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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Additional information

Bacterial reverse mutation assay (OECD 471)

The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1997), EU Method B.13/14 (2008) and according to GLP principles. The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a preincubation assay, both in the absence and presence of S9-mix. The concentrations were corrected for the solid content of the test material. To obtain more information about the possible mutagenicity, additional experiments were performed both for the direct plate and the preincubation assay. Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA, both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

In vitro mammalian chromosome aberration test (OECD 473)

A chromosome aberration study with the substance was performed in accordance with OECD 473 (1998), EU Method B.10 (2008) and according to GLP principles in human peripheral blood lymphocytes in two independent experiments, with and without metabolic activation. The dosing preparations were adjusted for the solid content of the substance and were set based on the observed toxicity in the dose range finding study (no precipitation was observed up to and including the top dose of 5000 µg/mL). In the first cytogenetic assay, the cells were treated for 4 hr (with and without S9-mix) and for 20 hr (without S9-mix) with the substance at doses ranging from 30 to 500 µg/mL. In the second cytogenetic assay, the cells were treated for 4 hr (with S9 -mix) with the substance at doses ranging from 75 to 500 µg/mL. Toxicity was reached at the dose levels selected for scoring. In all exposure groups, the percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to solvent control at any dose level (p > 0.05, Fisher's Exact test). Adequate negative and positive controls were included. No effects of the substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Based on the results of this study, it is concluded that the substance does not disturb mitotic processes and cell cycle progression and is not clastogenic in human lymphocytes.

Mammalian cell gene mutation assay with the registered substance (OECD 476)

The mutagenic activity of the substance has been evaluated in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells (with independent repeat) in accordance with OECD 476 (1997), EU Method B.17 (2008), EPA OPPTS 870.5300 (1998) and according to GLP principles. The dosing preparations were adjusted for the solid content of the registered substance and were set based on the observed toxicity in the dose range finding study. Two independent experiments (+/- S9), with an additional third experiment (+S9) to verify results, were performed. Adequate solvent and positive controls were included.

In the absence of S9-mix, the test substance did not induce a significant increase in the mutation frequency (MF) in the first experiment. This result was confirmed in the second experiment with modifications in the duration of treatment time. In the presence of S9-mix (8%), the test substance induced a 3.3-fold increase in the mutation frequency in the first mutation experiment at one of the high dose levels (300 µg solid/mL), in the presence of cytotoxicity (RTG of 16%). The mutation frequency of 192 x 10-6 was just above the global evaluation factor (GEF) plus mutation frequency of the negative controls (GEF + MF(controls): 184 x 10-6) and slightly outside the historical control data range. This response fulfilled the criteria for a positive response. The test substance increased the mutation frequency of both the small and large colonies. In the second experiment, no increase in mutation frequency was observed in the presence of S9 –mix (12%).

Therefore a 3rd experiment was performed, to verify the positive result obtained in the first experiment. The selected test concentrations were around the test concentration at which a positive response was observed in the first experiment. The test substance induced up to 2.5-fold increase in the mutation frequency at the TK locus, and slightly outside the range for historical data of the spontaneous mutation frequencies for the solvent control, with no clear dose-response. However, the mutation frequencies were all below the global evaluation factor (GEF+MF(controls): 205 x 10 -6), and the 2 highest positive concentrations showed some evidence of cytotoxicity (RTG of 13 and 16%). The test substance showed up to 2.7- and 2.3-fold increases in the mutation frequency of the small and large colonies, respectively.

As the responses observed in the presence of S9 -mix (first and third experiment) fulfilled the criteria for a positive response, it is concluded that the substance is mutagenic in this assay. The test substance increased the mutation frequency of both the small and large colonies, indicating increases in both chromosome aberrations and gene mutations.

Mammalian cell gene mutation assay with methanol (published results)

McGregor et al. (1988) investigated the optimal test conditions for the mutagenicity assay in mouse lymphoma cells system in a study prior to the validation of the method as a test guideline. Methanol was included in the test with several chemicals known to induce mutagenic response after metabolic activation, some of which are now used as positive controls in the current guideline.

The study indicates that different S9 concentrations may be required to detect the mutagenic response for different compounds.

Methanol treatment for 4 hours was found to induce a significant mutagenic response in mouse lymphoma cells under certain conditions of metabolic activation concentrations (10 to 30 µl S9/ml, i.e., 1 to 3% (v/v), corresponding to 2.5 to 7.5 mg Whole Liver Equivalent/ml), with associated RTG reduced at < 30%, or below.

With regard to methanol treatment, the conditions of 2.5 mg WLE/ml (10 µl S9/ml, or 1% v/v) showed evidence of increased mutagenic response with moderate cytotoxicity, but effects were not consistently reproduced in another experiment with the same S9 concentrations, whereas a higher concentration of S9 (7.5 mg WLE/ml, i.e., 30 µl S9/ml, or 3% v/v) was needed to induce MF, with a high associated toxicity (RTG < 15%).

Mammalian cell gene mutation assay with the solid content of the registered substance (OECD 476)

The mutagenic activity of a freeze dried sample of the substance, representing the solid content of the registered substance, was investigated in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells (with independent repeat) in accordance with OECD 476 (1997), EU Method B.17 (2008), EPA OPPTS 870.5300 (1998) and according to GLP principles. Adequate solvent and positive controls were included. In the absence of S9-mix, the test substance did not induce a significant increase in the mutation frequency (MF) in the first experiment. This result was confirmed in the second experiment with modifications in the duration of treatment time. In the presence of S9-mix (8%), the test substance did not induce a significant increase in the mutation frequency (MF) in the first experiment. In the second experiment, in the presence of 12% S9-mix, the test substance induced an up to 2.2-fold increase (at 366 μg/mL) in the mutation frequency. Although the increase (169 x 10-6 revertants) was slightly outside the historical control data range (167 x 10-6 revertants), the mutation frequency was not above the global evaluation factor plus mutation frequency of the negative controls (GEF+MEF(controls): 126+76=202 x 10-6), the criterion for a positive response, and it was observed in the presence of cytotoxicity.

Therefore it is concluded that the solid content of the registered substance is not mutagenic in the absence as well as in the presence of S9-mix in this assay.

Overview of the mutagenicity data

The effects in the Mouse Lymphoma assay observed with the registered substance as such in the presence of metabolic activation were marginal: slightly above historical controls but in most cases below the criteria GEF+ MF(controls), no clear dose-reponse relationship, effects close to toxic doses, not reproducible under different S9 conditions.

These characteristics are in fact consistent with effects previously observed with the methanol treatment of mouse lymphoma cells reported by McGregor et al. (1988), indicating that methanol can induce genetic mutation in this test system under certain conditions of high metabolic activation. However, the registration dossier for methanol and prior international evaluations provide further evidence that methanol is not considered a mutagenic substance based on weight of evidence from numerous in vitro and in vivo studies, most of which are negative. Overall there is a low concern for mutagenic potential related to methanol.

Furthermore the potential effects of the registered substance on chromosomes identified in the MLA assay (small colonies) were not confirmed in the chromosome aberration assay in human lymphocytes. No mutagenic effects were observed in the Ames test, with or without metabolic activation.

Taken together, the results support a low concern for the mutagenic potential of the registered substance. The surfactant content of the registered substance is not considered mutagenic and the effects observed in the MLA with the registered substance were marginal and are related to the low levels of methanol by-product. Methanol is not considered a mutagenic substance based on weight of evidence from numerous in vitro and in vivo studies. No further information is considered needed.


Short description of key information:
The substance as such has been tested in 3 in vitro GLP studies (OECD 471; OECD 473; OECD 476). In all 3 studies, the dosing preparations were adjusted for the solid content of the registered substance. The substance was not mutagenic with and without metabolic activation, in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay (OECD 471), and no chromosomal effects were detected in peripheral human lymphocytes (OECD 473), with and without metabolic activation. The mutagenic activity of the substance has been evaluated in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells (with independent repeat) in accordance with OECD 476 and GLP. It was concluded that the substance is not mutagenic in this assay without metabolic activation, but weakly mutagenic with metabolic activation (3.3-fold increase only at one cytotoxic concentration; weakly positive effects reproduced in a repeat experiment (up to 2.5-fold increase)).
Also an in vitro Mouse Lymphoma Assay with a freeze dried sample of the substance, representing the solid content of the registered substance, has been performed. Based on the results of this study, it was concluded that the solid content of the registered substance is not mutagenic in the absence as well as in the presence of S9-mix in this assay.
Taken together, the results support a low concern for the mutagenic potential of the registered substance. The surfactant content of the registered substance is not considered mutagenic and the effects observed in the MLA with the registered substance were marginal and are related to the low levels of methanol by-product. Methanol is not considered a mutagenic substance based on weight of evidence from numerous in vitro and in vivo studies. No further information is considered needed.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the current available data, the substance does not need to be classified for genetic toxicity in accordance with the CLP Regulation. The only effects that were observed were marginal and related to the low levels of methanol by-product. Methanol is not considered a mutagenic substance based on weight of evidence from numerous in vitro and in vivo studies.