Ethyl 3-[4-(hydroxymethyl)-2-methyl-1,3-dioxolan-2-yl]propanoate

Endpoint summary

Description of key information

non-skin irritant

eye irritant

Key value for chemical safety assessment

Skin irritation / corrosion

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

The toxic potential of the test substance to skin was measured in an in-vitro study based on the conversion of MTT by EpiDerm tissues after the substance exposure. The study was performed similar to the OECD Guideline 431. The ability of the test substance to directly reduce MTT was initially tested via incubating 100 µl with 1 ml MTT for approximately one hour, which showed that the substance can directly reduce MTT in the absence of viable cells. The EpiDerm cultures were tested in duplicated in the MTT assay with 100 µl test material at four exposure times – 1, 4, 8 and 24 hours. Positive control (1 % Triton-X-100) and negative control (sterile deionised water) run concurrently. Since the test substance was found to directly reduce MTT, a killed-control experiment was conducted, which revealed that there was little or no direct MTT reduction in the test article-treated killed control compared to the negative control-treated killed controls and the MTT reduction in the test article-treated viable tissue was ascribed to the viable cells.

The ET50 value recorded is 9.3 hours. Based on the EpiDerm (EPI-200) MatTek model, the substance is expected to be a moderate to mild in vivo irritant. The tissue viability obtained after 4 hours of exposure was 80.4 %. As the study was performed similar to the OECD Guideline 431 the criteria for classification indicated in the guideline can be considered: even if the tissue viability after 3 minutes and 1 hours is not known, is expected to be much higher than the tissue viability observed at 4 hours and thus it is much higher than the threshold for classification as skin corrosive. The substance is therefore not considered as skin corrosive.

Eye irritation

The potential of the substance to cause ocular irritancy to isolated bovine corneas was measured by the test article’s ability to induce opacity and permeability to fluorescein (relative to the control corneas) that can ultimately be used to determine an in vitro score. The study was performed similar to OECD Guideline 437. Bovine corneas obtained as a by-product from freshly slaughtered animals, were mounted in special holders and exposed to the test articles. Positive (ethanol) and negative control (sterile, deionised water) were tested concurrently. The corneas were incubated at 32 ± 1 °C for 10 minutes and the epithelial side was washed at least three times with Complete MEM (containing phenol red) to ensure total removal of test article or control and then finally rinsed with Complete MEM (without phenol red). An opacity measurement was taken and then the corneas were returned to the anterior chambers refilled with fresh Complete MEM (without phenol red) and incubated for approximately 2 hours before a final opacity measurement was taken. The posterior chamber was filled with fresh Complete MEM and fluorescein solution was added to the anterior chamber and incubated at 32 ± 1 °C for 90 minutes and then 360 µl aliquots were taken from the posterior chambers and the OD490 value was measured. A 1:5 dilution with Complete MEM was carried for any aliquot whose OD490 value exceeded 1.500 and the plate was read again. The permeability measurement and in vitro score was then calculated. The test substance, produced an in vitro score of 20.1, as measured by changes in opacity and permeability (to fluorescein).

The guideline followed was similar to the one of OECD Guideline 437 (2009) and thus based on its classification criteria, the IVIS Score obtained does not permit any prediction to be made since is lower than the corrosivity threshold 55.1; the substance cannot be defined as severe irritant or corrosive. For this reason, a further in vitro study was carried out.

The test item was evaluated for its potential to induce eye irritation or serious eye damage according to the OECD guideline 492.

The test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 28 minutes.

50 µL of the liquid test was applied to two tissue replicates.

After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution.

Demineralised water was used as negative control and methyl acetate was used as positive control.

The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD>0.8 and < 2.5, OD was 1.9. The positive control showed clear eye irritating effects, the mean value of the relative tissue viability was 34.7 % (< 50%).

Variation within tissue replicates of the controls and the test item was acceptable (< 20%).

After treatment with the test item, the mean value of relative tissue viability was 41.8 %.

This value is well below the threshold for eye irritation potential (≤ 60%). Test items that induce values below the threshold are either eye irritant or inducing serious eye damage.

According to the OECD Guideline 492, the EpiOcular^TMEye Irritation Test does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1). For this reason, this outcome has to be considered together with the results obtained in the study carried out according to the OECD guideline 437, in a weight of evidence approach.

Justification for classification or non-classification

Skin irritation

The evaluation for the classification or non-classification of the substance was based on the results of the in-vitro study on the substance.

According to the OECD 431 (2004):

The test substance is considered to be corrosive to skin:

i) if the viability after 3 minutes exposure is less than 50 %, or ii) if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15 %.

The test substance is considered to be non-corrosive to skin:

i) if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is greater than or equal to 15 %.

Considering the viability values obtained, the viabiliy after four hours is 80.4 %. No information is available for the viability after 3 minutes and after 1 hour but it is expected to be higher than the one obtained after 4 hour due to the fact that the viability seems to decrease over time. This suggests that the substance is not corrosive but it does not exclude the possibility to be irritant. However, all the other available data: no irritation effects were observed during the acute dermal toxicity study and the HPRT study nor it is a skin sensitiser, suggest that the substance is not expected to be a skin-irritant. It is therefore not classified for skin irritation according to the CLP Regulation (EC) No. 1272/2008.

Eye irritation

In vitro test methods to detect serious eye damage (Category 1 under CLP) and/or absence of effects requiring classification for serious eye damage/eye irritation (i.e. not classified under CLP) are accepted for REACH purposes.

This tests are used in an Integrated Approach on Testing Strategy (IATA) in order to determine the final toxicity of a substance.

An in vitro study carried out according to the OECD 492, is available, and indicated that the test item is irritant and/or able to cause serious eye damage. However, this test method cannot resolve between Categories 1 and 2 of the CLP Regulation.

For this reason a study according to the OECD guideline 437 is also available. Under test conditions the IVIS value obtained was 20.1, falling therefore in the range where no stand-alone prediction can be made. However, consdiering the results obtained in the two in vitro studies, it can be concluded that the test substance is irritant to the eyes Cat. 2, according to the CLP Regulation (EC) No. 1272/2008.