(Z)‐ethyl 2‐methylpent‐3‐enoate

Endpoint summary

Description of key information

DPRA: Negative in an OECD TG 442C test.

KeratinoSens: Negative in an OECD TG 442D test.

Key value for chemical safety assessment

Skin sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

DPRA

In an in chemico (DPRA) study, performed according to OECD guideline 442C and in accordance with GLP principles, the reactivity of the Substance towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) was determined. Isopropanol was found to be an appropriate solvent to dissolve the test substance in and was therefore used in this DPRA study. Cinnamic aldehyde was used as a positive control. Following incubation of the test substance with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and spectrophotometric detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in the prediction model. All acceptability criteria were met and therefore, the study was considered to be valid. No precipitate or phase separation was observed in any of the samples before and after the incubation. In the cysteine reactivity assay the test item showed 1.4% SPCC depletion and in the lysine reactivity assay the test item showed 2.8% SPCL depletion. The mean of the SPCC and SPCL depletion was 2.1% and as a result the test item was considered to be negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

KeratinoSens

In an in vitro (KeratinoSens) study, performed according to OECD guideline 442D and in accordance with GLP principles, the ability of the test substance to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway was determined. The test item was dissolved in dimethyl sulfoxide (DMSO) at 200 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 – 2000 μM (2-fold dilution series). The highest test concentration was the highest dose required in the current guideline. In experiment 2, precipitation was observed at the start of the incubation period in the 96-well plates, at the highest tested dose level of 2000 μM. Three independent experiments were performed. Overall it is concluded that the test conditions were adequate and that the test system functioned properly. In the first experiment, the test item showed toxicity (IC30 value of 26 μM). A biologically relevant induction of the luciferase activity (EC1.5 value of 1.1 μM) was measured. The maximum luciferase activity induction (Imax) was 3.10-fold leading to an individual run conclusion of positive since positive results (>1.5-fold induction) were observed at test concentrations < 1000 μM with a cell viability of >70% compared to the vehicle control. In the second experiment, the test item showed no toxicity (no IC30 and IC50 value). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations. The maximum luciferase activity induction (Imax) was 1.29-fold leading to an individual run conclusion of negative assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 μM. Since the first two experiments were not concordant, a third experiment was performed to provide a final conclusion. In the third experiment, the test item showed no toxicity (no IC30 and IC50 value). The test item showed no biologically relevant induction of the luciferase activity (no EC1.5 value) at any of the test concentrations. The maximum luciferase activity induction (Imax) was 1.45-fold leading to an individual run conclusion of negative assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 μM. Overall, the test item is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed in two out of three experiments at test concentrations up to 2000 μM with cell viability > 70% compared to the vehicle control. In conclusion, the test substance is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes).

Justification for classification or non-classification

The substance is not a skin sensitiser based on two negative in vitro skin sensitisation tests covering two key events of the Adverse Outcome Pathway (OECD TG 497) and therefore does not need to be classified for skin sensitisation according to EU CLP (EC No. 1272/2008 and its amendments).