3.1.1. Oral administration

Groups of 50–200 male and female B6C3F₁ mice (age, 7 weeks) were given drinking-water containing dichloromethane (purity, 99%) at a dose of 0 (first control group), 0 (second control group), 50, 125, 185, or 250 mg/kg body weight (bw) per day for 104 weeks (Serota et al., 1986a). Two vehicle-control groups were run simultaneously. 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, there was an increased incidence of hepatocellular carcinoma at the highest dose compared with the first control group. A dose-related increase in the incidence of hepatocellular adenoma or carcinoma (combined) was also observed.

Groups of 50 male and 50 female Swiss mice (age, 9 weeks) were given dichloromethane (purity, > 99.9%) at a dose of 100 or 500 mg/kg bw in olive oil by gavage once per day, for 4 or 5 days per week, for 64 weeks (Maltoni et al., 1988). Groups of 60 male and 60 female mice were given olive oil only (vehicle controls). The mice were then kept under observation for their lifespan. Excess mortality was observed in male and female mice exposed to dichloromethane at the lowest and the highest dose. An increase in mortality appeared after week 36 of treatment and led to withdrawal of the treatment at week 64. In mice that died by week 78, the incidence of pulmonary adenoma or adenocarcinoma (combined) was significantly increased in the group at the highest dose. At the end of the experiment, the cumulative incidences of pulmonary adenoma or carcinoma (combined) in males were 5/50, 5/50, and 9/50. No treatment-related increase in the incidence of any tumour type in females, or of any type of tumour other than pulmonary in males was reported. [The Working Group noted the short period of exposure and the high numbers of animals lost due to mortality and thus not available for examination at the end of the experiment.]

3.1.2. Inhalation

Groups of 50 male and 50 female B6C3F₁ mice (age, 8–9 weeks) were exposed to dichloromethane (purity, > 99%) at concentrations of 0, 2000, or 4000 ppm (0, 6940 or 13 900 mg/m³) by whole-body inhalation for 6 hours per day, 5 days per week, for up to 102 weeks and were killed after 104 weeks (NTP, 1986). The final body weights of male mice at the highest dose and of female mice at the lowest and highest dose were 10–17% lower than those of the respective controls. The survival of exposed male rats was comparable to that of the controls. The survival of exposed male mice and of female mice at the highest dose was reduced relative to that of the controls. The incidences of bronchiolo-alveolar adenoma and of bronchiolo-alveolar carcinoma were significantly increased in exposed males and females. The incidence of hepatocellular adenoma was significantly increased in females at the highest dose, and the incidence of hepatocellular carcinoma was significantly increased in males and females at the highest dose.

Groups of 68 female B6C3F₁ mice (age, 8–9 weeks) were given dichloromethane (purity, > 99%) at a concentration of 0 ppm (control) or 2000 ppm [6940 mg/m³] by whole-body inhalation for 6 hours per day on 5 days per week for various lengths of time over a 104-week period (Kari et al., 1993). Only the lung and liver were evaluated histopathologically. Survival was reduced compared with controls in groups exposed to dichloromethane for 52, 78, or 104 weeks. The incidences of bronchiolo-alveolar adenoma, bronchiolo-alveolar carcinoma, and adenoma or carcinoma (combined), and the incidences of hepatocellular adenoma, hepatocellular carcinoma, and adenoma or carcinoma (combined) were significantly increased in all groups in which exposure was begun during the first 26 weeks of the study. [The Working Group noted that statistical analyses were reported only for the combined tumour incidences.]

Groups of 50 male and 49 or 50 female Crj:BDF₁ mice (age, 6 weeks) were exposed to dichloromethane (purity, > 99.9%) at a concentration of 0, 1000, 2000, or 4000 ppm [0, 3470, 6940, or 13 900 mg/m³] by whole-body inhalation for 6 hours per day on 5 days per week for up to 104 weeks (JBRC, 2000a; Aiso et al., 2014). Survival rates and body weights of both males and females exposed to 2000 and 4000 ppm were decreased [no statistical analysis reported]. The incidences of bronchiolo-alveolar carcinoma were significantly increased in exposed males and females. The incidences of bronchiolo-alveolar adenoma or carcinoma (combined) were significantly increased in exposed males and females. The incidences of hepatocellular carcinoma were significantly increased in males and females at the highest dose. The incidence of hepatocellular carcinoma, hepatoblastoma, or hepatocellular adenoma (combined) was significantly increased in exposed males, and the incidence of hepatocellular adenoma or carcinoma (combined) was significantly increased in females at the highest dose. The incidence of liver haemangioma was increased in males at the highest dose. The incidence of liver haemangioma or haemangiosarcoma (combined) was significantly increased in females at the highest dose. In males, the incidence of pheochromocytoma of the adrenal gland was increased with a positive trend. Hyperplasia in the terminal bronchiole of the lung [this lesion may be classified as a preneoplastic lesion capable of developing into bronchiolo-alveolar adenoma and carcinoma] and peripheral vacuolar change in the liver were increased in males and females at 4000 ppm.

3.1.3. Intraperitoneal injection

In a screening assay based on the production of bronchiolo-alveolar adenoma in strain A mice, groups of 20 male mice (age, 6–8 weeks), were given reagent-grade dichloromethane (purity, > 95%; impurities unspecified) at a dose of 0, 160, 400, or 800 mg/kg bw in tricaprylin by intraperitoneal injection three times per week for a total of 16–17 injections (total doses, 2720, 6800, and 12 800 mg/kg bw in the treated groups, respectively) (Theiss et al., 1977). After 24 weeks, 18, 5, and 12 mice were still alive in the three treated groups, respectively; these and 15 out of 20 surviving mice in the vehicle-control group were killed. Lungs were examined for macroscopic nodules. No significant increase was found in the multiplicity of bronchiolo-alveolar adenoma in exposed mice. [The Working Group noted that histopathology was not performed on all of those nodules, and multiplicity was the only type of data reported in this study.]

3.2.1. Oral administration

Groups of 25–85 male and female Fischer 344 rats, (age, 7 weeks) were given drinking-water containing dichloromethane (purity, 99%) at a dose of 0 (control group 1), 0 (control group 2), 5, 50, 125, 250 (highest dose), or 250 (recovery group) mg/kg bw per day for 104 weeks (Serota et al., 1986b). Interim terminations were carried out at 26, 52, and 78 weeks in control group 1 and in the groups at the lowest, intermediate, and highest dose, such that 50 males and 50 females per group received treatment for 104 weeks. There was no significant difference in survival between the exposed and control groups. In females, the incidence of hepatocellular carcinoma after 104 weeks was: 0/85, 0/50, 0/85, 2/83, 0/85, 2/85, and 0/25; the incidence of neoplastic nodules [hepatocellular adenomas] was: 0/85, 0/50, 1/85, 2/83, 1/85, 4/85, and 2/25; and the incidence of neoplastic nodules [hepatocellular adenomas] or hepatocellular carcinoma (combined) was: 0/85, 0/50, 1/85, 4/83, 1/85, 6/85, and 2/25 in the seven groups, respectively. This increasing trend was statistically significant (the recovery group was excluded). In male rats, no increased incidence of hepatocellular tumours was observed at 104 weeks. No other significant increase in tumour incidence was found.

Groups of 50 male and 50 female Sprague-Dawley rats (age, 12 weeks), were given dichloromethane (purity, > 99.9%) at a dose of 100 or 500 mg/kg bw in olive oil by gavage once per day, 4 or 5 days per week, for 64 weeks (Maltoni et al., 1988). A group of 50 males and 50 females was given olive oil only (vehicle controls) and additional groups of 20 males and 26 females were kept untreated (controls). The rats were then kept under observation for their lifespan. Excess mortality was observed in male and female rats given dichloromethane at the highest 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. [The Working Group noted the short period of treatment and the inadequate reporting of the data.]

3.2.2. Inhalation

Groups of approximately 95 male and 95 female Sprague-Dawley rats (age, 8 weeks) were given dichloromethane (purity, > 99%) at a concentration of 0, 500, 1500, or 3500 ppm [0, 1740, 5200, or 12 100 mg/m³] by whole-body inhalation for 6 hours per day, 5 days per week, for 104 weeks (Burek et al., 1984; EPA, 1985). The numbers of rats per group still alive at the end of the study were 14, 14, 6, 7 for males, and 21, 24, 13, 4 for females, respectively. From the 18th month onwards, the mortality among females at the highest dose was significantly increased. There was no significant increase in the incidence of benign or malignant tumours of the mammary gland; however, the total number of benign tumours of the mammary gland [type not specified] showed a small dose-related increase in males, and a dose-related increase in females [statistics not reported]. The incidence of sarcoma located around the salivary glands was increased in males at the highest dose (1/92, 0/95, 5/95, and 11/97). [The Working Group noted the reported occurrence of sialodacryoadenitis of the salivary gland early in the study. The effect of this viral infection on tumour formation is unknown.]

Groups of 50 male and 50 female Fischer 344/N rats (age, 7–8 weeks) were exposed to dichloromethane (purity, 99%) at a concentration of 0, 1000, 2000, or 4000 ppm (0, 3470, 6940, or 13 900 mg/m³) by whole-body inhalation for 6 hours per day, 5 days per week, for 102 weeks and were killed after 104 weeks (NTP, 1986). Mean body weights of control and dosed males and females were similar throughout the study. Survival of treated males was similar to that of controls. Survival at termination of the study was reduced in females at the highest dose compared with controls. Significantly increased incidences of benign tumours of the mammary gland (all fibroadenoma, except for one adenoma in the group at the highest dose) were observed in treated females (5/50, 11/50, 13/50, 23/50). In males, there was a positive trend in the incidences of adenoma or fibroadenoma (combined) of the mammary gland, and of fibroma or sarcoma (combined) of the subcutis. There was no difference in the distribution of other types of tumours in the control and treated groups.

Groups of 54–70 male and female Sprague-Dawley rats (age, 13 weeks), were given dichloromethane (purity, > 99.9%) at a concentration of 100 ppm [347 mg/m³] or 60 ppm [208 mg/m³] by whole-body inhalation for 7 hours per day, 5 days per week (Maltoni et al., 1988). The exposure was started in female breeders, and male and female offspring (12-day embryos). The breeders and a first group of offspring were exposed for 104 weeks, and a second group of offspring was exposed for 15 weeks only. Control groups were composed of 60 female rats (untreated breeders controls), and 158 males and 149 females (untreated offspring controls). The rats 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. [The Working Group noted the low concentration of exposure.]

Groups of 90 male and 108 female Sprague-Dawley rats [age unspecified] were given dichloromethane (technical-grade; purity, > 99.5%) at a concentration of 0, 50, 200, or 500 ppm [0, 174, 694, or 1740 mg/m³] by whole-body inhalation for 6 hours per day, 5 days per week, for 20 (males) or 24 (females) months (Nitschke et al., 1988). An additional group of 30 female rats was exposed to dichloromethane at 500 ppm for the first 12 months and to room air for the last 12 months of the study. An additional group of 30 female rats was exposed to room air for the first 12 months, followed by dichloromethane at 500 ppm for the last 12 months of the study. Subgroups of five rats per sex per exposure level were scheduled for interim termination after 6, 12, 15, or 18 months of exposure to dichloromethane. No exposure-related adverse effect on body weight or mortality was observed. In females, the incidences of benign tumours of the mammary gland (adenomas and fibroadenomas, combined) were 52/70, 58/70, 61/70 [significant increase], and 55/70 in the control group, and the groups at the lowest, intermediate, and highest dose, respectively. No significant increase in the incidence of any other tumour type was seen in the exposed groups. There was no significant increase in the incidence of any tumour type in males.

Groups of 50 male and 50 female F344/DuCrj rats (age, 6 weeks) were exposed to dichloromethane (purity, 99.9%) at concentrations of 0, 1000, 2000, or 4000 ppm (0, 3470, 6940, or 13 900 mg/m³) by whole-body inhalation for 6 hours per day, 5 days per week, for 104 weeks (JBRC, 2000b; Aiso et al., 2014). Survival rates of females exposed to dichloromethane at 4000 ppm were decreased compared with the controls [no statistical analysis reported]. The incidence of fibroma of the subcutis was significantly increased in exposed males. The incidence of fibroadenoma of the mammary gland was significantly increased in males at the highest dose and with a positive trend in females. The incidence of peritoneal mesothelioma was significantly increased with a positive trend in males.