DICHLOROMETHANE
First draft prepared by
Dr J.C. Larsen
Institute of Toxicology
National Food Agency of Denmark
Soborg, Denmark
1. EXPLANATION
Dichloromethane has previously been evaluated for an acceptable
daily intake for humans at the fourteenth, twenty-third, and
twenty-seventh meetings of the Committee (Annex 1, references 22,
50, and 62). At the twenty-third meeting the Committee established a
temporary ADI of 0-0.5 mg/kg bw, and recommended that the solvent
should be used according to good manufacturing practice, which would
result in minimum residues and prevent any significant toxicological
effects. At its twenty-seventh meeting the Committee withdrew the
previously allocated ADI and recommended that the use of
dichloromethane as an extraction solvent should be limited in order
to ensure that its residues in foods are as low as practicable. The
Committee felt that the available lifetime studies in rats and mice,
due to a number of shortcomings, were inadequate for a complete
evaluation of the possible carcinogenicity of dichloromethane. The
Committee noted that ongoing studies on lifetime exposure of mice to
dichloromethane in drinking-water, and on exposure by inhalation in
mice and rats, might resolve the problems raised in the previous
studies.
Guidelines for the evaluation of solvents used in food
processing have been published by WHO under the International
Programme on Chemical Safety (IPCS) (Annex 1, reference 76, Annex
III)
At its thirty-fifth meeting (Annex 1, reference 88) the
Committee during its deliberations on specifications for spice
oleoresins considered solvent residues and expressed the opinion
that the use of dichloromethane as an extraction solvent should be
discouraged because of toxicological concerns. Because this and
other solvents had not been recently evaluated and new data had
become available, the Committee concluded that an overall review of
solvents used in food processing would be appropriate. The Committee
further stressed that levels of residues resulting from the use of
any solvent should be the minimum technically achievable and
toxicologically insignificant.
Since the last review, additional data on dichloromethane have
become available and are summarized and discussed in the following
monograph addendum.
Dichloromethane has been reviewed by the International Agency
for Research on Cancer (IARC, 1986).
2. BIOLOGICAL DATA
2.1 Biochemical aspects
2.1.1 Absorption, distribution, and excretion
Low temperature whole-body autoradiography of [14C]-labelled
dichloromethane in male mice after inhalation of 400 mg/kg bw
(during 10 minutes) showed a high uptake of radioactivity in the
white matter of the brain, spinal cord, spinal nerves, body fat,
blood, liver, lung, and kidney immediately after inhalation.
Autoradiograms obtained from dried and evaporated sections showed
high levels of radioactivity in the liver, bronchi, and kidney only.
No volatile radioactivity could be detected in the nervous system at
30 min, while volatile radioactivity was still present in body fat
after one hour. An accumulation of radioactivity in liver, kidney,
bronchi, and nasal mucosa could be detected at 30 min and one hour
after the inhalation. The levels of non-volatile radioactivity in
liver, kidney, bronchi, and nasal mucosa decreased between 2 and
8 h, after which an almost constant level was present in these
tissues up to 48 h following the administration. A high level of
radioactivity was registered only by low-temperature autoradiography
in the blood up to 4 h after the inhalation. This activity probably
represented 14C-carboxyhaemoglobin (Bergman, 1983).
The tissue distribution and metabolism of [14C]-labelled
dichloromethane was investigated in male B6C3F1 mice following
intravenous or oral administration. The route of exposure and the
composition of the dosing solution were found to have a significant
effect on the pharmacokinetics. Following single intravenous doses
of 10 or 50 mg [14C]-dichloromethane/kg bw to groups of 6 mice,
dose-dependent metabolism to 14CO2 and 14CO and rapid
pulmonary clearance of unchanged [14C]-dichloromethane
characterized the elimination of dicbloromethane from the body. The
highest concentrations of dichloromethane were found in the liver,
lung and kidney, with more than 50% of the total radioactivity in
these tissues represented by the parent compound. When
dichloromethane was administered orally in single gavage doses for
14 consecutive days at treatment levels of 50 mg/kg bw in water or
500 and 1000 mg/kg bw in corn oil, rapid absorption and elimination
of dichloromethane characterized the treatment in water while
distinctly slower trends were found for the doses in corn oil. No
observable pharmacokinetic or metabolic effect resulted from
repeated oral dosing over the two-week treatment period (Angelo
et al., 1986).
The tissue distribution and metabolism of [14C]-labelled
dichloromethane was investigated in male Fischer 344 rats following
either single intravenous or 14-day continuous oral administration.
In the experiment with intravenous administration doses of 10 and
50 mg/kg bw were used, and groups of 12 rats were used for blood
samples after 2, 5, 10, 15, 20, 30, and 40 min, and samples of
expired air after 20, 40, 60, and 240 min. A total of 73 (10 mg/kg
bw) and 75 (50 mg/kg bw) per cent of the administered radioactivity
was collected as expired gases during the first four hours, mainly
as [14C]-dichloromethane. The remainder was more slowly excreted,
mainly as [14C]-carbon dioxide and [14C]-carbon monoxide. A
two-compartment model was used to describe the elimination of
dichloromethane from blood following single intravenous doses. When
[14C]-dichloromethane was administered orally (by gavage in water)
in a daily dose of 50 or 200 mg/kg bw for 14 consecutive days, rapid
absorption and distribution to the tissues characterized the
disposition. Groups of 6 rats were used to sample blood, liver, and
carcass 10, 30, and 240 min after dosing on days 1, 7, and 14, and
to obtain expired air after 0.5, 1, 4, 6, and 24 h on days 1, 7, 10,
and 14. The average levels of dichloromethane decreased rapidly in
blood and liver between 10 and 240 min, while maximal carcass
concentrations occurred after 30 min. Dose-dependent metabolism to
14CO2 and 14CO and rapid pulmonary clearance of unchanged
[14C]-dichloromethane were the dominant routes of elimination from
the body. After 10 mg/kg bw 98% was recovered during 24 h as
dichloromethane (63%), CO2 (19%), and CO (16%). At the higher dose
(50 mg/kg bw) a proportionally higher level of dichloromethane was
expired. For oral administration the excretion rate of
dichloromethane apparently followed first order kinetics. Metabolism
was the rate-limiting mechanism that controlled the production of
CO2 and CO. No observed pharmacokinetic nor metabolic effect
resulted from repeated oral dosing (Angelo et al., 1986).
Based on the above-mentioned studies on the pharmacokinetics of
dichloromethane a physiologically based pharmacokinetic model was
developed to simulate the pharmacokinetic behaviour in mice and rats
following single and repeated oral exposures. Using this model it
was confirmed that corn oil can not only affect uptake, but can also
have an influence on the distribution of dichloromethane to target
tissues and on metabolism profiles (Angelo & Pritchard, 1984).
Dichloromethane, 125 mg/kg bw, administered by gavage to groups
of five male Wistar rats, was more easily absorbed from the
gastrointestinal tract when administered in aqueous solution than in
corn oil. Serial blood samples collected over a five-hour period
showed that the peak concentration of dichloromethane was about
three times higher following oral administration in water than when
given in corn oil. Furthermore, the time taken to reach the peak
level was approximately three times longer when the compound was
administered in corn oil as compared to water (Withey et al.,
1983).
In preparation for the design and performance of chronic
toxicity and carcinogenicity studies on dichloromethane in rats and
mice the following conclusions were made on the basis of biochemical
and short-term, metabolic feeding studies: the major metabolites of
dichloromethane in rats and mice are CO2 and CO, the same
metabolites that have been found in man. Saturation of metabolic
pathways was demonstrated in both rats and mice at oral doses of
approximately 100 mg/kg bw/day. There was a significant change in
the rate of metabolite expiration at approximately 100 mg of
dichloromethane/kg bw/day. The proportion of metabolites formed from
increasing doses above 100 mg/kg bw/day is significantly less than
that at and below 50 mg/kg bw/day. Absorption, metabolism, and
elimination is fairly rapid with little systemic accumulation of
dichloromethane or its metabolites. Systemic exposures to
dichloromethane by inhalation at 50, 500, and 1500 ppm were
equivalent to those achieved by oral gavage of 10, 150, and
450 mg/kg bw, respectively, in water. Studies also showed that
gastric intubation of doses of 5-1000 mg/kg bw/day to male mice for
3 days, or administration for 28 days with drinking-water, had no
measurable effect on liver microsomal enzyme activity (Kirschman
et al., 1986).
The rates of metabolism of dichloromethane in male Fischer rats
exposed to 50 and 1000 ppm were 0.20 and 3.3 nmol/min/g,
respectively. A pharmaco-kinetic model incorporating the metabolic
rate at steady state, blood concentration versus time, and
respiratory minute volume estimated the inhaled "effective" doses in
six-hour exposed rats to be 3.8 and 67 mg/kg bw, respectively
(Landry et al., 1983).
Groups of 5 female Sprague-Dawley rats, on the 17th day of
pregnancy, were exposed in inhalation chambers for five hours to
concentrations of dichloromethane ranging from about 107 to
2961 ppm. Immediately following exposure, the concentrations of the
compound were determined in each fetus and in the maternal blood.
Fetal weights and fetal concentrations were related to their
position on the two horns of the uterus. The mean concentrations in
fetuses were dose-related and ranged from 0.87 to 36.8 µg/g. The
results revealed a linear decrease in fetal concentration with the
location of the fetus from the ovarian to the cervical end of the
uterine horns. These relationships were consistent across doses.
Good linear relationships were observed between exposure level and
mean fetal concentrations and maternal blood concentrations (Withey
& Karpinski, 1985).
Absorption of dichloromethane through the skin of male Fischer
344 rats was shown using groups of six rats dermally exposed to
concentrations of 30 000, 60 000, and 100 000 ppm for four hours.
Dose-dependent blood concentrations ranging from 25 to 100 µg/ml
were obtained at two to four hours after the beginning of the
exposure (McDougal et al., 1986).
2.1.2 Biotransformation
Dichloromethane (CH236Cl2) was incubated with cytosolic
and microsomal fractions of livers and lungs from male Fischer 344
rats, B6C3F1 mice, Syrian Golden hamsters, and otherwise
healthy human accident victims. As regards the microsomal metabolism
of dichloromethane (1 to 10 mM) the highest activity (expressed as
nmol product formed/min/mg protein) was found in liver from the
hamster, followed by the mouse, rat and human. In lung microsomes
the highest activity was found in the mouse, and the lowest in the
human. The glutathione-S-transferase dependent metabolism in either
organ was highest in the mouse cytosol, followed by the rat, human,
and hamster. The in vitro rate constants for the two enzyme
systems were consistent with the hypothesis that metabolism of
dichloromethane occurs in vivo by two competing pathways: a
high-affinity saturable pathway, identified as mixed function
oxidases, and a low-affinity first-order pathway, identified as
glutathione-S-transferases. The data from the in vitro studies
were incorporated into a physiologically-based pharmacokinetic model
for dichloromethane in the mouse, rat, hamster, and man. Using this
model, internal doses in human liver and lung of 3.66 x 10-6 and
4.26 x 10-7 mg equivalents of dichloromethane metabolized to
supposedly reactive metabolites by the glutathione-S-transferase
dependent pathway per day per litre volume of tissue were calculated
assuming a concentration of 1 µg/litre of drinking water. For the
mouse it was calculated that the lowest (60 mg/kg bw/day) and the
highest (250 mg/kg bw/day) doses of dichloromethane given in the
drinking water in a long-term carcinogenicity study (Serota et al.,
1986) where no tumours were induced in the liver nor lung would lead
to concentrations of 3.0 and 16.0 (liver), and 0.4 and 2.2 (lung) mg
equivalents/day/litre tissue, respectively. When the same model was
applied to the concentrations of 2000 and 4000 ppm of
dichloromethane used in an long-term inhalation carcinogenicity
study in mice, in which tumours of liver and lung were induced
(Mennear et al., 1988), concentrations of 785 and 1670 (liver) and
321 and 482 (lung) mg equivalents/day/litre tissue were calculated,
respectively. In comparison, an inhalation concentration of 4000 ppm
used in a long-term rat study, in which no tumours of liver nor lung
were observed (Mennear et al., 1988; as cited in Reitz), would
lead to concentrations of 677 (liver) and 96 (lung) mg
equivalent/day/litre of tissue (Reitz et al., 1988, 1989).
When dichloromethane was incubated with primary cultures of
hepatocytes from adult male Sprague-Dawley rat livers the production
of carbon monoxide increased with time, increased cell number, and
concentration of dichloromethane. However, the carbon monoxide
production per hepatocyte decreased with increasing cell density.
When present at a concentration higher than 6 µl/ml in the media the
metabolism of dichloromethane to carbon monoxide was extensively
depressed, and total and mitochondrial glutamic-oxaloacetic
transaminase levels in the culture medium were extensively elevated,
and the cultured hepatocytes were destroyed by dichloromethane
(Mizutani et al., 1988).
Dichloromethane (0.4 ml/kg bw, intraperitoneally) metabolism to
carbon monoxide as measured as carboxyhaemoglobin formation was
induced in male Wistar rats (n = 6) by isoniazid, acetone and
fasting, all treatments known to lead to the induction of liver
microsomal cytochrome P-450j (Pankow & Hoffmann, 1989).
Groups of six male Wistar rats were given 527 mg
dichloromethane/kg bw by gavage. Prior oral administration of
benzene, toluene, or o-, m-, or p-xylene significantly enhanced
the production of carboxyhaemoglobin, while simultaneous
administration of the aromatic hydrocarbons inhibited
carboxyhaemoglobin formation (Pankow et al, 1991).
In rats a single high dose of ethanol (174 mmol/kg bw by
gavage) completely inhibited the increase in carboxyhaemoglobin
concentration due to dichloromethane (136, 527, and 1326 mg/kg bw by
garage), but did not prevent the dichloromethane-induced decrease in
nerve conduction velocity (Glatzel et al., 1987).
The uptake of dichloromethane by inhalation was greatly reduced
in groups of five male B6C3F1 mice exposed to 1000 and
3000 ppm of dichloromethane when the mice had been pretreated with
the hepatic microsomal oxidative pathway inhibitors pyrazole
(320 mg/kg bw) or diethyldithiocarbamate (300 mg/kg bw). The authors
concluded that the glutathione-dependent pathway of dichloromethane
metabolism is of minor importance in the mouse (Ottenwalder et al.,
1989).
A physiologically-based pharmacokinetic model was developed for
dichloromethane which describes the fate of the compound and its
metabolic products in the mouse, rat, hamster and human. The model
was used to predict specific tissue concentrations of critical
metabolic reaction products, related to the
glutathione-S-transferases pathway in target tissues of animals and
humans.
The model was validated for humans by comparing predicted blood
concentrations with measured concentrations in healthy human
volunteers exposed to 100 or 350 ppm for 6 h. Over a concentration
range of 1-4000 ppm the model predicted somewhat lower
concentrations of glutathione-dependent metabolites in liver and
lung of humans than of mice. Above 1000 ppm the concentration of
metabolites of the glutathione pathway could be calculated by linear
extrapolation, while deviation from linearity was apparent in the
region below 1000 ppm. The authors concluded that saturation of the
mixed function oxidases makes a larger percentage of dichloromethane
available for metabolism by the glutathione-dependent pathway,
resulting in a disproportionate increase in metabolites at exposure
concentrations above 1000 ppm (Watanabe et al., 1987).
As dichloromethane is metabolized to carbon monoxide which
binds reversibly to haemoglobin and is eliminated by exhalation, a
physiologically based pharmacokinetic model which describes the
combined kinetics of carbon monoxide, carboxyhaemoglobin, and parent
dichloromethane was developed and applied to examine the inhalation
kinetics of carbon monoxide and of dichloromethane in rats and
humans. Dichloromethane kinetics and metabolism had been described
previously (Watanabe et al., 1987). Physiological and biochemical
constants for carbon monoxide were estimated by exposing rats to
200 ppm carbon monoxide for 2 h and examining the time course of
carboxyhaemoglobin after cessation of exposure. The two models were
coupled to a physiologically based pharmacokinetic model for
dichloromethane to predict carboxyhaemoglobin time course behaviour
during and after dichloromethane exposures in rats. By coupling the
models it was possible to estimate the yield of carbon monoxide from
oxidation of dichloromethane. In rats only about 0.7 mol of carbon
monoxide are produced from 1 mol of dichloromethane during
oxidation. The combined model adequately represented
carboxyhaemoglobin and dichloromethane behaviour following 4 h
exposures to 200 or 1000 ppm dichloromethane, and 1/2-hour exposure
to 5160 ppm dichloromethane. The rat model was scaled to predict
kinetics in humans exposed either to dichloromethane or to carbon
monoxide. Three human data sets from the literature and an
additional data set from human volunteers exposed to 100 or 350 ppm
dichloromethane for 6 h were examined. The combined model gave a
good representation of the observed behaviour in all four human
studies (Andersen et al., 1991).
2.1.3 Effects on enzymes and other biochemical parameters
Groups of four male Sprague-Dawley rats were exposed by
inhalation to 0, 500, 1500 or 3000 ppm of dichloromethane for three
days. Dichloromethane did not increase the concentration of liver
microsomal cytochrome P-450. However, a dose-dependent increase in
the in vitro liver microsomal formation of several metabolites of
biphenyl and benzo(a)pyrene was observed (Toftgard et al., 1982).
Groups of 10 male Wistar rats were exposed to dichloromethane
vapours at concentrations of 500 ppm, 1000 ppm, or 1000 ppm as a
time-weighted average (100-2800 ppm). All of the exposures lasted
for six hours, five days a week for two weeks. Kidney microsomes
displayed a dose-dependent enhancement of the ethoxycoumarin
O-deethylase activity. After the second week the enhancement was
accompanied by an increase in the renal glutathione content. In the
liver, the UDP-glucuronyltransferase activity showed a
dose-dependent increase and the NADPH-cytochrome c reductase
activity decreased. The hepatic glutathione content remained
unchanged. Dichloromethane exposure did not affect the haemoglobin
concentration of the blood. An 8 to 9% carboxyhaemoglobin
concentration was found after exposure in all of the study groups.
The similarity in carboxyhaemoglobin concentrations suggest that the
metabolic pathway converting dichloromethane to carbon monoxide was
already saturated at the lowest exposure level studied (Kurppa &
Vainio, 1981).
Dichioromethane did not inhibit the ethoxycoumarin deethylase
activity of the major phenobarbital-inducible isozyme of rat liver
cytochrome P-450 in a reconstituted system (Halpert et al., 1986).
Serum transaminases (GOT and GPT) and ornithylcarbamyl
transferase (OCT) were increased in a group of 5 male Wistar rats
40 h after an intraperitoneal treatment with 20 µl of
dichloromethane in olive oil (Corsi et al., 1983).
Single administrations of 1326 mg dichloromethane/kg bw by
gavage to male Wistar rats induced increased serum levels of leucine
aminopeptidase and alanine aminotransferase activities after 2-4 h,
indicative of liver toxicity. The effect was reversible, the levels
of the enzyme activities being normal after 24-48 h (Pankow &
Marzotko, 1987).
Dichloromethane slightly inhibited protein synthesis but did
not affect lipid peroxidation in male Sprague-Dawley rat liver
slices. Protein synthesis by rat liver slices was evaluated by
[3H]leucine incorporation into the trichloroacetic acid-insoluble
material, and lipid peroxidation was evaluated by thiobarbituric
acid-reactive substances released into the incubation medium (Fraga
et al., 1989).
In contrast to a number of other halogenated compounds
dichloromethane did not induce lipid peroxidation in male
Sprague-Dawley rat liver, kidney, spleen, nor testis slices (Fraga
et al., 1987).
Dichloromethane was given by oral gavage at doses of 39, 425,
and 1275 mg/kg bw to groups of 22, 8, and 15 adult female
Sprague-Dawley rats, respectively, both 21 h and 4 h before
sacrifice. A dose of 1275 mg/kg bw of dichloromethane caused a
small, but significant amount of hepatic DNA damage as measured by
the alkaline elution technique. In three of the 15 rats in that
group an extremely high ornithine decarboxylase activity was
measured in the liver. No changes were observed in liver cytochrome
P-450 nor glutathione content nor in serum alanine aminotransferase
activity (Kitchin & Brown, 1989).
In contrast to such anaesthetics and solvents as chloroform,
halothane, and trichloroethylene dichloromethane was a very weak
depressor of the alpha1-adrenoreceptor in membranes prepared from
the rabbit myometrium as assayed by binding with the selective
radioligand 3H-prazosin (Wikberg et al., 1985).
Dichloromethane was less potent than carbon tetrachloride,
trichloroethylene, halothane, or chloroform in the ability to
inhibit the binding of 3H-clonidine to alpha2-adrenoceptors in
male NMRI mouse cerebral cortex membranes in vitro (Wikberg
et al., 1987).
Dichloromethane was able to activate protein kinase C in intact
rabbit platelets in vitro. In addition, dichloromethane stimulated
enzyme activity as well as 12-O-tetradecanoylphorbol-13-acetate
binding capacity in a cell-free system. Scatchard analysis of the
data indicated that dichloromethane increased the number of phorbol
ester binding sites (Roghani et al., 1987).
Dichloromethane was incubated with purified nerve myelin from
sciatic nerve from Sprague-Dawley rats at concentrations from 0.4 to
4% (v/v) together with [gamma-32P]ATP. The incubation resulted in
enhanced phosphorylation of PO, the major intrinsic membrane
glycoprotein from peripheral nerve myelin. Two per cent (v/v) of
dichloromethane was required to maximally stimulate phosphorylation
of PO, higher concentrations were inhibitory. It was postulated that
the increased phosphorylation of PO may result from the activation
of myelin associated protein kinase C (Agrawal & Agrawal, 1989).
2.2 Toxicological studies
2.2.1 Acute toxicity studies
Groups of adult male albino rats received single oral doses of
dichloromethane. The mean peroral LD50 was 2330 mg/kg bw. When
groups of 6-11 rats were dosed with 0, 3.1, 4.7, 6.2, 9.4, or
15.6 mmol of dichloromethane/kg bw urinary catecholamine excretion
increased significantly at 6.2 and at 9.4 mmol/kg bw, corresponding
to 527 and 799 mg/kg bw. At 4 and 48 h following single doses of
1236 mg/kg bw to groups of 10 rats, morphological investigations
revealed cytological changes and a distinct reduction of chromaffin
reaction in the adrenal medulla. The norepinephrine contents of
cells were decreased strongly four hours after administration and
increased weakly again, but were still distinctly reduced in
comparison with the control animals 48 h later (Marzotko & Pankow,
1987).
2.2.2 Short term studies
2.2.2.1 Mice
Groups of 20 male and 20 female B6C3F1 mice were
administered dichloromethane (purity > 99.0%) in the drinking water
for 90 days at levels of 0, 0.15, 0.45, and 1.50%. The calculated
intakes were 0, 226, 587, and 1911 mg/kg bw/day for males and 0,
231, 586, and 2030 mg/kg bw/day for females. Slightly lower body
weights were noted for the mid- and high-dose animals from week 6 to
termination. Lower liquid consumption in all compound-treated groups
suggested an effect on palatability. Increased reticulocyte counts
were recorded for all treated females at 1 month. Histopathological
evaluation of tissues from animals killed after 1 month revealed no
treatment-related effects. Livers from animals killed after 3 months
showed a subtle central lobular fatty change, which was most
prominent in the mid- and high-dose groups. No other
histopathological changes were observed. Also, no adverse effects
were seen on mortality, physical observations, food consumption,
feed efficiency, nor at gross necropsy (Kirschman et al., 1986).
Groups of 10 to 11 male and female NMRI mice were exposed by
inhalation to 37, 75, 150, or 300 ppm of dichloromethane for
different time periods, ranging from 4 days to 90 days. In some of
the experiments exposure free periods were incorporated as well as
periods with intermittent exposures. Exposure to dichloromethane
produced a time- and concentration-related increase in liver weight,
in all groups except the low-dose groups (37 ppm). The effect was
more prominent in female mice than in male. The activity of plasma
butyrylcholinesterase increased even more than the liver weight at
corresponding exposures, but only in the males. Fatty infiltration
was noticeable after exposure to 75 ppm and was more prominent in
the females than in the males. Thirty to 60 days of continuous
exposure were required to produce maximal effects. Intermittent
exposure was less effective than continuous exposure. Most effects
were fully reversible after exposure for both 30 and 90 days if the
animals were transferred to a solvent-free environment. However,
after exposure for 90 days, butyrylcholinesterase activity in the
males did not return to normal within 30 days but after 90 and 120
days free from exposure only slight if any effects on the activity
remained (Kjellstrand et al., 1986).
2.2.2.2 Rats
Dichloromethane was administered to groups of eight male CD1
mice by gavage in corn oil at dose levels of 0, 133, 333, and
665 mg/kg bw for 14 days. No treatment-related effects were seen on
body weight, blood urea nitrogen, serum creatinine, nor serum
glutamate-pyruvate transaminase levels. Dose-related effects on the
kidney were detected in the uptake of p-aminohippurate into renal
cortical slices, but no histopathological changes were observed in
that organ. The only histologic change that appeared to be related
to dichloromethane administration was minimal to slight hepatic
centrilobular cytoplasmic vacuolation (Condie et al., 1983).
Groups of 20 male and 20 female Fischer 344 rats were
administered dichloromethane (purity >99.0%) in the drinking water
for 90 days at levels of 0, 0.15, 0.45, and 1.50 per cent. The
calculated intakes were 0, 166, 420, and 1200 mg/kg bw/day for males
and 0, 209, 607, and 1469 mg/kg bw/day for females. Slightly
decreased body weights were noted for males at the mid-dose group
and the females of the high-dose group throughout the study but
reduction in the females did not exceed 6%. Elevations in mean
haemoglobin concentration were seen for both sexes of the mid- and
high-dose groups at 1 month, and for the males only at 3 months.
Higher mean erythrocyte counts for all compound-treated females were
also noted at 3 months. These higher counts were reflected in lower
mean corpuscular haemoglobin concentrations for all groups of
females. Mean serum glutamic-pyruvic transaminase values were
elevated for all treated males at 1 month and for all the high-dose
females at 3 months. The latter group also exhibited elevated
glutamic-oxalacetic transaminase values. Decreases in fasting
glucose, cholesterol, and triglyceride values were noted at 1 and 3
months for all treated groups. Total serum protein was also reduced
at 3 months in the high-dose group, while lactic dehydrogenase
values were elevated in the mid- and high-dose females at 3 months.
Histopathological evaluation of tissues from animals killed for an
interim necropsy at 1 month revealed no compound-related effects.
Tissues from animals examined at the 3-month terminal necropsy
showed hepatocellular changes. The high-dose animals and some
mid-dose animals exhibited central lobular necrosis and
granulomatous loci as well as ceroid or lipofuscin accumulation and
cytoplasmic eosinophilic bodies. An increased incidence of
hepatocyte vacuolation occurred in all compound-treated groups in a
dose-dependent pattern. The distribution of lipid was altered,
tending to be more generalized or concentrated in the central
lobular regions. No other compound-related changes were observed.
Also, no adverse effects were seen on mortality, physical
observations, food consumption, feed efficiency, nor at gross
neetopsy (Kirschman et al., 1986).
Groups of 20 male and 20 female Sprague-Dawley rats were
exposed to dichloromethane (purity 99.97%) by inhalation of
10 000 ppm, 6 h/day for 90 days. Apart from a slight redness of the
conjunctivae, no effects were reported on behaviour, faeces,
consumption of food and drinking water, body weight gain,
haematology, clinical biochemistry, composition of urine,
examination of sight, hearing and dentition nor macroscopical
examination during autopsy. Histological examinations revealed no
changes attributable to the treatment (Leuschner et al., 1984).
The effects of a 12-h exposure schedule and those of an 8-h
schedule on the carboxyhaemoglobin formation resulting from
dichloromethane inhalation were examined in groups of 5 male
Sprague-Dawley rats and 5 male Swiss-Webster mice exposed to 200,
500, or 1000 ppm dichloromethane for 8 h/day for 5 days or 12 h/day
for 4 days. The effect of the 12-h exposure schedule on
carboxyhaemoglobin levels was not significant. The metabolic pathway
for the formation of carboxyhaemoglobin appeared to be saturated
even at the lowest concentration of dichloromethane. To examine the
possible increase in the retention of inhaled dichloromethane in the
longer exposure schedule, single exposures for 8 and 12 h were
compared. The peak blood dichloromethane level was dependent upon
the exposure concentration, but the half-life was independent of the
duration of exposure and the concentration of dichloromethane. The
half-life of carboxyhaemoglobin in blood was prolonged by increasing
the dichloromethane concentration, but was not affected by the
exposure period (Kim & Carlson, 1986).
2.2.2.3 Dogs
Groups of 3 male and 3 female beagle dogs were exposed to
dichloromethane (purity 99.97%) by inhalation of 10 000 ppm, 6 h/day
for 90 days. Dichloromethane possibly induced slight sedation
throughout the exposure period. Furthermore all dogs exhibited
slight erythema. No effects were reported on behaviour, faeces,
consumption of food and drinking water, body weight gain,
haematology, clinical biochemistry, composition of urine,
electrocardiography, examination of circulatory functions, sight,
hearing and dentition nor macroscopical examination during autopsy.
Histological examinations revealed no changes attributable to the
treatment (Leuschner et al., 1984).
2.2.3 Long-term/carcinogenicity studies
2.2.3.1 Mice
Dichloromethane (food-grade, containing < 300 mg/kg
cyclohexane, < 20 mg/kg trans-1,2-dichloroethylene, < 10 mg/kg
chloroform, < 2 mg/kg vinyl chloride, and < 1 mg/kg each methyl
chloride, ethyl chloride, vinylidene chloride, carbon tetrachloride
and trichloroethylene) was administered at levels of 0, 0, 60, 125,
185 and 250 mg/kg bw/day to a total of 1000 B6C3F1 mice in
deionized drinking-water for 104 weeks. Control group 1 consisted of
60 male and 50 females, control group 2 of 65 males and 50 females,
the low-dose group of 200 males and 100 females, mid-dose group 1 of
100 males and 50 females, mid-dose group 2 of 100 males and 50
females, and the high-dose group of 125 males and 50 females. No
significant treatment-related changes in survival were found in
males; in females a statistically significant trend towards longer
survival in treated groups was reported. No treatment-related
effects on body weight nor water consumption were observed during
the study. The high-dose male and female mice showed a transitory
(week 52) increase in mean leucocyte counts. No treatment-related
histopathological effects were noted in any of the tissues examined,
except for the liver in which treatment-related changes consisting
of a marginal increase in the amount of Oil Red O-positive material
were noted in both male and female livers at the highest dose. There
was a slight elevation of proliferative hepatocellular lesions in
the treated males but no dose-related trend was apparent and the
effect was absent in the females. In male mice, the incidences of
hepatocellular adenoma were: 6/60, 4/65, 20/200, 14/100, 14/99 and
15/125; and the incidences of hepatocellular carcinomas were: 5/60,
9/65, 33/200, 18/100, 17/99 and 23/125 in the six groups,
respectively. A slight but statistically significant (p = 0.035)
dose-related increase in the incidence of hepatocellular adenomas
and/or carcinomas (combined) was observed in male mice: 11/60,
13/65, 51/200, 30/100, 31/99, 35/125. However, the authors note that
the tumour incidences in the dosed groups were within the range of
the incidence in historical controls (carcinomas, mean: 16.1% and
range: 5-34%; combined, mean: 17.8% and range: 5-40%; other authors
have reported mean: 32.1% and range: 7-58% on the combined
incidences in B6C3F1 mice) (Serota et al., 1986b).
Groups of 50 male and 50 female B6C3F1 mice, eight to
nine weeks of age, were exposed to 0, 2000 or 4000 ppm (0, 6940 or
13 880 mg/m3) dichloromethane (> 99% pure) by inhalation for six
hours per day during five days per week for 102 weeks and were
killed after 104 weeks of study. The survival rate in males was
39/50 in controls, 24/50 in the low-dose group, and 11/50 in the
high-dose group; and the survival rate in females was 25/50, 25/49
and 8/49, respectively. Non-neoplastic lesions considered to be
related to the treatment were testicular atrophy in male mice (0/50,
4/50, 31/50), and ovarian atrophy in female mice (6/50, 28/47,
32/43). Atrophy of the uterus was observed at an increased incidence
in the high-dose females. Significant dose-related increases in lung
and liver turnours were observed in treated mice. The incidence of
alveolar/bronchiolar adenomas in males was 3/50, 19/50 and 24/50;
and that in females 2/50, 23/48 and 28/48. The incidence of
alveolar/bronchiolar carcinomas in males was 2/50, 10/50 and 28/50;
and in females 1/50, 13/48 and 29/48. The incidences of
hepatocellular adenomas in males was: 10/50, 14/49 and 14/49 and in
females: 2/50, 6/48 and 22/48. The incidence of hepatocellular
carcinomas in males was: 13/50, 15/49 and 26/49 and that in females
was: 1/50, 11/48 and 32/48. The incidence of combined hepatocellular
adenomas/carcinomas in females was: 3/50, 16/48, and 40/48 (Mennear
et al., 1988; IARC, 1986).
Dichloromethane (99.973% pure; containing 250 ppm of
trans-1,2-dichloroethylene, 5 ppm of 1,2-dichloroethane, 10 ppm of
carbon tetrachloride, 5 ppm of trichloroethylene, and 0.2% ethanol
as a stabilizer) was administered to groups of 50 male and 50 female
Swiss mice by ingestion (stomach tube), in olive oil, at doses of
100 and 500 mg/kg bw, once daily, 4-5 days weekly, for 64 weeks. A
group of 60 male and 60 female mice served as vehicle controls. An
excess in mortality was observed in male and female mice exposed to
dichloromethane at both doses. The increase in mortality in the
treated groups started to appear after 36 weeks of treatment and
became more evident within the following weeks; for this reason the
treatment was withdrawn after 64 weeks. In the exposed male and
female mice a decrease in body weight was observed. This effect
appeared after 36-40 weeks from the start of the experiment and
became more evident throughout the course of the experiment. No
treatment-related increase was observed in the percentage of animals
bearing benign and malignant turnours nor of animals bearing
malignant turnouts, nor the number of total malignant turnours per
100 animals. Among the most frequently observed tumours,
dichloromethane did not increase the incidence of mammary
carcinomas, leukaemias nor hepatomas. A dose-related increase in
pulmonary tumours was observed in male mice (8.3, 12.0 and 18.0% in
the control, low-dose and high-dose males, respectively). Taking
into account the mortality, the higher incidence of pulmonary
turnours was significant for the males treated at the high-dose
level that died in the period ranging from 52 to 78 weeks (Maltoni
et al., 1988).
2.2.3.2 Rats
The two-year drinking-water study of dichloromethane in rats
evaluated by JECFA in 1982 (Annex 1, reference 63) has been
published (Serota et al., 1986a).
Dichloromethane (99.973% pure; containing 250 ppm of
trans-1,2-dichloroethylene, 5 ppm of 1,2-dichloroethane, 10 ppm of
carbon tetrachloride, 5 ppm of trichloroethylene, and 0.2% ethanol
as a stabilizer) was administered to groups of 50 male and 50 female
Sprague-Dawley rats by ingestion (stomach tube), in olive oil, at
doses of 0, 100, and 500 mg/kg bw, once daily, 4-5 days weekly, for
64 weeks. An additional control group of 20 male and 26 female rats
did not receive any treatment. An excess mortality was observed in
male and female rats administered dichloromethane at 500 mg/kg
bw/day. The increase in mortality in the affected group started to
appear after 36 weeks of treatment and became more evident within
the following weeks; this was the reason why the treatment was
withdrawn after 64 weeks. No effect of dichloromethane on body
weight was observed during the experiment. Dichloromethane did not
affect the percentage of animals bearing benign and malignant
tumours, nor of animals bearing malignant turnours, nor the number
of total malignant turnours per 100 animals. Among the most
frequently observed turnouts dichloromethane did not affect the
incidence of pheochromocytomas and pheochromoblastomas. A higher
incidence of malignant mammary tumours, which was not statistically
significant, and was mainly due to adenocarcinomas, was observed in
the females exposed to the dose of 500 mg dichloromethane/kg bw/day
(18% versus 6% in the low-dose females, and 8% in the vehicle
controls) (Maltoni et al., 1988).
The two-year inhalation toxicity and oncogenicity study of
dichloromethane in rats and hamsters evaluated by JECFA in 1982
(Annex 1, reference 63) has been published (Burek et al., 1984).
Groups of 90 male and 108 female Sprague-Dawley rats were
exposed to 0, 50, 200, or 500 ppm dichloromethane (> 99.5% pure)
for 6 h/day, 5 days/week for 20 (males) and 24 (females) months. In
addition 30 female rats were exposed to 500 ppm dichloromethane for
the first 12 months and to room air for the last 12 months of the
study. Thirty additional female rats were exposed to room air for
the first 12 months and 500 ppm of dichloromethane for the last 12
months of the study. The mortality rates for the various groups of
treated male and female rats were comparable to control values.
Absolute and relative organ weights, clinical chemistry values, and
plasma hormone levels were not altered in rats exposed to
dichloromethane. Blood carboxyhaemoglobin levels were elevated in a
dose-dependent (less than linear) manner in rats exposed to 50-500
ppm methylene chloride. The percentage carboxyhaemoglobin was
similar within exposure groups after 6, 12, or 20-24 months,
indicating a lack of accumulation with repeated exposure. There were
no detectable alterations in the rate of DNA synthesis measured as
[3H]thymidine incorporation into liver DNA of female rats exposed
to 50-500 ppm dichloromethane. Histopathologic lesions related to
dichloromethane exposure were confined to the liver and mammary
tissue. An increased incidence of hepatocellular vacuolization was
observed in male and female rats exposed to 500 ppm dichloromethane.
Female rats exposed to 500 ppm dichloromethane also had an increased
incidence of multinucleated hepatocytes and number of spontaneous
benign mammary tumours per turnour-bearing rat (2.7) (adenomas,
fibromas, and fibroadenomas with no progression toward malignancy);
the incidence of benign mammary tumours in female rats exposed to 50
or 200 ppm was comparable to historical control values (2.1). No
increase in the number of any malignant tumour type was observed in
the treated rats. The response observed in female rats exposed to
500 ppm for the first 12 months only was the same as that observed
in female rats exposed to 500 ppm for 2 years. Conversely, the
response observed in female rats exposed to 500 ppm during the last
12 months of the study was similar to that observed in control
animals (Nitschke et al., 1988).
Dichloromethane (99.973% pure; containing 250 ppm of
trans-1,2-dichloroethylene, 5 ppm of 1,2-dichloroethane, 10 ppm of
carbon tetrachloride, 5 ppm of trichloroethylene, and 0.2% ethanol
as a stabilizer) was administered to groups of Sprague-Dawley rats
by inhalation, at concentrations of 0 (60 female breeders) and
100 ppm (54 female breeders), 7 h daily, for 5 days weekly. The
inhalatory treatment was started on 13-week-old breeders, and male
and female offspring (12-day embryos). The breeders and part of the
offspring were exposed for 104 weeks. The offspring were exposed to
0 (58 males and 49 females) or 100 ppm (60 males or 69 females) of
dichloromethane. The other part of the offspring, 60 males and 70
females, was exposed for 15 weeks only. Neither excess in mortality
nor any effects on body weight was found in the exposed groups. In
exposed breeders or offspring rats, dichloromethane did not affect
the percentage of animals bearing benign and malignant tumours, nor
of animals bearing malignant turnours, nor the incidence of total
mammary turnours, leukaemias, pheochromocytomas and
pheochromoblastomas. A higher, not statistically significant,
incidence of malignant tumours per 100 animals was observed among
the breeders (16.7% in controls; 24.1% in exposed females) and
offspring exposed to dichloromethane for 104 weeks (18.3% in control
males; 10.0% in 15-week exposed males; 23.3% in 104-week exposed
males; 17.4% in control females; 24.3% in 15-week exposed females;
29.0% in 104-week exposed females). A slight, not statistically
significant increase in the percentage of malignant mammary tumours
was observed in female offspring exposed for 15 weeks (5.4% in
control females; 10.0% in 15-week exposed females; 4.3% in 104-week
exposed females (Maltoni et al., 1988).
Groups of 50 male and 50 female Fischer 344/N rats, seven to
eight weeks of age, were exposed by inhalation to 0, 1000, 2000 or
4000 ppm (0, 3470, 6940 or 13 880 mg/m3) dichloromethane (> 99 %
pure) for six hours per day during five days per week for 102 weeks
and were killed after 104 weeks on the study. The survival of the
treated males was comparable with that of the controls. Survival in
high-dose females was reduced at termination of the study as
compared to controls: controls, 30/50; low-dose, 22/50; mid-dose,
22/50; high-dose, 15/50. Non-neoplastic changes considered to be
treatment-related included squamous metaplasia of the nasal cavity
in high-dose females, degeneration of kidney tubules, and fibrosis
of the spleen in animals of both sexes at all treatment levels.
Increased incidences of mammary-gland tumours (all fibroadenomas or
adenocarcinomas, except for one adenoma in the high-dose group) were
observed in treated females (5/50, 11/50, 13/50 and 23/50). There
was a positive trend in the incidence of benign tumours in the
mammary gland area (adenomas, fibroadenomas, and subcutaneous tissue
fibroma or sarcoma of mammary origin) in males (1/50, 1/50, 4/50,
9/50). No nasal tumours were observed, and there was no increase in
the incidence of respiratory-tract tumours, and there was no
difference considered biologically significant in the distribution
of other types of turnours between the control and treated groups
(Mennear et al., 1988; IARC, 1986).
2.2.4 Reproduction studies
Reproductive parameters in Fischer 344 rats were evaluated
following inhalation of dichloromethane for two successive
generations. Thirty male and female rats were exposed to 0, 100,
500, or 1500 ppm dichloromethane (> 99.86% pure) for 6 h/day, 5
days/week for 14 weeks and then mated to produce F1 litters. After
weaning, 30 randomly selected F1 pups/sex/group were exposed to
dichloromethane for 17 weeks and subsequently mated to produce F2
litters. Reproductive parameters examined included fertility, litter
size and neonatal growth, and survival. All adults and selected
weanlings were examined for grossly visible lesions. Tissues from
selected weanlings were examined histopathologically. No adverse
effects on reproductive parameters, neonatal survival, nor neonatal
growth were noted in animals exposed to dichloromethane in either
the F0 or F1 generations. Similarly, there were no
treatment-related gross pathologic observations in F0 or F1
adults nor F1 and F2 weanlings. Histopathologic examination of
tissues from F1 and F2 weanlings did not reveal any lesions
attributed to dichloromethane. It was concluded that exposure of
rats to concentrations as high as 1500 ppm dichloromethane did not
affect any reproductive parameters (Nitschke et al., 1988).
2.2.5 Special studies on cardiovascular effects
A review on cardiovascular disease resulting from solvent
exposure has been published. Due to its metabolism to carbon
monoxide, a suspected cause of cardiovascular disease,
dichloromethane is a biologically plausible cardiovascular toxicant.
However, none of the available cohort studies has associated
dichloromethane with increased cardiovascular disease (Wilcosky &
Simonsen, 1991).
2.2.6 Special studies on genotoxicity
The results of genotoxicity assays on dichloromethane are
summarized in Table 1.
2.2.7 Special studies on immune responses
The effects of single (50 and 100 ppm) and multiple (50 ppm, 5
days) 3-hour inhalation exposures to dichloromethane were evaluated
in female CD1 mice by monitoring changes in their susceptibility to
experimentally induced Streptococcus aerosol infection and
pulmonary bactericidal activity to inhaled Klebsiella pneurnoniae.
Significant increases in susceptibility to respiratory
Streptococcus infection were observed after a single 3 h exposure
to 100 ppm of methylene chloride. This exposure condition also
resulted in significantly decreased pulmonary bactericidal activity.
The treatment with 50 ppm did not induce any changes (Aranyi
et al., 1986).
2.2.8 Special studies on neurotoxicity and behavioural effects
The neurotoxicity of dichloromethane has been reviewed. The
neurotoxicity of dichloromethane depends both on a direct,
non-specific narcotic action on the central nervous system (CNS), as
well as an equally non-specific carbon monoxide-induced hypoxic
effect (Winneke, 1981).
The intraperitoneal administration of dichloromethane
(0.5 ml/kg bw) produced increases in male Swiss albino mouse
striatal concentrations of p-tyramine and m-tyramine. The effect was
much less pronounced after dichloromethane than after benzene
(Juorio & Yu, 1985).
Dichloromethane had a depressive effect on the vestibulo-ocular
reflex in female Sprague-Dawley rats. Nystagmus, induced by
accelerated rotation, was recorded by electronystagmography in 15
female rats continuously intravenously infused during 60 min with
concentrations of dichloromethane ranging from 0.1 to 10% in an
emulsion of lipids. The effect was related to the blood levels of
the solvent. The threshold limit for effect was observed at a blood
level of 0.7 mM/L (60 ppm) at an infusion rate of 60 µM/kg bw/min.
In contrast, such solvents as benzene compounds like xylene,
toluene, styrene and cumene and halogenated unsaturated hydrocarbons
like trichloroethylene caused an excitation of the vestibulo-ocular
reflex. It was suggested that the depression was caused by
interaction with central pathways in the reticular formation and the
cerebellum (Tham et al., 1984).
Groups of male Swiss Webster mice of different ages at start of
the experiment were exposed via inhalation to a high concentration
of dichloromethane (168 mg/l) (pesticide grade). The mice were
either 3 (41 mice), 5 (45 mice), or 8 (75 mice) weeks old and were
exposed until loss of their righting reflex, usually 20 seconds.
After either 1, 2 or 4 days the mice were tested for learning
ability using a passive-avoidance conditioning task. Exposed animals
were found to have a significantly decreased ability to learn when
compared with controls. The 3 week-old mice were more affected than
the 5 week-old and the 8 week-old mice. The exposed animals were
indistinguishable from controls in terms of motor activity, weight
gain, and absence of analgesia (Alexeeff & Kilgore, 1983).
Table 1. Results of genotoxicity assays on dichloromethane
Test system Test object Concentration of Results Reference
dichloromethane
Ames test1 S. typhimurium 10-100 mg/l in Nestmann et al. (1981)
TA 98 chamber Positive
TA 100 Positive
TA 1535 Positive
TA 1537 Negative
TA 1538 Negative
Ames test S. typhimurium 0.5-1.4% (v/v) in Jongen et al. (1982)
TA 100 chamber Positive2
Ames test S. typhimurium 2.8-8.4% (v/v) in Positive2 Green (1983)
TA 100 chamber
Ames test S. typhimurium 500-10 000 µg/ Positive Hughes et al. (1987)
TA 100 plate, in chamber (weak)3
Ames test S. typhimurium 5-100 µl/3m in Mersch Sundermann
TA 97 chamber Negative (1989)
TA 98 positive4
TA 100 positive4
Ames test1 S. typhimurium Osterman Golkar et al.
TA 1535 10 µl/plate Negative (1983)
TA 1950 10 µl/plate Positive
TA 100 20-80 mM (weak)
Positive
(weak)
Streptomycin E. coli Sd-4 10 µl/plate Negative Osterman Golkar et al.
locus test1 (1983)
Table 1. cont'd
Test system Test object Concentration of Results Reference
dichloromethane
Reverse E. coli 10 µl/plate Positive Osterman Golkar et al.
mutation1 WU361089 (weak) (1983)
Prophage E. coli K 39 10 µl/plate Positive Osterman Golkar et al.
induction1 (lambda) (1983)
Somatic Aspergillus nidulans, Positive8 Crebelli et al. (1988)
segregation diploid strain P1
Enhancement of Syrian Golden 0.31-5 ml/ Positive Hatch et al. (1983)
viral5 cell hamster embryo chamber6
transformation cells in vitro
Sister chromatid Chinese hamster 2-15 µl/ml Negative1,4 Thilagar & Kumaroo
exchange (SCE) ovary (CHO) cells, (1983)
in vitro
Sister chromatid Chinese hamster 1-5% (v/v) in Positive Jongen et al. (1981)
exchange (SCE) V79 cells in vitro chamber (weak)
Chromosomal Chinese hamstcr 2-15 µl/ml Positive1,4 Thilagar & Kumaroo
aberrations ovary (CHO) cells, (1983)
in vitro
Forward mutation Chinese hamster 1-5% (v/v) in Negative Jongen et al., 1981)
(HGPRT) V79 and Chinese chamber
hamster ovary cells, Negative
in vitro
Table 1. cont'd
Test system Test object Concentration of Results Reference
dichloromethane
Unscheduled Chinese hamster 1-5% (v/v) in Negative Jongen et al. (1981)
DNA synthesis V79 cells and chamber
(UDS) human fibroblasts, Negative
in vitro
Inhibition of Chinese Hamster 1-5% (v/v) in Inhibition, Jongen et al. (1981)
DNA synthesis V79 cells and chamber aspecific not
human fibroblasts, indicative of
in vitro DNA damage
Sex-linked D. melanogaster 8-125 mg/m3 for Negative Kramers et al. (1991)
recessive male 96 h
lethal assay
Unscheduled Alpk:AP, male rats, 100-1000 mg/kg Negative Trueman & Ashby (1987)
DNA synthesis in vivo, in vitro bw p.o.,
(UDS)
Fischer 344, male 2000 or 4000 ppm Negative
rats,
B6C3F1, male mice, inhalation, Negative
in vivo, in vitro 2-6 h
Unscheduled Fischer 344, male 10.400 mg/kg Negative Mirsalis et al. (1989)
DNA synthesis rats, in vivo, bw p.o.
(UDS) in vitro
Unscheduled B6C3F1 male mice, 1000 mg/kg bw p.o. Negative Lefevre & Ashby (1989)
DNA synthesis in vivo, in vitro 4000 ppm,
(UDS) inhalation Positive
(very weak)
Table 1. cont'd
Test system Test object Concentration of Results Reference
dichloromethane
Sister chromatid C57B1/6J male 100-2000 mg/kg bw Negative Westbrook Collins et al.,
exchange (SCE) mice, in vivo, i.p. (1990)
and bone marrow cells
chromosomal
aberrations
Sister chromatid B6C3F1 female 2500 and 5000 mg/kg Negative Allen et al. (1990)
exchange (SCE) mice, in vivo, bw s.c.
and Bone marrow cells 4000 and 8000 ppm, Positive
chromosomal 10 days inh.
aberrations 2000 ppm, 3 mo. inh. Negative
Sister chromatid B6C3F1 female 4000 and 8000 ppm, Positive Allen et al. (1990)
exchange (SCE) mice, in vivo, 10 days inh.
and lung cells 2000 ppm, 3 mo. inh. Positive
chromosomal (weak)7
aberrations
Sister chromatid B6C3P1 female 4000 and 8000 ppm, Positive Allen et al. (1990)
exchange (SCE) mice, in vivo, 10 days inh.
lymphocytes 2000 ppm, 3 mo. inh. Negative
Micronucleous B6C3F1 female 4000 and 8000 ppm, Positive Allen et al. (1990)
test mice, in vivo, 10 days inh.
erythrocytes 2000 ppm, 3 mo. inh. Positive
(weak)
Micronucleous C57BL/6J/Alpk 1250-4000 mg/kg bw Negative Sheldon et al. (1987)
test male and female p.o.
mice,
in vivo,
bone marrow
Table 1 (continued)
1 Without rat liver S-9 fraction
2 Enhanced by the addition of either rat-liver microsomes or the cytosolic
3 Negative in plate assay and in preincubation assay
4 With the addition of rat liver S-9 fraction
5 SAT adenovirus
6 ml dichloromethane added per 4.6 litre chamber
7 SCE only
8 Increased frequency of haploid sectors and diploid non-disjunctional sectors
Groups of 24 male NMRI mice were exposed via inhalation to 400,
500, 600, or 750 ppm and groups of 13 mice were exposed to 850,
1100, 2200, or 2500 ppm of dichloromethane for one hour. Motor
activity of the animals during the exposures was measured with a
Doppler radar. No effects were observed at 600 ppm and lower
concentrations. Concentrations of 750 ppm and higher increased the
motor activity during the exposure. When the generation of vapour
was terminated and the concentration started to decline, the motor
activity decreased reaching a minimum at two hours and had returned
to normal six hours after the termination of the exposure
(Kjellstrand et al., 1985).
Groups of 10 male NMRI mice were exposed by inhalation to
different concentrations of dichloromethane for up to four hours. An
inhalation concentration of 4500 ppm was needed to significantly
stimulate motor activity in the mice. During prolonged exposure
acute tolerance developed to dichloromethane. It was speculated that
the formation of metabolites, e.g., carbon monoxide, with sedative
effects counteracted the stimulating effect of the pure solvent
(Kjellstrand et al., 1990).
Male Sprague-Dawley rats in groups of 6 were exposed by
inhalation to 0, 70, 300, or 1000 ppm of dichloromethane, 6 h/day
for 3 consecutive days. Dichloromethane produced a selective
reduction in dopamine levels without a change of dopamine turnover
in certain types of forebrain dopamine nerve terminal systems. In
the low concentration group a selective reduction in dopamine
turnover was observed in the medial palisade zone of the median
eminence. Dichloromethane also produced a discrete dose-dependent
increase in noradrenaline turnover within the anterior
periventricular hypothalamic area and, with the highest
concentration, an increase in noradrenaline turnover in the
anteromedial frontal cortex. Dichloromethane reduced noradrenaline
levels dose-dependently in the posterior periventricular
hypothalamic area and also in the dorsomedial hypothalamic nucleus
(1000 ppm). Following tyrosine hydroxylase inhibition
dichloromethane produced an inversely dose-related increase in serum
LH levels and, at the highest concentration, an increase of ACTH
secretion was observed. It is suggested that dichloromethane can
produce discrete changes in amine storage and turnover in
catecholamine nerve terminal systems of the tel- and diencephalon,
some of which may contribute to the dichloromethane-induced
disturbances in the secretion of anterior pituitary hormones (Fuxe
et al., 1984).
Groups of male and female Fischer 344 rats were exposed to
dichloromethane or carbon monoxide for 6 h/day, 5 days/week, for 13
weeks. Since oxidative metabolism of dichloromethane to carbon
monoxide and carbon dioxide is a saturable process, dichloromethane
exposure concentrations were selected clearly below saturation
(50 ppm), just below saturation (200 ppm), and well above saturation
(2000 ppm). At saturation of metabolism, metabolic carbon monoxide
causes about 10% carboxyhaemoglobinaemia. Therefore, as a control
for carbon monoxide effects, a separate group of rats was exposed to
135 ppm carbon monoxide to induce approximately 10%
carboxyhaemoglobinemia. Post-exposure functional tests included an
observational battery, hindlimb grip strength, and a battery of
evoked potentials (flash, auditory brainstem, somatosensory, caudal
nerve). After functional tests were completed, rats from all groups
were perfused with fixative and a comprehensive set of nervous
tissues from the high dichloromethane exposure group and from
controls were examined by light microscopy. Although some
miscellaneous functional and morphologic variations were recorded,
none were related to treatment. The authors conclude that subchronic
exposures as high as 2000 ppm dichloromethane or 135 ppm carbon
monoxide had no deleterious effects on any of the parameters
examined (Mattsson et al., 1990).
Groups of 10 male and 10 female Mongolian gerbils were exposed
to dichloromethane at concentrations of 210, 350, and 700 ppm for 3
months. Because of a high mortality rate, however, the 700 ppm
groups were terminated after 7 weeks. In the 350 ppm experiment half
the exposed animals died and the exposure period was terminated
after 10 weeks. After the exposure period, the surviving gerbils in
the 350 ppm exposure group and those from the 210 ppm group were
allowed a post-exposure solvent-free period of 4 months. Two
astroglial proteins S-100 and GFA, as well as DNA, were
quantitatively determined in different regions of the gerbil brain.
After exposure to 350 ppm, increased concentrations of the two
astroglial proteins were found in the frontal and sensory motor
cerebral cortex, compatible with astrogliosis in these regions.
Exposure to 350 ppm and 210 ppm decreased the concentrations of DNA
in the hippocampus. Moreover, after exposure at 350 ppm, DNA
concentrations were also decreased in the cerebellar hemispheres. It
was concluded that these results indicate a decreased cell density
in these brain regions, probably due to cell loss. The neurotoxic
effects were not found to correlate with the endogenous formation of
carbon monoxide (Rosengren et al., 1986).
Groups of 8 male Mongolian gerbils were exposed to
dichloromethane for either three weeks or three months by continuous
inhalation at 210 ppm. The body and whole brain weights as well as
the weights of the dissected brain regions did not differ between
control and exposed groups. In the group exposed for three weeks the
carboxyhaemoglobin percent saturation was significantly increased
(11.5%) when compared to that of controls (0.43%). In the animals
exposed for three months total free tissue amino acids, glutathione,
and phosphoethanolamine were determined in the vermis posterior of
the cerebellum and the frontal cerebral cortex. These two brain
areas were chosen because humans occupationally exposed to
dichloromethane have shown abnormalities in the electroencephalogram
of the frontal part of the cerebral cortex. This study showed that
long-term exposure of gerbils to dichloromethane (210 ppm) for three
months led to decreased levels of glutamate, gamma-aminobutyric
acid, and phosphoethanolamine in the frontal cerebral cortex, while
glutamine and gamma-aminobutyric acid were elevated in the posterior
cerebellar vermis (Briving et al., 1986).
Groups of 4 male and 4 female adult Mongolian gerbils were
continuously exposed by inhalation to dichloromethane (0.3-0.5%
stabilizers (butylene oxide and antioxidants)) at 210 ppm, for three
months, followed by a four-month post-exposure solvent-free period.
No animals died during the experiment, and no changes were observed
in body weight, weight of brain, nor weight of dissected brain
regions. The total protein concentrations per wet weight in the
different brain regions were not changed. The DNA concentrations per
wet weight were slightly decreased in the hippocampus after exposure
to dichloromethane (Karlsson et al., 1987).
2.2.9 Special studies on macromolecular binding
The in vivo interaction of dichloromethane and its
metabolites with male F-344 rat (n=2) and male B6C3F1 mouse
(n=10) lung and liver DNA was measured after inhalation exposure to
4000 ppm [14C]-dichloromethane for 3 h. DNA was isolated from the
tissues 6, 12, and 24 h after the start of exposure and analyzed for
total radioactivity and the distribution of radioactivity within
enzymatically hydrolysed DNA samples. Covalent binding to hepatic
protein was also measured. Low levels of radioactivity were found in
DNA from lungs and livers of both rats and mice exposed to
[14C]-dichloromethane. Two- to fourfold higher levels were found
in mouse DNA and protein than in rat. Chromatographic analysis of
the DNA nucleosides showed the radioactivity to be associated with
the normal constituents of DNA. No peaks of radioactivity were found
that did not coincide with peaks of radioactivity present in
hydrolyzed DNA from formate-treated rats and mice. It was concluded
that there was no evidence for alkylation of DNA by dichloromethane
in either rats or mice (Green et al., 1988).
Groups of 5 female F-344 rats or 25 female B6C3F1 mice
were exposed by inhalation to [14C]-dichloromethane of very high
specific radioactivity (13.4 mCi/mmol). The exposure concentrations
were between 581 and 785 ppm of dichloromethane at the start of the
experiments and were then allowed to decline to insignificant
levels. Incorporation of radioactivity into DNA of liver, kidneys,
and lung was measured in each species. The radioactivity in DNA
which was derived from [14C]-dichloromethane was exclusively
confined to the physiological deoxyribonucleosides. Radioactive
alkylation products were not found. It was concluded that
dichloromethane is not a systemic genotoxic carcinogen on the target
tissues examined (Ottenwalder & Peter, 1989).
2.3 Observations in humans
The odour threshold for dichloromethane is 214 ppm
(743 mg/m3). Fatalities have been associated with acute or
prolonged exposure to dichloromethane. Dichloromethane acts
primarily on the central nervous system, causing narcosis at high
doses. Temporary neurobehavioural effects have been reported after
exposure to doses as low as 200 ppm (700 mg/m3) by some authors,
but not by others (IARC, 1986).
Exposure to dichloromethane levels in the range of 28 to
173 ppm during an eight-hour working-day caused increased sleepiness
and physical and mental exhaustion (Cherry et al., 1983).
A case of delirium resulting from exposure to dichloromethane
has been reported (Tariot, 1983).
A case of acute renal failure, myoglobinuria,
hypocomplementaemia, and liver enzyme elevation after two days of
working with dichloromethane in a poorly ventilated room has been
reported (Miller et al., 1985).
A case of paint remover inhalation causing pulmonary oedema and
pleural effusions has been described. Dichloromethane, a major
ingredient of paint removers, was suspected as the causative agent
due to its conversion to carbon monoxide (Buie et al., 1986).
Two cases of transient hepatitis induced after inhaling organic
solvents were primarily ascribed to dichloromethane. Elevated serum
levels of aspartate aminotransferase, alanine aminotransferase, and
alkaline phosphatase were reported (Cordes et al., 1988).
Carboxyhaemoglobin concentrations at peak levels of 11.5 and
13% were observed in two cases of dichloromethane poisoning. Both
patients were found unconscious in an occupational setting (Rioux &
Myers, 1989).
In a patient complaining of upper respiratory irritation,
fatigue, and lightheadedness occurring on a daily basis after using
a dichloromethane-containing paint stripper, determinations of blood
carboxyhaemoglobin on three occasions showed an apparently linear
elevation of carboxyhaemoglobin as a function of hours worked on the
day of sampling (Shusterman et al., 1990).
The cause of death in two patients who arrived in cardiac
arrest following exposure in an enclosed space to dichloromethane
was not carbon monoxide poisoning, but solvent-induced narcosis. It
was noted that carboxyhaemoglobin levels continue to rise following
cessation of exposure, despite administration of high flow oxygen
(Leikin et al., 1990).
Based on a review of 26 cases of dichloromethane exposures
reported in the literature from 1936 to 1986, various symptoms were
identified. These include, in the central nervous system -
cognitive, sensory, motor, and behavioural symptoms, headache,
reduced activity, drowsiness and unconsciousness; in the respiratory
system - pulmonary oedema/dyspnoea; gastrointestinal symptoms;
hepatic/renal; dermatologic; musculoskeletal; haematologic, cardiac;
others - chills, shock, and death (Rioux & Myers, 1988).
For the period 1961-80, 33 cases of industrial gassings caused
by dichloromethane were reported in the United Kingdom. One of the
persons died. Symptoms were most commonly attributable to the
nervous and gastrointestinal systems, and in four cases to the
respiratory system. The clinical symptoms most often reported were
headache, dizziness, unconsciousness, nausea, and vomiting (Bakinson
& Jones, 1985).
Mortality was studied among 1271 employees of a cellulose fibre
production plant in the USA in which dichloromethane had been used
as a general-purpose solvent. Particular attention was given to
evaluating possible direct and carboxyhaemoglobin-mediated effects
on the haematopoietic and circulatory systems. Each subject had been
employed for at least three months between 1 January 1954 and 1
January 1977 in jobs that entailed exposure to high concentrations
of methylene chloride and was followed up to 30 June 1977. The study
design included a retrospective cohort mortality study and several
health evaluation studies. Industrial hygiene monitoring indicated
that typical dichloromethane exposures ranged from an 8 h
time-weighted average of 140 ppm in areas of low dichloromethane use
to a corresponding average of 475 ppm in areas of high
dichloromethane use and that methanol was present in about a one to
ten ratio to dichloromethane. Acetone exposures ranged from 100 to
over 1000 ppm (time-weighted average). The observed numbers were
compared with the expected numbers from an internal referent cohort
of 948 acetone-exposed employees and with mortality data for US
white males, non-white males and white females. No significant
increase in overall mortality or deaths due to ischaemic heart
disease was found when compared to the mortality of the general
population. An exposure-related increase in serum bilirubin was
observed, but no signs of liver injury nor haemolysis were reported.
A cross-sectional study of 24 employees showed no excess of
electro-cardiographic abnormalities among those exposed to 60-475
ppm dichloromethane and monitored for 24 h. Among the exposed white
men and women seven deaths due to malignant neoplasms were observed
compared to 10.1 expected in the general population. No specific
cancer sites were over-represented. Seven malignant neoplasms were
observed in the referent cohort compared to 12.3 expected (Ott
et al., 1983; IARC, 1986).
At follow-up of the cohort 122 deaths were identified through
September 1986, and mortality rates for the cohort were compared
with mortality rates for York County, South Carolina, USA. Deficit
mortality was observed for cancers of the respiratory system,
breast, and pancreas and from ischaemic heart disease. Excess
mortality was observed for cancers of the buccal cavity and pharynx
(2 versus 0.87) and the liver and biliary tract (4 versus 0.7), and
for melanoma as well (2 versus 0.88). The largest relative excess
was for liver and biliary tract cancers. There were only four deaths
in this category; however, three of the four deaths were cancer of
the biliary tract (3 observed, 0.15 expected, standardized mortality
ratio 20) (Lanes et al., 1990).
The mortality of a 1964 to 1970 cohort of 1013 full-time,
hourly men who had been employed at Eastman Kodak Corporation, USA,
was evaluated through 1984. On average, employees were exposed to
dichloromethane at a rate of 26 ppm (eight-hour time-weighted
average) for 22 years; median latency was 30 years. Compared with
the general population, no statistically significant excesses in
mortality were observed for such hypothesized causes of mortality as
lung cancer (14 observed versus 21.0 expected), liver cancer
(0 versus 0.8), and ischaemic heart disease (69 versus 98.1).
Dose-response relationships based on dichloromethane exposure and
latency were not demonstrated. Among non-hypothesized mortality
causes, a significant deficit was reported for total deaths
(176 versus 253.2). It was stated that sufficient power was
available to detect relative risks of 1.6 for lung malignancy and
1.3 for ischaemic heart disease. The cohort had a higher mortality
from pancreatic cancer than their controls (8 versus 3.2), but this
difference was interpreted as being unrelated to dichloromethane
exposure (Hearne et al., 1987).
The interpretation of the absence of a significant increased
incidence of pancreatic cancer in the Eastman Kodak cohort has been
questioned (Mirer et al., 1988).
At follow-up in the Eastman Kodak cohort, mortality findings
were substantially unchanged after 4 additional years of observation
through 1988. Mean exposure was 26 ppm (8 h time-weighted average)
for 23 years and median follow-up from first exposure was 33 years.
A comparison with death rates in both general population and
industrial referents showed non-significant deficits in
observed-expected ratios for such hypothesized causes of death as
lung and liver cancer and ischaemic heart disease. Overall mortality
from 1964 to 1988 (n = 238) was significantly decreased versus both
referent groups. The study had 90% power to detect relative risks of
1.7 and 1.3 for lung cancer and ischaemic heart disease,
respectively; power was inadequate for hepatic cancer. No pancreatic
cancer deaths had occurred since the 1984 follow-up (8 observed
versus 4.2 expected (P = 0.13) (Hearne et al., 1990).
3. COMMENTS
Dichloromethane is readily absorbed from the gastrointestinal
tract and distributed to the blood, liver, lung, kidneys, body fat,
and nervous tissues of animals and humans. The absorption proceeds
at a faster rate when the compound is ingested in water solution
than in oil solution. The compound is readily cleared from the
organism, mainly by exhalation of the parent compound and the
metabolites carbon dioxide and carbon monoxide. The formation of
these metabolites is dose-dependent; at higher doses (exceeding
100 mg/kg bw), a proportionally higher level is expired as the
parent compound. Systemic accumulation of dichloromethane does not
occur.
Dichloromethane is able to cross the placental barrier in
pregnant rats, but no reproductive effects have been observed even
at high doses.
Dichloromethane is metabolized to carbon monoxide and carbon
dioxide by two pathways, one dependent on oxidation by
mixed-function oxidases and the other on glutathione S-transferases.
The mixed-function oxidase pathway seems to be the preferred
metabolic route at low concentrations of dichloromethane, while at
higher concentrations this pathway becomes saturated, making a
larger percentage of dichloromethane available for metabolism by the
glutathione-dependent pathway.
The metabolic production of carbon monoxide from
dichloromethane leads to the formation of carboxyhaemoglobin, the
cause of the hypoxic state commonly seen in accidental poisoning by
dichloromethane.
Single and repeated doses of dichloromethane have produced
elevations in serum enzyme levels indicative of kidney and, in
particular, liver toxicity. No adverse effects were seen in rats in
a 3-month study when approximately 230 mg/kg bw/day were given
orally. Slight hepatocellular changes were noted in rats at oral
doses administered via the drinking-water of 420-607 mg/kg bw/day
for 90 days.
High doses of dichloromethane are neurotoxic, the neurotoxicity
depending on both a direct, non-specific narcotic action on the
central nervous system, and an equally non-specific carbon
monoxide-induced hypoxic effect. A variety of behavioural effects,
such as increased motor activity and decreased learning ability,
have been observed in experimental animals after inhalation exposure
to high concentrations of dichloromethane.
Dichloromethane is weakly mutagenic in Salmonella typhimurium
tester strain TA100, while mainly negative results have been
produced in other tester strains. Weak clastogenic effects have been
recorded in mammalian cell-culture systems in vitro, while tests
for point mutations and DNA interactions have mainly failed to show
any effects, in agreement with DNA-binding studies in rodents, in
which dichloromethane-DNA adducts have not been detected after
in vivo treatment with dichloromethane. When in vivo systems
have been used, e.g., tests for unscheduled DNA synthesis in liver,
sister chromatid exchange, and chromosomal aberrations in bone
marrow of mice, dichloromethane has not produced effects when given
orally but has done so in a number of studies after inhalation of
high doses.
In a long-term study in mice in which dichloromethane was
administered in the drinking-water, there was a slight dose-related
increase in fatty infiltrations in the liver of male and female mice
given the highest dose of 250 mg/kg bw/day. This effect was not
reported in mice receiving a dose of 185 mg/kg bw/day. A slight, but
statistically significant, dose-related increase in combined
hepatocellular carcinomas and adenomas in male mice was found to be
within the incidence range of historical controls. In another
long-term study in mice, in which dichloromethane was administered
by gavage in olive oil at daily doses up to 500 mg/kg bw, no liver
carcinogenicity was observed. However, the treatment produced excess
mortality, and the exposure had to be terminated after 64 weeks.
When the mortality was taken into account, there was a slight but
significant increase in the incidence of pulmonary tumours in the
males given the highest dose. In a study of similar design in rats
in which dichloromethane was administered by gavage in olive oil at
daily doses up to 500 mg/kg bw, the exposure also had to be
terminated after 64 weeks because of excess mortality. In this
study, no statistically significant increases in tumour incidences
were seen.
When high doses of dichloromethane have been administered by
inhalation to mice over the entire lifetime, increased incidences of
lung tumours (alveolar/brochiolar adenomas and carcinomas) and liver
tumours (hepato-cellular adenomas and carcinomas) have been
reported, and in three different lifetime studies where
dichloromethane was administered to rats by inhalation exposure at
high concentrations (500 mg/l or higher), an increased incidence of
benign mammary-gland tumours (adenomas, fibromas, fibroadenomas) was
seen in females. In one of the studies, adenocarcinomas were also
found, together with a positive trend in the incidence of benign
tumours in the mammary-gland area in males.
New long-term studies in mice and rats on the carcinogenicity
of dichloromethane administered by the oral route were either
negative or inconclusive because of premature deaths. As regards the
administration of dichloromethane by the inhalation route, the
available studies in mice and rats point to a carcinogenic effect on
the liver and lung of mice and the mammary gland of rats receiving
higher doses.
A physiologically based pharmacokinetic model that provides
quantitative data on the rates of metabolism and levels of
dichloromethane in various organs was applied to the dose levels
used in the above-mentioned long-term studies in mice and rats. It
was calculated that the concentrations of glutathione-dependent
metabolites in the liver and lungs of mice that received the
material in the drinking-water study were several orders of
magnitude lower than those in the mice receiving dichloromethane in
the inhalation study. In addition, the model predicted that
considerably lower concentrations of these metabolites would be
present in the liver and lungs of the rats exposed to
dichloromethane in the inhalation studies than in the mice similarly
exposed. This may provide an explanation for the differences in the
results obtained in carcinogenicity studies in which different
routes of administration are used. Epidemiological studies have not
shown any carcinogenic effect of dichloromethane after occupational
exposure. However, the Committee noted that the power to detect
excess risk in these studies was limited.
4. EVALUATION
On the basis of the available data, the Committee concluded
that the use of dichloromethane as an extraction solvent in food
processing should be limited to use for spice oleoresins and the
decaffeination of tea and coffee, and for food additives in which
previous specifications drawn up by the Committee included residues
of dichloromethane. The Committee was made aware that stabilizers
may be used in dichloromethane, and was of the opinion that only
those that are toxicologically acceptable, and therefore not
expected to lead to toxicologically significant residues, should be
used in food-grade dichloromethane.
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See Also:
Toxicological Abbreviations
Dichloromethane (ICSC)
Dichloromethane (FAO Nutrition Meetings Report Series 48a)
DICHLOROMETHANE (JECFA Evaluation)
Dichloromethane (IARC Summary & Evaluation, Volume 71, 1999)