
INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
ENVIRONMENTAL HEALTH CRITERIA 32
METHYLENE CHLORIDE
This report contains the collective views of an international group of
experts and does not necessarily represent the decisions or the stated
policy of the United Nations Environment Programme, the International
Labour Organisation, or the World Health Organization.
Published under the joint sponsorship of
the United Nations Environment Programme,
the International Labour Organisation,
and the World Health Organization
World Health Orgnization
Geneva, 1984
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joint venture of the United Nations Environment Programme, the
International Labour Organisation, and the World Health
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and the quality of the environment. Supporting activities include
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toxicology. Other activities carried out by the IPCS include the
development of know-how for coping with chemical accidents,
coordination of laboratory testing and epidemiological studies, and
promotion of research on the mechanisms of the biological action of
chemicals.
ISBN 92 4 154092 3
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CONTENTS
ENVIRONMENTAL HEALTH CRITERIA FOR METHYLENE CHLORIDE
PREFACE
1. SUMMARY
2. PROPERTIES AND ANALYTICAL METHODS
2.1. Chemical and physical properites of methylene chloride
2.2. Analytical methods
3. PRODUCTION, USES, DISPOSAL OF WASTES; ENVIRONMENTAL
TRANSPORT AND DISTRIBUTION
3.1. Production, uses, disposal of wastes
3.1.1. Production levels and processes
3.1.2. Uses
3.1.3. Disposal of wastes
3.2. Environmental transport and distribution
4. ENVIRONMENTAL LEVELS AND EXPOSURES
4.1. Air
4.2. Water
4.3. Food
4.4. Occupational exposure
4.5. Controlled exposure
5. CHEMOBIOKINETICS AND METABOLISM
5.1. Absorption
5.1.1. Animal studies
5.1.2. Human studies
5.2. Distribution
5.2.1. Animal studies
5.2.2. Human studies
5.3. Metabolic transformation
5.3.1. Animal studies
5.3.1.1 Enzyme pathway
5.3.2. Human studies
5.4. Excretion
5.4.1. Animal studies
5.4.2. Human studies
6. EFFECTS ON ORGANISMS IN THE ENVIRONMENT
7. EFFECTS ON ANIMALS
7.1. Short-term exposures
7.1.1. Inhalation exposure
7.1.2. Oral exposure
7.1.3. Intraperitoneal exposure
7.1.4. Effects on the eye and skin
7.2. Long-term exposure and carcinogenicity
7.2.1. Inhalation exposure
7.2.2. Oral exposure
7.3. Mutagenicity
7.4. Effects on reproduction and teratogenicity
8. EFFECTS ON MAN
8.1. Short-term exposures
8.1.1. Controlled studies
8.1.2. Accidental exposures
8.1.3. Effects on the skin and eyes
8.2. Long-term exposure
8.2.1. Occupational exposure
8.2.2. Mortality studies
9. EVALUATION OF HEALTH RISKS FOR MAN
10. SOME CURRENT REGULATIONS, GUIDELINES, AND STANDARDS
10.1. Occupational exposure
10.2. Food
10.3. Storage and transport
REFERENCES
WHO TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR METHYLENE CHLORIDE
Members
Dr C.M. Bishop, Health and Safety Executive, London, England
Dr V. Hristeva-Mirtcheva, Institute of Hygiene and
Occupational Health, Sofia, Bulgaria
Dr R. Lonngren, National Products Control Board, Solna, Sweden
(Chairman)
Dr M. Martens, Institute of Hygiene and Epidemiology,
Brussels, Belgium
Dr W.O. Phoon, Department of Social Medicine & Public Health,
Faculty of Medicine, University of Singapore, National
Republic of Singapore
Dr L. Rosenstein, Assessment Division, Office of Toxic
Substances, US Environmental Protection Agency, Washington
DC, USA
Mr C. Satkunananthan, Consultant, Colombo, Sri Lanka
(Rapporteur)
Dr G.O. Sofoluwe, Oyo State Institute of Occupational Health,
Ibadan, Nigeria
Dr A. Takanaka, Division of Pharmacology, Biological Safety
Research Center, National Institute of Hygienic Sciences,
Tokyo, Japan
Dr R.G. Tardiff, Life Systems, Inc., Arlington, VA, USA
Representatives of Other Organizations
Dr J.P. Tassignon, European Chemical Industry Ecology and
Toxicology Centre, Brussels, Belgium
Observers
Dr M. Nakadate, Division of Information on Chemical Safety,
National Institute of Hygienic Sciences, Tokyo, Japan
Dr R. McGaughy, Carcinogen Assessment Division, US
Environmental Protection Agency, Washington, DC, USA
Secretariat
Dr M. Gilbert, International Register of Potentially Toxic
Chemicals, United Nations Environment Programme, Geneva,
Switzerland
Dr K.W. Jager, Scientist, International Programme on Chemical
Safety, World Health Organization, Geneva, Switzerland
Dr M. Mercier, Manager, International Programme on Chemical
Safety, World Health Organization, Geneva, Switzerland
Dr F. Valic, Scientist, International Programme on Chemical
Safety, World Health Organization, Geneva, Switzerland
(Secretary)
Dr G.J. Van Esch, National Institute for Public Health,
Bilthoven, Netherlands (Temporary Adviser)
Dr T. Vermeire, National Institute for Public Health,
Bilthoven, Netherlands, (Temporary Adviser)
The WHO Task Group for the Environmental Health Criteria for
Methylene Chloride met in Brussels from 19 to 22 September, 1983.
Professor A. Lafontaine opened the meeting and welcomed the
participants on behalf of the host government, and Dr M. Mercier,
Manager, IPCS, on behalf of the heads of the three IPCS co-
sponsoring organizations (ILO/WHO/UNEP). The Group reviewed and
revised the second draft criteria document and made an evaluation
of the health risks of exposure to methylene chloride.
The efforts of Dr G.J. Van Esch and Dr T. Vermeire, who were
responsible for the preparation of the draft, and of all who helped
in the preparation and the finalization of the document are
gratefully acknowledged.
* * *
Partial financial support for the publication of this criteria
document was kindly provided by the United States Department of
Health and Human Services, through a contract from the National
Institute of Environmental Health Sciences, Research Triangle Park,
North Carolina, USA - a WHO Collaborating Centre for Environmental
Health Effects.
PREFACE
A partly-new approach to develop more concise Environmental
Health Criteria documents has been adopted with this issue. While
the document is based on a comprehensive search of the available,
original, scientific literature, only key references have been
cited. A detailed data profile and a legal file on methylene
chloride can be obtained from the International Register of
Potentially Toxic Chemicals, Palais des Nations, 1211 Geneva 10,
Switzerland (Telephone No. 988400 - 985850).
The document focuses on describing and evaluating the risks of
methylene chloride for human health and the environment.
While every effort has been made to present information in the
criteria documents as accurately as possible without unduly
delaying their publication, mistakes might have occurred and are
likely to occur in the future. In the interest of all users of the
environmental health criteria documents, readers are kindly
requested to communicate any errors found to the Manager,
International Programme on Chemical Safety, World Health
Organization, Geneva, Switzerland, in order that they may be
included in corrigenda, which will appear in subsequent volumes.
1. SUMMARY
Methylene chloride (dichloromethane) is widely used as a multi-
purpose solvent and paint remover. The assessment of its toxicity
can be complicated by the presence of stabilizers and other
solvents, frequently found in commercial products. Methylene
chloride can be measured by gas chromatographic techniques at
minimum concentrations of approximately 0.02 µg/m3 in air and
0.1 µg/litre in water. Exposure to methylene chloride can be
roughly estimated by the determination of its levels in blood or
expired air. Exposure to methylene chloride will result in
elevated carboxyhaemoglobin levels in blood, which can be measured.
However, blood carboxyhaemoglobin levels can give a false picture
of exposure when either exercise or smoking is involved.
High concentrations have been measured in industrial indoor
environments and during the use of methylene chloride as a paint
remover. The general population is exposed to much lower levels of
the solvent in ambient air, drinking-water, and food.
About 80% of the world production of methylene chloride is
estimated to be released into the atmosphere, but photodegradation
takes place at a rate that makes accumulation in the atmosphere
unlikely. Initial products are phosgene and carbon monoxide, which
are transformed into carbon dioxide and hydrochloric acid. In
surface water, volatilization is the major process of removal,
hydrolysis and photodegradation being insignificant. The solvent
is readily biodegradable, aerobically. Bioaccumulation seems
unlikely in the environment. The behaviour of the compound in soil
has yet to be determined.
The major route of human exposure is through inhalation.
Methylene chloride vapour is rapidly absorbed via the lungs and the
gastrointestinal tract, uptake being directly proportional to
exposure. It also increases with exercise and with the amount of
body fat. The absorbed compound, which is distributed to all body
tisses the placenta and blood-brain barrier. Absorption of liquid
methylene chloride via the skin is slow. At current exposure
levels, most of the methylene chloride taken up is metabolized to
carbon monoxide and probably carbon dioxide, mainly in the liver,
kidneys, and lungs. With high exposures, the microsomal cytochrome
P-450 enzyme system becomes saturated and some partitioning of
unmetabolized methylene chloride may occur in fat. Even at low
exposure levels, carboxyhaemoglobin levels in the blood can be
sustained for many hours after exposure, because of delayed
conversion of methylene chloride from fat.
The toxicity of methylene chloride may be influenced by factors
such as exposure to exogenous carbon monoxide, obesity, and an
increased workload. The predominant effects of methylene chloride
on human beings are elevated carboxyhaemoglobin saturation of the
blood and central nervous system depression. The normal levels of
blood carboxyhaemoglobin in man are exceeded in non-smoking,
sedentary individuals after inhalation exposure to a methylene
chloride concentration of 400 mg/m3 for 7.5 h. The lowest-observed
adverse acute effect level, for inhalation exposure of non-smoking,
healthy individuals, was approximately 694 mg/m3 with 1.5 - 3 h
of exposure. Some neurobehavioural changes were observed at this
exposure level. Two cases of permanent damage to the central
nervous system in high, long-term occupational exposures (5 years
at 2290 - 12 500 mg/m3 and 3 years at 1735 - 3470 mg/m3,
respectively) have been reported. In spite of many reports of
fatty degeneration in the liver and tubular degeneration in the
kidneys of animals, there is no clear evidence of liver or kidney
damage in human beings.
The vapour is moderately irritating to the eyes and respiratory
tract while the liquid is irritating to the skin. Patients with
heart disease may be at increased risk if exposed to high levels of
methylene chloride; this may particularly occur during the use of
paint removers.
Results of in vitro studies showed that methylene chloride
was weakly mutagenic in bacteria and fungi. Some mutagenic effects
were also observed in Drosophila melanogaster, but the results of
most tests on mammalian somatic cells, including human cells, were
negative.
In 2 inhalation studies on rats, the incidence of benign
mammary tumours was not increased in exposed male or female rats
compared with controls, but the total number of mammary tumours in
treated animals increased in a dose-related manner. In the study,
an increase in salivary gland region sarcomas was found in male
rats. In Golden Syrian hamsters, no significant increases in
tumour incidences were found. In a drinking-water study with rats
and mice, no significant increases in tumour incidences were found,
while an increased incidence of foci or areas of altered liver
cells was observed.
In 2 human epidemiological mortality studies, there was no
excess mortality due to cancer compared with control populations.
Animal experimental data and human epidemiological data are
inadequate for assessing whether or not methylene chloride should
be considered carcinogenic for animals and man.
There is only limited evidence that methylene chloride is
teratogenic in animals.
2. PROPERTIES AND ANALYTICAL METHODS
2.1. Chemical and Physical Properties of Methylene Chloride
Methylene chloride (CH2Cl2) is nonflammable and nonexplosive
when mixed with air. It hydrolyses very slowly in the presence of
moisture. It also reacts with hydroxyl radicals. No appreciable
decomposition occurs at room temperature when the dry compound
comes into contact with common metals. Phosgene and hydrochloric
acid are formed by contact with hot surfaces or flames.
H
|
Cl-----C-----Cl
|
H
CAS registry number: 75-09-2
RTECS registry number: PA 8050000
Common synonyms: DCM, dichloromethane, methane dichloride,
methylene bichloride, methylene dichloride,
methylenum chloratum
Trade names: Aerothene MM, Freon 30, Narkotil,
Solaesthin, Solmethine
Some physical data on methylene chloride
physical state liquid
colour colourless
odour ethereal
relative molecular mass 84.93
melting point -95 °C
boiling point 40 °C
water solubility 20 g/litre, 20 °C
log n-octanol-water partition 1.25
coefficient
density 1.33 g/ml, 20 °C
relative vapour density 2.93
vapour pressure 46.52 kPa (349 mm Hg) at 20 °C
surface tension 28.12 dyne/cm at 20 °C
flame limits 0.5-2.3 g/litre in oxygen
0.5-0.8 g/litre in air
Conversion factors for methylene chloride and carbon monoxide:
methylene chloride 1 ppm = 3.47 mg/m3 air
carbon monoxide 1 ppm = 1.14 mg/m3 air
2.2. Analytical Methods
A summary of methods for the sampling and determination of
methylene chloride in air, water, sediments, food, breath, blood,
and urine is presented in Table 1.
Table 1. Sampling, preparation, analysis
---------------------------------------------------------------------------------------------------------
Medium Specifi- Sampling Analytical Detection Comments References
cation method method limit
---------------------------------------------------------------------------------------------------------
air occupa- on charcoal de- gas chromatography 10 µg per recommended range White et al.
tional sorption with with flame ioniza- sample (1 350 - 10 400 mg/m3 (1970)
carbon disul- tion detection litre (1 litre sample) NIOSH (1984)
fide sample)
air occupa- direct reading a non-specific Saltzman
tional detector tube cheap method to (1972)
estimate exposure
air occupa- infra-red spectros- continuous monito- Baretta et al.
tional copy ring and breath (1969)
analysis
air occupa- photodetection 3-6 mg/m3 suitable for con- Nelson &
tional (for CCl4) tinuous monito- Shapiro (1971)
ring when methy-
lene chloride is
the only contam-
inant
air ambient gas chromatography 0.017 direct analysis Grimsrud &
and mass spectro- µg/m3 Rasmussen
metry (1975)
---------------------------------------------------------------------------------------------------------
Table 1. (contd.)
---------------------------------------------------------------------------------------------------------
Medium Specifi- Sampling Analytical Detection Comments References
cation method method limit
---------------------------------------------------------------------------------------------------------
water drinking- gas chromatography 0.1 µg/ sparging, trap- Nicholson et
water with electrolyte litre ping in line on al. (1977)
conductivity detec- gas chromatography
tion column
water drinking- gas chromatography 0.2 µg/ direct aqueous in- Fujii (1977)
water and mass spectro- litre jection, diglycerol
metry as liquid phase on
precolumn
sediment cooling, extrac- gas chromatography 0.1-1 mg/ also used for Dietz &
tion with pen- with flame ioniza- litre drinking-water Traud (1973)
tane tion detection analysis
food extraction by gas chromatography 1 mg/kg analysis of spice Page &
vacuum distil- with electron cap- oleoresins Kennedy (1975)
lation washing ture detection
blood gas chromatography head-space analysis Di Vincenzo et
urine with flame-ioniza- (blood, urine), al. (1971)
breath tion detection 0.7 mg/m3 direct analysis
(breath)
---------------------------------------------------------------------------------------------------------
3. PRODUCTION, USES, DISPOSAL OF WASTES; ENVIRONMENTAL TRANSPORT
AND DISTRIBUTION
3.1. Production, Uses, Disposal of Wastes
3.1.1. Production levels and processes
World production in 1980 amounted to 570 kilotonnes of which
270 kilotonnes were produced in Western Europe (CEFIC, 1983). In
the USA, production increased from 180 kilotonnes in 1971 (Gordon,
1976) to 254 kilotonnes in 1980 (IRPTC; 1984) and 269 kilotonnes in
1981 (USITC, 1982). In Japan, 35 kilotonnes were produced in 1980
(IRPTC, 1984).
Two processes are important: the chlorination of methyl
chloride, obtained from the reaction of methanol and hydrogen
chloride, and the direct chlorination of methane (IARC, 1979).
Additives may include 0.0001 - 1% of stabilizers such as:
amines, 4-cresol, hydroquinone, methanol, 2-methylbut-2-ene,
1-naphthol, nitromethane + 1,4 dioxane, phenol, resorcinol, and
thymol.
3.1.2. Uses
Methylene chloride is used as: a solvent, a blowing agent for
polyurethane, a component of paint remover, a degreasing solvent,
and as a propellant in aerosols such as insecticides, hair sprays,
shampoos, and paints. Methylene chloride is being increasingly
used as a replacement for fluorocarbons in aerosols. As a solvent,
it is used in pharmaceutical applications, in the manufacture of
photographic and synthetic fibres, and, as an extraction solvent,
for naturally-occurring, heat-sensitive substances such as edible
fats, cocoa, butter, caffeine, and beer flavouring in hops. It is
also used as a component in fire-extinquishing products, as an
insecticidal fumigant for grains, and as a coolant and refrigerant
(Gordon, 1976; IARC, 1979).
3.1.3. Disposal of wastes
Methylene chloride can be destroyed by incineration, sometimes
after adsorption by activated carbon. End products are carbon
dioxide, water, and hydrochloric acid, which can be recovered
(Gordon, 1976).
3.2. Environmental Transport and Distribution
Approximately 80% of the world production of methylene
chloride is emitted into the atmosphere during its use as a
solvent, and in paint removers, aerosols, solvent degreasers,
and fumigants. Minor losses occur during production and shipping.
In the USA alone, emissions in 1975 amounted to 177 kilotonnes
(Gordon, 1976). Volatilization also appears to be the major
process by which methylene chloride is lost from water (Dilling
et al., 1975). Under field conditions, half-lives of 33 and 38
days were estimated for river water (Zoeteman et al., 1980).
Once in the troposphere, hydroxyl radicals can attack the
compound yielding mainly carbon dioxide and hydrogen chloride and
minor quantities of carbon monoxide and phosgene (Pearson &
McConnell, 1975; Cox et al., 1976; Spence et al., 1976). Phosgene
readily hydrolises to hydrochloric acid and carbon dioxide. Cox et
al. (1976) estimated a lifetime for methylene chloride in the
troposphere of 0.3 years, with respect to oxidation by hydroxyl
radicals. Photodegradation and hydrolysis in water do not seem to
take place to any significant extent (Dilling et al., 1975).
Methylene chloride is absorbed on dry bentonite clay and peat moss
but not significantly on limestone and silica sand (Dilling et al.,
1975).
The available reports show that methylene chloride is readily
biodegradable. The compound was rapidly degraded aerobically by
microorganisms from settled domestic waste water containing
methylene chloride concentrations of 5 and 10 mg/litre (Tabak et
al., 1981). After adaptation, sewage microorganisms and a
Pseudomonas species were found to degrade methylene chloride
aerobically and to use it for growth at concentrations below 425
mg/litre (Brunner et al., 1980; Rittman & McCarty, 1980). Similar
results were obtained by Stucki et al. (1981) using hyphomicrobium
species. Methylene chloride was dehalogenated by aerobic
microorganisms from municipal activated sludge yielding carbon
dioxide and chloride, after adaptation only. The compound was
toxic above a concentration of 1000 mg/litre (Klechka, 1982).
4. ENVIRONMENTAL LEVELS AND EXPOSURES
It can be seen from the uses and the physical and chemical
properties of methylene chloride that the main route of human
exposure is through vapour inhalation, sometimes accompanied by
direct skin and eye contact, both at the place of work and at home.
Much lower levels of human exposure can occur through inhalation of
methylene chloride in ambient air and through its ingestion via
drinking-water, food, and beverages.
4.1. Air
The background concentration of methylene chloride at surface
level at 40 °N latitude was found to be about 0.12 µg/m3 by Cox et
al., (1976) and 0.17 µg/m3 by Singh et al., (1982). In the air of
7 cities in the USA during 24-h sampling periods, concentrations
ranged between 0.17 and 196.75 µg/m3, while average concentrations
varied from 1.35 to 6.76 µg/m3 (Singh et al., 1982). The highest
detected concentrations in drinking-water have been less than 5
µg/litre (Saunders et al., 1975; Fujii, 1977; US National Academy
of Science, 1977).
4.2. Water
Few reports contain data concerning the occurrence of
methylene chloride in natural waters. In a survey in the
USA, 8% of finished-water supplies tested contained methylene
chloride, but only 1% of the raw-water supplies (US National
Academy of Sciences, 1977). Water from a sewage treatment plant
contained a methylene chloride concentration of 8.2 mg/litre before
treatment, 2.9 µg/litre after treatment but before chlorination,
and 3.4 µg/litre after chlorination (Bellar et al., 1974). These
results show that methylene chloride is formed during the
chlorination of water. Concentrations of 1 - 2 µg/litre (Bauer,
1978) and 5 µg/litre (Zoeteman et al., 1980) were reported at the
same point in the river Rhine.
4.3. Food
One report is available describing the occurrence of the
extractant methylene chloride in 15 out of 17 spice-oleoresins at
levels between 1 and 83 mg/kg wet weight (Page & Kennedy, 1975).
4.4. Occupational Exposure
Methylene chloride exposure was investigated in a variety of
jobs in the USA including: servicing of diesel engines, spray-
painting of booths, plastic tank construction, ski manufacture,
cleaning foam heads, and cleaning nozzles in plastic manufacture.
The concentrations ranged from below the detection limit to 257
mg/m3 air. In a chemical plant, an 8 h, time-weighted average
exposure was measured of 3040 mg/m3 with an exposure range from
below the detection limit to 19 150 mg/m3 (NIOSH, 1976). In
cellulose-acetate-fibre-producing plants in Czechoslovakia and the
USA, methylene chloride concentrations in air in a total of 335
samples ranged from 100 to 17 000 mg/m3 (Kuzelova & Vlasak, 1966;
NIOSH, 1976). The median 8-h, time-weighted average concentrations
in another plant producing cellulose acetate fibre in the USA,
ranged from 280 to 1650 mg/m3 (Ott et al., 1983). In a beauty
salon, where methylene chloride exposure stemmed from its use as an
aerosol propellant in sprays, daily mean background concentrations
were below 6.9 mg/m3, while peak concentrations of 451 mg/m3 were
reached, directly after spraying coiffures (Hoffman, 1973).
4.5. Controlled Exposure
Methylene chloride is widely used at home in paint removers and
aerosol sprays. Most paint-stripping formulations contain about
80% by weight of methylene chloride, often in combination with
methanol. Breathing zone concentrations were measured during the
use of a paint remover under controlled conditions with normal
ventilation, (70 m3/h). The maximum concentration was 4430 mg/m3
during the 3 h studies, the averages, in various experiments, ranging
between 2270 and 2730 mg/m3 (Stewart & Hake, 1976).
In a detailed study on exposure levels during the use of paint
removers, the time-weighted averages in a room without ventilation
varied between 460 and 2980 mg/m3 during the first 6 h following
application. Grab samples showed levels of up to 3410 mg/m3, 30
min after application. In studies with the door open, the levels
were between 60 and 490 mg/m3 (Otson et al., 1981).
5. CHEMOBIOKINETICS AND METABOLISM
5.1. Absorption
5.1.1. Animal studies
From the moment of application, dermal absorption of liquid
methylene chloride in mice increased linearly with time at a rate
of 0.1 mg per cm2 per min (Tsuruta, l975). Rapid absorption
occurred in rats after both oral ingestion and inhalation of
methylene chloride. A steady state plasma concentration, attained
after 2 h of vapour exposure, was not proportional to the exposure
level at low concentrations (McKenna et al., 1982). Almost
directly after oral application, peak concentrations of methylene
chloride could be detected in expired air (McKenna & Zempel, 1981).
In rats, methylene chloride readily passed the placenta (Anders &
Sunram, 1982) and the blood-brain barrier (e.g., Fodor & Winneke,
1971).
5.1.2. Human studies
Dermal exposure of volunteers to liquid methylene chloride
resulted in maximum levels of the compound in expired air, 30 min
after exposure (Stewart & Dodd, 1964). After 0.5 - 8 h of
inhalation exposure to concentrations ranging from 173 to 1740
mg/m3, blood and expired air concentrations and thus, total
uptake of methylene chloride, were found to be directly
proportional to the magnitude of exposure, in sedentary
individuals. The concentration in blood increased gradually and
appeared to be slowly reaching a plateau over 8 h. In the lungs,
the uptake was rapid and remained almost constant after 1 h of
exposure. The concentration in expired air at the end of the
respiration cycle was 2.3 - 2.8 times lower than that in inspired
air. Repeated exposures did not result in higher blood or expired-
air levels. The uptake varied between 55 and 75% of the total
exposure in sedentary individuals. The absolute uptake increased
during exercise, but uptake relative to total exposure decreased
(DiVincenzo et al., l972; Astrand et al., 1975; DiVincenzo &
Kaplan, 1981a,b). In another study on human volunteers, the uptake
of methylene chloride, at steady-state with respect to expired air
within 2.5 - 3 h in 6-h exposures, seemed to deviate from a linear
increase when the exposure level was above 690 - 870 mg/m3
(McKenna et al., 1980). The absolute uptake in a 1-h exposure to
2600 mg/m3 was proportional to the body weight and to the amount of
body fat. The uptake in relation to total exposure was almost the
same for both slim and obese persons, reflecting an increase in
respiratory volume with body weight (Engström & Bjurström, 1977).
Methylene chloride was found to cross the placenta (Vosovaja et
al., 1974) and the blood-brain barrier (e.g., Putz et al,. 1976).
5.2. Distribution
5.2.1. Animal studies
Forty-eight hours after a single oral ingestion of labelled
methylene chloride at 1 mg/kg body weight or inhalation during 6 h
at 170 mg/m3 by rats, the percentages of radioactivity in the skin
and carcass were 7.5% and 30% of the body burden, respectively.
At higher exposures, retention was less. Unmetabolized
methylene chloride was not detected. In rats and mice, most
radioactivity was retained in the liver, kidneys, and lung
(Bergman, 1979; McKenna & Zempel, 1981; McKenna et al., 1982).
Body autoradiography in mice also indicated metabolites in tissues
with a high rate of protein synthesis such as the pancreas, thymus,
and salivary glands (Bergman, 1979). Directly after short vapour
exposures, the highest amounts of volatile radioactivity, which
probably represented unmetabolized methylene chloride and were
found in the adipose tissue, brain, and blood, diminished rapidly
within 2 h (Carlsson & Hultengren, 1975; Bergman, 1979). After
repeated exposure of rats to methylene chloride concentrations in
air of 1735 mg/m3 and 3470 mg/m3 during a 2-week period, the
concentration of the compound in perirenal fat increased, while
that in the brain decreased. Exposure to a time-weighted average
concentration of 3470 mg/m3, involving short intervals at high
concentrations, resulted in greater accumulation than exposure to a
constant concentration of 3470 mg/m3 (Savolainen et al., 1981).
In liver microsomes of rat, methylene chloride was found to be
bound to lipids and proteins, but only after metabolic activation
in the presence of NADPH and oxygen. Pretreatment of rats with
phenobarbital increased the binding (Anders et al., 1977). In
whole liver cells, this binding was enhanced by oxygen and
decreased by phenobarbital pretreatment or glutathione depletion.
Nucleic acids were not alkylated (Cunningham et al., 1981). In
vivo binding of labelled metabolites was observed in rat liver.
Radioactivity was mainly found in the acid-soluble and protein
fractions and, to a lesser extent in the lipid and nucleic acids
fractions. The labelling pattern was similar to that of
formaldehyde (Reynolds & Yee, 1967).
5.2.2. Human studies
The average concentration of methylene chloride in the adipose
tissue of obese men was found to decline from 10.2 to 1.6 mg/kg,
between 1 and 22 h after a single, 1-h exposure to 2600 mg/m3. The
concentration was not measured just after exposure. Even though
obese subjects had lower concentrations of methylene chloride in
adipose tissue than slim subjects, the former had a greater
fraction of the uptake in their adipose tissue (Engström &
Bjurström, 1977).
5.3. Metabolic Transformation
5.3.1. Animal studies
The metabolism of methylene chloride was found to be a
saturable process. Forty-eight hours after inhalation, 55% of the
uptake was expired unchanged at 4920 mg/m3, 30% at 1700 mg/m3,
and 5% at 170 mg/m3, respectively (McKenna et al., 1982).
In these studies, the major metabolites were carbon monoxide
and carbon dioxide found in expired air. This was also observed in
other studies with rats, mice, and rabbits (e.g., Kubic et al.,
1974; Roth et al., 1975; McKenna & Zempel, 1981). McKenna et al
(1982) reported that, after inhalation exposure, about 60% of all
metabolites represented these 2 compounds at all exposures, without
a clear predominance of either one of them. After oral
application, this value was about 80% (McKenna & Zempel, 1981).
The balance was mostly retained as unidentified metabolites in the
carcass and skin, while small quantities of unidentified
metabolites were recovered in the urine and faeces.
Rats inhaling a low dose of labelled methylene chloride in a
closed rebreathing system excreted 47% of the administered label as
carbon monoxide and 29% as carbon dioxide. No radioactivity was
detected in the carcass. The initial rate of carbon monoxide
production was constant at all exposures; this also points to a
saturable enzymatic conversion (Rodkey & Collison, 1977a,b).
Endogenous carbon monoxide production following exposure to
methylene chloride leads to accumulation of carboxyhaemoglobin in
blood. In a study on rats, McKenna et al. (1982) measured steady-
state carboxyhaemoglobin levels of 3 and 10 -13% of saturation,
respectively, after 1 h of exposure to 173 mg/m3 and after 2.5 -
3 h of exposure to 1730 and 5200 mg/m3. The carboxyhaemoglobin
levels in the blood of rats rose with increasing exposure until a
plateau was reached at about 12% of saturation (Fodor et al.,
1973). This was also found during long-term exposures (Burek et
al., 1984).
5.3.1.1. Enzyme pathway
Two pathways that have been proposed on the basis of in vitro
experiments are:
(a) The conversion of methylene chloride to carbon monoxide by
the hepatic microsomal cytochrome P-450 1-dependent mixed
function oxidase system (Kubic & Anders, 1975, 1978; Jongen et
al., 1982). It is proposed that the metabolism starts by a
rate-limiting oxygen insertion, followed by rearrangement to
formyl chloride, which decomposes to carbon monoxide. The
formyl chloride may be involved in macromolecular binding.
Phenobarbital pretreatment increases binding to cytochrome
P-450 and the rate of conversion in vitro, but not in vivo
(Haun et al., 1972; Kubic et al., 1974; Roth et al., 1975).
(b) The conversion of methylene chloride to formaldehyde,
formic acid, and chloride by the hepatic cytosol fraction
(Ahmed & Anders, 1976, 1978; Jongen et al., 1982). In this
conversion, it is proposed that binding of glutathione to
methylene chloride is followed by hydrolysis via glutathione
transferase (EC 2.5.1.18). Carbon dioxide will be the main
product. The resulting S-hydroxymethyl glutathione may yield
formaldehyde or formic acid. Formic acid may inhibit
cytochrome c oxidase (EC 1.9.3.1) (Nicholls, 1975).
5.3.2. Human studies
DiVincenzo & Kaplan (1981a,b) exposed volunteers for 7.5 h to
concentrations of methylene chloride up to 694 mg/m3. Less than 5%
of the uptake was excreted unchanged via the lungs, while 30% of
the metabolized methylene chloride was converted to carbon
monoxide. It was suggested that the rest was converted to carbon
dioxide. Exercise increased both the biotransformation to carbon
monoxide and the blood carboxyhaemoglobin levels. An increased
workload did not further elevate the carboxyhaemoglobin levels,
because of increased excretion of carbon monoxide. Smoking had a
additive effect on the carboxyhaemoglobin values, as was found with
carbon monoxide exposure by Fodor et al. (1973).
Blood carboxyhaemoglobin levels increased in direct proportion
to the level and the duration of exposure up to 694 mg/m3. There-
after, a plateau seemed to be reached. Peak carboxyhaemoglobin
saturation was reached, either at the end of the exposure or, at
high uptakes, shortly afterwards. Nonsmoking subjects have control
values between 0.4 and 2.0% of saturation, while smokers generally
have control values between 2 and 8%. An 8-h exposure of non-
smokers to a methylene chloride concentration of 350 mg/m3
appeared equivalent to an 8-h exposure to a carbon monoxide
concentration of 43 mg/m3, leading to a carboxyhaemoglobin level
of 5% of saturation (Stewart et al., 1972; Fodor & Roscavanu, 1976;
Stewart 1981a). During repeated exposures to methylene chloride
over 5 working days, carboxyhaemoglobin levels did not show any,
or only a slight increase, compared with levels following single
exposures, and returned to pre-exposure levels over the weekend.
Occupational exposure of non-smokers to a time-weighted average of
114 mg/m3 resulted in carboxyhaemoglobin levels of between 0.8 and
2.5% of saturation (DiVincenzo & Kaplan, 1981a; Fodor & Roscavanu,
1976). Residual elevated carboxyhaemoglobin levels associated
with, and proportional to, the level of previous-day exposure to
methylene chloride was found both in smoking and non-smoking
industrial workers, in equal measure. In this epidemiological
study, the dose-related increases in the carboxyhaemoglobin levels
and alveolar carbon monoxide concentration were correlated with a
decrease in the oxyge pressure. Sex, race, and age of the subjects
were found to be unimportant in predicting carboxyhaemoglobin
levels in contrast to smoking and time of venipuncture (Ott et al.,
1983). In the blood of workers exposed to methylene chloride
concentrations ranging from 552 to 760 mg/m3, but not to carbon
monoxide, the carboxyhaemoglobin levels rose to 8.3% after 8 h and
dropped to base-line values of about 4.5% at the start of the new
work day (Ratney et al., 1974). Kuzelova & Vlasak (1966) detected
formic acid in the urine of workers exposed to methylene chloride
for long periods.
5.4. Excretion
5.4.1. Animal studies
The concentrations of methylene chloride in the blood and
expired air of rats and dogs, after exposure to methylene chloride,
declined exponentially and were directly proportional to the level
of exposure (DiVincenzo et al., 1972; McKenna & Zempel, 1981;
McKenna et al., 1982). In rats, the excretion from blood after
inhalation was resolved in a fast and a slow first order process
with half-lives of respectively 2 and l5 min. The fast and slow
processes in the excretion in expired air of rats, after oral
ingestion, had half-lives of 13 and 46 min. After a high oral dose
of 50 mg/kg body weight, the concentration of methylene chloride in
the expired air was constant for 1 h and then declined. The
excretion of carbon dioxide and carbon monoxide after oral intake
and inhalation also followed 2 first order processes with half-
lives of 1.4 - 2.7 h for the first 24 h after exposure and of 6.7 -
17.3 h, thereafter.
The disappearance of carboxyhaemoglobin from the blood was
exponential with a half-life of 23 - 35 min. At high exposures,
blood carboxyhaemoglobin levels remained elevated for 60 - 90 min
following exposure (McKenna & Zempel, 1981; McKenna et al., 1982).
5.4.2. Human studies
After exposure, expired air and blood concentrations of
methylene chloride measured were directly proportional to the
concentration of the vapour. Elimination of methylene chloride
occurred rapidly, mainly through expiration. An initially rapid
phase of elimination had a half-life of less than 1 min. The
elimination of carbon monoxide in the expired air and of
carboxyhaemoglobin from blood was more gradual and returned to
pre-exposure values about 24 h after exposure to up to 694 mg/m3
for 7.5 h. The half-lives for the observed 2 phases of elimination
were reported to be 1.5 h and 10 - 15 h, respectively (McKenna et
al., 1980; DiVincenzo & Kaplan, 1981b). Pulmonary excretion of
carbon monoxide increased when exposure was continued with
exercise. Excretion of methylene chloride via the urine was
negligible (DiVincenzo & Kaplan, 1981b). In contrast, Stewart et
al. (1976) reported a biological half-life following exogenous
carbon monoxide exposure of only 5 h. Moreover, after short
exposures to 1740 mg/m3, blood carboxyhaemoglobin levels continued
to rise for several hours (Astrand et al., 1975). These data
suggest a delayed conversion of methylene chloride from fat
(Engström & Bjurström, 1977). Methylene chloride has been found in
breast milk (Vosovaja et al., 1974).
6. EFFECTS ON ORGANISMS IN THE ENVIRONMENT
A summary of studies on the acute toxicity of methylene
chloride in aquatic organisms is presented in Table 2.
No experimental bioconcentration factor was available, but the
low log n-octanol-water partition coefficient of 1.25 (Hansch et
al., 1975) and the rather high water solubility suggest that
bioaccumulation is very limited. A bioconcentration factor in fish
of 5 can be calculated according to the method of Veith et al.
(1980).
Table 2. Acute aquatic toxicity
---------------------------------------------------------------------------------------------------------
Organism Description t pH Dissolved Hardness Flow/1 Parameter Concen- Reference
(°C) oxygen (mgCaCO3/ stat tration
(mg/ litre) mg/litre
litre)
---------------------------------------------------------------------------------------------------------
algae Chlorella vul- 19 6.5 3-h EC50 27 000 Hutchinson et
garis, Chlamy- & 17 400 al. (1978)2
domonas angulosa respec-
tively
crustacea water flea 22 7.4- 6.5-9.1 173 stat 48-h LC50 220 Le Blanc
(Daphnia magna) 9.4 no-observed 68 (1980)3
adverse-
effect level
fish fathead minnow 12 7.8- >5.0 stat 96-h LC50 310 Alexander et
(Pimephales) 8.0 al. (1978)4
promelas
fish fathead minnow 12 7.8- >5.0 flow 96-h LC50 193 Alexander et
(Pimephales 8.0 96-h EC50 99 al. (1978)4
promelas)
fish bluegill sunfish 21- 7.9- 9.7-3.0 32-48 stat 96-h LC50 220 Buccafusco et
(Lepomis macro- 23 6.5 al. (1981)5
chirus)
fish sheepshead 25- stat 96-h LC50 330 Heitmuller et
minnow 31 no-observed 130 al. (1981)6
(Cyprinodon adverse-
variegatus) effect level
---------------------------------------------------------------------------------------------------------
Notes
1) Flow through or static method.
2) EC50 for growth inhibition by determination of 14CO2 uptake, no analysis for methylene chloride
reported.
3) 15 daphnias/concentration, < 24 h of age, no analysis for methylene chloride reported.
4) Dechlorinated, sterilized lake water, analysis for methylene chloride by gas chromatography.
5) 10 juvenile fish/concentration, deionized reconstituted water; no aeration, no analysis for
methylene chloride reported.
6) 10 juvenile fish/concentration, sea water with salinity of 1.0 - 3.1%, no aeration, no analysis for
methylene chloride reported.
7. EFFECTS ON ANIMALS
7.1. Short-Term Exposures
In short-term exposure studies, effects on organs after
inhalation of methylene chloride are mainly limited to the liver,
kidneys, and heart but central nervous system depression also
occurs. There are not sufficient data to indicate definitely the
effects after oral and dermal exposure. Acute mortality data are
shown in Table 3.
Table 3. Acute mortality after oral intake or inhalation of methylene
chloride
--------------------------------------------------------------------------
Species Route Vehicle Parameter Value Reference
studied
--------------------------------------------------------------------------
rat oral none LD50 3000 mg/kg Kimura et al.
body weight (1971)
rat inhalation - 2-h LC50 79 000 mg/m3 Kashin et al.
(1980)
rat inhalation - 6-h LC50 52 000 mg/m3 Bonnet et al.
(1980)
mouse inhalation - 7-h LC50 56 230 mg/m3 Svirbely et
al. (1947)
mouse inhalation - 6-h LC50 49 100 mg/m3 Gradiski et
al. (1978)
mouse inhalation - 2-h LC50 51 500 mg/m3 Kashin et al.
(1980)
dog oral mucil- LD50 3000 mg/kg Barsoum &
age of body weight Saad (1934)
acacia
guinea- inhalation - 6-h LC50 40 200 mg/m3 Balmer et
pig al. (1976)
--------------------------------------------------------------------------
The slope of the regression line giving the probability units
of the percentage mortality as a function of the logarithm of the
concentration is rather steep for both rats and mice, the
difference between the LC10 and the LC90 being less than 14 000
mg/m3 (Gradiski et al., 1978; Bonnet et al., 1980).
7.1.1. Inhalation exposure
No macroscopic lesions were found in rats at the 6-h LC50 of
52 000 mg/m3 (Bonnet et al., 1980). After 6 h of exposure to
17 350 mg/m3, the concentration of triglycerides was increased in
the liver of guinea-pigs, and reduced in the serum (Balmer et al.,
1976; Morris et al., 1979). Histopathological liver changes,
consisting of the appearance of lipid droplets, were first seen
in guinea-pigs at 18 000 mg/m3 (Morris et al., 1979). Slight
to moderate vacuolization in the liver was seen after 6 h at
38 520 mg/m3. In addition, lungs showed congestion and
haemorrhage; behavioural changes were also noted (Balmer et al.,
1976). Heppel et al. (1944) did not find organ lesions related
to exposure at 17 350 mg/m3 in studies on dogs, monkeys, rats,
rabbits, and guinea-pigs, with the exception of moderate
centrilobular fatty degeneration of the liver and pneumonia in
3 out of 14 guinea-pigs. At 34 700 mg/m3, dogs also showed
fatty degeneration.
After continuous exposure to methylene chloride concentrations
of 87 and 347 mg/m3 for 100 days, slight cytoplasmatic vacuolization
with positive fat stains were noted in livers of rats as well as
tubular degeneration in kidneys. Similar changes and a decrease in
the microsomal cytochrome P-450 content were found in the livers of
mice exposed to a concentration of 347 mg/m3 (Haun et al., 1972).
Similar changes in the liver were found in dogs and monkeys
exposed continuously to a methylene chloride concentration of
3470 mg/m3. The dogs also showed vacuolar changes in the renal
tubules. After 4 weeks, they exhibited abnormal haematology,
increased activities of serum glutamic pyruvic transaminase
(SGPT) (EC 2.6.1.2), isocitric dehydrogenase (EC 1.1.1.41),
bromosulphthalein (BSP) retention. Additional effects at 17 350
mg/m3 were oedema of the brain in dogs and encephalomalacia in
monkeys (Haun et al., 1972).
The effects on the mouse liver were studied microscopically
by Weinstein & Diamond (l972) and Weinstein et al. (1972) during
continuous exposures. At 347 mg/m3, fatty infiltration,
vacuolization, and enlarged nuclei persisted up to the end of the
10-week exposure, while an increase in triglycerides concentration
was reversible. At 17 350 mg/m3, body weights fell, and relative
liver weights increased up to the end of the 168-h exposure. Fatty
infiltration, an increase in the triglycerides concentration, and
hydropic degeneration of the endoplasmic reticulum gradually
disappeared. Protein synthesis was depressed. Necrosis was
observed in a few hepatocytes.
No consistent increase of the total liver microsomal concentration
of cytochrome P-450 was found after repeated exposure of rats, but
the metabolic activity of liver microsomal enzymes increased in
vitro and in vivo (Norpoth et al., 1974; Toftgard et al., 1982;
Kurppa & Vainio, 1981).
Cardiac effects such as arrhythmia, tachycardia, and hypotension
was found in monkeys and rabbits exposed for 1 - 5 min to levels of
methylene chloride exceeding 60 000 mg/m3 (Belej et al., 1974;
Taylor et al., 1976).
Central nervous system depression was noted in dogs, monkeys,
rats, rabbits, and guinea-pigs during each daily session of
repeated exposure to a methylene chloride concentration of 34 700
mg/m3 for 7 h/day, 5 days per week, for 6 months. All animals
became inactive, some time after initial excitement (Heppel et al.,
1944). Mice continuously exposed to a level of 17 350 mg/m3 showed
decreased activity, and water and food intake, and changes in
appearance, which disappeared after 168 h (Weinstein & Diamond,
1972).
Central nervous system depression resulting in reversible
narcosis occurred in dogs, mice, and guinea-pigs after 2 - 6 h of
exposure to levels of methylene chloride between 13 900 and 20 800
mg/m3 (Flury & Zernick, 1931). During exposure for 1.5 h to
17 350 mg/m3, rats showed decreased running activity (Heppel &
Neal, 1944). The sleep-wakefulness patterns were disturbed in rats
from a level of 3470 mg/m3 upwards, as shown mainly by a reduction
in Rapid-Eye Movement sleep (Fodor & Winneke, 1971).
7.1.2. Oral exposure
A single oral dose of 1000 mg/kg body weight resulted in a
decreased cytochrome P-450 content in liver microsomes of rats
(Moody et al., 1981).
Rats, receiving methylene chloride in the drinking-water at a
concentration of 125 mg/litre for 13 weeks, did not show any
effects on behaviour, body weight, haematology, urinalysis, blood
glucose, plasma-free fatty acids, and the estrus cycle (Bornmann &
Loeser, 1967).
7.1.3. Intraperitoneal exposure
One intraperitonal injection of methylene chloride at 510 mg/kg
body weight in rats, slowed down the sciatic motor conduction
velocity by 11%, and gave rise to a carboxy-haemoglobin level of
6.8% of saturation (Pankow et al., 1979).
7.1.4. Effects on the eye and skin
Duprat et al. (1976) and Ballantyne et al. (1976) exposed
rabbits once to 0.5 ml of methylene chloride by ocular instillation.
Moderate to severe changes were seen in the conjunctiva, together
with increased corneal thickness and intraocular tension. All
effects were reversible. Vapour exposure of the eyes caused slight
increases in corneal thickness and intraocular tension.
Application of methylene chloride to the skin of rabbits caused
severe erythema and oedema with necrosis and acanthosis (Duprat et
al., 1976).
7.2. Long-Term Exposure and Carcinogenicity
7.2.1. Inhalation exposure
Groups of 129 male and 129 female Sprague Dawley rats and 107 -
109 male and 107 - 109 female Golden Syrian hamsters were exposed
to methylene chloride (99% pure) at 0, 1730, 5200,and 12 100 mg/m3
for 2 years, 6 h/day and 5 days per week (Burek et al., 1984). The
survival rate of high-exposure female rats was reduced. Slight
exposure-related effects consistent with fatty infiltration were
seen in the livers of both sexes at all exposures. Mean corpuscular
volume, mean corpuscular haemoglobin, and carboxyhaemoglobin were
increased in both sexes at all exposures. An increased, dose-
related incidence of salivary gland region sarcomas was observed in
males at the 2 highest exposures (the authors assume that the
effect might have been due to the combination of viral infection
and methylene chloride exposure). The total number of benign
mammary tumours in the rats increased in a dose-related manner in
both sexes, most pronouncedly in females. It was noted that the
Sprague-Dawley rats used in this study have a very high incidence
of spontaneous mammary tumours. This incidence was not increased
in exposed rats compared to controls.
In hamsters, elevated haematocrit, haemoglobin levels (both
dose-related), mean corpuscular volume, mean corpuscular
haemoglobin, and carboxyhaemoglobin were found in both sexes.
There was no significant increase in the incidence of tumours.
Groups of 90 male and 90 female Sprague Dawley rats were
exposed to 99.5% pure methylene chloride at 0, 173, 694, and 1730
mg/m3 for 20 (male) or 24 (female) months, 6 h per day and 5 days
per week. Two additional groups of 30 female rats were each
exposed for 6 months to 1730 mg/m3 followed by 6 months without
exposure and vice versa.
There was an increase in the incidence of foci of altered
hepatocytes at 1730 mg/m3, in surviving females, and at 694 and
1730 mg/m3, in surviving males. When these liver alterations in
interim kills and end kills were combined, no increase was found.
Hepatocellular vacuolization was noted in males and females
receiving high doses and multinucleated hepatocytes in females
receiving high doses. The number of female rats with benign
mammary tumours was not increased, but the total number of mammary
tumours in the female rats was increased at 1730 mg/m3 (Nitschke
et al., 1983).
7.2.2. Oral exposure
Groups of 85 male Fischer 344 rats received 99% pure methylene
chloride in the drinking-water for 24 months at levels of 6, 52,
125, and 235 mg/kg body weight per day. Groups of 85 female rats
received 6, 58, 136, and 263 mg/kg body weight per day. Duplicate
control groups comprised a total of 135 rats of each sex. A high
dose recovery group was only treated for 18 months.
Slight reductions in weight gain, water intake, and food
consumption were found at the 2 highest doses. Survival was not
affected. Dose-related increases were noted in mean haematocrit,
haemoglobin levels, and red blood cell counts at the 3 highest
doses. Decreases in serum alkaline phosphatase (EC 3.1.3.1)
activity in males and in creatinine, blood urea nitrogen, serum-
protein, and cholesterol in both sexes were also dose-related.
At the 2 highest doses, a dose-related increased incidence of
fatty livers was reversible. At all levels, except the lowest, the
incidence of foci or areas of altered hepatocytes was increased in
a dose-related manner.
In the liver of females, a total of 0, 1, 2, 1 and 4 neoplastic
nodules were observed at 0, 6, 58, 136, and 263 mg/kg, respectively,
while 2 hepatocellular carcinomas were observed at both 58 and 263
mg/kg, against none in the control groups. This incidence of
carcinoma was within the range of historical control values. In
treated males, these incidences were comparable to those of the
controls. No earlier onset of nodules or carcinoma was observed
(NCA, 1982),
In the same project, groups of 100 - 200 male and 50 - 100
female B6C3F1 mice received food grade methylene chloride in the
drinking-water for 24 months at levels of approximately 60, 125,
175, and 235 mg/kg body weight per day. Duplicate control groups
comprised a total of 125 male and 100 female mice.
The survival of the treated female mice was better than that of
the controls. At the highest dose level in both sexes, the
leukocyte count was increased at week 52, but not at the end of the
study.
A very slightly increased incidence of hepatocellular adenomas
and carcinomas, alone or combined, was found in the treated males.
There was no dose relation. The only significant increase was
found for carcinomas in males at 235 mg/kg, which was nevertheless
very close to the average historical incidence for B6C3F1 mice in
the performing laboratory. A slightly increased incidence of
Harderian gland neoplasms were observed in males at 125 and 235
mg/kg. The significance of this finding is unclear (NCA, 1983).
At the time of the evaluation of this document, the results of
an inhalation study in progress in the US National Institute of
Environmental Health Sciences were not available (NTP, 1984).
7.3. Mutagenicity
The numbers of revertants of Salmonella typhimurium TA98,
TA100, and TA1535 were increased 3- to 7-fold in a dose-related
manner, when plates were exposed to the vapour of methylene
chloride of undisclosed purity at levels up to 200 g/m3 air.
Metabolic activation by either induced rat liver S9 fraction,
cytosol fraction, or microsomal fraction increased the mutagenicity
(Simmon et al., 1977; Jongen et al., 1978, 1982; Nestmann et al.,
1980, 1981; Gocke et al., 1981). It was shown that the direct
mutagenicity of methylene chloride could be attributed to bacterial
metabolic pathways similar to those in the rat (Green, 1983).
A dose-related increase in the frequency of gene conversions,
mitotic recombinations, and reversions was found for cultures of
Saccharomyces cerevisiae strain D7, but not for strains D4 and D3,
exposed to methylene chloride of undisclosed purity. Strain D7
contains more cytochrome P-450 than strain D4 and could, perhaps,
activate methylene chloride (Simmon et al., 1977; Callen et al.,
1980).
No mutagenicity was detected in the recessive lethal test on
Drosophila melanogaster fed, or injected with, 1 - 2% methylene
chloride (Abrahamson & Valencia, 1980). A 2-fold increase in the
number of recessive lethals was found after the feeding of 1 - 5%
methylene chloride in 2% dimethylsulfoxide (Gocke et al., 1981).
Methylene chloride was not mutagenic in several tests in which
mammalian somatic cells, including human cells were used (Gocke et
al., 1981; Jongen et al., 1981; Perocco & Prodi, 1981; Andrae &
Wolff, 1983; Burek et al., 1984). A weakly positive effect on SCEs
was observed in vapour-exposed Chinese hamster V79 cells (Jongen et
al., 1981). The same test was negative with and without metabolic
activation in a suspension of Chinese hamster ovary cells, while
mitotic delays and chromosome aberrations were found (Thilagar &
Kumaroo, 1983). Transformation of vapour-exposed Syrian hamster
embryo cells by SA7 adenovirus was enhanced in a dose-related
manner (Hatch et al., 1983).
7.4. Effects on Reproduction and Teratogenicity
Rats received methylene chloride in the drinking-water at a
level of 125 mg/litre during a period of 13 weeks before mating.
No effects were found on the female fertility index, litter size,
survival of pups at 4 weeks, and the number of resorptions
(Bornmann & Loeser, 1967). Fetuses of 19 rats exposed to a
methylene chloride concentration of 4340 mg/m3 air on days 6 - 15
of pregnancy, for 7 h/day, showed an increased incidence of dilated
renal pelvis. Fetuses of 12 mice, exposed similarly, showed an
increased incidence of extra sternebrae. The maternal weight of
mice increased. Rat and mice dams had carboxyhaemoglobin levels as
high as 12.5% during exposure (Schwetz et al., 1975). Groups of 18
rats were exposed before and during, or only during 17 days of
pregnancy to a methylene chloride concentration of 15 600 mg/m3
air for 6 h/day. Blood carboxyhaemoglobin levels of dams ranged
from 7.2 to 10.1% of saturation. Relative and absolute liver
weights were increased. Fetal body weights decreased (Hardin &
Manson, 1980). In the same study, 4 groups of 10 rats each were
exposed under the same conditions and were allowed to deliver their
litters for neurobehavioural testing. Changes in the general
activity of pups were found from the age of 10 days in both sexes,
to 150 days in males only (Bornschein et al., 1980).
Exposure of pregnant rats to methylene chloride may lead to
exposure of the fetus to both methylene chloride and carbon
monoxide (Anders & Sunram, 1982).
8. EFFECTS ON MAN
8.1. Short-Term Exposures
8.1.1. Controlled studies
Neurobehavioural changes were observed at low exposures.
After 1.5 - 3 h of exposure to 694 mg/m3, vigilance disturbance
and an impaired combined tracking monitoring performance were found
(Putz et al., 1976). The critical flicker frequency, a measure for
sensory function, was reduced after 95 min of exposure to 1040 mg/m3
(Fodor & Winneke, 1971). After 4 h of exposure to 2610 mg/m3,
psychomotor performance was decreased (Winneke, 1974). Visually
evoked response alterations, also a measure of sensory function,
were seen after 1 h of exposure to 2400 mg/m3, while exposed
subjects experienced lightheadedness. Blood and urine variables,
except carboxyhaemoglobin levels, were normal in this study after
1 - 2 h of exposure to levels of methylene chloride between 739 and
3420 mg/m3. No eye, nose, or throat irritation was observed
(Stewart et al., 1972). Most neurobehavioural effects observed
were less pronounced or absent with carbon monoxide exposures
resulting in comparable carboxyhaemoglobin levels (Winneke, 1974;
Putz et al., 1976). The odour threshold for methylene chloride is
743 mg/m3 (Leonardos et al., 1969).
8.1.2. Accidental exposures
The increase in blood carboxyhaemoglobin saturation following
methylene chloride exposure has already been discussed. The most
prominent effect of methylene chloride exposure is a reversible
central nervous system (CNS) depression, ultimately resulting in
narcosis, for example, after 30 min of exposure to 69 000 mg/m3
(Moskowitz & Shapiro, 1952). High carboxyhaemoglobin levels (up to
50%) have been measured in the blood of unconscious subjects (Fagin
et al., 1980).
Signs of CNS-depression, narcosis, irritation of the eyes and
respiratory tract, lung oedema, and sometimes death were found
after accidental exposures to methylene chloride or paint remover
containing this compound (Moskowitz & Shapiro, 1952; Hughes, 1954;
Bonventre et al., 1977; Fagin et al., 1980). Three myocardial
infarctions in one subject were reported to have followed 3
exposures to a paint remover containing methylene chloride over a
period of approximately 8 months. The subject was exposed in a
poorly ventilated room, and concentrations may have been very high
(Stewart & Hake, 1976). Electrocardiographic changes resembling
those after carbon monoxide poisoning were found in an exposed man
with a history suggesting ischaemic heart disease (Benzon et al.,
1978). Three probable cases of phosgene poisoning occurred after
the use of methylene chloride-based paint remover near a source of
heat (Gerritsen & Buschmann, 1960; English, 1964).
8.1.3. Effects on the skin and eyes
Several reports already discussed indicate the irritative
action of methylene chloride on the eyes and skin.
Slight erythema was found, when methylene chloride-containing,
aerosol-spray deodorants were used twice a day for 12 weeks by 75
men and women (Meltzer et al., 1977). On direct contact, methylene
chloride caused a burning sensation and pain (Stewart & Dodd,
1964).
8.2. Long-Term Exposure
8.2.1. Occupational exposure
The few reports available deal with small groups of
occupationally-exposed subjects. Workers exposed occupationally to
a time-weighted average of 114 mg/m3 had carboxyhaemoglobin levels
of between 0.8 and 2.5%. No effects were found on clinical
chemistry, haematology, or electrocardiograms (DiVincenzo & Kaplan,
1981a). Cherry et al. (1981) did not find any exposure-related,
long-term damage in 29 subjects as evidenced by subjective
symptoms, neurobehavioural tests, motor nerve conduction velocity,
electrocardiograms, and clinical examinations. The men had been
exposed for several years to levels of methylene chloride ranging
from 260 to 347 mg/m3. Age-matched controls were used. In a
study without a control group, neurasthenic disorders and
irritation of the eyes and respiratory passages were experienced by
half of the 33 workers exposed to methylene chloride for an average
of 2 years. Digestive disorders were reported by one-third of
these workers. Formic acid was found in the urine. No other
deviations were found during the internal, nervous, eye, and
laboratory examinations. The methylene chloride concentrations
measured varied between 100 and 17 000 mg/m3 (Kuzelova & Vlasak,
1966). Irreversible damage to the central nervous system with
acoustic and optical illusions and hallucinations was diagnosed in
1 man, who had been exposed for 5 years to methylene chloride at
levels ranged from 2290 to 12 500 mg/m3 (Weiss, 1969). Another
man, exposed for 3 years to levels of methylene chloride ranging
from 1735 to 3470 mg/m3 showed a bilateral temporal lobe
degeneration (Barrowcliff & Knell, 1979). A case of delirium and
seizures was reported of a man who was exposed to methylene
chloride during 4 years. The man reported a 12-month history of
intermittent headache, nausea, blurred vision, shortness of breath,
and transient memory disturbances. Neuro-psychological and EEG
examinations revealed a dysfunction of the right hemisphere. All
symptoms and signs cleared with removal from the workplace (Tariot,
1983). In 46 subjects exposed to methylene chloride concentrations
of 6 - 34 mg/m3 for several years, an excess (not significant) of
digestive disorders and hypotonia was found over controls, while
symptoms of gall bladder pathology and swollen liver were frequent.
No details were given concerning drinking or smoking habits (Kashin
et al., 1980).
A clinical laboratory evaluation of 266 exposed volunteer
workers and 251 reference volunteer workers from two cellulose
di- and tri-acetate plants in the USA, taking into account smoking
habits, race, sex, age, intensity of exposure, and time of
venepuncture, revealed increases in red cell counts, haemoglobin
levels and haematocrit among white women exposed to a methylene
chloride level of approximately 1650 mg/m3. Carboxyhaemoglobin
levels were elevated in all exposed groups at all exposure levels
(section 5.3). A dose-related increase was observed in serum
bilirubin for exposed subjects of both sexes. A total of 24
exposed male volunteers and 26 reference male volunteers from the
above 2 industries were also selected for 24-h electrocardiographic
monitoring. Three exposed and 8 reference workers had reported a
history of heart disease. Neither increased ventricular or
supraventricular ectopic activity nor increased episodic ST-segment
depression was found to be associated with methylene chloride
exposure (Ott et al., 1983).
In women occupationally exposed to an average methylene
chloride concentration of 86 mg/m3, the compound was found in the
placenta, fetus, and breast milk (0.07 mg/litre milk average)
(Vosovaja et al., 1974).
8.2.2. Mortality studies
When the mortality experience of 334 deceased male industrial
workers, who had been exposed to levels of methylene chloride up to
1210 mg/m3, was compared with that of the non-exposed industrial
workers and the New York State male populations, no excess age-and
cause-specific mortality was found. A total of 751 male workers
with exposures for up to 30 years were subsequently followed up for
mortality for 13 years. No excess mortality was found compared
with 2 internal and 2 external control groups (Friedlander et al.,
1978). The data on this cohort have been updated with 4 more
years. The mortality was consistent with that of industrial
controls and less than that expected of the New York State controls
(Hearne & Friedlander, 1981).
The mortality experience of 1271 male and female workers of a
cellulose di- and tri-acetate plant with time-weighted-average
exposures to methylene chloride of between 486 and 1648 mg/m3,
for a period up to 23 years, was compared with that of corresponding
USA populations and a reference cohort of 948 workers in a
cellulose diacetate plant, where no methylene chloride was used.
The mortality, specified by cause (with a focus on ischaemic heart
disease), sex, race, and for each cause, duration of exposure,
length of follow-up, and employment status was comparable to that
of the USA populations. The mortality rate among white men was
higher than that of the reference cohort for the following
categories: all causes, diseases of the circulatory system,
ischemic heart disease, and all external causes. Observed deaths
in the reference group were considerably fewer than the USA
experience for each of these categories. According to the authors,
the mortality trends for cardiovascular disease can be explained by
expected geographical differences (Ott et al., 1983).
9. EVALUATION OF HEALTH RISKS FOR MAN
Human exposure is mainly through inhalation. Absorption via
the skin is slow. Methylene chloride is rapidly absorbed via the
gastrointestinal tract and crosses the placenta and blood-brain
barrier.
Acute effects
The odour threshold for methylene chloride is 743 mg/m3
(Leonardos et al., 1969). The predominant effects of methylene
chloride in human beings are central nervous system depression and
the production of elevated carboxyhaemoglobin levels in the blood.
These effects are reversible. Mild behavioural disturbances (e.g.,
disturbances of vigilance) have been reported following exposure to
694 mg/m3 of methylene chloride in air for 1.5 - 3 h (Putz et al.,
1976) and impairment of psychomotor performance after a 4-h
exposure to 2610 mg/m3 (Winneke, 1974). Narcosis occurred
following exposure to 69 000 mg/m3 for 30 min (Moskowitz & Shapiro,
1952). Individuals with heart disease may be especially at risk,
when exposed to methylene chloride, because of the hypoxia induced.
The minimum observed effect level for short-term inhalation
exposure was approximately 690 mg/m3 (Putz et al., 1976).
Chronic effects
The predominant chronic effects in human beings are nervous
system depression and an elevated carboxyhaemoglobin saturation of
the blood. However, no exposure-related subjective symptoms,
neurobehavioural effects, motor nerve conduction velocity changes,
electrocardiogram changes, or clinical effects were noted in
workers exposed to methylene chloride levels of between 260 -347
mg/m3, compared with age-matched controls (Cherry et al., 1981).
Methylene chloride has been shown to cross the placenta and has
been found to accumulate in fetal tissue and breast milk (Vosovaja
et al., 1974). In one study designed to assess teratogenic
potential (Schwetz et al., 1975), rats and mice were exposed by
inhalation to 4340 mg/m3 during days 6 - 15 of gestation. The
results of this study indicated an increased incidence of extra
sternebrae in mice as well as a greater incidence of dilated renal
pelvis in rats. In another study (Hardin & Manson, 1980), rats
were exposed by inhalation to a methylene chloride concentration of
15 600 mg/m3 for 6 h/day, before and/or during 17 days of pregnancy.
The purpose of this study was to ascertain whether exposure before
and during gestation has a greater effect on the conceptus than
exposure only before gestation. No significant effects were
reported, except that the pups of rats exposed during gestation had
lower fetal body weights. In the same study, additional groups of
rats were similarly exposed and allowed to litter, in order to
evaluate the potential for behavioural teratogenic effects
(Bornschein et al., 1980). As early as 10 days of age, both male
and female pups exhibited treatment-related effects in general
activity tests. Changes in the general activity of male pups were
still demonstrable 150 days post partum. While results of the
previous studies (Schwetz et al., 1975; Hardin & Manson, 1980) tend
to indicate that the teratogenic hazard is minimal, the results of
the neurobehavioural study of Bornschein et al. (1980) suggest the
possiblity of delayed behavioural effects.
Only one reproduction study was available, which precludes
assessment of a potential reproduction hazard. In this study
(Bornmann & Loeser, 1967), no reproductive impairment was found
when rats were allowed to mate after receiving 125 mg of methylene
chloride per litre of drinking-water for 13 weeks.
Results of in vitro studies showed that methylene chloride
was mutagenic in bacteria and fungi. However, most tests on
mammalian somatic cells, including human cells, were negative.
In 2 inhalation studies in rats the incidence of benign mammary
tumours was elevated neither in male nor in female rats, but the
total number of mammary tumours was increased in a dose-related
manner (Nitschke et al., 1983; Burek et al., 1984). In the study
of Burek et al. (1984), an increase of salivary gland region
sarcomas was found in male rats. In Golden Syrian hamsters, no
significant increases in tumour incidences were found. In a
drinking-water study with rats and mice, no significant increases
in tumour incidences were found, while an increased incidence of
foci or areas of altered liver cells was observed (NCA, 1982,
1983). In two epidemiological mortality studies, there was no
excess mortality due to cancer compared with control populations
(Friedlander et al., 1978; Ott et al., 1983). The data are
inadequate for assessing whether or not methylene chloride is to be
considered carcinogenic for animals and man. According to a
previous reevaluation of all available published epidemiological,
experimental, and short-term test data, an IARC Working Group
concluded that methylene chloride could not be classified as to its
carcinogenicity for human beings (IARC, 1982).
A no-observed-adverse-effect level for long-term inhalation
exposure was of the order of 260 - 350 mg/m3 (Cherry et al., 1981).
A lowest-observed-effect level could not be derived from the
available human data.
10. SOME CURRENT REGULATIONS, GUIDELINES, AND STANDARDS
10.1. Occupational Exposure
Maximum allowable concentrationsa are 49 mg/m3 (14 ppm, ceiling
value) in the USSR, 250 mg/m3 (70 ppm, TWA) in Sweden, 360 mg/m3
(100 ppm, TWA) in the Federal Republic of Germany, 700 mg/m3 (200 ppm,
TWA) in the Netherlands, 360 mg/m3 (100 ppm, TWA) in the USA.
10.2. Food
The Council of Europe (1978) recommends a maximum of 5 mg/kg
wet weight in food. The Food and Drug Administration (1977) in the
USA allows maxima of 30 mg/kg wet weight in spice oleoresins, 2.2%
in hops, and 10 mg/kg wet weight in coffee.
10.3. Storage and Transport
The United Nations Committee of Experts on the Transport of
Dangerous Goods (1984) qualifies methylene chloride as a toxic
substance (Class 6.1) with minor danger for packing purposes
(Packing Group III). Packing methods and a label are recommended.
The Inter-Governmental Maritime Consultative Organizationb (1981)
also qualifies methylene chloride as a toxic substance (Class 6.1)
and recommends packing, stowage, and labelling methods for maritime
transport in glass bottles, cans, and drums. It is stressed, that
phosgene fumes are formed when methylene chloride is involved in a
fire and that stowage should be under shaded conditions away from
radiant heat.
The label recommended by both organizations is:
---------------------------------------------------------------------------
a Values quoted in national lists.
b Now the International Maritime Organization.
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