1.1.1. Nomenclature

• Chem. Abstr. Serv. Reg. No.: 75-09-2
• Chem. Abstr. Name: Dichloromethane
• IUPAC Systematic Name: Dichloromethane
• Synonyms: Methane dichloride; methylene bichloride; methylene chloride; methylene dichloride

1.1.2. Structural and molecular formulae and relative molecular mass

1.1.3. Chemical and physical properties of the pure substance

• (a) Description: Colourless liquid with penetrating ether-like odour (Lewis, 1993; Budavari, 1996; Verschueren, 1996)
• (b) Boiling-point: 40°C (Lide, 1995)
• (c) Melting-point: −95.1°C (Lide, 1995)
• (d) Density:d²⁰₄ 1.3266 (Lide, 1995)
• (e) Spectroscopy data: Ultraviolet (Grasselli & Ritchey, 1975), infrared (Sadtler Research Laboratories, 1995; prism [6620 (gas), 1011], grating [28523]), nuclear magnetic resonance (Sadtler Research Laboratories, 1995; proton [6401], ¹³C [167]) and mass spectral data (Grasselli & Ritchey, 1975) have been reported.
• (f) Solubility: Slightly soluble (1.38 g/100 mL) in water at 20°C; soluble in carbon tetrachloride; miscible in ethanol, diethyl ether and dimethylformamide (Lide, 1995; Budavari, 1996)
• (g) Volatility: Vapour pressure, 58 kPa at 25°C (Lide, 1995); relative vapour density (air = 1), 2.93 (Verschueren, 1996)
• (h) Stability: Vapour is nonflammable and is not explosive when mixed with air (Budavari, 1996) but may form explosive mixtures in atmospheres with higher oxygen content (Sax, 1984)
• (i) Reactivity: Reacts vigorously with active metals (lithium, sodium, potassium) and with strong bases (potassium tert-butoxide) (Sax, 1984)
• (j) Octanol/water partition coefficient (P): log P, 1.25 (Hansch et al., 1995)
• (k) Conversion factor: mg/m³ = 3.47 × ppm¹

1.1.4. Technical products and impurities

Dichloromethane is available in several grades based on its intended end use: technical; aerosol; vapour degreasing; special; urethane; and decaffeination or Food Chemicals Codex/National Formula (food and pharmaceutical applications). Purity, when reported, ranges from 99 to 99.99%. Acidity (as hydrochloric acid) may be up to 5 mg/kg. The maximum concentration of water in these grades of dichloromethane is 100 mg/kg (Rossberg et al., 1986; Holbrook, 1993; Dow Chemical Co., 1995; Vulcan Chemicals, 1995, 1996a,b,c,d).

Small amounts of stabilizers are often added to dichloromethane at the time of manufacture to protect against degradation by air and moisture. The following substances in the listed concentration ranges are the preferred additives (wt %): ethanol, 0.1–0.2; methanol, 0.1–0.2; cyclohexane, 0.01–0.03; and amylene (2-methyl-2-butene), 0.001–0.01. Other substances have also been described as being effective stabilizers, including phenols (phenol, hydroquinone, para-cresol, resorcinol, thymol, 1-naphthol), amines, nitroalkanes (nitromethane), aliphatic and cyclic ethers, epoxides, esters and nitriles (Rossberg et al., 1986; Holbrook, 1993).

Trade names for dichloromethane include Aerothene MM, Narkotil, R30, Solaesthin, and Solmethine.

1.1.5. Analysis

Analytical methods are available for determination of dichloromethane in biological media and environmental samples. All methods involve gas chromatography in combination with a suitable detector. Very low detection limits have been achieved for most media (e.g., in food, 7 ng/sample; water, 0.01 µg/L; air, 1.76 µg/m³; and blood, 0.022 mg/L) (WHO, 1996).

Selected methods for the analysis of dichloromethane in various matrices are identified in Table 1. The United States Environmental Protection Agency methods for analysing water (Methods 8010 and 8240) have also been applied to liquid and solid wastes (United States Environmental Protection Agency, 1982a,b). Volatile components of solid-waste samples are first extracted with polyethylene glycol or methanol before purge/trap concentration and analysis (United States Environmental Protection Agency, 1982b).

Table 1

Methods for analysis of dichloromethane.

Exposures to dichloromethane can also be monitored in air using a direct-reading infrared analyser, with minimum concentrations of 0.7 mg/m³ (0.2 ppm) (Goelzer & O'Neill, 1985).

1.2.1. Production

Dichloromethane was first prepared by Regnault in 1840 by the chlorination of methyl chloride in sunlight. It became an industrial chemical of importance during the Second World War. Two commercial processes are currently used for the production of dichloromethane—hydrochlorination of methanol and direct chlorination of methane (Rossberg et al., 1986; Holbrook, 1993).

The predominant method of manufacturing dichloromethane uses as a first step the reaction of hydrogen chloride and methanol to give methyl chloride. Excess methyl chloride is then mixed with chlorine and reacts to give dichloromethane, with chloroform and carbon tetrachloride as co-products. This reaction is usually carried out in the gas phase thermally but can also be performed catalytically or photolytically. At low temperature and high pressure, the liquid-phase process is capable of giving high selectivity for dichloromethane (Rossberg et al., 1986; Holbrook, 1993).

The older and currently less used production method for dichloromethane involves direct reaction of excess methane with chlorine at high temperatures (400–500°C), or at somewhat lower temperatures either catalytically or photolytically. Methyl chloride, chloroform and carbon tetrachloride are also produced as co-products (Rossberg et al., 1986; Holbrook, 1993).

World production of dichloromethane increased from 93 thousand tonnes in 1960 to an estimated 570 thousand tonnes in 1980 (Edwards et al., 1982) and is believed to be still several hundred thousand tonnes. Production in the United States has shown a steady decline from 1981 to 1993, as shown by the following figures (thousand tonnes): 1981, 404; 1984, 275; 1987, 234; 1990, 209; 1993, 160 (Anon., 1994, 1997). The total amount produced in western Europe ranged from 331 500 tonnes in 1986 to 254 200 tonnes in 1991 (WHO, 1996).

1.2.2. Use

Most of the current applications of dichloromethane are based on its solvent properties. For use in paint strippers, one of its first applications, dichloromethane is blended with other chemical components to maximize its effectiveness against specific coatings. Typical additives include alcohols, acids, amines or ammonium hydroxide, detergents and paraffin wax (Rossberg et al., 1986; Holbrook, 1993; WHO, 1996).

Dichloromethane has been used as an extraction solvent for spices and beer hops and for decaffeination of coffee. It has also found use as a carrier solvent in the textile industry, in the manufacture of photographic film and as a blowing agent for polymer foams. Dichloromethane is used as a solvent for vapour degreasing of metal parts and may also be blended with petroleum distillates and other chlorinated hydrocarbons for use as a dip-type cleaner in the metal-working industry, although consumption by this industry is declining because of recycling and recovery efforts on the part of end users. The reduction in use of 1,1,1-trichloroethane because of the Montreal Protocol and clean air legislation may increase the use of dichloromethane. It is also used as a component of low-pressure refrigerants, in air-conditioning installations, and as a low-temperature heat-transfer medium (Rossberg et al., 1986; Holbrook, 1993; WHO, 1996).

In chemical processing, dichloromethane is used in the manufacture of polycarbonate plastic from bisphenol and phosgene, the manufacture of photoresist coatings, and as a solvent carrier for the manufacture of insecticide and herbicide chemicals. It is used by the pharmaceutical industry as a process solvent in the manufacture of steroids, antibiotics, vitamins and, to a lesser extent, as a solvent in the coating of tablets. Other uses include grain fumigation, oil dewaxing, in inks and adhesives and in plastics manufacture (Rossberg et al., 1986; Holbrook, 1993).

The use of dichloromethane in western Europe has shown a decrease from 200 thousand tonnes in 1975–85 to 175 thousand tonnes per year in 1989 and to 150 thousand tonnes per year in 1992 (WHO, 1996). Estimated use patterns for dichloromethane in the United States are presented in Table 2.

Table 2

Estimated use patterns (%) for dichloromethane in the United States.

1.3.1. Natural occurrence

Dichloromethane is not known to occur as a natural product.

1.3.2. Occupational exposure

The uses of dichloromethane reviewed in Section 1.2 can lead to human exposure.

According to the 1990–93 CAREX database for 15 countries of the European Union (Kauppinen et al., 1998) and the 1981–83 United States National Occupational Exposure Survey (NOES, 1997), approximately 250 000 workers in Europe and as many as 1.4 million workers in the United States were potentially exposed to dichloromethane (see General Remarks).

Information on numbers of workers potentially exposed in other countries was not available to the Working Group.

Concentrations of dichloromethane measured in 1968–73 in a plant producing plastic films in the United States were 458–2060 mg/m³ in the casting area, 583–3350 mg/m³ in the filtration area, 625–659 mg/m³ in the winding area and 160–1130 mg/m³ in offices (United States National Institute for Occupational Safety and Health, 1976).

Table 3 summarizes personal occupational exposures measured in various industries using dichloromethane. The levels vary widely by operation and within operations. Concentrations exceeding 1000 mg/m³ have been measured, e.g., in paint stripping, in the printing industry and in the manufacture of plastics and synthetic fibres. Full-shift exposures to levels above 100 mg/m³ of dichloromethane are possible, e.g., in furniture-stripping shops and in certain jobs in aeronautical, pharmaceutical, plastic and footwear industries.

Table 3

Personal occupational exposures to dichloromethane.

Workers exposed to dichloromethane may be exposed also to various other agents, depending on their specific tasks and working environments.

1.3.3. Air

The principal route of exposure to dichloromethane for the general population is inhalation of ambient air. Average daily intake of dichloromethane from urban air has been estimated to range from about 33 to 309 µg. Exposure to dichloromethane in indoor air may be much higher, especially from spray painting or other aerosol uses and from paint removal and metal degreasing. Dichloromethane is degraded in the atmosphere by reaction with hydroxyl radicals, with an atmospheric lifetime of less than one year. The compound is highly mobile in soil and volatilizes rapidly from surface water to the atmosphere (Agency for Toxic Substances and Disease Registry, 1993).

Because dichloromethane is highly volatile, most environmental releases are into the atmosphere. It is released to the atmosphere during its production, storage and transport, but most (more than 99%) of the atmospheric releases result from industrial and consumer uses. Estimates of annual global emissions of 500 thousand tonnes have been reported for dichloromethane. It has been estimated that 85% of the total amount of dichloromethane produced in the United States is released to the environment, mostly to the atmosphere. Industrial dichloromethane emissions to the atmosphere in the United States fell from about 58 thousand tonnes in 1988 to approximately 25 thousand tonnes in 1995 (United States National Library of Medicine, 1997a). The total emission into the air in western Europe was estimated to be 173 thousand tonnes for 1989 and 180 thousand tonnes in 1991 (Agency for Toxic Substances and Disease Registry, 1993; WHO, 1996).

Dichloromethane has been detected in ambient air samples taken around the world, with background levels usually at about 0.17 µg/m³. Concentrations in urban areas and in the vicinity of hazardous waste sites may be one to two orders of magnitude higher (up to 43 µg/m³). Even higher levels (mean, 670 µg/m³; peak level, 5000 µg/m³) have been found in the indoor air of residences (WHO, 1996).

A large do-it-yourself consumer population uses paint strippers containing dichloromethane on furniture and woodwork. Formulations are available mainly in liquid form, but also, occasionally, as an aerosol. Exposures have been estimated on the basis of investigations of the use of household liquid products in the United States. The estimated levels ranged from less than 35 mg/m³ to a few short-term exposures of 14 100–21 200 mg/m³. The majority of the concentration estimates were below 1770 mg/m³ (WHO, 1996).

Dichloromethane is also formed during water chlorination and is emitted into the air from wastewater in treatment plants (Agency for Toxic Substances and Disease Registry, 1993).

1.3.4. Water

About 2% of environmental releases of dichloromethane are to water. Industrial releases of dichloromethane to surface water and underground injection (potential groundwater release) reported to the United States Toxic Chemical Release Inventory in 1988 totalled 158 tonnes. Dichloromethane has been identified in industrial and municipal wastewaters from several sources at concentrations ranging from 0.08 µg/L to 3.4 g/L (Agency for Toxic Substances and Disease Registry, 1993; WHO, 1996).

Dichloromethane has been detected in surface water, groundwater and finished drinking-water throughout the United States. It was detected in 30% of 8917 surface water samples recorded in the STORET database of the United States Environmental Protection Agency, at a median concentration of 0.1 µg/L. In a New Jersey survey, dichloromethane was found in 45% of 605 surface water samples, with a maximum concentration of 743 µg/L. Dichloromethane has also been identified in surface waters in Maryland, in Lakes Erie and Michigan, and at hazardous waste sites (Agency for Toxic Substances and Disease Registry, 1993; WHO, 1996).

Dichloromethane is also present in small amounts in seawater. It has been found at up to 2.6 µg/L in coastal waters of the Baltic Sea. Levels up to 0.20 µg/L have been found in North Sea coastal waters. Dichloromethane is generally not detected in open ocean; a mean concentration of 2.2 ng/L has been reported in the southern Pacific Ocean (WHO, 1996).

Since volatilization is restricted in groundwater, concentrations of dichloromethane are often higher there than in surface water. Occurrence of dichloromethane in groundwater has been reported in several surveys across the United States, with concentrations ranging from 0 to 3600 µg/L (Agency for Toxic Substances and Disease Registry, 1993).

Dichloromethane has been detected in drinking-water supplies in numerous cities in the United States, with reported mean concentrations generally below 1 µg/L. It has also been identified in commercially bottled artesian water. Water chlorination in treatment plants appears to increase both the concentration and the frequency of occurrence of dichloromethane in drinking water supplies (Agency for Toxic Substances and Disease Registry, 1993; WHO, 1996).

Samples from 128 drinking-water wells in the United States showed that 3.1% of them had dichloromethane levels of 1–5 µg/L. Dichloromethane was detected in 98.4% of drinking-water samples from Santiago de Compostela, Spain, in 1987; the average concentration was 14.1 µg/L, with a range of 1.2–93.2 µg/L. A sampling of 630 public community water supplies (serving 690 million people in New Jersey, United States) in 1984 and 1985 detected dichloromethane in 2.6–7.1% of the samples; the median concentration ranged from 1.1 to 2.0 µg/L and the range for the whole sampling period was 0.5–39.6 µg/L (WHO, 1996).

Dichloromethane has been detected in both surface water and groundwater samples taken at hazardous waste sites. Data from the Contract Laboratory Program Statistical Database of the United States Environmental Protection Agency indicate that dichloromethane was present at geometric mean concentrations of 68 and 98 µg/L in surface water and groundwater samples, respectively, at about 30% of the sites sampled (Agency for Toxic Substances and Disease Registry, 1993).

Wastewater from certain industries has been reported to contain dichloromethane at average concentrations in excess of 1000 µg/L. Such industries include coal mining, aluminium forming, photographic equipment and supplies, pharmaceutical manufacture, organic chemical/plastics manufacture, paint and ink formulation, rubber processing, foundries and laundries. The maximal concentration measured was 210 mg/L in wastewater from the paint and ink industry and the aluminium-forming industry. In leachate from industrial and municipal landfills, dichloromethane concentrations were reported to range up to 184 mg/L (WHO, 1996).

1.3.5. Soil/sediment

The principal sources of dichloromethane releases to land are disposal of dichloromethane products and containers to landfills. Industrial releases of dichloromethane to land and off-site transfers to landfills reported to the Toxic Chemical Release Inventory in 1988 totalled about 71 tonnes. It is estimated that about 12% of dichloromethane releases to the environment are to land (Agency for Toxic Substances and Disease Registry, 1993).

Dichloromethane has been detected in soil and sediment samples taken at 36% of the hazardous waste sites included in the Contract Laboratory Program Statistical Database at a geometric mean concentration of 104 µg/kg (Agency for Toxic Substances and Disease Registry, 1993).

The levels of dichloromethane found in samples of sediment from Lake Pontchartrain, Louisiana, United States, ranged from not detectable to 3.2 µg/kg wet weight. In Germany, levels found in sediments from the River Rhine in 1987–88 varied from not detectable to 30–40 µg/kg. At one site, concentrations of 220–2200 µg/kg were measured (WHO, 1996).

1.3.6. Aquatic organisms

Concentrations of dichloromethane in freshwater organisms have been reported for oysters and clams from Lake Ponchartrain, Louisiana, United States; levels ranging from 4.5 to 27 µg/kg wet weight were detected. Levels up to 700 µg/kg wet weight were found in marine bottom fish taken from Commencement Bay, Washington, United States. Data on biota collected in the STORET database of the United States Environmental Protection Agency showed an average level of 660 µg/kg in the 28% of the samples in which dichloromethane was detected (WHO, 1996).

1.3.7. Foodstuffs

Although dichloromethane has been used as a grain fumigant and in processing certain raw food commodities, there is little information on residual levels in food. At a large decaffeinating plant in the United States in 1978, monthly average residues in dichloromethane-decaffeinated coffee beans ranged from 0.32 to 0.42 mg/kg dichloromethane (115–295 samples analysed per month) (Cohen et al., 1980). In seven types of decaffeinated ground coffee, the dichloromethane content ranged from < 0.05 to 4.04 mg/kg; in eight instant coffee samples, it ranged from < 0.05 to 0.91 mg/kg; and in 10 decaffeinated tea samples, it ranged from < 0.05 to 15.9 mg/kg (Page & Charbonneau, 1984). Dichloromethane apparently is no longer used for decaffeination in the United States (Agency for Toxic Substances and Disease Registry, 1993).

In an investigation of several halocarbons in table-ready foods, eight of the 19 foods examined contained dichloromethane levels above the quantification limit (not given). The following ranges were reported (µg/kg): butter, 1.1–280; margarine, 1.2–81; ready-to-eat cereal, 1.6–300; cheese, 3.9–98; peanut butter, 26–49; and highly processed foods (frozen chicken dinner, fish sticks, pot pie), 5–310 (Heikes, 1987).

3.1.1. Mouse

Groups of male and female B6C3F₁ mice, seven weeks of age, were administered dichloromethane (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) in the drinking-water for 104 weeks according to the study design shown in Table 6. No significant exposure-related trend in survival was found in males; in females, a significant trend towards longer survival in exposed groups was reported. In male mice, the incidences of hepatocellular adenoma were: 6/60 (10%), 4/65 (8%), 20/200 (10%), 14/100 (14%), 14/99 (14%) and 15/125 (12%); and the incidences of hepatocellular carcinomas were: 5/60 (8%), 9/65 (14%), 33/200 (17%), 18/100 (18%), 17/99 (17%) and 23/125 (18%) in control 1, control 2, low-dose, mid-dose 1, mid-dose 2 and high-dose groups, respectively. A slight but significant [p = 0.035] dose-related increase in the incidence of hepatocellular adenomas and/or carcinomas (combined) was observed in male mice: 11/60 (18%), 13/65 (20%), 51/200 (25%), 30/100 (30%), 31/99 (31%) and 35/125 (28%). However, the authors noted that tumour incidences in exposed groups were similar to those reported in historical controls (mean, 32.1%; range, 7–58%) (Serota et al., 1986a).

Table 6

Design of studies of dichloromethane in drinking-water.

Groups of 50 male and 50 female Swiss mice, nine weeks of age, were administered 100 (low-dose) or 500 (high-dose) mg/kg bw dichloromethane (purity, > 99.9%) in olive oil by gavage once per day on four to five days per week for 64 weeks. Groups of 60 mice of each sex were given olive oil (vehicle-control). Animals were then kept under observation for their lifespan. Excess mortality was observed in male and female mice exposed to the high dose (p < 0.01). An increase in mortality appeared after 36 weeks of treatment and led to withdrawal of the treatment at 64 weeks. In mice that died by 78 weeks, the incidence of lung tumours in males was 1/14 control, 4/21 low-dose and 7/24 high-dose (p < 0.05) mice, respectively. At the end of the experiment, the cumulative incidence of lung tumours in males was 5/47, 5/28 and 9/36. No treatment-related increase in the incidence of any tumour in females or other type of tumour in males was reported (Maltoni et al., 1988). [The Working Group noted the short period of exposure and the high numbers of animals lost for examination.]

3.1.2. Rat

Groups of male and female Fischer 344 rats, seven weeks of age, were administered dichloromethane (containing < 300 mg/kg cyclohexane, < 20 mg/kg trans-1,2-dichloroethylene, 26 mg/kg chloroform and < 1 mg/kg each methyl chloride, vinyl chloride, ethyl chloride, vinylidene chloride and trichloroethylene) in the drinking-water for 104 weeks according to the study design shown in Table 6. Interim terminations were carried out at 26, 52 and 78 weeks in control group 1 and in the low-, mid-1 and -2 and high-dose groups, such that 50 males and 50 females per group received the treatment for 104 weeks. There was no significant difference in survival between the exposed and control groups. In females, the incidences of hepatocellular carcinomas after 104 weeks were: 0/85, 0/50, 0/85, 2/83, 0/85 and 2/85; those of neoplastic nodules [now classified as hepatocellular adenomas] were: 0/85, 0/50, 1/85, 2/83, 1/85 and 4/85; and those of neoplastic nodules and/or hepatocellular carcinomas (combined) were: 0/85, 0/50, 1/85, 4/83, 1/85 and 6/85 in the six groups, respectively. This increasing trend was significant [p < 0.01]; however, tumour incidences in exposed groups were similar to those reported in historical controls in this laboratory (mean, 8%; range, 0–16%). In male rats, no increased incidence of liver tumours was observed at 104 weeks (neoplastic nodules: 4/85, 5/50, 2/85, 3/84, 3/85 and 1/85; carcinomas: 2/85, 2/50, 0/85, 0/84, 0/85 and 1/85; neoplastic nodules and/or carcinomas combined: 6/85, 7/50, 2/85, 3/84, 3/85 and 2/85). No other significant increase in tumour incidence was found (Serota et al., 1986b).

Groups of 50 male and 50 female Sprague-Dawley rats, 12 weeks of age, were administered 100 (low-dose) or 500 (high-dose) mg/kg bw dichloromethane (purity, > 99.9%) in olive oil by gavage once per day on four or five days per week for 64 weeks. A group of 50 rats of each sex was given olive oil (vehicle controls) and an additional group of 20 males and 26 females was kept untreated (untreated controls). Animals were then kept under observation for their lifespan. Excess mortality was observed in male and female rats administered dichloromethane at the high dose. An increase in mortality started to appear after 36 weeks of treatment and led to cessation of exposure after 64 weeks [details on mortality not reported]. There was no significant increase in tumour incidence associated with exposure (Maltoni et al., 1988). [The Working Group noted the short period of treatment and the inadequate reporting of the data.]

3.2.1. Mouse

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 13 900 mg/m³] dichloromethane (> 99% pure) by whole-body inhalation for 6 h per day on five days per week for 102 weeks and were killed after 104 weeks on study. The mean body weight of the high-dose male mice was generally comparable to that of controls until week 90, and that of high-dose females was somewhat lower from weeks 51 to 95. Survival to the end of the study period in males was: control, 39/50; low-dose, 24/50; and high-dose, 11/50; and that in females was: 25/50, 25/49 and 8/49. Significant dose-related increases in the incidence of lung and liver tumours were observed in exposed mice. The incidences of alveolar/bronchiolar adenomas were: males—3/50, 19/50 and 24/50 (p < 0.001); and females—2/50, 23/48 and 28/48 (p < 0.001). Those of alveolar/bronchiolar carcinomas were: males—2/50, 10/50 and 28/50 (p < 0.001); and females—1/50, 13/48 and 29/48 (p < 0.001). The incidences of hepatocellular adenomas were: males—10/50, 14/49 and 14/49 (p = 0.075); and females — 2/50, 6/48 and 22/48 (p < 0.001). The incidences of hepatocellular carcinomas were: males—13/50, 15/49 and 26/49 (p = 0.016); and females—1/50, 11/48 and 32/48 (p < 0.001) (United States National Toxicology Program, 1986).

Groups of 68 female B6C3F₁ mice, eight to nine weeks of age, were administered dichloromethane (> 99% pure) by whole-body inhalation at concentrations of 0 ppm (control) or 2000 ppm [6940 mg/m³] for various lengths of time over a 104-week period (Table 7). Lung and liver were evaluated histopathologically. Survival was reduced compared with controls in groups exposed to dichloromethane for the first 52, 78 or the complete 104 weeks of the study. The incidences of mice with lung adenomas, carcinomas or adenomas and carcinomas combined and the incidences of mice with hepatocellular adenomas, carcinomas or adenomas and carcinomas combined were increased in all groups in which exposure was begun during the first 26 weeks of the study [statistical analyses were reported only for the combined tumour incidences] (Table 7) (Kari et al., 1993).

Table 7

Effect of various dichloromethane inhalation exposure regimens on survival and pulmonary and liver tumours in female B6C3F₁ mice.

3.2.2. Rat

Groups of approximately 95 male and 95 female Sprague-Dawley rats, eight weeks of age, were administered dichloromethane by whole-body inhalation at concentrations of 0, 500, 1500 or 3500 ppm [1740, 5200 or 12 100 mg/m³] for 6 h per day on five days per week for two years. The dichloromethane was > 99% pure, with ≤ 706 mg/kg (ppm) trans-1,2-dichloroethylene, ≤ 467 mg/kg cyclohexane, ≤ 576 mg/kg chloroform, ≤ 90 mg/kg vinylidene chloride, ≤ 20 mg/kg carbon tetrachloride, ≤ 23 mg/kg methyl bromide, ≤ 11 mg/kg ethyl chloride, ≤ 4.5 mg/kg methyl chloride and ≤ 1 mg/kg vinyl chloride. The numbers of animals still alive at the end of the study were 14, 14, 6 and 7 control, low-, mid- and high-dose males and 21, 24, 13 and 4 females, respectively. Mortality among high-dose females was significantly increased from the 18th month onwards. Non-neoplastic pathological changes in the liver and kidney were more frequently observed in treated animals. There was no significant increase in the incidence of benign or malignant mammary tumours; however, the total number of benign mammary tumours [type not specified] showed a slight dose-related increase in males (control, 8/95; low-dose, 6/95; mid-dose, 11/95; and high-dose, 17/97; p = 0.046), and a dose-related increase [p < 0.001] in the total number of benign mammary tumours [type not specified] was observed in females (165/96, 218/95, 245/95 and 287/97). The incidence of sarcomas located around the salivary glands was increased in mid- and high-dose males (1/93, 0/94, 5/91 and 11/88; p = 0.002 [p < 0.001, trend test]) (Burek et al., 1984; United States Environmental Protection Agency, 1985). [The Working Group noted the reported occurrence of salivary gland sialodacryoadenitis early in the study.]

Groups of 50 male and 50 female Fischer 344/N rats, seven to eight weeks of age, were administered dichloromethane (> 99% pure) by whole-body inhalation at concentrations of 0, 1000, 2000 or 4000 ppm [0, 3470, 6940 or 13 900 mg/m³] for 6 h per day on five days per week for 102 weeks and were killed after 104 weeks on study. Mean body weights of control and dosed rats of both sexes were comparable throughout the study. Survival of treated males was similar to that of controls. Survival at termination of the study was reduced in high-dose females compared with controls: control, 30/50; low-dose, 22/50; mid-dose, 22/50; and high-dose, 15/50. Increased incidences of benign mammary gland tumours (all fibroadenomas, except for one adenoma in the high-dose group) were observed in treated females (5/50, 11/50, 13/50 and 23/50; p < 0.001). There was a positive trend in the incidence of mammary gland adenoma or fibroadenoma combined in males (0/50, 0/50, 2/50 and 5/50; p < 0.01). There was no difference in the distribution of other types of tumour between the control and treated groups (United States National Toxicology Program, 1986).

Groups of 54–70 male and female Sprague-Dawley rats, 13 weeks old, were administered 100 ppm [347 mg/m³] dichloromethane (purity, > 99.9%) by whole-body inhalation for 7 h per day on five days per week. The exposure was started on breeders, and male and female offspring (12-day embryos). The breeders and a group of offspring were exposed for 104 weeks, another group of offspring was exposed for 15 weeks only. Control groups were composed of 60 female rats (breeder controls) and 158 males and 149 females (untreated controls). Animals were observed for their lifespan. No excess in mortality was found in the exposed groups. No significant increase in the incidence of any tumour type was noted (Maltoni et al., 1988). [The Working Group noted the low exposure concentration.]

Groups of 90 male and 108 female Sprague-Dawley rats [age unspecified] were administered 0, 50, 200 or 500 ppm [0, 174, 694 or 1740 mg/m³] dichloromethane (technical-grade; purity, > 99.5%) by whole-body inhalation for 6 h per day on five days per week for 20 (males) or 24 (females) months. A further group of 30 female rats was exposed to 500 ppm dichloromethane for the first 12 months and to room air for the last 12 months of the study (denoted 500/air). An additional group of 30 female rats was exposed to room air for the first 12 months followed by 500 ppm dichloromethane for the last 12 months of the study (denoted air/500). Subgroups of five rats per sex per exposure level were scheduled for interim terminations after 6, 12, 15 and 18 months of exposure to dichloromethane. No exposure-related adverse effect on body weight or mortality was observed. In females, the incidence of benign mammary tumours (adenomas and fibroadenomas combined) was 52/70, 58/70, 61/70 (p < 0.05, Fisher's exact test) and 55/70 in control, low-, mid- and high-dose groups, respectively. The multiplicity of benign mammary tumours was 1.8, 2.1, 2.0 and 2.2 (p < 0.05) in the control, low-, mid- and high-dose groups, respectively, and 2.3 (p < 0.05) and 2.7 (p < 0.05) in the air/500 and 500/air groups. No significant increase in the incidence of any other tumour type was seen in the exposed groups (Nitschke et al., 1988).

3.2.3. Hamster

Groups of 95 male and 95 female Syrian golden hamsters, eight weeks of age, were administered dichloromethane by whole-body inhalation at concentrations of 0, 500, 1500 or 3500 ppm [0, 1740, 5200 or 12 100 mg/m³] for 6 h per day on five days per week for two years. The dichloromethane was > 99% pure, with ≤ 706 mg/kg trans-1,2-dichloroethylene, ≤ 467 mg/kg cyclohexane, ≤ 576 mg/kg chloroform, ≤ 90 mg/kg vinylidene chloride, ≤ 20 mg/kg carbon tetrachloride, ≤ 23 mg/kg methyl bromide, ≤ 11 mg/kg ethyl chloride, ≤ 4.5 mg/kg methyl chloride and ≤ 1 mg/kg vinyl chloride. The numbers of animals surviving to the end of the study were 16, 20, 11 and 14 in males and 0, 4, 10 and 9 in females. The incidence of lymphosarcomas was slightly higher in exposed females than in controls: control, 1/91; low-dose, 6/92; mid-dose, 3/91; and high-dose, 7/91 (p = 0.032) (Burek et al., 1984; United States Environmental Protection Agency, 1985). [The Working Group noted that the higher survival in treated animals may have contributed to this non-dose-dependent result for which historical control data were not available.]

4.1.1. Humans

Liquid dichloromethane is absorbed through human skin, with maximum concentrations in expired air being reached 30 min after exposure (Stewart & Dodd, 1964). After 0.5–8 h inhalation exposure, concentrations of dichloromethane in the blood and expired air were directly proportional to dose, over the concentration range 173–1740 mg/m³ (DiVincenzo et al., 1972). It is distributed principally to adipose tissue. In male volunteers exposed to 2600 mg/m³ for 1 h at a work intensity of 50 W, mean adipose tissue concentrations after 1, 4 and 22 h were 10.2, 8.4 and 1.7 mg/kg, respectively (Engström & Bjurström, 1977). Elevated carboxyhaemoglobin saturation and increased urinary formic acid concentrations have been found in exposed workers (Ku elová & Vlasák, 1966; DiVincenzo & Kaplan, 1981), but inhaled dichloromethane is excreted principally unchanged in expired air (Riley et al., 1966).

Dichloromethane can be conjugated with glutathione by human θ-class glutathione S-transferase (GST) T1-1, which is expressed in many human organs. However, the tissue-specific expression pattern differs from that of the α-, µ- and π-forms of GST, the θ-class being expressed only at very low levels in a small number of Clara cells and ciliated cells at the alveolar/bronchiolar junctions in human lung (Mainwaring et al., 1996a; Sherratt et al., 1997).

4.1.2. Experimental systems

Dichloromethane is rapidly absorbed through the lungs and distributed throughout the body reaching all organs, including the brain. Dichloromethane has a particular affinity for fat; concentrations in adipose tissues may be seven- to eight-fold higher than in other tissues (Savolainen et al., 1977). After inhalation or oral exposure of rats, the majority of the dose is exhaled unchanged (McKenna & Zempel, 1981; McKenna et al., 1982). Dichloromethane metabolites are also largely excreted via the lungs as carbon monoxide and carbon dioxide (DiVincenzo & Hamilton, 1975).

Two pathways for the metabolism of dichloromethane are known, one catalysed by a cytochrome P450 and the other by a GST (Kubic & Anders, 1975; Ahmed & Anders, 1976, 1978; Kubic & Anders, 1978; Gargas et al., 1986). The P450-mediated oxidative pathway produces carbon monoxide and inorganic chloride, presumably via the highly unstable intermediate, formyl chloride. The glutathione pathway produces carbon dioxide after the formation of a postulated glutathione conjugate and formaldehyde (Figure 1).

Figure 1

Metabolic pathways for dichloromethane.

More recently the specific isoenzymes involved in the metabolism of dichloromethane have been identified as the cytochrome CYP2E1 (Kim & Kim, 1996) and the θ-class GST, GSTT1-1 (Meyer et al., 1991; Mainwaring et al., 1996b).

Early studies suggested that the relative activities of each pathway could be determined by measuring exhaled carbon monoxide (or carboxyhaemoglobin in blood) and carbon dioxide, resulting from the P450 and glutathione pathways respectively. However, it is now known that this is an unreliable method, since significant quantities of carbon dioxide are also derived from the oxidative pathway (Gargas et al., 1986).

The metabolism of dichloromethane is a saturable process. For example, 48 h after inhalation exposure of rats to 50 ppm, 500 ppm or 1500 ppm [74, 1740 or 5200 mg/m³] dichloromethane, 5, 30 and 55% respectively of the chemical was expired unchanged (McKenna et al., 1982). Detailed investigations, in both rats and mice, have indicated that the cytochrome P450-mediated metabolism is a saturable high-affinity/low-capacity pathway. Saturation occurs at relatively low dose levels (< 500 ppm) in both rats and mice and results in similar levels of carboxyhaemoglobin in the blood (12–15%). Conversely, the GST-mediated pathway is low-affinity/high-capacity and exhibits dose-dependent linear kinetics (Gargas et al., 1986; ECETOC, 1987). This pathway is particularly active in mice; indeed it is the major pathway at the dose levels (2000 and 4000 ppm [6940 and 13 900 mg/m³]) used in the carcinogenicity bioassays. In vitro, the relative rates for this pathway (as indicated by the yield of RNA–formaldehyde adducts) are 1, 2, 4 and 14 in Syrian hamster hepatocytes, in human hepatocytes with functional GSTT1 genes and in rat and mouse hepatocytes, respectively (Casanova et al., 1997). Marked human inter-individual differences have been described for dichloromethane metabolism via the GST pathway. In 39 human samples, this activity varied between 0 and 3 nmol/min/mg cytosolic protein (Green, 1989; Reitz et al., 1989; Bogaards et al., 1993; Graves et al., 1995). Indeed a genetic polymorphism has been described for a θ-class GST found in erythrocytes (Bogaards et al., 1993; Hallier et al., 1994; Schroder et al., 1996). Some 10–40% of human samples studied appear to be deficient in this transferase activity.

The distribution of GSTT1-1 has been examined in mouse, rat and human liver and lung (Pemble et al., 1994; Mainwaring et al., 1996a; Sherratt et al., 1997; Mainwaring et al., 1998). GSTT1-1 mRNA and protein were visualized by in-situ hybridization and immunocytochemistry, respectively. High levels of both GSTT1-1 protein and mRNA were observed in mouse hepatocytes and mouse Clara cells, while much less was seen in rat or human liver and lung (Mainwaring et al., 1996a, 1998). Additionally, GSTT1-1 mRNA and protein were concentrated preferentially in certain cell types (mouse lung Clara cells, limiting plate hepatocytes) and, particularly in the nuclei of these cells in mice, whereas in rat and man, the distribution was more generalized.

A number of physiological toxicokinetic models have been developed to describe the metabolism of dichloromethane in mouse, rat, Syrian hamster and man following exposure either by inhalation or in drinking-water (Andersen et al., 1987; ECETOC, 1988; Reitz et al., 1989; Andersen et al., 1991).

4.2.1. Humans

The odour threshold of dichloromethane is about 200 ppm [694 mg/m³] (Stahl, 1973). One of the products of dichloromethane metabolism is carbon monoxide, and the acute effects of dichloromethane poisoning are mainly due to carbon monoxide, which binds to haemoglobin and thus decreases the oxygen-transporting capacity of blood, and simultaneously increases the affinity of haemoglobin toward oxygen, thereby decreasing the liberation of oxygen to tissues (Shusterman et al., 1990; Dhillon & Von Burg, 1995).

Fatalities have been associated with acute or prolonged exposure to dichloromethane (Moskowitz & Shapiro, 1952; Ku elová et al., 1975; Stewart & Hake, 1976; Bonventre et al., 1977; Bakinson & Jones, 1985; Manno et al., 1989). Temporary neurobehavioural effects have been reported after exposure to doses as low as 200 ppm [694 mg/m³] by some (Winnekke, 1974; Putz et al., 1976) but not by others (Gamberale et al., 1975).

An exposure-related increase in serum bilirubin was observed in workers exposed to dichloromethane, but no other sign of liver injury or haemolysis was reported (Ott et al., 1983a). A cross-sectional study of 24 employees at a fibre production plant showed no excess of electrocardiographic abnormalities among those exposed to 60–475 ppm [208–1650 mg/m³] (time-weighted average) dichloromethane and monitored for 24 h (Ott et al., 1983b).

The following mortality studies are described in more detail in Section 2.1. No significant increase in overall mortality or deaths due to ischaemic heart disease was found among 1271 male and female employees exposed to 140–475 ppm [486–1650 mg/m³] dichloromethane compared to the mortality of the general United States population (Ott et al., 1983c). An increased risk of ischaemic heart disease was found in comparison with an internal reference group. In another study in the United States of America on cellulose triacetate fibre workers (Gibbs et al., 1996), mortality from cardiovascular disease as a whole was not elevated, and in addition showed an inverse relationship with duration of exposure. In a further study on cellulose triacetate fibre workers in the United Kingdom (Tomenson et al., 1997), mortality from ischaemic heart disease was similarly less than expected from national rates. However, it was higher among the exposed than in the non-exposed cohort, and showed an association with the estimated lifetime exposure to dichloromethane. There was a statistically significant deficit in the mortality from nonmalignant lung disease and cerebrovascular disease. In a third cohort study on dichloromethane-exposed workers (Hearne et al., 1990), which had a 90% probability of detecting an 1.3-fold risk of ischaemic heart disease, no elevated risk was observed. The risk of non-malignant pulmonary disease was not elevated either, and there was a (non-significant) deficit of cerebrovascular disease.

4.2.2. Experimental systems

Intraperitoneal LD₅₀ values for dichloromethane are approximately 1.5 mL/kg bw (2000 mg/kg bw) in mice (Klaassen & Plaa, 1966) and 0.95 mL/kg bw (1300 mg/kg bw) in dogs (Klaassen & Plaa, 1967); the oral LD₅₀ in rats ranges from 1.6 to 2.3 mL/kg bw (2100–3000 mg/kg bw) (Kimura et al., 1971); and the subcutaneous LD₅₀ in mice is approximately 76 mmol/kg bw (6400 mg/kg bw) (Kutob & Plaa, 1962). The LC₅₀ values in mice, rats and guinea-pigs are 16 000 ppm [55 500 mg/m³] (7-h exposure plus 1-h observation), 5.7% [200 000 mg/m³] (15-min exposure) and 11 600 ppm [40 300 mg/m³] (6-h exposure plus 18-h observation), respectively (Sviberly et al., 1947; Balmer et al., 1976; Clark & Tinston, 1982).

The acute toxicity of dichloromethane is expressed mainly as disturbances of the central nervous system, involving sleep disturbance and reductions in spontaneous activity (Heppel & Neal, 1944; Schumacher & Grandjean, 1960; Fodor & Winneke, 1971; United States National Institute for Occupational Safety and Health, 1976).

Hepatotoxic effects are seen after exposure to near-lethal concentrations of dichloromethane (Gehring, 1968). Inhalation exposure of guinea-pigs to 5200 ppm [18 000 mg/m³] dichloromethane for 6 h increased hepatic triglyceride concentrations (Morris et al., 1979). Exposure of guinea-pigs to approximately 11 000 ppm [38 200 mg/m³] dichloromethane for 6 h also increased hepatic triglyceride concentrations, but concomitant exposure to 21 400–24 100 ppm [40 200–45 300 mg/m³] ethanol blocked this effect (Balmer et al., 1976). Continuous exposure of mice by inhalation to 5000 ppm [17 400 mg/m³] dichloromethane caused swelling of the rough endoplasmic reticulum, fatty changes in the liver and necrosis of individual hepatocytes (Weinstein et al., 1972). Slight liver damage was also observed following administration of dichloromethane by gavage (133–665 mg/kg bw) to mice (Condie et al., 1983). In Sprague-Dawley rats, two doses of 1250 mg/kg dichloromethane by gavage 21 and 4 h before killing the animals did not affect serum alanine aminotransferase, or hepatic glutathione or cytochrome P450 content, but increased the hepatic ornithine decarboxylase activity in 3/15 animals (Kitchin & Brown, 1989). In a long-term bioassay of dichloromethane, increased incidences of haemosiderosis, cytomegaly, cytoplasmic vacuolation, necrosis, granulomatous inflammation and bile-duct fibrosis were observed in the livers of treated male and female Fischer 344/N rats (United States National Toxicology Program, 1986). Increased liver weight associated with glycogen accumulation in the hepatocytes, but no hepatotoxicity, was observed in another carcinogenicity study in mice, in which an elevated frequency of hepatic tumours was observed (Kari et al., 1993). The proportion of S-phase cells was frequently higher in altered foci than in cells from the areas of the liver with normal architecture, but similar to that of the altered foci from non-treated animals (Foley et al., 1993). Administration of dichloromethane to B6C3F₁ mice by gavage (1000 mg/kg, once) or inhalation (4000 ppm [13 900 mg/m³] dichloromethane for 2 h) did not induce DNA synthesis, as measured by the number of cells in S-phase ([³H]thymidine incorporation) (Lefevre & Ashby, 1989) and, when female B6C3F₁ mice were exposed to 1000, 2000, 4000 or 8000 ppm [3470, 6940, 13 900 or 27 800 mg/m³] dichloromethane for 6 h per day on five days per week for up to four weeks, followed by a recovery period of 1–2 weeks (Foley et al., 1993), the hepatocyte labelling index was mostly decreased. There were, however, transient increases in the labelling index in the 4000- and 8000-ppm groups at two weeks and in the 1000-ppm group at one week. The labelling index of bronchiolar epithelium (in two branches proximal to the terminal bronchiole and in the terminal bronchioles themselves) of female B6C3F₁ mice exposed to 2000 ppm dichloromethane for 2–26 weeks decreased to 40–60% of the value found in unexposed control mice. Exposure to 8000 ppm dichloromethane led to a smaller decrease in labelling index. No pathological change was found in the exposed lungs (Kanno et al., 1993).

Inhalation exposure of male B6C3F₁ mice to dichloromethane (6 h, once) led to vacuolation of bronchiolar cells at exposure levels ≥ 2000 ppm [6940 mg/m³], while no effect was observed at levels ≤ 1000 ppm [3470 mg/m³] (Foster et al., 1994). Pretreatment with the cytochrome P450 inhibitor piperonyl butoxide (300 mg/kg intraperitoneally) 1 h before the exposure practically abolished the toxic effect upon bronchiolar cells, while buthionine sulfoximine (1 g/kg intraperitoneally), which decreased the pulmonary glutathione content by 50%, had no such protective effect. In Clara cells isolated after exposure to dichloromethane exposure (≥ 1000 ppm), the proportion of cells in the S-phase was increased.

Following intraperitoneal administration of dichloromethane at near-lethal doses, hydropic degeneration was observed in the kidneys of mice (Klaassen & Plaa, 1966), while no kidney damage was observed following administration of dichloromethane by gavage at dose levels of 133–665 mg/kg bw (Condie et al., 1983). Slight calcification of the renal tubules in dogs was seen after intraperitoneal administration of dichloromethane at near-lethal doses (Klaassen & Plaa, 1967). In rats, intraperitoneal administration of 1330 mg/kg bw dichloromethane produced renal proximal tubular swelling (Kluwe et al., 1982). After a similar dose by gavage, a transient elevation of blood urea nitrogen and decreased urine output, coinciding with cloudy swelling of tubular cells, were observed (Marzotko & Pankow, 1988). Urinary flow was already decreased at the lowest dose tested, 3.1 mmol/kg bw (263 mg/kg bw).

In gerbils exposed continuously by inhalation to 350 ppm [1210 mg/m³], but not in those exposed to 210 ppm [730 mg/m³], dichloromethane for up to three months, increased brain concentrations of two astroglial proteins (S-100 and GFA) and decreased cerebellar DNA concentrations were observed. Decreased hippocampal DNA concentrations were observed at both exposure levels (Rosengren et al., 1986).

Dichloromethane (≥ 6.3 mmol/kg bw) administered to rats by gavage induced increased urinary excretion of catecholamines in the urine in rats; cytomorphological changes and a decrease in chromaffin reaction were observed in the adrenal medulla at a dose level of 15.6 mmol/kg bw (1330 mg/kg) (Marzotko & Pankow, 1987).

In a two-year carcinogenicity test of inhaled dichloromethane, an increased incidence of testicular atrophy was observed in B6C3F₁ mice exposed to 4000 ppm [13 900 mg/m³] for 6 h per day on five days a week (United States National Toxicology Program, 1986).

4.3.1. Humans

A case–control study on 44 women who had had a spontaneous abortion was performed within a cohort of female workers employed in Finnish pharmaceutical factories during 1973 or 1975–80. Three controls matched for age at conception within 2.5 years were chosen for each case (except two). Information about pregnancy outcome was collected from hospital data, and data on exposures from health personnel at the factories. The odds ratio for dichloromethane exposure, based on 11 exposed cases, was 2.3 (95% CI, 1.0–5.7); the odds ratio was also increased for exposure to many other solvents. The odds ratio for those exposed once a week or more during the first trimester of pregnancy was 2.8, and that for those exposed less often was 2.0 (Taskinen et al., 1986).

4.3.2. Experimental systems

In a teratology study, groups of Swiss Webster mice and Sprague-Dawley rats were exposed by inhalation to 0 or 1225 ppm [0 or 4250 mg/m³] dichloromethane (purity, 97.9%) for 7 h per day on gestation days 6–15. Exposure of female mice resulted in a significant increase in body weight during and after exposure, while absolute but not relative liver weights were increased in both species. There was no significant increase in visceral anomalies in the fetuses of either species, but skeletal anomalies included decreased incidence of lumbar spurs and delayed ossification of the sternebrae in rats and increased incidence of a single extra sternal ossification centre in mice (Schwetz et al., 1975).

Female Long-Evans rats were exposed to 0 or 4500 ppm [0 or 15 600 mg/m³] dichloromethane (> 97% pure) during either a three-week pregestational period or during the first 17 days of gestation or both. Ten females per group were allowed to give birth, and the offspring were examined for abnormal growth and behaviour. Dams exposed to dichloromethane during gestation had increased absolute and relative liver weights. There was no effect on litter size or viability, but fetal weight was reduced in both groups exposed during gestation. No treatment-related visceral or skeletal abnormality was detected in the fetuses of any exposure group, but a greater proportion of litters exposed during both the pregestational and gestational periods had fetuses with rudimentary lumbar ribs (Hardin & Manson, 1980). No difference in pup birth weight, viability or growth rate was observed, but alterations in spontaneous locomotor activities were seen in all exposure groups. No change was observed in running-wheel activity or acquisition of an avoidance response (Bornschein et al., 1980).

In a two-generation reproduction study (Nitschke et al., 1988), male and female Fischer 344 rats were exposed to 0, 100, 500 or 1500 ppm [0, 347, 1740 or 5200 mg/m³] dichloromethane for 6 h per day on five days per week for 14 weeks and then mated to produce F₁ litters. After weaning, 30 randomly selected pups of each sex and dosage were exposed to dichloromethane for 17 weeks and subsequently mated to produce F₂ litters. Reproductive parameters examined included fertility, litter size, neonatal growth and survival. All adults and selected (10 per group) weanlings were examined for grossly visible lesions. Tissues from the selected weanlings were examined histopathologically. No adverse effects were observed on reproductive parameters, neonatal survival or neonatal growth; there were no gross or histopathological lesions.

4.4.1. Humans

No data were available to the Working Group.

4.4.2. Experimental systems (see Table 8 for references)

Table 8

Genetic and related effects of dichloromethane.

Gene mutations were induced in Salmonella typhimurium strains TA100, TA1535 and TA98 exposed to dichloromethane vapour in a closed chamber with or without the addition of exogenous metabolic activation. Glutathione-deficient strains of TA100 (NG 11 and NG 54) were less responsive to the effects of dichloromethane than were the parent strains. Studies using the liquid plate incorporation assay were negative, with the exception of one study which reported positive results in strain TA1535 transfected with rat θ-class GST 5-5+. Dichloromethane also induced mutation in Escherichia coli and gene conversion and mutation in Saccharomyces cerevisiae. In Drosophila melanogaster it did not induce sex-linked recessive lethal mutations.

Dichloromethane induced DNA–protein cross-links in vitro in hepatocytes of male B6C3F₁ mice but not in hepatocytes of Fischer 344 rats, Syrian hamsters or in human hepatocytes with functional GSTT1 genes. DNA–protein cross-links were also induced in Chinese hamster ovary CHO cells exposed to dichloromethane with or without exogenous metabolic activation. DNA damage was greater, however, in the presence of metabolic activation.

Dichloromethane induced DNA single-strand breaks in AP rat primary hepatocytes and B6C3F₁ mouse hepatocytes and Clara cells, but not in Syrian hamster hepatocytes in vitro. DNA damage was reduced in Clara cells co-treated with buthionine sulfoximine, a glutathione-depleting agent. In one study, DNA single-strand breaks were increased in CHO cells cultured with dichloromethane in the presence, but not in the absence, of an exogenous metabolic activation system.

When tested in Chinese hamster lung V79 cells in the absence of exogenous metabolic activation, dichloromethane did not induce unscheduled DNA synthesis or hprt locus gene mutations but did induce a slight increase in sister chromatid exchange frequencies. It was mutagenic in CHO cells at the hprt locus in one study, in the presence of exogenous metabolic activation, and gave equivocal results in the mouse lymphoma tk^+/− assay in another study. DNA sequence analysis of the hprt mutants of CHO cells treated with dichloromethane indicated that most were GC→AT transitions (4/8), with two GC→CG transversions and two AT→TA transversions. This pattern was more similar to that of 1,2-dibromoethane (ethylene dibromide) (see this volume) (7/9 being GC→AT transitions) than that of formaldehyde, a metabolite of dichloromethane that has been identified in vitro (see Section 4.1), for which all mutations were single-base trans-versions and 5/6 arose from AT base pairs (Graves et al., 1996). Dichloromethane induced chromosomal aberrations in CHO cells in the presence and absence of an exogenous metabolic system in one of two studies, but did not increase sister chromatid exchange frequencies. Virus-infected Fischer rat and Syrian hamster embryo cells were transformed after treatment with dichloromethane in vitro. Neither DNA single-strand breaks nor unscheduled DNA synthesis were induced in human primary hepatocytes or AH fibroblasts, respectively, following dichloromethane treatment. Sister chromatid exchanges were induced in human peripheral blood lymphocyte cultures but only in those from donors lacking GST activity towards methyl bromide. In a single study, dichloromethane induced kinetochore-staining micronuclei (which are indicative of aneuploidy) and kinetochore-negative micronuclei in human MCL-5 cells that stably express cDNA encoding human CYP1A2, CYP2A6, CYP3A4, CYP2E1 and epoxide hydrolase and in h2E1 cells, which contains a cDNA for CYP2E1. AHH-1 cells constitutively expressing CYP1A1 showed neither an increase in total micronucleus frequencies nor kinetochore-staining micronuclei.

DNA–protein cross-links were induced in the liver but not the lung of B6C3F₁/CrlBR mice exposed to dichloromethane. No DNA–protein cross-links were detected in Syrian hamster liver or lung after inhalation. Inhalation exposure of B6C3F₁ mice to dichloromethane induced DNA single-strand breaks in both lung and liver. Prior treatment of the mice with buthionine sulfoximine immediately before dichloromethane exposure reduced the amount of DNA damage to control levels. DNA single-strand breaks were not induced in liver or lung of AP rats but were seen in liver of CD rats treated by gavage. Dichloromethane did not induce unscheduled DNA synthesis in Fischer 344 rat or B6C3F₁ mouse hepatocytes in vivo. In a single study, mice treated with 2000 ppm [6940 mg/m³] dichloromethane for 6 h per day on five days per week for 12 weeks showed an increased sister chromatid exchange frequency in lung cells and an increased frequency of micronuclei in peripheral blood erythrocytes. Exposure to higher concentrations (8000 ppm [27 800 mg/m³] for two weeks) also induced an increase in sister chromatid exchange frequency in peripheral blood erythrocytes. Dichloromethane did not induce sister chromatid exchanges, micronuclei or chromosomal aberrations in bone marrow of mice treated by gavage or intraperitoneal or subcutaneous injection. A small increase in chromosomal aberrations in mouse bone marrow and lung cells was reported in one study following inhalation exposure to 8000 ppm dichloromethane for 6 h per day on five days per week for two weeks. Dichloromethane gave negative results in the rat bone-marrow chromosomal aberration assay. Covalent binding of dichloromethane to DNA was not observed in liver, kidney or lung of rats or mice exposed by inhalation, although metabolic incorporation of ¹⁴C was found in normal deoxyribonucleosides in both species.