First draft prepared by
    Dr J.C. Larsen
    Institute of Toxicology
    National Food Agency of Denmark
    Soborg, Denmark


         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

         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

         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).


    2.1  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.,

         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.,

         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

         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,

    2.2.2  Short term studies  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).  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).  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  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).  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

    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)

    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

    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

    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.,
                          Fischer 344, male        2000 or 4000 ppm         Negative
                          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

    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

    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

    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

    Micronucleous         C57BL/6J/Alpk            1250-4000 mg/kg bw       Negative           Sheldon et al. (1987)
    test                  male and female          p.o.
                          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).


         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

         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

         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.


         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.


    AGRAWAL, H.C. & AGRAWAL, D. (1989). Tumor promoters accentuate
    phosphorylation of PO: evidence for the presence of protein kinase C
    in purified PNS myelin.  Neurochem. Res., 14: 409-413.

    ALEXEEFF, G.V. & KILGORE, W.W. (1983). Learning impairment in mice
    following acute exposure to dichloromethane and carbon
    tetrachloride. J.  Toxicol. Environ. Health, 11: 569-581.

    EREXSON, G., KARI, F. & ZEIGER, E. (1990). Cytogenetic analyses of
    mice exposed to dichloromethane.  Environ. Mol. Mutagen., 15:

    REITZ, R.H., NOLAN, R.J. & McKENNA, M.J. (1991). Physiologically
    based pharmacokinetic modeling with dichloromethane, its metabolite
    carbon monoxide, and blood carboxyhemoglobin in rats and humans.
     Toxicol. Appl. Pharmacol., 108: 14-27.

    R.H. (1987). Physiologically based pharmacokinetics and the risk
    assessment process for methylene chloride.  Toxicol. Appl.
     Pharmacol., 87: 185-205.

    ANGELO, M.J. & PRITCHARD, A.B. (1984). Simulations of methylene
    chloride pharmacokinetics using a physiologically based model.
     Regul. Toxicol. Pharmacol., 4: 329-339.

    ROBERTS, A. (1986a). The pharmacokinetics of dichloromethane. I.
    Disposition in B6C3F1 mice following intravenous and oral
    administration.  Food. Chem. Toxicol., 24: 965-974.

    ROBERTS, A. (1986). The pharmacokinetics of dichloromethane. II.
    Disposition in Fischer 344 rats following intravenous and oral
    administration.  Food. Chem. Toxicol., 24: 975-980.

    ARANYI, C., O'SHEA, W.J., GRAHAM, J.A. & MILLER, F.J. (1986). The
    effects of inhalation of organic chemical air contaminants on murine
    lung host defenses.  Fundam. Appl. Toxicol., 6: 713-720.

    BAKINSON, M.A. & JONES, R.D. (1985). Gassings due to methylene
    chloride, xylene, toluene, and styrene reported to Her Majesty's
    Factory Inspectorate 1961-80.  Br. J. Ind. Med., 42: 184-190.

    BERGMAN, K. (1983). Application and results of whole-body
    autoradiography in distribution studies of organic solvents.  Crit.
     Rev. Toxicol., 12: 59-118.

    BOGEN, K.T. (1990). Risk extrapolation for chlorinated methanes as
    promoters vs initiators of multistage carcinogenesis.  Fundam. Appl.
     Toxicol., 15: 536-557.

    KARLSSON, J.E. & HAGLID, K.G. (1986). Chronic effects of
    dichloromethane on amino acids, glutathione and phosphoethanolamine
    in gerbil brain.  Scand. J. Work. Environ. Health., 12: 216-220.

    BUIE, S.E., PRATF, D.S. & MAY, J.J. (1986). Diffuse pulmonary injury
    following paint remover exposure.  Am. J. Med., 81: 702-704.

    (1984). Methylene chloride: a two-year inhalation toxicity and
    oncogenicity study in rats and hamsters.  Fundam. Appl. Toxicol., 4:

    CHERRY, N., VENABLES, H. & WALDRON, H.A. (1983). The acute
    behavioural effects of solvent exposure.  J. Soc. Occup. Med., 33:

    CONDIE, L.W., SMALLWOOD, C.L. & LAURIE, R.D. (1983). Comparative
    renal and hepatotoxicity of halomethanes: bromodichloromethane,
    bromoform, chloroform, dibromochloromethane and methylene chloride.
     Drug. Chem. Toxicol, 6: 563-578.

    CORDES, D.H., BROWN, W.D. & QUINN, K.M. (1988). Chemically-induced
    hepatitis after inhaling organic solvents.  West. J. Med., 148:

    CORSI, G.C., VALENTINI, F. & BERTAZZON, A. (1983). Effect of
    subtoxic amounts of furan, acetylfuran and methylene chloride on
    some serum enzymes of rat.  Boll. Soc. Ital. Biol. Sper., 59:

    CARERE, A. (1988). Induction of chromosome malsegregation by
    halogenated organic solvents in  Aspergillus nidulans: unspecific
    or specific mechanism?  Mutat. Res., 201: 401-411.

    FRAGA, C.G., LEIBOVITZ, B.E. & TAPPEL, A.L. (1987). Halogenated
    compounds as inducers of lipid peroxidation in tissue slices.  Free.
     Radic. Biol. Med., 3: 119-123.

    FRAGA, C.G., ZAMORA, R. & TAPPEL, A.L. (1989). Damage to protein
    synthesis concurrent with lipid peroxidation in rat liver slices:
    effect of halogenareal compounds, peroxides, and vitamin E1.  Arch.
     Biochem. Biophys., 270: 84-91.

    GUSTAFSSON, J.A. (1984). Central catecholamine neurons and exposure
    to dichloromethane. Selective changes in amine levels and turnover
    in tel- and diencephalic DA and NA nerve terminal systems and in the
    secretion of anterior pituitary hormones in the male rat.
     Toxicology, 29: 293-305.

    GLATZEL, W., TIETZE, K., GUTEWORT, R. & PANKOW, D. (1987).
    Interaction of dichloromethane and ethanol in rats: toxicokinetics
    and nerve conduction velocity.  Alcohol. Clin. Exp. Res., 11:

    GREEN, T. (1983). The metabolic activation of dichloromethane and
    chlorofiuoromethane in a bacterial mutation assay using  Salmonella
     typhimurium. Mutat. Res., 118: 277-288.

    GREEN, T., PROVAN, W.M., COLLINGE, D.C. & GUEST, A.E. (1988).
    Macromolecular interactions of inhaled methylene chloride in rats
    and mice.  Toxicol. Appl. Pharmacol., 93: 1-10.

    Dichloromethyl compounds as mechanism-based inactivators of rat
    liver cytochromes P-450  in vitro. Mol. Pharmacol., 30: 19-24.

    (1983). Chemical enhancement of viral transformation in Syrian
    hamster embryo cells by gaseous and volatile chlorinated methanes
    and ethanes.  Cancer. Res., 43: 1945-1950.

    R.L. (1987). Methylene chloride mortality study: dose-response
    characterization and animal model comparison. J.  Occup. Med., 29:

    HEARNE, F.T., PIFER, J.W. & GROSE, F. (1990). Absence of adverse
    mortality effects in workers exposed to methylene chloride: an
    update.  J. Occup. Med., 32: 234-240.

    Vaporization technique to measure mutagenic activity of volatiles
    organic chemicals in the Ames/ Salmonella assay.  Environ.
     Mutagen., 9: 421-441.

    IARC (1986). IARC (International Agency for Research on Cancer)
    monographs on the evaluation of the carcinogenic risk of chemicals
    to humans: Some halogenated hydrocarbons and pesticide exposures.
     Lyon, 41: 43-85.

    JONGEN, W.M., HARMSEN, E.G., ALINK, G.M. & KOEMAN, J.H. (1982). The
    effect of glutathione conjugation and microsomal oxidation on the
    mutagenicity of dichloromethane in  S. typhimurium. Mutat. Res.,
    95: 183-189.

    F. & KOEMAN, J.H. (1981). Mutagenicity testing of dichloromethane in
    short-term mammalian test systems.  Mutat. Res., 81: 203-213.

    JUORIO, A.V. & YU, P.H. (1985). Effects of benzene and other organic
    solvents on the decarboxylation of some brain aromatic-L-amino
    acids.  Biochem. Pharmacol., 34: 1381-1387.

    (1987). Effects of low-dose inhalation of three chlorinated
    aliphatic organic solvents on deoxyribonucleic acid in gerbil brain.
     Scand. J. Work. Environ Health, 13: 453-458.

    KIM, Y.C. & CARLSON, G.P. (1986). The effect of an unusual workshift
    on chemical toxicity. I. Studies on the exposure of rats and mice to
    dichloromethane.  Fundam. Appl. Toxicol., 6: 162-171.

    Review of investigations of dichloromethane metabolism and
    subchronic oral toxicity as the basis for the design of chronic oral
    studies in rats and mice.  Food. Chem. Toxicol., 24: 943-949.

    KITCHIN, K.T. & BROWN, J.L. (1989). Biochemical effects of three
    carcinogenic chlorinated methanes in rat liver.  Teratogenesis.
     Carcinog. Mutagen, 9: 61-69.

    (1986). Effects of methylene chloride on body and organ weight and
    plasma butyrylcholinesterase activity in mice.  Acta. Pharmacol.
     Toxicol. Copenh., 59: 73-79.

    L. (1985). Effects of organic solvents on motor activity in mice.
     Toxicology, 35: 35-46.

    Tolerance during inhalation of organic solvents.  Pharmacol.
     Toxicol., 66:409-414.

    KODELL, R.L., CHEN, J.J. & GAYLOR, D.W. (1989). A note on the role
    of background tumor incidence in risk assessment for carcinogens.
     Regul. Toxicol. Pharmacol., 9: 141-146.

    KRAMERS, P.G., MOUT, H.C., BISSUMBHAR, B. & MULDER, C.R. (1991).
    Inhalation exposure in  Drosophila mutagenesis assays: experiments
    with aliphatic halogenated hydrocarbons, with emphasis on the
    genetic activity profile of 1,2-dichloroethane.  Mutat. Res., 252:

    KREWSKI, D., MURDOCH, D. & WITHEY, J.R. (1989). Recent developments
    in carcinogenic risk assessment.  Health. Phys., 57 Suppl 1:

    KURPPA, K. & VAINIO, H. (1981). Effects of intermittent
    dichloromethane inhalation on blood carboxyhemoglobin concentration
    and drug metabolizing enzymes in rat.  Res. Commun. Chem. Pathol.
     Pharmacol., 32: 535-544.

    LANDRY, T.D., RAMSEY, J.C. & McKENNA, M.J. (1983). Pulmonary
    physiology and inhalation dosimetry in rats: development of a method
    and two examples.  Toxicol. Appl. Pharmacol., 71: 72-83.

    (1990). Mortality of cellulose fiber production workers.  Scand. J.
     Work. Environ. Health, 16: 247-251.

    LEFEVRE, P.A. & ASHBY, J. (1989). Evaluation of dichloromethane as
    an inducer of DNA synthesis in the B6C3F1 mouse liver.
     Carcinogenesis, 10: 1067-1072.

    D.O. (1990). Methylene chloride: report of five exposures and two
    deaths.  Am. J. Emerg. Med., 8: 534-537.

    LEUSCHNER, F., NEUMANN, B.W. & HUBSCHER, F. (1984). Report on
    subacute toxicological studies with dichloromethane in rats and dogs
    by inhalation.  Arzneimittelforschung, 34: 1772-1774.

    MALTONI, C., COTTI, G. & PERINO, G. (1988). Long-term
    carcinogenicity bioassays on methylene chloride administered by
    ingestion to Sprague-Dawley rats and Swiss mice and by inhalation to
    Sprague-Dawley rats.  Ann. N.Y. Acad. Sci., 534: 352-366.

    MARZOTKO, D. & PANKOW, D. (1987). Effect of single dichloromethane
    administration on the adrenal medulla of male albino rats.  Acta.
     Histochem. Jena., 82: 177-183.

    Neurotoxicologic evaluation of rats after 13 weeks of inhalation
    exposure to dichloromethane or carbon monoxide.  Pharmacol.
     Biochem. Behav., 36: 671-681.

    ANDERSEN, M.E. (1986). A physiological pharmacokinetic model for
    dermal absorption of vapors in the rat.  Toxicol. Appl. Pharmacol.,
    85: 286-294.

    E. (1988). Inhalation toxicity and carcinogenesis studies of
    methylene chloride (dichloromethane) in F344/N rats and B6C3F1
    mice.  Ann. N.Y Acad. Sci., 534: 343-351.

    MERSCH SUNDERMANN, V. (1989). Untersuchungen zur mutagenitfit
    organisher mikrokontaminationen in der umwelt. II. Die mutagenität
    leichtflüchtiger organohalogene im  Salmonella-mikrosomen-test
    (Ames-Test) unter berücksichtigung der kontaminationen der grund-
    und trinkwässern.  Zentralbl. Bakteriol. Mikrobiol. Hyg. B., 187:

    MILLER, L., PATERAS, V., FRIEDERICI, H. & ENGEL, G. (1985). Acute
    tubular necrosis after inhalation exposure to methylene chloride.
    Report of a case.  Arch. Intern. Med., 145: 145-146.

    MIRER, F.E., SILVERSTEIN, M. & PARK, R. (1988). Methylene chloride
    and cancer of the pancreas.  J. Occup. Med., 30: 475-6, 478, 48.

    C.M., BAKKE, J.P. & SPALDING, J.W. (1989). Measurement of
    unscheduled DNA synthesis and S-phase synthesis in rodent
    hepatocytes following  in vivo treatment: testing of 24 compounds.
     Environ. Mol. Mutagen., 14: 155-164.

    MIZUTANI, K., SHINOMIYA, K. & SHINOMIYA, T. (1988). Hepatotoxicity
    of dichloromethane.  Forensic. Sci. Int., 38:113-128.

    NESTMANN, E.R., OTSON, R., WILLIAMS, D.T. & KOWBEL, D.J. (1981).
    Mutagenicity of paint removers containing dichloromethane.  Cancer.
     Lett., 11: 295-302.

    McKENNA, M.J. (1988). Methylene chloride: a 2-year inhalation
    toxicity and oncogenicity study in rats.  Fundam. Appl. Toxicol.,
    11: 48-59.

    NITSCHKE, K.D., EISENBRANDT, D.L., LOMAX, L.G. & RAO, K.S. (1988).
    Methylene chloride: two-generation inhalation reproductive study in
    rats.  Fundam. Appl. Toxicol., 11: 60-67.

    SIGVARDSSON, K. (1983). Chemical reactivity and mutagenicity of some
    dihalomethanes.  Chem. Biol. Interact., 46: 121-130.

    (1983). Health evaluation of employees occupationally exposed to
    methylene chloride.  Scand. J. Work: Environ. Health, 9 Suppl 1:

    OTTENWALDER, H., JAGER, R., THIER, R. & BOLT, H.M. (1989). Influence
    of cytochrome P-450 inhibitors on the inhalative uptake of methyl
    chloride and methylene chloride in male B6C3F1 mice.  Arch. Toxicol.
     Suppl., 13: 258-261.

    OTTENWALDER, H. & PETER, H. (1989). DNA binding assay of methylene
    chloride in rats and mice.  Arch. Toxicol., 63: 162-163.

    PANKOW, D. & HOFFMANN, P. (1989). Dichloromethane metabolism to
    carbon monoxide can be induced by isoniazid, acetone and fasting.
     Arch. Toxicol. Suppl, 13: 302-303.

    PANKOW, D. & MARZOTKO, D. (1987). Zur akuten lebertoxizität von
    dichlorrnethan.  Z. Gesamte. Hyg., 33: 518-519.

    PANKOW, D., MATSCHINER, F. & WEIGMANN, H-J. (1991). Influence of
    aromatic hydrocarbons on the metabolism of dichloromethane to carbon
    monoxide in rats.  Toxicology, 68: 89-100.

    REITZ, R.H., MENDRALA, A.L. & GUENGERICH, F.P. (1989).  In vitro
    metabolism of methylene chloride in human and animal tissues: use in
    physiologically based pharmacokinetic models.  Toxicol. Appl.
     Pharmacol., 97: 230-246.

    GUENGERICH, F.P. (1988). Incorporation of  in vitro enzyme data
    into the physiologically-based pharmacokinetic (PB-PK) model for
    methylene chloride: implications for risk assessment.  Toxicol.
     Lett., 43: 97-116.

    RIOUX, J.P. & MYERS, R.A. (1988). Methylene chloride poisoning: a
    paradigmatic review.  J. Emerg. Med., 6: 227-238.

    RIOUX, J.P. & MYERS, R.A. (1989). Hyperbaric oxygen for methylene
    chloride poisoning: report on two cases.  Ann. Emerg. Med., 18:

    ROGHANI, M., DA SILVA, C. & CASTAGNA, M. (1987). Tumor promoter
    chloroform is a potent protein kinase C activator.  Biochem.
     Biophys. Res. Commun., 142: 738-744.

    Irreversible effects of dichloromethane on the brain after long-term
    exposure: a quantitative study of DNA and the glial cell marker
    proteins S-100 and GFA.  Br. J. Ind. Med, 43: 291-299.

    N.M., COOTS, R.H. & MORGAREIDGE, K. (1986a). A two-year
    drinking-water study of dichloromethane in rodents. I. Rats.
     Food. Chem. Toxicol, 24: 951-958.

    N.M., COOTS, R.H. & MORGAREIDGE, K. (1986b). A two-year
    drinking-water study of dichloromethane in rodents. II. Mice.
     Food. Chem. Toxicol, 24: 959-963.

    SHELDON, T., RICHARDSON, C.R. & ELLIOTT, B.M. (1987). Inactivity of
    methylene chloride in the mouse bone marrow micronucleus assay.
     Mutagenesis, 2: 57-59.

    Methylene chloride intoxication in a furniture refinisher. A
    comparison of exposure estimates utilizing workplace air sampling
    and blood carboxyhemoglobin measurements.  J. Occup. Med., 32:

    TARIOT, P.N. (1983). Delirium resulting from methylene chloride
    exposure: case report.  J. Clin. Psychiatry, 44: 340-342.

    ODKVIST, L.M. (1984). Vestibulo-ocular disturbances in rats exposed
    to organic solvents.  Acta. Pharmacol. Toxicol. Copenh., 54: 58-63.

    THILAGAR, A.K. & KUMAROO, V. (1983). Induction of chromosome damage
    by methylene chloride in CHO cells.  Mutat. Res., 116: 361-367.

    TOFTGARD, R., NILSEN, O.G. & GUSTAFSSON, J.A. (1982). Dose-dependent
    induction of rat liver microsomal cytochrome P-450 and microsomal
    enzymatic activities after inhalation of toluene and
    dichloromethane.  Acta: Pharmacol. Toxicol. Copenh., 51: 108-114.

    Comparison of the cancer risk of methylene chloride predicted from
    animal bioassay data with the epidemiologic evidence.  Risk. Anal.,
    10: 429-435.

    TRUEMAN, R.W. & ASHBY, J. (1987). Lack of UDS activity in the livers
    of mice and rats exposed to dichloromethane.  Environ. Mol.
     Mutagen., 10: 189-195.

    M.E. (1987). Research strategy in industrial toxicology.
     J. Toxicol. Sci., 12: 223-233.

    (1990). Further evidence that dichloromethane does not induce
    chromosome damage.  J. Appl. Toxicol., 10: 79-81.

    WIKBERG, J.E., HEDE, A.R. & LINDAHL, M. (1985). Effect of general
    anaesthetics and organic solvents on alpha 1-adrenoceptors in the
    myometrium.  Acta Pharmacol. Toxicol. Copenh., 57: 53-59.

    WIKBERG, J.E., HEDE, A.R. & POST, C. (1987). Effects of halothane
    and other chlorinated hydrocarbons on alpha 2-adrenoceptors in the
    mouse cortex.  Pharmacol. Toxicol., 61: 271-277.

    WILCOSKY, T.C. & SIMONSEN, N.R. (1991). Solvent exposure and
    cardiovascular disease.  Am. J. Ind. Med, 19: 569-586.

    WINNEKE, G. (1981). The neurotoxicity of dichloromethane.
     Neurobehav. Toxicol. Teratol., 3: 391-395.

    WITHEY, J.R., COLLINS, B.T. & COLLINS, P.G. (1983). Effect of
    vehicle on the pharmacokinetics and uptake of four halogenated
    hydrocarbons from the gastrointestinal tract of the rat.  J. Appl.
     Toxicol., 3: 249-253.

    WITHEY, J.R. & KARPINSKI, K. (1985). The fetal distribution of some
    aliphatic chlorinated hydrocarbons in the rat after vapor phase
    exposure.  Biol. Res. Pregnancy. Perinatol., 6: 79-88.

    See Also:
       Toxicological Abbreviations
       Dichloromethane (ICSC)
       Dichloromethane (FAO Nutrition Meetings Report Series 48a)
       Dichloromethane (IARC Summary & Evaluation, Volume 71, 1999)