(a) Humans

Dichloromethane is a lipophilic solvent of low relative molecular mass, which can readily cross biological membranes. Pulmonary uptake is rapid, approaching steady state within a few hours after the start of exposure (Riley et al., 1966; DiVincenzo et al., 1971, 1972; Astrand et al., 1975; DiVincenzo & Kaplan, 1981). Measured values of pulmonary uptake are about 55–75% at rest and 30–40% during physical exercise (Astrand et al., 1975; DiVincenzo & Kaplan, 1981). The blood:air partition coefficient for dichloromethane describes the ratio of the concentrations in the two media at steady state, and is a factor in determining pulmonary uptake. The partition coefficient has been measured in vitro using vial equilibrium methods. Mean reported values range from around 8 to 10 for humans (Sato & Nakajima, 1979; Gargas et al., 1989; Meulenberg & Vijverberg, 2000). However, these data might have been influenced by the presence of glutathione S-transferase T1 (GSTT1) in human erythrocytes (Schröder et al., 1996).

Data on oral absorption in humans are limited to case reports of accidental ingestion, and suggest that dichloromethane is also readily absorbed by this route of exposure (Hughes & Tracey, 1993; Vetro et al., 2012). Quantitative estimates of oral bioavailability in humans are not available because the ingested amounts are not known precisely.

Ursin et al. (1995) report that the permeability of human skin to dichloromethane is 24 g/m² per hour. No other information on human dermal absorption of dichloromethane was available to the Working Group.

(b) Experimental systems

Inhalation studies in experimental animals provide clear evidence that dichloromethane is readily absorbed via the lungs into the systemic circulation (Carlsson & Hultengren, 1975; Anders & Sunram, 1982; McKenna et al., 1982; Andersen et al., 1991). The blood:air partition coefficient for dichloromethane, measured in vitro using vial equilibrium methods, has been reported to range from 19 to 23 for rodents (Gargas et al., 1989; Marino et al., 2006).

Absorption from the gut after oral doses is rapid and nearly complete, according to reports of several studies with radiolabel in mice and rats (McKenna & Zempel, 1981; Angelo et al., 1986a, b). For instance, Angelo et al. (1986b) reported that on average 97% of the radiolabel was recovered in expired air as dichloromethane, CO, and carbon dioxide (CO₂) in the 24 hours after each repeated oral dose of 50 or 200 mg/kg per day in rats. Angelo et al. (1986a) reported absorption in mice to be more rapid (but equally extensive) with an aqueous vehicle than with an oil-based vehicle, consistent with studies on other chlorinated solvents.

No studies of dermal uptake of dichloromethane in experimental animals were available to the Working Group.

(a) Humans

Once absorbed, dichloromethane enters blood circulation and undergoes rapid systemic distribution to tissues. The highest concentrations are expected in adipose tissue and other fatty tissues, due to the lipophilicity of the compound. Engström & Bjurström (1977) detected dichloromethane in fat biopsy specimens obtained from men exposed to dichloromethane for 1 hour during light exercise. Other data in humans on tissue distribution in vivo are limited to tissues taken from autopsies after accidental fatalities, which showed wide systemic distribution in blood and across all tested tissues, including the fat, lung, liver, heart, kidney, spleen, and brain (Moskowitz & Shapiro, 1952; Winek et al., 1981; Shinomiya & Shinomiya, 1985; Manno et al., 1989; Leikin et al., 1990; Kim et al., 1996; Goullé et al., 1999). Goullé et al. (1999) and Leikin et al. (1990) measured the largest number of tissues, and found the highest concentrations in brain, spleen, and fat.

Engström & Bjurström (1977) also measured an in-vitro partition coefficient of 51 between adipose tissue and air using a vial equilibrium method. This value is about five times the blood:air partition coefficient, consistent with the lipophilicity of dichloromethane. Partition-coefficient measurements for other human tissues were not available to the Working Group.

(b) Experimental systems

Studies in experimental animals provide clear evidence that dichloromethane distributes widely to all tissues of the body. After in-vivo oral and/or intravenous exposures in mice and/or rats, dichloromethane has been measured in the liver, kidney, lung, and whole carcass, with the highest concentrations in the liver (Angelo et al., 1986a, b). Several inhalation experiments with radiolabeled dichloromethane detected the presence of radiolabel in all tissues, including the liver, kidney, adrenals, brain, fat, lung, muscle, and testes (Carlsson & Hultengren, 1975; McKenna et al., 1982). While part of the radiolabel is likely to be metabolites, it is likely that a substantial portion also represents dichloromethane. Experiments in animals show that dichloromethane readily crosses the blood–brain barrier and the placenta (Savolainen et al., 1981; Anders & Sunram, 1982).

Tissue:air partition coefficients have also been measured in vitro for several tissues in rats and mice, including fat, liver, muscle, skin, kidney, and brain (Andersen et al., 1987; Gargas et al., 1989; Clewell et al., 1993). The highest reported values are for fat (60–120), with values for the remaining tissues ranging from 8 to 40, as compared with blood:air partition coefficients of around 20.

(a) Overview

The pathways for metabolism of dichloromethane were initially characterized nearly 40 years ago in the mid-1970s and are widely considered to be well established (Kubic & Anders, 1975, 1978; Ahmed & Anders, 1976, 1978). Dichloromethane is metabolized by either of two pathways, as summarized in Fig. 4.1.

Fig. 4.1

Pathways for the metabolism of dichloromethane

One pathway, a reductive dehalogenation that is a mixed-function oxygenation, was subsequently shown to be catalysed by cytochrome P450 2E1 (CYP2E1) (Guengerich et al., 1991), and ultimately generates CO and CO₂ as stable end products. The initial product of the reaction, chloromethanol, spontaneously rearranges to form formyl chloride, which is reactive and can spontaneously generate CO or react with nucleophiles such as glutathione (GSH) to generate formylglutathione; the latter rearranges to release CO₂. CO avidly reacts with haemoglobin, displacing oxygen and forming COHb.

The other pathway for dichloromethane metabolism involves conjugation with GSH, forming S-chloromethyl GSH. The conjugation is catalysed by GSTs, with the GSTT1 isoform being the most active (Mainwaring et al., 1996; Sherratt et al., 1997). S-Chloromethyl GSH is reactive and is believed to be one of the dichloromethane metabolites responsible for DNA binding and mutagenicity (Graves & Green, 1996). Alternatively, S-chloromethyl GSH can be hydrolysed to form hydroxymethyl GSH, which can either decompose to release formaldehyde or be oxidized by formaldehyde dehydrogenase to form S-formyl GSH. The latter is subsequently hydrolysed to release formic acid and GSH. Formic acid further decomposes to release CO₂. Thus, while both the CYP and GST pathways can generate CO₂, only the CYP pathway produces CO from dichloromethane. Although both pathways can generate reactive and unstable metabolites that are mechanistically linked to dichloromethane-induced genotoxicity and carcinogenesis, it is thought that these come primarily from the GST pathway (Andersen et al., 1987).

Despite the wealth of data over more than three decades from in-vivo and in-vitro studies in humans and experimental animals, which supports the function of both CYP2E1 and GSTT in dichloromethane metabolism, Evans & Caldwell (2010a) proposed a different explanation for dichloromethane metabolism that involves only CYP2E1. As precedent for this alternative metabolic pathway, the authors cited studies by Harrelson and colleagues (Harrelson et al., 2007, 2008) and Tracy (2006) and two studies by Guengerich and colleagues (Watanabe & Guengerich, 2006; Watanabe et al., 2007). Their conclusion was that the available data support a limited role for GST-dependent metabolism. Anders et al. (2010) criticized this proposal by noting that the authors misinterpreted the data from Watanabe & Guengerich (2006) and Watanabe et al. (2007), for the limited in-vitro data supporting the alternative mechanism, for dismissing the wealth of data on the role of GSTT in dichloromethane metabolism, mutagenicity, and carcinogenicity, and for rejecting without any sound basis the several well-established and validated physiologically based pharmacokinetic models of dichloromethane metabolism in humans and rodents. Although Evans & Caldwell (2010b) maintained the validity of their interpretations, no data supporting metabolism of dichloromethane that exclude GST, particularly at higher dichloromethane concentrations, were identified by the Working Group.

Specific studies on dichloromethane metabolism and mechanisms in humans and human-derived tissues and in experimental systems are summarized below.

(b) Humans or human-derived tissues

Oxidative metabolism of dichloromethane to CO was first demonstrated in occupationally exposed humans (Stewart et al., 1972a, b; Ratney et al., 1974; Astrand et al., 1975). DiVincenzo & Kaplan (1981) measured dichloromethane metabolism using COHb in nonsmoking volunteers exposed to dichloromethane vapour at concentrations of up to 200 ppm for 7.5 hours (once, or daily for 5 days). Dose-dependent COHb formation was readily demonstrated, with the single-day exposures resulting in peak CoHb saturations of 1.9%, 3.4%, 5.3%, and 6.8%, respectively, at 0, 50, 100, and 200 ppm. A comparative study of the effects in humans exposed to either CO or dichloromethane up to concentrations that produce 5% COHb saturation was performed; both substances impaired performance (Putz et al., 1979), this was consistent with evidence that about 70% of dichloromethane at relatively low doses is metabolized to CO (Andersen et al., 1991).

Metabolic parameter estimates made by Clewell (1995) show that the oxidative pathway in human liver has a capacity of 100- to 200-fold that of the GST pathway, although in-vitro studies by Reitz et al. (1989) generally showed a much more modest difference in capacity of the two pathways, with the CYP pathway having a two- to fourfold higher capacity than the GST pathway in most of the human liver samples studied.

Bogaards et al. (1993) measured GST activity with dichloromethane and 1-chloro-2,4-dinitrobenzene (CDNB) in nine human liver cytosol samples, finding three distinct activity groups. Specifically, with dichloromethane, two exhibited no detectable activity, four exhibited relatively low activity (0.2–0.4 nmol/min per mg protein), and three exhibited relatively high activity (0.9–1.1 nmol/min per mg protein). Interestingly, although metabolic activity with CDNB as substrate also exhibited an approximately fivefold variation among the nine samples, there were no apparent null variants and the pattern of metabolism with CDNB and dichloromethane did not coincide. While CDNB is a substrate for multiple GST isoforms (Habig et al., 1974), it is now widely accepted that dichloromethane is selectively metabolized by GSTT (see below).

Mainwaring et al. (1996) determined mRNA and protein expression of GSTT1 in cells from human liver and lung, both of which are target organs for dichloromethane in the mouse. While expression of GSTT1 was readily detected in the liver, very low levels were detected in the lungs. Furthermore, GSTT1 activity with dichloromethane was measured in three samples of lung at 0.06, 0.21, and 0.23 nmol/min per mg protein, which was about one order of magnitude less than that in human liver.

Casanova et al. (1997) detected RNA–formaldehyde adducts in human hepatocytes with functional GST genes and incubated with dichloromethane, which is evidence that formaldehyde is formed in human cells as a metabolite of dichloromethane.

GST activity in human liver was further related to carcinogenic risk with dichloromethane in studies of GSTT1 polymorphism (El-Masri et al., 1999; Sherratt et al., 2002; Olvera-Bello et al., 2010). Although the importance of genetic polymorphisms in determining carcinogenic risk is discussed elsewhere (see Section 4.5.1), it is mentioned here as providing further evidence of the presence and importance of GST activity in dichloromethane metabolism.

In addition to absolute levels of GSTT protein expression in target organs, another important issue is the subcellular localization of the expressed enzyme. While GSTT11 in mouse liver is readily found in cytoplasm and nuclei of hepatocytes, it is found at lower levels in nuclei of bile-duct epithelial cells, and in cytoplasm and nuclei of some human hepatocytes (Sherratt et al., 2002). This less intense nuclear localization is thought to be of significance for carcinogenic risk because less S-chloromethyl GSH and formaldehyde will be generated near DNA.

GST is also present in human erythrocytes and is thought to play a role in toxicity of dichloromethane in lymphocytes (Hallier et al., 1993, 1994). Erythrocyte GSTT is polymorphic, as further discussed in Section 4.5.

(c) Experimental systems

(ii) Mouse

Reitz and colleagues analysed dichloromethane metabolism by the CYP and GST pathways in the liver and lung of male B6C3F₁ mice, F344 rats, Syrian golden hamsters, and humans (Table 4.1) (Reitz et al., 1988). Several striking species-dependent differences are clearly evident from the data. First, mice exhibit similar rates of CYP-dependent metabolism in liver as hamsters and nearly threefold higher rates than rats or humans. Second, in lung tissue CYP-dependent metabolism of dichloromethane in mice was ~30-fold higher than in rats and ~5-fold higher than in hamsters. No CYP-dependent metabolism was detected in the human lung sample. Third, even greater species-dependent differences in addition to interindividual differences were observed in the liver and lung for GST-dependent dichloromethane metabolism. In this case, rates of GSH conjugation in mouse liver were ~4-fold faster than in rats, ~20-fold faster than in hamsters, and ~10-fold faster than in humans. Finally, perhaps the greatest species-dependent metabolic difference was observed for GST metabolism in the lung. Here, rates in mice were ~7-fold faster than in rats and ~20-fold faster than in humans. These metabolic differences have been interpreted to explain species-dependent differences as well as interindividual differences in target-organ specificity and sensitivity to dichloromethane-induced mutagenesis and carcinogenicity (Green, 1990; Starr et al., 2006). Furthermore, data on tumour incidence across species show a correlation with the amount of dichloromethane metabolized by GST but not by CYP (Andersen et al., 1987).

Table 4.1

Reaction rates for dichloromethane metabolism by CYP and GST in liver and lung tissue from different species.

Ottenwälder et al. (1989) gave two specific CYP inhibitors (i.e. pyrazole, 320 mg/kg, and diethyldithiocarbamate, 300 mg/kg) to male B6C3F₁ mice also exposed to dichloromethane at 1000 or 3000 ppm, or a mixture of dichloromethane at 1000 ppm and methyl chloride at 1000 ppm. For those mice given only dichloromethane, uptake by inhalation was markedly decreased by the CYP inhibitors. In contrast, CYP inhibitors had no effect on the uptake of methyl chloride by inhalation. Because methyl chloride is metabolized solely by GSTs, these results showed that even at relatively high exposures, dichloromethane is predominantly metabolized by CYP. These results contrasted with those of Andersen et al. (1987) described above, who concluded that GST-dependent, rather than CYP-dependent, metabolism was critical for dichloromethane-induced liver tumorigenesis.

The in-vivo metabolism of dichloromethane by CYP was further demonstrated by Casanova et al. (1992), who pre-exposed male B6C3F₁ mice to dichloromethane at 4000 ppm for 6 hours per day for 2 days, and then on day 3 to ¹⁴C-labelled dichloromethane at a declining concentration (4500–2500 ppm). DNA–protein cross-links and incorporation of ¹⁴C derived from dichloromethane into DNA was observed in the liver of these mice.

Foster et al. (1994) also showed that modulation of pulmonary CYP activity can also alter responses of the lung to dichloromethane.

(a) Humans

In humans, the main route of excretion of dichloromethane is by exhalation of the parent compound and its primary metabolites CO₂ and CO, with lesser amounts as dichloromethane excreted in the urine (DiVincenzo et al., 1971, 1972; DiVincenzo & Kaplan, 1981). DiVincenzo & Kaplan (1981) estimated that only 5% of absorbed dichloromethane is exhaled unchanged, 25–34% excreted converted as CO, and the balance excreted as CO₂. After cessation of exposure, the half-life of dichloromethane in the blood has been estimated to be about 40 minutes, with concentrations of parent and metabolites returning the preexposure levels within a few days (DiVincenzo et al., 1972; DiVincenzo & Kaplan, 1981). Urinary excretion occurs mostly during and/or within the first hour after cessation of exposure, and in total accounts for less than 0.1% of uptake (DiVincenzo et al., 1971, 1972).

(b) Experimental systems

As in humans, the main route of excretion of dichloromethane in experimental animals is by exhalation of the parent compound and its primary metabolites CO₂ and CO, with lesser amounts excreted in the urine. As exposure levels increase, the percentage excreted as unchanged parent compound increases, reflecting saturation of metabolism. For instance, McKenna et al. (1982) reported that in rats exposed to dichloromethane at 50 ppm via inhalation, elimination in expired air consists of about 5% parent compound, and 26% and 27% CO₂ and CO, respectively. At 500 and 1500 ppm, elimination of parent compound increased to 30% and 55%, respectively, with declines in the amount of CO₂ and CO expired. Similarly, for oral doses of 1 mg/kg, McKenna & Zempel (1981) reported that rats exhaled 12% of the administered dose as parent compound, and 35% and 31% as CO₂ and CO, respectively. At higher oral doses (50 mg/kg or greater), rats and mice exhale greater amounts as parent compound (60–80%), and lesser amounts as CO₂ and CO (McKenna & Zempel, 1981; Angelo et al., 1986a, b).

Overall, experimental studies in rodents have found that > 90% of absorbed dichloromethane is eliminated within 24 or 48 hours of exposure, regardless of dose. McKenna et al. (1982) reported that after inhalation exposure in rats, a low percentage of the initial body burden of dichloromethane remained at 48 hours. After a single intravenous dose in mice, Angelo et al. (1986a) reported 92–94% recovery within 4 hours after dosing. After repeated oral exposures in mice, Angelo et al. (1986a) reported 90–96% recovery of within 24 hours after each dose.

(a) In vivo

No data were available to the Working Group.

(b) In vitro

See Table 4.2

Table 4.2

Studies of genotoxicity with dichloromethane in human cell lines in vitro.

Dichloromethane did not induce DNA single-strand breaks in human primary hepatocytes (Graves et al., 1995). There was no induction of DNA–protein cross-links in vitro in human hepatocytes with functional GSTT1 genes (Casanova et al., 1997) or unscheduled DNA synthesis in AH fibroblasts (Jongen et al., 1981) after treatment with dichloromethane in vitro.

In a study by Doherty et al. (1996), dichloromethane induced the formation of 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. The increased frequency of micronucleus formation is combined with the fact that MCL-5 and h2El cell lines showed the capacity to produce metabolites in the presence of dichloromethane. AHH-1 cells, constitutively expressing CYP1A1, showed no increase in the total frequency of micronucleus formation or in the frequency of kinetochore-staining micronuclei.

Hallier et al. (1993) showed that sister-chromatid exchanges were induced in human peripheral blood lymphocyte cultures from non-conjugator donors lacking GST activity, but not in those from conjugators. This study did not provide details on the type of GST activity that was monitored. Sister-chromatid exchanges were also induced by dichloromethane in vitro in human peripheral blood mononuclear cells (Olvera-Bello et al., 2010). This study also demonstrated that the group with high GSTT1 activity showed a larger increase in the frequency of sister-chromatid exchanges induced by dichloromethane than did the groups with low and medium GSTT1 activity.

(a) Mammalian systems

See Tables 4.3 and 4.4

Table 4.3

Studies of genotoxicity with dichloromethane in mammalian systems in vivo.

Table 4.4

Studies of genotoxicity with dichloromethane in mammalian systems in vitro.

(b) Bacterial and other systems

See Table 4.5

Table 4.5

Studies of genotoxicity with dichloromethane in non-mammalian systems in vitro.

(a) Humans

Temporary neurobehavioural effects have been reported (Putz et al., 1979; Winneke, 1981), or not (Gamberale et al., 1975) after exposure to dichloromethane at doses as low as 200 ppm [694 mg/m³]. Cerebral damage after exposure to dichloromethane has been reported (Barrowcliff & Knell, 1979).

(b) Experimental systems

Increase in concentrations of astroglial proteins S-100 and glial fibrillary acidic protein was found in the frontal and sensory motor cerebral cortex of gerbils exposed to dichloromethane at 210 or 350 ppm for 3 months (Rosengren et al., 1986). DNA concentration was also measured as a possible index of astroglial proliferation. DNA concentration was not increased in the frontal and sensory motor cerebral cortex, but was decreased in the hippocampus at 210 and 350 ppm, and in the cerebellar hemispheres (Rosengren et al., 1986).

(a) Humans

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., 1983)

(b) Experimental systems

A 2-year study of exposure to dichloromethane by inhalation in F344 rats reported that the incidence of some non-neoplastic liver lesions was significantly elevated in response to treatment when compared with concurrent controls (NTP, 1986). These liver lesions were haemosiderosis, focal necrosis, cytoplasmic vacuolization, and bile duct fibrosis in males, and focal granulomatous inflammation, haemosiderosis and cytoplasmic vacuolization in females. In the same study, liver cytological degeneration was observed in female B6C3F₁ mice.

A 2-year study of exposure to dichloromethane by inhalation in F344 rats reported that the incidence some non-neoplastic liver lesions (acidophilic, basophilic and vacuolated cell foci in males) was significantly elevated in response to treatment when compared with controls (JISHA, 2000a). In the same study, liver granulation and peripheral vacuolation were observed in male and female BDF1 mice.

Increased liver weight associated with glycogen accumulation in the hepatocytes, but no hepatotoxicity, was observed in another study of carcinogenicity in mice, in which an elevated incidence of hepatic tumours was observed (Kari et al., 1993). An experiment in female B6C3F₁ mice showed that 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 in the altered foci from non-treated mice (Foley et al., 1993). Administration of dichloromethane to B6C3F₁ mice by gavage (1000 mg/kg, single dose) or inhalation (4000 ppm [13 900 mg/m³] dichloromethane for 2 hours) did not induce DNA synthesis, as measured by the number of cells in S-phase ([³H]thymidine incorporation) (Lefevre & Ashby, 1989). When female B6C3F₁ mice were exposed to dichloromethane at 1000, 2000, 4000, or 8000 ppm [3470, 6940, 13 900 or 27 800 mg/m³] for 6 hours per day, 5 days per week, for up to 4 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 groups at 4000 and 8000 ppm at 2 weeks and in the group at 1000 ppm at 1 week.

In Sprague-Dawley rats, two doses of dichloromethane at 1250 mg/kg given by gavage for 4 and 21 hours, there was no effect on serum alanine aminotransferase levels, or hepatic GSH or CYP content, but hepatic ornithine decarboxylase activity increased in 3 out of 15 rats (Kitchin & Brown, 1989).

Hepatotoxic effects were seen after exposure to near-lethal concentrations of dichloromethane in mice (Gehring, 1968). Continuous exposure of mice to dichloromethane at 5000 ppm [17 400 mg/m³] by inhalation 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 after administration of dichloromethane (133–665 mg/kg bw) by gavage in mice (Condie et al., 1983).

Exposure of guinea-pigs to dichloromethane at 5200 ppm [18 000 mg/m³] by inhalation for 6 hours increased hepatic concentrations of triglyceride (Morris et al., 1979). Exposure of guinea-pigs to dichloromethane at approximately 11 000 ppm [38 200 mg/m³] for 6 hours also increased hepatic concentrations of triglyceride, but concomitant exposure to ethanol at 21 400–24 100 ppm [40 200–45 300 mg/m³] blocked this effect (Balmer et al., 1976).

(a) Humans

Of four epidemiological studies on mortality from cardiovascular disease, two studies showed increased mortality from ischaemic heart disease in workers exposed to dichloromethane, compared with an internal reference group or a non-exposed cohort, although mortality did not increase compared with the general population (Tomenson et al., 1997; Tomenson, 2011).

(b) Experimental systems

No data were available to the Working Group.

(a) Humans

No data were available to the Working Group.

(b) Experimental systems

Nasal cavity lesions of olfactory epithelium and hyperplasia of the terminal bronchiole have been reported in male and female BDF1 mice in a 2-year study of exposure to dichloromethane by inhalation (JISHA, 2000b). The incidence of eosinophilic changes in the respiratory epithelium was also elevated in female mice in this study.

F344/N rats were exposed to dichoromethane at a concentration of 0, 1000, 2000, or 4000 ppm by inhalation for 6 hours per day, 5 days per week, for 102 weeks. Squamous metaplasia of the nasal cavity was observed as a treatment-related non-neoplastic change in rats (Mennear et al., 1988).

The labelling index in bronchiolar epithelium (in two branches proximal to the terminal bronchiole and in the terminal bronchioles themselves) in female B6C3F₁ mice exposed to dichloromethane at 2000 ppm for 2–26 weeks decreased to 40–60% of the value for control mice. Exposure to dichloromethane at 8000 ppm led to a smaller decrease in labelling index. No pathological changes were found in the exposed lungs (Kanno et al., 1993). In male B6C3F₁ mice exposed to dichloromethane by inhalation (6 hours, single dose), vacuolation of bronchiolar cells was observed 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 CYP inhibitor piperonyl butoxide (300 mg/kg, administered intraperitoneally) 1 hour before exposure abolished the toxic effect in bronchiolar cells, while buthionine sulfoximine (1 g/kg, administered intraperitoneally), which decreased the pulmonary GSH content by 50%, had no protective effect. In Clara cells isolated after exposure to dichloromethane (≥ 1000 ppm), the proportion of cells in S-phase was increased.

(a) Humans

No data were available to the Working Group.

(b) Experimental systems

In a 2-year study in female F344 rats exposed to dichloromethane by inhalation, kidney tubular degeneration was reported to be significantly elevated in response to treatment when compared with controls (NTP, 1986). In the same study, kidney tubule casts were observed in male and female B6C3F₁ mice.

In a 2-year study in female F344 rats exposed to dichloromethane by inhalation, the incidence of chronic nephropathy was significantly elevated in response to treatment when compared with controls (JISHA, 2000a). In a study in similarly exposed BDF1 mice, basophilic change, lymphocytic infiltration and proximal tubule vacuolation were observed (JISHA, 2000b).

After intraperitoneal administration of dichloromethane at near-lethal doses, hydropic degeneration was observed in the mouse kidney (Klaassen & Plaa, 1966), no kidney damage was observed after administration of dichloromethane at doses of 133–665 mg/kg bw by gavage (Condie et al., 1983). Slight calcification of the renal tubules in mongrel dogs was seen after intraperitoneal administration of dichloromethane at near-lethal doses (Klaassen & Plaa, 1967).

In rats, intraperitoneal administration of dichloromethane at 1330 mg/kg bw produced renal proximal tubular swelling (Kluwe et al., 1982). After a similar dose administered by gavage, a transient elevation in blood urea nitrogen levels 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 F344/N rats exposed to dichloromethane at 0, 1000, 2000, or 4000 ppm by inhalation, for 6 hours per day, 5 days per week, for 102 weeks, treatment-related degeneration of kidney tubules was reported (Mennear et al., 1988).

(a) Humans

No data were available to the Working Group.

(b) Experimental systems

In F344/N rats were exposed by inhalation to dichloromethane at 0, 1000, 2000, or 4000 ppm, for 6 hours per day, 5 days per week, for 102 weeks, fibrosis of the spleen was observed as a treatment-related non-neoplastic change (Mennear et al., 1988).

(a) CYP2E1

The association between exposure to organic solvents including dichloromethane and NHL was investigated in relation to different genetic variations in four metabolic genes – CYP2E1, microsomal epoxide hydrolase (EPHX1), myeloperoxidase (MPO), and quinone oxidoreductase (NQO1) – using unconditional logistic regression models based on data collected from women in Connecticut, USA, in 1996–2000 (Barry et al., 2011). Overall associations between total NHL and dichloromethane (OR, 1.69; 95% CI, 1.06–2.69), carbon tetrachloride (OR, 2.33; 95% CI, 1.23–4.40), and methyl chloride (OR, 1.44; 95% CI, 0.94–2.20) were increased among women of genotype TT for rs2070673 in the CYP2E1 gene (dichloromethane: OR, 4.42; 95% CI, 2.03–9.62; P interaction < 0.01; carbon tetrachloride: OR, 5.08; 95% CI, 1.82–14.15; P interaction = 0.04; and methyl chloride: OR, 2.37; 95% CI, 1.24–4.51; P interaction = 0.03). In contrast, no effects of these solvents were observed among women of genotype TA/AA. Similar patterns were observed for dichloromethane and diffuse large B-cell lymphoma, follicular lymphoma, and marginal zone lymphoma (Barry et al., 2011). [The Working Group noted that the functional significance of this polymorphism was unknown.]

(b) GSTT1

GSTT1 polymorphisms may result in interindividual variation in the ability to metabolize dichloromethane by GSH conjugation; some individuals (non-conjugators) completely lack GSH conjugation activity. Because GSH conjugation of dichloromethane leads to formation of reactive and genotoxic metabolites, it is plausible that diminished or lack of GSH conjugation activity will lead to reduced risk of carcinogenesis. For instance, in the absence of GSTT1, exposure to dichloromethane did not lead to formaldehyde production in human erythrocytes (Hallier et al., 1994), and DNA–protein-cross-links were not detected in human liver cells (Casanova et al., 1997). This could be relevant to multiple target tissues that express GSTs, including the liver, kidney, brain, and lung (Sherratt et al., 1997, 2002).

Interindividual variation in the conjugation of dichloromethane with GSH by cytosolic GST in vitro was investigated in 22 samples of human liver (Bogaards et al., 1993). In nine of the liver samples, the α-, mu-, and pi-class GST subunits were quantified. In two of these samples, no activity was observed towards dichloromethane, while α-, mu-, and pi-class subunits were expressed in these human liver cytosolic samples, suggesting no relationship between enzymatic activities and dichloromethane with these classes of GST.

Hallier et al. (1993) found that dichloromethane induced sister-chromatid exchange in the human lymphocytes of non-conjugators donors lacking GST activity, but not in those of conjugators. However, Olvera-Bello et al. (2010) demonstrated that the group with high GSTT1 activity showed a larger increase in the frequency of sister-chromatid exchange induced by dichloromethane than did the groups with low and medium GSTT1 activity.

Garte et al. (2001) showed major and significant differences in the allele and genotypes frequencies between ethnic groups, especially between Asians and Caucasians (Table 4.6).

Table 4.6

Frequencies of GSTT1*0 gene polymorphism in Caucasians and Asians.