dichloromethane; methylene chloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In general dichloromethane induces gene mutations in bacteria, but not in mammalian cells in vitro, whereas dichloromethane is clastogenic in vitro at high concentrations.

Genetic toxicity in vivo

Description of key information

From the large number of tests performed, it can be concluded that, in vivo, dichloromethane is not clastogenic via several routes of exposure, whereas also no indications for gene mutations (via UDS testing) were found. It should be noted that toxicokinetic studies show that dichloromethane is distributed to many organs, including liver, kidney, lungs, brain, muscle and adipose tissue, after inhalation and oral exposure, assuring that the target organs investigated in the available in vivo genotoxicity studies will have been reached.This means that dichloromethane is not genotoxic in vivo.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

C57BL

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

corn oil

Duration of treatment / exposure:

single application

Frequency of treatment:

once

Post exposure period:

24, 36, 48 or 72 hours

Remarks:

Doses / Concentrations:
1250, 2500, 4000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Statistics:

One-sided Student's t test

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

death (females at 2500 and 4000 mg/kg bw); decreased number of PCE's (males, 2500 and 4000 mg/kg bw)

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Table: mean incidence of PCE

 

Compound	Dose (mg/kg)	Sex	Incidence of MPCEs / 1000 PCEs
			24 h	36 h	48 h	72 h
Corn oil (control)	10 mL/kg	M	1.6±1.7	1.6±0.5	3.6±3.1	2.4±1.7
		F	0.6±0.9	0.8±0.4	0.8±1.3	0.6±0.9
Cyclophosphamide	65	M	16.2±8.6**	18.6±4.3**	17.0±5.7**	6.0±4.6*
		F	10.0±3.5**	14.2±2.2**	7.0±1.4**	3.0±2.3*
Methylene chloride	1250	M	1.4±1.7	2.2±1.3	2.6±3.6	2.0±1.0
		F	0.2±0.4	1.0±1.0	1.2±0.8	1.0±1.0
Methylene chloride	2500	M	1.2±1.3	1.2±0.8	1.8±2.5	1.6±1.5
		F	0.8±1.8	0.8±1.3	1.3±1.3 (4)	1.5±1.3 (4)
Methylene chloride	4000	M	2.2±1.9	2.0±1.7	2.0±0.8 (4)	2.2±1.9
		F	1.0±0.8 (4)	1.3±1.3 (4)	2.0±2.8 (4)	0.5±0.6 (4)

N=5, unless where indicated in parentheses; *p<0.05; **p<0.01

 

 

Table: mean percentage PCE (500 cells counted PCEs + NCSs)

 

Compound	Dose (mg/kg)	Sex	% PCE
			24 h	36 h	48 h	72 h
Corn oil (control)	10 mL/kg	M	35.8±15.1	35.2±9.1	32.3±8.5	28.7±9.6
		F	40.0±8.5	33.1±6.3	35.5±10.3	32.7±7.4
Cyclophosphamide	65	M	21.4±8.3*	35.4±6.3	27.1±9.3	24.4±8.2
		F	23.3±13.2*	28.0±5.7	21.4±10.7*	32.1±10.8
Methylene chloride	1250	M	35.0±9.2	43.1±4.9	31.7±14.7	27.6±13.0
		F	43.6±11.9	34.2±6.1	40.7±18.0	31.6±8.7
Methylene chloride	2500	M	19.1±8.1*	38.2±10.3	25.2±5.5	27.9±6.8
		F	35.2±16.2	32.5±16.4	29.8±11.5 (4)	31.6±5.4(4)
Methylene chloride	4000	M	24.9±11.3	38.9±10.5	38.1±17.0 (4)	37.7±10.6
		F	33.5±11.5 (4)	32.5±7.4(4)	33.2±2.9 (4)	40.0±14.5 (4)

N=5, unless where indicated in parentheses; *p<0.05

 

Conclusions:

Interpretation of results of MN test in vivo: negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

In a key study similar to OECD 471, dichloromethane was mutagenic in S.typhimurium strains TA 98 and TA 100 with and without metabolic activation, but not in strains TA 1535, 1537, and 1538. In addition, in one study also strain TA 1535 was positive. Of particular note is that depletion of glutathione (Salmonella typhimuriumand CHO cells) or expression of a rat GST (S. typhimurium) decreased or increased the mutagenicity respectively when exposed to dichloromethane. No increase in the mutant frequency was found in Chinese hamster epithelial (V79) or ovary cells in a HPGRT assay after 1 hour exposure to 0.5 -5% (v/v) dichloromethane without metabolic activation. The short exposure and lack of metabolic activation are short-comings of this study. In a chromosome aberration test similar to OECD 473 using Chinese hamster ovary cells, dichloromethane was found to be positive with and without metabolic activation at 5 µl/ml and above in a dose-related manner (concentrations were ≥25% cytotoxic). Supporting studies evaluated by WHO (1996) and US EPA (2011) confirmed the above results.

Increased sister chromatid exchanges were found in lung cells and peripheral lymphocytes from B6C3F1 mice exposed by inhalation for 2 weeks to 27760 mg/m3 or for 12 weeks to 6940 mg/m3. Under the same exposure conditions, increased chromosomal aberrations in lung and bone cells and micronuclei in peripheral red blood cells were also found, except for one study where no micronuclei were found. During a 3 -day inhalation exposure to either 13800 mg/m3 or between 1735 and 13800 mg/m3 dichloromethane DNA-protein cross-links were found in mouse hepatocytes but not lung cells (and in neither liver nor lung cells in hamsters) and DNA damage (increased DNA SSBs) was observed in liver and lung tissue of B6C3F1 mice immediately following 3 -h exposures, with a recovery to no detectable damage by 2 hours later. A comet assay detected DNA damage in liver and lung tissues from male CD-1 mice sacrificed 24 hours after administration of a single oral dose of 1720 mg/kg of dichloromethane, however, DNA damage in liver and lung was not detected 3 hours after dose administration and no DNA damage occurred at either time point in several other tissues (e.g., stomach, kidney, bone marrow). Several studies in B6C3F1 mice have also looked for mutations in a specific oncogene (H-ras) or in a tumour suppressor gene (p53). Similar frequencies of activated H-rasgenes and inactivation of the tumour suppressor genep53 in the liver tumours were seen in nonexposed and dichloromethane-exposed mice. 

Of note, studies of genotoxicity in rats were negative. These include studies of unscheduled DNA synthesis in rat hepatocytes following inhalation of dichloromethane for 2 -6 h at 6940 -13880 mg/m3, or by gavage up to 1000 mg/kg, or by intraperitoneal exposure of 400 mg/kg, which showed no increase. In addition, DNA adducts were not detected in the livers or kidneys of male F344 rats dosed with 5 mg/kg dichloromethane intraperitoneally. Finally, single-strand breaks (SSB) were increased in hepatocytes from B6C3F1 mice exposed to 16763 mg/m3 dichloromethane for 6 hours, but were not increased in hepatocytes from Sprague-Dawley rats exposed to 15709 mg/m3 for the same time-frame.

From these, and mechanistic data (see also at the toxicokinetics section), it is probable that genotoxic results in mice are based on a metabolism less relevant for humans and other species (the GST-pathway). In fact, cells depleted of glutathione, either in vitro or in vivo, had decreased DNA damage, strengthening the link with the GST pathway.

Concluding, in general dichloromethane induces gene mutations in bacteria, but not in mammalian cells in vitro, whereas dichloromethane is clastogenic in vitro at high concentrations. In vivo, dichloromethane is not clastogenic via several routes of exposure, whereas also no indications for gene mutations (via UDS testing) were found. It should be noted that toxicokinetic studies show that dichloromethane is distributed to many organs, including liver, kidney, lungs, brain, muscle and adipose tissue, after inhalation and oral exposure, assuring that the target organs investigated in the available in vivo genotoxicity studies will have been reached. This means that dichloromethane is not genotoxic in vivo.

According to MAK (2016): In vitro, dichloromethane was found to be mutagenic in bacteria. In vivo, most tests yielded negative results, only the results of tests on liver cells and the bone marrow of mice, but not of rats and hamsters, were positive at high exposure concentrations. Studies of germ cells are not available. The GSTT1 enzyme of the mouse is 5 times more efficient than the GSTT1 enzyme of humans as a result of its higher substrate specificity for dichloromethane. Also, the formation of RNA–formaldehyde adducts in humans is 7 times lower than that in mice. In addition, it is known that the prostate, ovaries and placenta in humans have moderate to low levels of the mRNA for GSTT1. This means that germ cell mutagenicity is not to be expected. 

Justification for selection of genetic toxicity endpoint

All available in vitro and in vivo studies were evaluated in combination.

Justification for classification or non-classification

From the large number of tests performed it can be concluded that, in general, dichloromethane was found to be mutagenic in bacteria and not mutagenic in mammalian cells in vitro. It was found to be clastogenic in vitro. Dichloromethane did not induce DNA damage in rats in vivo. The increases in chromosomal damage (aberrations and micronuclei) seen in mice in vivo is thought to be related to unusually high use of the GST pathway for dichloromethane metabolism in B6C3F1 mice. Overall, the data indicate that dichloromethane is not genotoxic in vivo. Also, the formation of RNA–formaldehyde adducts in humans is 7 times lower than that in mice. In addition, it is known that the prostate, ovaries and placenta in humans have moderate to low levels of the mRNA for GSTT1. This means that germ cell mutagenicity is not to be expected. 

Classification for genotoxicity, therefore, is not warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.