naturally-occurring halocarbons in foodstuffs has been noted. Limu kohu
(Hawaiian for supreme seaweed), a highly prized edible seaweed in Hawaii, has
been found to contain a variety of halocarbons. Burreson _e_t _al_. (543) iden-
tified 42 organohalogen compounds in the essential oil of the seaweed. The
principal (80% by weight) constituent of the oil is tribromomethane (bromo-
form). Other prevalent haloalkanes and haloalkenes are dibromoiodomethane
(5%), bromodiiodomethane (2%) and 1,1,3,3-tetrabromopropene (2%). The
presence of these and other halogenated compounds suggests that limu kohu
should be a poisonous seaweed to eat; however, to the knowledge of the authors
(543), not a simple case of illness has so far been attributed to ingestion of
the alga. A prospective epidemiological study should probably be undertaken
to assess the long-term health hazard of the seaweed.
(b) Indirect environmental contamination probably plays a major contribu-
tory role in the occurrence of halocarbons in foodstuffs. McConnell et al.
100
-------
(542) and Pearson and McConnell (544) have surveyed the extent of contamina-
tion by chlorinated aliphatic hydrocarbons in various foodstuffs in England
and edible marine organisms obtained from Liverpool Bay. The results of their
studies are summarized in Table XXVI. Consistent with their occurrence in the
atmosphere and the water, chloroform, carbon tetrachloride, 1,1,1-trichloro-
ethane, • trichloroethylene, and tetrachloroethylene are frequently detected;
the highest concentrations observed were: 33 ppb (in Cheshire cheese), 19.7
ppb (in black grapes), 47 ppb (in fish), 60 ppb (in packeted coffee), and 41
ppb (in fish), respectively.
Hexachloro-1,3-butadiene (HCBD) contamination of foodstuffs appears to be
universal (see Table XXVI). In England, HCBD residues (in ppb) were detected
in milk (0.08), butter (2.0), vegetable cooking oil (0.2), light ale (0.2),
tomatoes (0.8), and imported black grapes (3.7) in.the study of McConnell e t
al. (542). The tomatoes were harvested from plants grown on a reclaimed
lagoon contaminated with HCBD. The presence of HCBD in grapes was attributed
to its use as an insecticide for vineyards in some countries. In Germany,
Kotzias _e_t _al_. (545) found HCBD in milk, egg yolk, and margarine at levels of
4,242, and 32 ppb, respectively. In a study of foodstuffs collected from
Lower Mississippi River basin within a 25-mile radius of trichloroethylene and
tetrachloroethylene producers (who also produce HCBD as waste product), no
HCBD residues were consistently found in samples of milk, egg, and vegetables
(546). However, in the same area, freshwater fish were found to contain as
much as 4.65 ppm (547) and 1.2 ppm HCBD (546) while crayfish had 11-70 ppb
HCBD (524). Freshwater fish fed by the Rhine Water also contained HCBD
ranging from 110 ppb to 2.04 ppm (548).
The bioconcentration of haloalkanes and haloalkenes has not been
thoroughly investigated. Based on comparison of the concentrations of halo-
101
-------
Table XXVI
Concentrations of Haloalkanes and Haloalkenes Frequently Detected in Foodstuffs (ppb)a
Foodstuff CHC13 CC14 CH3CC13 CHC1=CC12 CC12=CC12 HCBDb
Dairy produce 1.4-33° 0.2-14° — 0.3-10° ND-13° ND-2°; NDd; 4-42e
Meat 1-4° 7-9° 3-6° 12-22° 0.9-5° ND°
Oils & Fats 2-10° 0.7-18° 5-10° ND-19° 0.01-7° ND-0.2e; 33e
Beverages 0.4-18° 0.2-4° 7° ND-60° ND-3° ND-0.2°
Fruits & vegetables ND-18° 3-19.7° 1-4° ND-7° ND-2° ND-3.7°; NDd
Bread 2C 5° 2° 7° 1° ND°
Fish & shellfish 3-180f 0.1-6f 0.03-47f — 0.05-41f 10-1200d; ND-7f;
»
.1 trace-465CP; 11-70 :
110-20401
aln parts per billion, equivalent to pjglliter or itg/kg
Abbreviations used: HCBD = Hexachloro-l,3-butadiene; ND = not detectable
°In England; summarized from G. McConnell, D.M. Ferguson, and C.R. Pearson [Endeavour, 34
13 (1975)]
dln U.S.A.; summarized from G. Yip [J. Assoc. Off. Anal. Chem., 59, 559 (1976)]
A
In Germany, summarized from D. Kotzias, J.P. Lay, W. Klein, and F. Korte [Chemosphere,
_4, 247 (1975)]
In U.K.; summarized from C.R. Pearson and G. McConnell [Proc. R. Soc. Lond. B., 189, 305
(1975)] •
&In U.S.A.; summarized from M.P. Yurawecz, P.A. Dreifuss, and L.R. Kamps [J. Assoc. Off.
Anal. Chem., 59, 552 (1976)]
In U.S.A.; summarized from J.L. Laseter et al., "An Ecological Study of
Hexachlorobutadiene," EPA 560/6-76-010, Environmental Protection Agency, 1976
•'•In Europe; R.W. Goldback, H. van Genderen, and P. Leeuwangh [Sci. Total Environ., 6. 31
(1976)]
-------
carbons in aquatic organisms and in water, McConnell _e_t _al_. (542) concluded
that tire bioconcentration of most haloalkanes and haloalkenes is at least a
thousand times lower than that of DDT or polychlorinated biphenyls. The U.S.
Environmental Protection Agency's calculated "weighted average" bioconcentra-
tion factors into the "edible portion of all freshwater and estuarine aquatic
organisms consumed by Americans" are: chloroform, 3.75 (7); carbon tetra-
chloride, 18.75 (10); 1,2-dichloroethane, 1.2 (519); 1,1,1-trichloroethane,
5.6 (519); vinyl chloride, 1.17 (25); vinylidene chloride, 5.6 (30); tri-
chloroethylene, 10.6 (33); tetrachloroethylene, 30.6 (36); and hexachlorobuta-
diehe, 2.78 (39). The calculation of most of these values is based on their
partition coefficients and have not been experimentally confirmed.
(c) Fumigation of foodstuffs may be a possible source of halocarbon
contamination. Halocarbons used (either alone or in mixture) in the fumiga-
tion of grain, fruit, or soil are methyl bromide, methyl chloride, chloroform,
carbon tetrachloride, 1,2-dichloroethane, 1,2-dibromoethane, 1,1,1-trichloro-
ethane, l,2-dibromo-3-chloropropane, trichloroethylene, tetrachloroethylene,
and 1,3-dichloropropene (549). In general, the amount of fumigant residues
depends on the nature of the fumigant, the fumigant dosage, storage condi-
tions, length of aeration, and extent of processing (58, 549). A National
Academy of Sciences panel (58) has recently reviewed the literature on the use
of halomethanes as fumigants. Usually, proper storage and aeration reduce
fumigant residues to trace levels; however, some fumigants (e.g., carbon
tetrachloride) may persist at low levels for as much as a year. For example,
Scudamore and Heuser (550) found that the residues in wheat after 3-6 days of
fumigation with 80 mg/1 CCl^ decreased from 200 to 400 ppm initially to 1-10
ppm after 6 months of aeration, but persisted with levels up to 4.7 ppm after
12 months. Berck (551) detected CCl^ in fumigated wheat in concentrations
102
-------
ranging from 72.6 ppm (1 week aeration) to 3.2 ppm (7 weeks aeration).. Bread
made from the latter sample had residues of about 0.02 ppm (the figure of 0.2
ppm in the original article appears to be a misprint) in the lower crust and
0.01 ppm in the crumb. In apples fumigated with 12 or 24 mg/1 1,2-dibromo-
ethane, the respective residues after 1 day were 36 and 75 ppm and decreased
to 1.2 and 1.6 ppm after 6 days (552). Newsome _et__al_. (553) reported that
1,2-dibromo-3-chloropropane was found in radish and carrot crops after appli-
cation of the fumigant to soil at a rate of 12.3 Ib/acre. Residues were
higher in carrots (up to 1.5 ppm), most of it in the root, and persisted for
16 weeks when applied at seeding. One-third of the residues still remained
after cooking unpeeled carrots in boiling water for 5 minutes.
(d) Chlorinated aliphatic hydrocarbons have been used as extraction
solvents in food processing. Trichloroethylene was widely used to decaf-
feinate coffee until its removal from use in 1977. To replace trichloro-
ethylene, dichloromethane is now being used; the maximum level of residue
allowed is 10 ppm. Dichloromethane, 1,2-dichloroethane, and trichloroethylene
have been employed as extractants in the preparation of spice oleoresins.
Page and Kennedy (554) analyzed 17 different samples of spice oleoresins from
three different manufacturers. No trichloroethylene residues were detected;
the residues of dichloromethane and 1,2-dichloroethane ranged from 1-83 ppm
•
and 2-23 ppm, respectively. -»
(e) Migration of residual vinyl chloride or vinylidene chloride monomer
from plastic food packaging film or containers into foods is another potential
source of contamination. Polyvinyl chloride (PVC) products may contain vinyl
chloride monomer ranging from 5-800 ppm. Depending on the type of food and
storage time,- some migration has been observed. As an indication of the
extent of migration, Van Esch and Van Logten (555) reported that from PVC
103
-------
bottles containing 30 ppm residual monomer, migration into water, soft drink
and blood after 40 days produced vinyl chloride concentrations of 20-50 ppb,
0.2 ppb, and 14-80 ppb, respectively. A sample of beer was found to contain 2
ppm vinyl chloride after 6 years storage in PVC boo tie containing 70 ppm
residual monomer. Williams and Miles (556) detected vinyl chloride in alco-
holic beverages, peanut oil, and vinegar in PVC bottles at levels of < 0.025-
1.6 yixg/ml, 0.3-3.29 ppm, and 0-8.4 ug/ml, respectively. Migration of
vinylidene chloride monomer from household or industrial food packaging film
is also a potential problem. U.S. Environmental Protection Agency (501)
reported findings of 4.9 and 58 ppm vinylideoe chloride in two samples of
Saran Wrap. Birkel _££_£!_• (557) analyzed several lots of plastic food
packaging films (Saran Wrap type) and detected vinylidene chloride with
concentrations ranging from 6 ' to 30 ppm. The extent of migration of the
monomer from the packaging film into foods has not been assessed.
104
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REFERENCES TO SECTION 5.2.2.1
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105
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12. NIOSH: "Criteria for a Recommended Standard: Occupational Exposure
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106
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21. NIOSH: "Criteria for a Recommended Standard: Occupational Exposure
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107
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32. IARC: "Cadmium, Nickel, Some Epoxides, Miscellaneous Industrial
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525. Ewing, B.B., Chian, E.S.K., Cook, J.C., Dewalle, F.B., Evans, C.A.,
Hopke, P.K., Kim, J.H., Means, J.C., Milberg, R., Perkins, E.G.,
Sherwood, J.D., and Wadlin, W.H.: "Monitoring to Detect Previously
Unrecognized Pollutants in Surface Waters," EPA 560/6-77-015, U.S.
Environmental Protection Agency, Washington, D.C., 1977.
i
526. Neely, W.B., Blau, G.E., and Alfrey, T. Jr.: Environ. Scf. Tech.
72 (1976).
527. Jolley, R.L. (ed.): "Water Chlorination. Environmental Impact [ n
I)
Health Effects," Volume 1, Ann Arbor Sci. Publisher, Ann Arbor, ' ,'
>' '•
Michigan, 1978.
528. Jolley, R.L., Gorchev, H., and Hamilton^ D.H. Jr. (eds.): "Water
Chlorination. Environmental Impact and. Health Effects," Volume 2, Ann
Arbor Sci. Publisher, Ann Arbor, Michigan, 1979.
529. Jolley, R.L., Brungs, W.A., and Gumming, R.B. (eds.): "Water
Chlorination. Environmental Impact and Health Effects," Volume 3, Ann
Arbor Sci. Publisher, Ann Arbor, Michigan, 1980.
530. Rook, J.J.: Water Treatment Exam. 23, 234 (1974).
531. Bellar, T.A., Litchenberg, J.J., and Kroner, R.C.: "The Occurrence of
Organohalides in Chlorinated Drinking Water," EPA 670/4-74-008, U.S.
Environmental Protection Agency, Washington, D.C., 1974.
532. Morris, J.C., and McKay, G.: "Formation of Halogenated Organics by
Chlorination of Water Supplies (A Review)," EPA 600/1-75-002, U.S.
Environmental Protection Agency, Washington, D.C., 1978.
145
-------
533. Arguello, M.D., Chriswell, C.D., Fritz, J.S., Kissinger, L.D., Lee,
K.W., Richard, J.J., and Svec, H.J.: Am. Water Works Assoc. J. 71,
504 (1979).
534. Morris, J.C., and Baum, B.: Water Chlorination 2, 29 (1979).
535. Dressman, R.C., and McFarren, E.F.: Am. Water Works Assoc. J. 70, 29
(1978).
536. USEPA: "Preliminary Assessment of Suspected Carcinogens in Drinking
Water. Report to Congress," U.S. Environmental Protection Agency,
Washington, D.C., 1975.
537. Keith, L.H., Garrison, A.W., Allen, F.R., Carter, M.H., Floyd, T.L.,
Pope, J.D., and Thruston, A.D. Jr.: Identification of Organic
Compounds in Drinking Water from Thirteen U.S. Cities. In
"Identification and Analysis of Organic Pollutants of Water" (L.H.
Keith, ed.), Ann Arbor Sci. Publ., Ann Arbor, Michigan, 1976, p. 329.
538. Coleman, W.E., Lingg, R.D., Melton, R.G., and Kopfler, F.C.: The
Occurrence of Volatile Organics in Five Drinking Water Supplies Using
Gas Chromatography/Mass Spectrometry. In "Identification and Analysis
of Organic Pollutants of Water" (L.H. Keith, ed.), Ann Arbor Sci.
Publ., Ann Arbor, Michigan, 1976, p. 305.
539. Bush, B., Narang, R.S., and Syrotynski, S.: Bull. Environ. Contain.
Toxicol. L8, 436 (1977).
540. Glaze, W.H., and Rawley, R.: Am. Water Works Assoc. J. 71, 509
(1979).
541. Sonneborn, M., and Bohn, B.: Water Chlorination 2, 537 (1979).
542. McConnell, G., Ferguson, D.M., and Pearson, G.R.: Endeavour 34, 13
(1975).
146
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543. Burreson, B.J., Moore, R.E., and Roller, P.P.: J. Agric. Food Chem.
_24_, 856 (1976).
544. Pearson, C.R., and McConnell, G.: Proc. R. Soc. London B 189, 305
(1975).
545. Kotzias, D., Lay, J.P., Klein, W., and Korte, F.: Chemosphere 4, 247
(1975).
546. Yip, G.: J. Assoc. Off. Anal. Chem. 59, 559 (1976).
547. Yurawecz, M.P., Dreifuss, P.A., and Kamps, L.R.: J. Assoc. Off. Anal.
Chem. 59, 552 (1976).
548. Goldbach, R.W., Van Genderen, H., and Leeuwangh, P.: Sci. Total
. Environ. _6, 31 (1976).
549. Fishbein, L.: Environ. Health Persp. 14, 39 (1976).
550. Scudamore, K.A., and Heuser, S.G.: Pesticide Sci. _4_, 1 (1973).
551. Berck, B.: J. Agric. Food Chem. 22, 977 (1974).
552. Dumas, T.: J. Agric. Food Chem. 21, 433 (1973).
553. Newsone, W.H., Iverson, F., Panopio, L.G., and Hierlihy, S.L.: J.
Agric. Food Chem. 25, 684 (1977).
554. Page, B.D,, and Kennedy, B.P.C.: J. Assoc. Off. Anal. Chem. 58, 1062
(1975).
555. Van Esch, G.J., and Van Logten, M.J.: Food Cosmet. Toxicol. 13, 121
(1975).
556. Williams, D.T., and Miles, W.F.: J. Assoc. Off. Anal. Chem. 58, 272
(1975).
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Chem. 60, 1210 (1977).
147
-------
Notes Added After Completion of Section 5.2.2.1
Many new findings on haloalkanes and haloalkenes have been reported since
the completion of Section 5.2.2.1. The vigorpus ongoing research is reflected
by a plethora of recent reviews or monographs on the toxicological, metabolic
and environmental aspects of chloroform (1), carbon tetrachloride (2), di-
bromochloropropane (3), vinyl chloride (4-6), tetrachloroethylene (7), and
various haloalkanes and haloalkenes (8-12).
MUTAGENICITY
As an update to Tables VI and VII, Ames mutagenicity data of a variety of
haloalkanes and arylalkyl halides are summarized in Update Table I and com-
pared to their in vitro cell transformation and in vivo carcinogenic activi-
ties. In agreement with previous findings, the data indicate that all fully
halogenated alkanes and most highly halogenated alkanes are not mutagenic in
the Ames test; most are also inactive in the cell transformation assay. There
is a poor correlation between bacterial mutagenicity and animal carcinogenic-
ity of highly halogenated alkanes (e.g., chloroform, 1,1,l-trifluoro-2-chloro-
ethane, 1,1,1,2-/1,1,2,2-tetrachloroethane, pentachloroethane). The bacterial
mutagenicity of chloromethane and bromomethane has been confirmed; the former
compound is also positive in cell transformation assay while the latter is a
potent carcinogen in the rat. With the exception of difluoromethane, all
dihalomethanes are mutagenic. A comparative study by Ostertnan-Golkar et al.
(19) showed that diiodomethane and dibromomethane are approximately equipotent
in mutagenicity and are about 3 times more active than chlorobromomethane
which, in turn, is about 20 time more potent than dichloromethane. In
contrast to the lack of mutagenicity of difluoromethane, chlorofluoromethane
(FC-31) is an active mutagen (13, 17); FC-31 is more mutagenic than vinyl
-------
p. 1 of 3
Update Table I
Mutagenicity of Haloalkanes and Arylalkyl Halides in the Ames Test
and Comparison to Their In Vitro Cell Transformation
and In Vivo
Compound
(A) Halomethanes
Chloromethane
Broraomethanec
lodomethane
Dif luoromethane (FC-32)
Chlorof luoromethane (FC-31)C
Dichloromethanec
Ch lor obromome thane
Dibromome thane
Diiodomethane
Tr if luoromethane (FC-23)
Chlorodifluoromethane (FC-22)C
Dichlorof luoromethane (FC-21)
Chloroform
Chlorodibromomethanec
Tribromomethane
Triiodome thane
Chlorotr if luoromethane (FC-13)
Dichlorodif luoromethane (FC-12)
Trichlorof luoromethane (FC-11)
Tetrabromome thane
Carcinogenic Activities
Chronic
Bioassay
Ames Test3 C.T. Mouse Rat
+ (13) + (13)
+ (14, 15) +
+d - (16) + +
- (13) - (13)
+ (13, 17) + (13) +
+ (13, 17-20) + (13) - (+)
+ (19)
+ (19)
+ (19)
- (13)
+ (13) - (13) -/(+)
- (13) - (13)
- (13, 21) - (13) + +
- (22) +/±
- (22, 24); (+)
+ (24)
+ (24)
- (13)
- (13) - (13)
- (13)
- (23) (-)
-------
Update Table I (continued)
p. 2 of 3
Compound
(B) Haloethanes
Bromoe thane
1,1-Difluoroethane (FC-152a)
1,1,1-Trifluoroethane (FC-143a)c
1,1,2-Trifluoroethane (FC-143)
l-Chloro-l,l-difluoroethane
(FC-142b)
1 , 1 , l-Trichloroethanec
Chronic
Bioassay
Ames Test* C.T. Mouse Rat
- (24)
- (13)
+ (13) - (13)
+ (13)
+ (13) + (13)
- (13, 24); - (13) +/±
+ (18, 26)
1,1,1,2-Tetrafluoroethane0
(FC-134a)
1,1,2,2-Tetrafluoroethane
(FC-134)
1,1,1-Trifluoro-2-chloroethanec
(FC-133a)
1,1,1,2-Tetrachloroethanec
1,1,2,2-Tetrachloroethane
Pentafluoroethane (FC-125)
1,1,1,2-Tetrafluoro-2-chloro-
ethane (FC-124)
1,1,1-Trifluoro-2,2-dichloro-
ethane (FC-123)
Pentachloroethanec
1,1,1,2,2-Pentafluoro-2-chloro-
ethane (FC-115)
1,1,2,2-Tetrafluoro-1,2-dichloro-
ethane (FC-114)
1,1,2-Trifluoro-1,2,2-trichloro-
ethane (FC-113)
+ (13)
- (13, 24);
+ (18, 26)
- (13)
- (13)
- (13)
- (24)
- (24)
- (13)
- (13)
+ (13)
- (13)
- (13)
- (13)
- (13)
- (24)
- (13)
- (13)
- (13)
- (13)
- (13)
-------
Update Table I (continued)
p. 3 of 3
Compound
Ames Test3
C.T.1
Chronic
Bi oa'ss ay
Mouse
Rat
(C) Higher Haloalkanes
1,2-Dichloropropanec
1,2,3-Trichloropropane
(D) Arylalkyl Halides
Benzyl chloride0
jr-Methylbenzyl chloride
jv-Nitrobenzyl chloride
jv-Phenylbenzyl chloride
Benzyl bromide
Benzal chloride0
Benzotrichloride0
jr-Chlorobenzotrifluoride
(24);
(27)
(24)
- (28);
+ (29, 30)
+ (29)
+ (29)
* (28)
+ (29)
+ (30)
+ (30)
- (31)
- (28)
+ (28)
aUpdate to Tables VI and VII. Additional mutagenicity data on some of the
compounds listed here have been summarized in Table VI.
bln vitro cell transformation assay using BHK or 3T3 cells.
cRecent carcinogenicity data on these compounds are discussed in this Update
(see Update Table II).
dSee Table VI. •
-------
chloride and is about 25-30 times more active than dichloromethane (13).
Green (17) attributed the higher mutagenicity of FC-31 (after mammalian
metabolic activation) to the formation of reactive intermediates (e.g., formyl
fluoride), stable enough to bind to cellular macromolecules (before being
hydrolyzed). In accord with bacterial mutagenicity data, FC-31 is active in
cell transformation assay and is a potent carcinogen.
Mixed results have been obtained with trihalomethanes. There is some
evidence for the mutagenicity of triiodomethane and tribromomethane, but
trichloromethane and trifluoromethane are clearly inactive. Chlorodifluoro-
tnethane (FC-22) is a weak to moderately active mutagen whereas dichlorofluoro-
methane (FC-21) is inactive. Both compounds are negative in cell transforma-
tion assay. In carcinogenicity bioassays, FC-22 is negative in one study but
active (weakly) in another. No convincing evidence of bacterial mutagenicity
of chlorodibromomethane has been found in a recent study by the U.S. National
Toxicology Program (22); however, there is some evidence that the compound is
carcinogenic in mice.
Among haloethanes, three fluorinated compounds were shown to be mutagenic
in the Ames test (13). The mutagenicity of 1,1,1-trifluoroethane (FC-143a)
and 1,1,2-trifluoroethane (FC-143) is of particular concern in view of the
e
generally held assumption that fluorinatd compounds are biologically almost
^
inert by virtue of their strong C-F bonds. Despite the positive mutagenicity
of FC-143a, however, the compound is not carcinogenic in the rat. In con-
trast, the nonmutagenic 1,1,1-trifluoro-2-chloroethane (FC-133a) is a potent
carcinogen in the rat (see Update Table II). 1-Chloro-l,1-difluoroethane
(FC-=-142b) gives positive response in both the Ames test and the cell transfor-
mation assay; its carcinogenicity remains to be tested. The mutagenicity of
1,1,1-trichloroethane has been confirmed (18, 26); its potency is, however,
-------
quite low (26). A variety of haloethanes with more than four halogens has
been tested and found negative in the Ames test and the cell transformation
assay. For these highly halogenated alkanes, short-term mutagenicity tests
appear to have little value in predicting potential carcinogenicity.
A series of arylalkyl halides has been tested for mutagenicity. Yasuda
jjt_jil_. (30) reported that the mutagenic potency of three arylalkyl halides
follows the order: benzotrichloride > benzal chloride > benzyl chloride. The
same ranking of carcinogenic potency has been observed for these compounds
(see Update Table II). Ashby^t_jl_. (28) showed that ring substitution with a
phenyl group at the para position of benzyl chloride yields a highly potent,
direct-acting mutagenic compound (jv-phenylbenzyl chloride or 4-chloromethyl-
biphenyl) which is also positive in the cell transformation assay. The intro-
duction of phenyl ring is believed to enhance the mutagenicity of benzyl
chloride by increasing its lipophilicity, diminishing its enzymatic detoxifi-
cation and helping to stabilize the reactive carbonium ion derived from it.
Hemminki et al. (29) compared the bacterial mutagenicity, and the sister
chromatid exchange (SCE) inducing activity of four arylalkyl halides with
their chemical reactivity. Using 4-(jv-nitrobenyl)pyridine (NBP) as the
nucleophile, the aralkylating activity follows the order: benzyl bromide >
jv-methylbenzyl chloride > benzyl chloride > jv-nitrobenzyl chloride. The
mutagenic potency in the Ames test follows the order: jv-nitrobenzyl chloride
» benzyl bromide > benzyl chloride = £-methylbenzyl chloride while the SCE-
inducing activity follows the order: benzyl bromide > benzyl chloride =
_p_-nitrobenzyl chloride > £-raethylbenzyl chloride. The particularly high
bacterial mutagenicity of jv-nitrobenzyl chloride is attributed to reactions
other than direct aralkylation whereas _p_-methylbenzyl chloride appears to be
exceptionally weak because of its preferential binding to the N-2 position of
guanine in DNA.
-------
TERATOGENICITY
Barlow and Sullivan (32) have recently reviewed the data on reproductive
hazards of a number of industrial chemicals which include 14 haloalkanes and
haloalkenes. The final results of a teratogenicity study of 1,1,1-trichloro-
ethane in Long-Evans rats by York _et_£l_. (33) have been published. No signi-
ficant malformations or neurobehavioral abnormalities have been found in the
offspring of female rats exposed to vapor containing 2,100 ppm of the compound
either before or during gestation. Ruddick and Newsome (34) gave pregnant
rats daily doses of 12.5, 25 or 50 mg/kg l,2-dibromo-3-chloropropane (DBCP)
orally from day 6 through 15 of gestation. No teratogenic effects were
observed. The two higher doses were slightly maternally toxic and reduced
fetal body weight. John et al. (35) found vinyl chloride (VC) not teratogenic
in the offspring of CF-1 mice exposed to 50 or 500 ppm VC and Sprague-Dawley
rats and New Zealand rabbits exposed to 500 or 2,500 ppm VC. Exposure of the
pregnant animals simultaneously to VC (by inhalation) and 15% ethanol (in
drinking water) failed to elicit additional fetal effects other than those
normally associated with ethanol consumption. Reevaluation by Clemmesen (6)
of the epidemiological evidence of teratogenicity or embryotoxicity of vinyl
chloride in humans casts doubt on earlier conclusions. Eliminating parental
age as a confounding factor, there seems to be no convincing evidence that
vinyl chloride may present a significant reproductive hazard to workers
employed in PVC-producing facilities.
CARCINOGENICITY
Recent carcinogenicity studies of haloalkanes and haloalkenes are sum-
marized in Update Table II. The highlights of these are discussed below.
-------
p. 1 of 5
Update Table II
Recent Carcinogenicity Studies on Haloalkanes and Haloalkenes3
Compound
Bromome thane
Dichlorome thane
Chlorof luoro-
methane (FC-31)
Dichlorof luoro-
methane (FC-22)
Species and
strain Route
Rat, Wistar Oral
Mouse, B6C3Fj Oral
Rat, S.-D. Inhalation
Hamster, Syrian Inhalation
Rat, Alpk/Ak Oral
Mouse, — Inhalation
Rat, Alpk/Ak Oral
Rat, — Inhalation
Dose and duration
0.4, 2, 10, or 50
mg/kg/ for 13 wk
60-250 mg/kg for
2 yr
500, 1,500 or 3,500
ppm for 2 yr
500, 1,500 or 3,500
ppm for 2 yr
300 mg/kg for
52 wk; observed
73 wk
50,000 ppm for 2 yr
300 mg/kg for
52 wk; observed
73 wk
50,000 ppm for
2 yr
Significant
neoplasm
Forestomach
carcinoma
None
Benign mammary
tumor
S.c. carcinoma
None
Forestomach
carcinoma
None
None
S.c. carcinoma
Incidence , References
65% at 50 mg/kg (36)
(37)
— c (38)
11.3% at 3,500 ppm
(38)
93% (13)
— (cited in
13)
(13)
"low" (cited in
13)
Chlorodibromo-
methane
Mouse, B6C3FJ Oral
50 or 100 mg/kg
for 105 wk
Hepatocellular
carcinoma
Hepatocellular
carcinoma or
adenoma
M: 38% at 100 mg/kg (22)
F: 12%, 20%,
-------
p. 2 of 5
\tuui. j.nued)
Compound
Ch lorodibrotno-
methane (cont'd)
1 , 2-Dibromo-
Species and
strain
Rat, F344/N
Rat, S.-D.
Route
Oral
Inhalation
Dose and duration
40 or 80 mg/kg
for 104 wk
20 ppm for 78 wk
Significant
neoplasm Incidence
None —
Spleen hemangio- M: 21%; F: 13%
References
(22)
(39)
ethane
1,1,1-Trifluoro- Rat, Alpk/Ar
ethane (FC-143a)
1,1,1-Trichloro-
ethane
1,1,1,2-Tetra-
fluoroethane
(FC-134a)
1,1,1-Trifluoro-
2-chloroethane
(FC-133a)
Oral
Mouse, B6C3FJ Oral
Rat, F344/N Oral
Rat, Alpk/Ak Oral
Rat, Alpk/Ak Oral
300 mg/kg for 52
wk; observed
73 wk
1,500 or 3,000
mg/kg for 103
wk
375 or 750 mg/kg
for 103 wk
300 mg/kg for 52
wk; observed
73 wk
300 mg/kg for 52
wk; observed
73 wk
sarcoma
Adrenal tumors
S.c. mesen-
chymal tumors
Mammary tumors
None
M: 23%; F: 13%
M: 23%
F: 52%
Hepatocellular F: 6%, 10%,
carcinoma
None*
None
Uterine carci- F: 43%
noma
Interstitial M: 81%
cell adenoma of
testis
(13)
(40)
(40)
(13)
(13)
-------
Update Table II (continued)
p. 3 of 5
Species and
Compound strain
1,1,1,2-Tetra- Mouse, B6C3FJ
chloroethane
Rat, F344/N
Pentachloroethane Mouse, B6C3F^
Rat, F344/N
1,2-Dichloro- Mouse, B6C3Fj
propane
Rat, F344/N
Vinyl chloride Mouse, ICR
or A/J
Mouse, CD-I
Rat, Wistar
Route Dose and duration
Oral 250 or 500 mg/kg
for 65-103 wk
Oral 125 or 250 mg/kg
for 103 wk
Oral 250 or 500 mg/kg
for 41-103 wk
Oral 75 or 150 mg/kg
for 103 wk
Oral 125 or 250 mg/kg
for 103 wk
Oral 62 or 125 mg/kg
for 103 weeks
Inhalation 50-50,000 ppm for
1 hour, single or
repeated
Inhalation 1-600 ppm for 4 wk
Oral 1.7, 5.0 or 14.1
Significant
neoplasm
Hepatocellular
carcinoma
Hepatocellular
adenoma
None6
Hepatocellular
carcinoma
None6
Hepatocellular
adenoma
Mammary adeno-
carcinoma
(marginal )
Pulmonary tumors
Pulmonary tumors
Hepatocellular
F:
M:
F:
M:
F:
M:
F:
F:
Incidence
2%, 11%, 13%d
13%, 30%, .42%d
8%, 17%, 50%d
—
8%, 59%, 16%d»e
2%, 67%, 29%d»e
—
14%, 20%, 32%d
0%, 8%, 10%d
2%, 4%, 10%d
Low incidences
See text
M:
14% at 14.1 mg/kg
References
(41)
(41)
(42, 43)
(42, 43)
(27)
(27)
(44)
(45)
(46)
mg/kg for up to
2.7 yr
carcinoma
Liver angio-
sarcomas
F: 32% at 5.0 mg/kg;
57% at 14.1 mg/kg
M: 11% at 5.0 mg/kg;
46% at 14.1 mg/kg
-------
Update Table II (continued)
p. 4 of 5
Compound
Vinyl chloride
(cont'd)
Vinyl bromide
Vinylidene
fluoride
Species and
strain
Rat, S.-D.
Rat, S.-D.
Rat, S.-D.
Route Dose and
duration
Inhalation 600 ppm for 1 yr
Inhalation 9.7-1,235
2 yr
Oral 4.12 or 8
for 52 wk
ppm for
.25 mg/kg
•
>
Significant
neoplasm
Hepatocellular
carcinoma
Liver angio-
sarcoma
Liver, Zymbal
gland tumors
Fat tissue
tumors
Incidence References
44% (47)
23%
See text (48)
8.6% at 8.25 mg/kg (49)
Trichloro-
ethylene (epi-
chlorohydrin-
free)
Mouse, B6C3F, Oral
Rat, F344/N
Benzyl chloride Mouse, ICR
(C6H5CH2Cl)
Benzal chloride Mouse, ICR
(C6H5CHC12)
Benzotrichloride Mouse, ICR
(C6H5CC13)
Oral
Topical
Topical
Topical
Topical
observed 89 wk
1,000 mg/kg for
103 wk
500 or 1,000 mg/kg
for 103 wk
2.3 /il/mouse,
2x/week for 50 wk
2.3 jul/mouse,
2x/week for 50 wk
2.3 ul/mouse,
2x/week for 50 wk
5 ^il/mouse, 2 or
3x/week for 9.8
months
Hepatocellular
carcinoma
Harderian gland
adenoma
Renal tubular-
cell adenocar-
cinoma
Skin carcinoma
Skin carcinoma
Skin carcinoma
Lung tumors
Digestive tract
tumors
Skin carcinoma
Lung tumors
Lymphoma
M: 60%; F: 27%
M: 8%; F: 6%
M: 0%, 0%, 6%d
15%
58%
68%
58%
100%
70%
100%
30%
(50)
(50)
(51)
(51)
(51)
(51)
-------
Update Table II (continued)
p. 5 of 5
Species and
Compound strain Route
Benzotrichloride Mouse, ICR- Topical
(cont'd) SLC
jv-Chlorobenzo- Mouse, ICR- Oral
trichloride SLC
(C1C6H4CC13)
Topical
Dose and duration
5 jul/mouse,
2x/week for 30 wk;
observed 10 wk
0.05-2 ^il/tnouse,
2x/week for 17.5
wk; observed
78 wk
5 jul/mouse,
2x/week for 30
wk; observed
10 wk
Significant
neoplasm
Skin carcinoma
or sarcoma
Lung tumors
Digestive tract
tumors
Fores tomach,
lung, skin cancer,
lymphoma, thymoma
Skin carcinoma
or sarcoma
Digestive tract
tumors
Incidence
48%
14%
10%
See text
64%
36%
References
(52)
(52)
(52)
Update to Tables VII, XIV, XV, XVI, XVII and XVIII.
Except where indicated, the doses were administered daily, 5 days per week for the period specified.
clncrease in the number of tumors per rat.
Tumor incidences for control, low-dose and high-dose groups.
eEarly mortality in the high-dose group might have reduced the sensitivity of the bioassay to detect a carcinogenic
response.
-------
Five halomethanes have been shown to be carcinogenic in at least one
animal species. In a 90-day subchronic toxicity study of a widely used soil
fumigant, bromomethane (methyl bromide), Danse et_ &\. (36) unexpectedly found
squamous cell carcinomas of the forestomach in 13 of 20 rats fed 50 mg/kg
bromomethane in arachis oil. All 20 animals showed marked diffuse hyperplasia
of the epithelium of the forestomach. Lower doses produced no tumors and much
less pronounced or no hyperplasia within this short period of time. Dichloro-
methane (methylene chloride) has been tested in three different animal
species. A preliminary communication by Serata et al. (37) indicated no
evidence of carcinogenicity in B6C3Fi mice ingesting dichloromethane via
drinking water. Burek et al. (38) found some evidence of a weak or marginally
active carcinogenic activity in rats exposed to dichloromethane vapor. In
female rats, only a dose-dependent small increase in the multiplicity of
benign mammary tumors (spontaneously occurring in this strain) was observed.
On the other hand, male rats exposed to 3,500 ppm dichloromethane had a signi-
ficant increase in the incidence of sarcomas (11.3% vs. 1% control) in the
ventral neck region located in or around salivary glands. In contrast to
rats, Syrian golden hamsters exposed to the same concentrations of dichloro-
methane showed no evidence of carcinogenicity. Longstaff _et_ jal.. (13) found
two fluorinated halomethanes carcinogenic in the rat. Fluorochloromethane
(FC-31) is a highly active carcinogen by oral administration inducing squamous
cell carcinoma and/or fibrosarcoma of the forestomach in 67 of 72 dosed rats
compared with only 1 of 208 controls. Dichlorofluoromethane (FC-22) is not
carcinogenic in fats by oral administration but induces a low incidence of
subcutaneous fibrosarcomas in the region of the salivary gland in male rats
exposed to 50,000 ppm FC-22 vapor for 2 years (Litchfield and Longstaff, cited
_in_ ref. 13). A 2-year carcinogenicity bioassay of chlorodibromomethane by the
-------
.1
U.S. National Toxicology Program (22) showed no evidence of carcinogenicity in
rats. There was equivocal evidence of carcinogenicity for male mice in which
chlorodibromomethane caused a significant increase in the incidence-of hepato-
cellular carcinomas in the high dose group. Some evidence of carcinogenicity
was observed for female mice since chlorodibromomethane caused a significant
increase in the combined incidence of hepatocellular adenomas and carcinomas.
Wong et al. (39) provided additional data for the potent carcinogenicity
of 1,2-dibromoethane (ethylene dibromide). Sprague-Dawley rats exposed to
vapor containing only 20 ppm 1,2-dibromoethane for 18 months developed a
variety of tumors (see Update Table II). A combined treatment of 1,2-dibromo-
ethane vapor and disulfiram in diet showed a marked potentiation of the car-
cinogenic effects. Significant increases in the incidences of hepatocellular.
splenic, mesentary, renal and thyroid tumors were observed. Disulfiram, an
inhibitor of aldehyde dehydrogenase, is believed to potentiate the carcino-
genic action of 1,2-dibromoethane by prolonging the lifetime of its putative
reactive intermediate, bromoacetaldehyde (see Section 5.2.2.1.4.1.2). Three
fluorinated haloethanes have been tested for carcinogenic activity in the rat
by Longstaf f j2t_jil_. (13). Both 1,1,1-trifluoroethane (FC-143a) and
1,1,1,2-tetrafluoroethane (FC-134a) are not carcinogenic. In contrast,
1,1,1-trifluoro-2-chloroethane (FC-133a) has been found to be a fairly potent
carcinogen. Female rats dosed with FC-133a showed an increased incidence of
uterine carcinomas whereas males had a significantly higher incidence of
benign interstitial cell tumors of the testis. The demonstration of car-
cinogenicity of FC-133a is somewhat surprising in view of its lack of activity
in the Ames test and the in vitro cell transformation assay. The carcino-
genicity studies of three chlorinated derivatives (1,1,1-tri-, 1,1,1,2-tetra-
and penta-) of ethane have been completed by the U.S. National Toxicology
-------
Program (40-43). Consistent with previous findings of the refractoriness of
rats to chlorinated ethanes, none of the three chlorinated ethanes showed any
evidence of carcinogenicity in the rat. However, in B6C3Fj mice, all three
chlorinated ethanes showed some evidence of carcinogenicity. 1,1,1-Trichloro-
ethane was active in female mice causing a significant increase in the inci-
dence of hepatocellular carcinoma. The compound also increased the incidence
of hepatocellular carcinomas in male mice but the evidence was considered
equivocal. In the study on 1,1,1,2-tetrachloroethane, the survival rate of
high-dose groups was poor because of toxicity. Nevertheless, 1,1,1,2-tetra-
chloroethane clearly increased the incidence of hepatocellular carcinomas in
female mice and of hepatocellular adenomas in mice of either sex. Technical
grade pentachloroethane (contains 4.2% hexachloroethane) significantly
elevated the incidence of hepatocellular carcinoma in all groups of dosed
mice. There was also a significant dose-related increase in hepatocellular
adenoma in female mice. Thus, among the eight chloroethanes tested, six are
hepatocarcinogenic in female mice inducing hepatocellular carcinoma. Based on
comparison of dosage and tumor incidence in the low-dose groups, the relative
hepatocarcinogenic potency follows the order: 1,1,2,2-tetra- > penta- >
1,1,2-tri- > hexa- > 1,1,1,2-tetra- > 1,1,1-tri- (see Update Table III).
The finding of potent carcinogenicity of l,2-dibromo-3-chloropropane has
prompted the U.S. National Toxicology Program to investigate the carcinogenic
potential of other halopropanes. A carcinogenesis bioassay (27) of 1,2-di-
chloropropane showed some evidence of carcinogenicity in mice of either sex as
indicated by an increased incidence of hepatocellular adenomas. There was
equivocal evidence of carcinogenicity in female F344/N rats as 1,2-dichloro-
propane caused a slight dose-related increase in the incidence of adenocar-
cinoma of the mammary gland. No increases in spontaneous tumor incidences
-------
Update Table III
Relative Potency of Chloroethanes in the Induction of
Hepatocellular Carcinoma in Female F6C3Fj Mice3
Dosage (mmol/kg)
Chloroethane
1,1,1-tri-
1,1,2-tri-
1,1,1,2-tetra-
1,1,2,2-tetra-
penta-
hexa-
Low
dose
11.2
1.46
1.49
0.85
1.23
2.49
High
dose
22.4
2.91
2.98
1.68
2.46
5.02
% Incidence
Low
dose
10
33
11
63
67
40
High
dose
20
89
(13)b
91
(29)b
31
Relative
Low
dose
1.2
30
10
100
74
22
potenency
High
dose
1.6
56
(8.0)b
100
(22)b
11
Calculated from U.S. National Cancer Institute/National Toxicology Program
data (see Table VII and Update Table II).
Early mortalities in these high dose groups precluded an accurate evaluation
of the lifetime incidence of hepatocellular carcinoma.
Calculated from the ratio of the percent incidence to dosage and assigning a
value of 100 to 1,1,2,2-tetrachloroethane.
-------
were observed in male F344/N rats. Another halopropane, 1,2,3-trichloropro-
pane, was being tested at the time of this writing.
A number of carcinogenicity studies of vinyl chloride (VC) have recently
been published. Hehir £t_£.l_. (44) reported that a single 1-hour exposure to
50,000 ppm VC was sufficient to elicit a positive carcinogenic response in
mice; lower concentrations brought about borderline (at 5,000 ppm) or negative
(at 50 or 500 ppm) responses. For repeated short-term exposures, the concen-,
tration of VC appeared to play a more important role than the cumulative
dose. Mice subjected to 10 1-hour exposures to 500 ppm VC had a positive
carcinogenic response whereas those subjected to 100 1-hour exposures to 50
ppm VC did not. Suzuki (45) exposed CD-I mice to low concentrations of VC (1,
10, 100, 300 and 600 ppm) for 4 weeks; at 41 weeks after exposure, a dose-
response relationship in the incidence of alveologenic tumors (11.1, 33.3,
66.7, 71.4 and 85.7%, respectively, compared with 0% in controls) was
observed. The latency period was 10 weeks in the 600 ppm group, 12 weeks in
the 300 ppm group and 40 weeks in the 100, 10 and 1 ppm groups. Feron et al.
(46) maintained Wistar rats on diets containing VC-contaminated polyvinyl
chloride powder giving daily intakes of 1.7, 5.0 and 14.1 mg VC/kg body
weight. Significant increases in the incidences of hepatocellular carcinoma
and angiosarcoma (see Update Table II) were observed. Even a low dose of 1.7
rag/kg was potentially carcinogenic as evidenced by an increased incidence of
neoplastic nodules in female mice. Further studies on even lower doses were
being conducted by the investigators at the time of this writing. Radike et
al. (47) found that rats exposed to 600 ppm VC in air for 1 year developed
hepatocellular carcinomas (44%) and liver angiosarcomas (23%). Simultaneous
exposure of the VC-treated rats to ethanol significantly enhanced the inci-
dences of hepatocellular carcinomas (60%) as well as angiosarcomas (50%). The
-------
authors (47) suggested that ethanol potentiates VC carcinogenesis by generat-
ing acetaldehyde which prolongs the half-life of one of the putative reactive
intermediates of VC (chloroacetaldehyde) by competing for oxidation by alde-
hyde dehydrogenase.
The final results of an inhalation study of vinyl bromide have been
published (48). Sprague-Dawley rats were exposed for 2 years to air contain-
ing 9.7, 52, 247 and 1,236 ppm vinyl bromide. Angiosarcomas, primarily of the
liver, were induced in both male (5.8%, 30%, 51%, 36% compared with 0%
controls) and female (8.3%, 42%, 51%, 34% compared with 1% controls) rats. A
significant increase in the number of Zymbal's gland carcinoma was seen in
males exposed to 247 and 1,235 ppm and females exposed to 1,235 ppm vinyl
bromide. An increased incidence of hepatocellular neoplasms (carcinomas or
"neoplastic nodules") was also found in males exposed to 247 ppm and females
exposed to 9.7, 52 and 247 ppm vinyl bromide. These results indicate that
vinyl bromide is at least as potent as or possibly more potent than vinyl
chloride as a carcinogen.
A long-term carcinogenicity bioassay.on vinylidene fluoride (1,1-di-
A
f luoroethylene) showed a slight but significant increase in the incidence of
tumors in fat tissue (lipomas and liposarcomas) in a high-dose group (8. 7% vs.
1.8% controls). Trichloroethylene (epichlorohydrin-free) was retested by U.S.
National Toxicology Program (50) because a previous bioassay used a technical
grade (epoxide-stabilized) sample. In agreement with the previous study,
trichloroethylene induced hepatocellular carcinomas in B6C3Fj mice. In addi-
tion, an increase in the incidence of Harderian gland adenomas was observed.
There was also some evidence that trichloroethylene is carcinogenic in male
(but not female) F344/N rats, inducing adenocarcinomas in renal tubular cells.
-------
Four arylalkyl halides have been tested for carcinogenic activity in ICR
;
mice. Fukuda et_ al. (51) found benzotrichloride to be a potent, multi-target
carcinogen. Topical application of 2.3 ul benzotrichloride to the skin of
female ICR mice led to high incidences of skin carcinomas, lung tumors and
tumors of the upper digestive tract. Topical application of higher amounts of
benzotrichloride induced lymphomas as well. The induction of digestive tract
tumors was attributed to a direct action of the chemical ingested as a result
of licking the treated skin. Two closely related compounds, benzal chloride
and benzyl chloride, were found to be less carcinogenic than benzotrichloride
(see Update Table II). The relative order of carcinogenic potency of these
three compounds correlates well their mutagenic activity in the Ames test
(30). The carcinogenicity of benzotrichloride was confirmed in a study by the
Hooker Chemical Co. (52). In addition, _p_-chlorobenzotrichloride is carcino-
genic both by topical and oral administration; its potency appears to be
higher than that of benzotrichloride (see Update Table II). Oral administra-
tion (0.05, 0.13, 0.32, 0.8 or 2 ul, twice weekly for 17.5 weeks) led to dose-
related increases in the incidences of tumors of the forestoraach, lung, skin
and lymphatic systems. The overall tumor incidences were 6/22, 10/28, 17/22,
27/29 and 25/29 compared with 2/26 controls.
METABOLISM AND MECHANISM OF ACTION <
The metabolism and mechanism of action of haloalkanes and haloalkenes
have been reviewed in several recent publications (2, 3, 7, 9, 11, 12). Among
halomethanes, Dodd et al. (53) reported that chloromethane (methyl chloride)
is metabolized mainly by GSH-S-alkyltransferase-catalyzed conjugation with
reduced GSH. Preliminary data indicate that the chloromethane-GSH conjugate
thus formed may be further metabolized to a toxic product (probably formalde-
hyde) suggesting that this pathway represents a toxication rather than detoxi-
10
-------
J.
cation metabolic route. Green (17) observed that rat liver S9 mix substan-
tially enhances the mutagenic activity of chlorofluorome thane (FC-31) but has
no effect on dich lor ome thane. The difference was attributed to differential
stability of formyl halide, the postulated reactive intermediate in the oxida-
tive metabolism of dihalomethane (see Fig. 1 in Section 5.2.2.1.4.1.1).
Formyl fluoride is apparently stable enough to reach and interact with target
macromolecules to exert its mutagenic action, whereas formyl chloride is not.
Dichloromethyl carbene (iCC^) was postulated as a possible reactive
intermediate in the reductive metabolism of carbon tetrachloride (see Fig. 3,
Section 5.2.2.1.4.1.1). Direct evidence for a carbene intermediate was
recently provided by Pohl and George (54) who trapped the intermediate
generated during the reductive metabolism of carbon tetrachloride with 2,3-di-
methyl-2-butene to form l,l-dichloro-2,2,3,3-tetramethylcyclopropane. Using
2,6-dimethylphenol as a nucleophilic trapping agent, Pohl, Mico and coworkers
(55, 56) found evidence for another reactive intermediate, electrophilic
halogen, in the metabolism of carbon tetrachloride, trichlorobromotnethane and
tetrabromomethane. They (56) postulated a reductive-oxygenation pathway in
which tetrahalomethane is first reductively dehalogenated to yield a trihalo-
methyl radical, which reacts with oxygen to form a trihalomethylperoxyl radi-
cal; this, in turn, decomposes to yield a dihalocarbonyl and an electrophilic
halogen. The carbene, the trihalomethyl radical, the peroxyl radical, the
electrophilic halogen and the dihalocarbonyl are all potentially toxic meta-
bolites. There is some evidence that trichloromethyl radical, chemically
produced from carbon tetrachloride by benzoyl peroxide catalysis, may interact
with. bases of DNA (57). DiRenzo .et^ jJU (58) showed that microsomes from
phenobarbital-pretreated rats can bioactivate bromotrichloromethane (0.51
nmol/mg DNA/h), chloroform (0.46) and carbon tetrachloride (0.39) to bind
11
-------
covalently to calf thymus DNA, a substantially lower level of binding (0.11)
was found for dichloromethane. There is growing evidence that a disturbance
in hepatocellular Ca++ homeostasis may be involved in triggering the hepato-
toxic actions of carbon tetrachloride (2); this new direction of research is
worthy of further exploration.
The structure-activity relationship in the microsomal dechlorination of a
series of 12 chloroethanes (including some fluorochloroethanes) was studied by
Salmon et_ a\_. (59). Comparison of the dechlorination rate with electron
densities showed poor correlation. However, substantially better correlation
was achieved if the compounds were separated into three structural classes
(RCt^Cl, RCHC12 and RCCl^). Among these three classes, the dechlorination •
rate and reactivities decrease in the order: RCHC^ » RCI^Cl > RCCl^. The
kinetic parameters of microsomal dechlorination of five chloroethanes were
measured; their respective V (in nmol/min/mg protein) and Km (in raM)
niciX
were: 1,1-dichloroethane (41.7; 0.36), 1,1,2,2-tetrachloroethane (18.2;
0.55), 1,2-dichloroethane (0.24; 0.14), 1,1,1-trichloroethane (0.2; 0.273) and
hexachloroethane (0.91; 2.73). Consistent with the above data, McCall et al.
(60) found that 1,1-dichloroethane was dechlorinated by rat hepatic microsomes
at an approximately 10-fold greater rate than was 1,2-dichloroethane. The
microsomal metabolism of 1,1-dichloroethane appeared to be mainly detoxifying,
with acetic acid as the major metabolite. On the other hand, microsomal
metabolism of 1,2-dichloroethane yielded chloroacetaldehyde as the major
metabolite, a mutagenic and potentially carcinogenic intermediate. DiRenzo et
al. (58) have compared the extent of microsome-catalyzed covalent binding of
several haloethanes to calf thymus DNA. Both 1,2-dibromoethane and 1,1,2-tri-
chloroethane are bound to an appreciable extent whereas low level of binding
was noted for halothane, 1,2-dichloroethane and 1,1,1-trichloroethane. The
12
-------
nature of covalent binding of 1,2-dibromoethane to DNA was investigated (61);
the compound binds to DNA probably as a monofunctional intermediate (possibly
as episulfonium ion) rather than as a bifunctional intermediate (such as
bromoacetaldehyde).
A quantum chemical structure-activity study intended to correlate oxida-
tive metabolism with carcinogenic potency of chloroethanes has been carried
out by Loew et al. (62). Assuming chlorinated aldehydes and acylchlorides as
the ultimate carcinogen(s), several molecular properties — possibly useful as
indicators of the relative oxidative metabolism of chloroethanes to reactive
intermediates and of the electrophilicity of reactive intermediates — were
calculated and compared to the known hepatocarcinogenic potency of seven
chloroethanes in mice. The molecular properties that gave the best correla-
tion were: the enthalpy of hydroxylation of chloroethanes by radical oxygen,
the heat of formation of chlorinated aldehydes and the energy of the lowest
unoccupied molecular orbital (LUMO; an indicator of electrophilicity) in
chlorinated aldehydes. Based on these considerations, 1,1,1,2-tetrachloro-
ethane was predicted to be an active carcinogen of similar potency as its
1,1,2,2-isomer, whereas monochloroethane was predicted to be inactive. A
recent carcinogenesis bioassay by U.S. National Toxicology Program (41) con-
firmed the carcinogenicity of 1,1,1,2-tetrachloroethane in mice; however, its
potency appeared to be substantially lower than that of its 1,1,2,2-isomer
(see Update Table III).
The role of epoxides, generated during the metabolic activation, in the
o
mechanism of genotoxic action of haloalkafhes has been further investigated.
Scherer et al. (63) showed that chloroethylene oxide (produced by UV-induced
chlorination of ethylene oxide by _t^butyl hypochlorite) binds covalently to
deoxyguanosine yielding 7-(2-oxoethyl)guanine as the major adduct. The ter-
13
-------
minal aldehyde group of the 7-N-(2-oxoethyl)guanine reacts reversibly with the
oxygen at the 6-position of guanine to form a hemiacetal, 0 ,7-(l'-hydroxy-
ethano)guanine, which is expected to cause faulty base pairing during DNA
replication. 7-(2-Oxoethyl)guanine is the major product of base alkylation in
liver DNA of rats exposed to vinyl chloride (64). Van Duuren et_ a±. (65)
showed that the cis and trans isomers of epoxides of 1-chloropropene and
1,3-dichloropropene are all carcinogenic in mice after topical or subcutaneous
administration, consistent with their putative role as the carcinogenic inter-
mediates of the parent chloropropenes. On the other hand, the epoxides of
trichloroethylene and tetrachloroethylene are both inactive in these skin
carcinogene'sis studies casting doubt on the carcinogenicity of the parent
compounds (see Section 5.2.2.1.3.5.2) or suggesting alternative carcinogenic
intermediates.
As discussed in Section 5.2.2.1.4.2.1, the stability and reactivity of
the epoxides of halogenated ethylenes have a profound effect in determining
the carcinogenic potential of the parent compounds. The key factor is an
optimum balance between the stability and the reactivity of the epoxide to
both reach and react with the DNA target (66). Several quantum chemical
structure-activity studies have been conducted using different computational
methods to address this issue (67-70). Jones and Mackrodt (67, 68) calculated
the "two-center bond energy" of the weakest C-0 bond of the epoxides of a
series of halogenated ethylenes and compared to their mutagenicity (in E. coli
K12) and oncogenicity (ability to induce preneoplastic hepatocellular foci).
A striking similarity between the patterns of these two activities was
observed. The observed "threshold band" for oncogenicity ranged from -14.1 to
-12.9 eV, compared with -14.5 to -12.8 eV for mutagenicity. It was suggested
that epoxides which fall within these limits are potentially hazardous while
14
-------
those outside may be either too unstable to reach target oV too stable and
therefore not reactive enough. Politzer and Proctor (69) found that oxygen
protonation weakens the C-0 bonds of chloroethylene oxide facilitating ring
opening and carbocation, the effect being substantially greater for the C-0
bond involving the carbon bearing the chlorine. Correlative studies with an
extended series of epoxides of haloalkenes (P. Politzer, personal communica-
tion) suggested that the ease of protonation (as measured by the electrostatic
potential around the oxygen of the epoxide group) may prove to be a useful
predictive tool in assessing the carcinogenic potential of epoxides and their
parent haloalkenes. Considering chlorinated aldehydes and acylchlorides as
well as epoxides as possible ultimate carcinogens, Loew^t_j^. (70) calculated
various molecular properties that can serve as indicators of the relative
metabolism of six chlorinated ethylenes to reactive intermediates and of the
electrophilicity of reactive intermediates and compared to the known carcino-
genic potency of four of these compounds. The results suggested that the
chlorinated aldehydes and acylchlorides may be the more likely ultimate car-
cinogens. The relative extent of metabolism of these carbonyl products,
rather than their electrophilicity, is a determinant of the relative carcino-
genic activity of the parent compound. 1,2-Dichloroethylene was predicted to
be carcinogenic with an activity intermediate between vinylidene chloride and
tetrachloroethylene.
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