Reaction mass of ethane-1,2-diol and 2-hydroxyethyl formate and ethylene diformate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A bacterial reverse mutation test (Ames) was conducted with the reaction mass yielding a negative result. Based on the read across from its hydrolysis products ethan-1,2 -diol and formic acid, the reaction mass does not induce chromosomal damage (negative in the in vitro

chromosome aberration assay) and is not mutagenic in mammalian cells (negative in the HPRT and mouse lymphoma assay).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The reaction mass is considered to be non-genotoxic based on the available in vitro genotoxicity tests. Thus, genetic toxicity testing in vivo is not needed.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial Reverse Mutation Assay

The reaction mass was tested with regard to a potential mutagenic activity in the Bacterial Reverse Mutation Assay. The experiments were carried out using Salmonella typhimurium TA98, TA100, TA1535 and TA1537, and Escherichia coli WP2uvrA with and without metabolic activation. The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Pre-Incubation Test).

Based on the results of the Solubility and the Range Finding Tests the reaction mass was dissolved in ultrapure water and tested at 5000; 1600; 500; 160; 50 and 16 μg/plate.

The vehicle control and positive controls showed the expected results. No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study. The test item did not show inhibitory, cytotoxic effects in the performed experiments.

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix).

In conclusion, the reaction mass is not mutagenic.

Gene mutation and chromosomal damage in mammalian cells

In the absence of tests conducted with the reaction mass, the potential to induce mammalian cell mutagenicity or chromosomal damage is evaluated on the basis of the data available on the hydrolysis products formic acid and ethane-1,2 -diol.

 

Chromosome aberration assay

Ethane-1,2 -diol was tested in Chinese hamster ovary cells with and without metabolic activation for its potential to induce chromosomal aberrations. Cells were incubated in culture medium with ethane-1,2 -diol at 160, 500, 1600, 5000 µg/mL. Appropriate negative and positive controls were used in parallel.

No cytotoxicity was seen even at the highest dose tested. The negative and positive controls showed the expected results. The number of aberrations/cell and percent cells with aberrations were not increased.

It can be concluded that ethane-1,2 -diol is not clastogenic.

Formic acid was tested in Chinese hamster ovary cells for its potential to induce chromosome aberrations with and without metabolic activation.

The cells were exposed to 276, 368, 460, 552, 644, 920, 1150, 1266, and 138 µg/mL (6 to 30 mM) for 6 hours with metabolic activation and continuously for 24 hours without metabolic activation. Chromosomes were prepared after 24 hours and evaluated for chromosomal damage. An appropriate negative control was used in parallel and cytotoxicity examined by counting surviving cells.

Acidic conditions were responsible for the chromosome aberrations observed. Likewise, the cytotoxicity depended on the pH value when formic acid was tested up to cytotoxic concentrations. Formic acid did not induce chromosomal damage in neutralized culture medium. Thus, formic acid was not itself clastogenic to the CHO cells.

It is concluded that formic acid is not clastogenic.

 

Mammalian cell gene mutation

In a mouse lymphoma assay, tk⁺/tk^- L5178Y mouse lymphoma cells were exposed to ethane-1,2 -diol with and without metabolic activation. At least five concentrations with the highest being 5000 µg/mL in duplicate cultures and in at least two independent experiments were tested. Appropriate vehicle control and positive control groups were incubated concurrently.

No cytotoxicity was observed at any of the tested concentrations. In one experiment without metabolic activation, a statistically significantly increased mutation frequency was obtained at the two highest dose levels. Although the significant result was acceptable based on the study criteria, the cloning efficiencies were low in all dose groups (<50%) and the vehicle control (59%). Moreover, the increase did not exceed the GEF of 90 x 10^6 for the agar version of the MLA and the result was not reproduced in the other two experiments without metabolic activation. In the two experiments with metabolic activation, no significant mutagenicity was observed.

It was concluded that ethane-1,2 -diol is not mutagenic.

In a mammalian cell gene mutation assay (HPRT locus), Chinese Hamster ovary cells cultured in vitro were exposed to formic acid (85.3%) at concentrations of 0, 31.25, 62.5, 125, 250, and 500μg/mL in the presence, and of 0, 25, 50, 100, 200, and 400 μg/mL in the absence of mammalian metabolic activation. 

Formic acid was tested up to cytotoxic concentrations (i.e. 200 to 400 µg/mL in the absence and 400 to 500 µg/mL in the presence of metabolic activation) without increasing mutation frequency at any concentration. The positive controls did induce the appropriate response as did the vehicle control. There was no evidence of induced mutant colonies over background.

Formic acid did not induce forward mutations in vitro in the CHO/HPRT assay, with or without metabolic activation.

Conclusion on the potential of the reaction mass to induce mammalian gene mutation and/or chromosomal damage

Due to the rapid hydrolysis of the reaction mass, the above endpoints can be assessed on the basis of data on the hydrolysis products formic acid and ethane-1,2 -diol. The available data for both substances do not indicate genotoxic potential on mammalian cells in the in vitro HPRT or mouse lymphoma test and the in vitro chromosomal aberration test. These results are further supported by the fact that both substances are not classified for effects on reproduction in Annex VI of Regulation (EC) No 1272/2008. Thus, it can be concluded that - based on read-across - the reaction mass does not induce gene mutation or chromosomal damage in mammalian cells.

Justification for classification or non-classification

The reaction mass did not show any potential for genotoxicity in an in vitro bacterial mutation test. Based on the read across from ethan-1,2 -diol and formic acid, the reaction mass can also be considered to be non-mutagenic in mammalian cells and not to induce chromosome aberrations.

As a result the reaction mass does not require classification for genotoxicity under Regulation (EC) No 1272/2008, as amended for the eighth time in Regulation (EU) No 2016/918.