2-methylpentyl 2-hydroxybenzoate

Phototoxicity in vitro

Description of key information

Phototoxicity: Not phototoxic, PIF could not be determined, however the test item curves of the presence (+Irr) and absence (-Irr) of irradiation showed similar progressions indicating that the test item has no phototoxic potential in the assay, OECD TG 432, 2021

Key value for chemical safety assessment

Results:

no phototoxicity

Additional information

Key study, OECD TG 432, 2021 : The phototoxicity of the substance was evaluated using OECD TG 432 : in vitro 3t3 NRU phototoxicity test method under GLP. A solubility test was performed based on visual assessment. The test item was dissolved in DMSO to a final concentration of 100 mg/mL. The test item was observed to be insoluble in EBSS at a concentration of 10 mg/mL. The 100-fold dilution of 100 mg/mL in DMSO stock solution showed no precipitation. This concentration: 1000 μg/mL, was selected as the highest test concentration for the main assay (the highest concentration in the OECD TG 432 guideline). For the main experiment the test item was dissolved in DMSO at a concentration of 100 mg/mL. From this stock, 8 concentrations of the test item were tested (8-replicates) in the main experiment: 1000, 316, 100, 31.6, 10.0, 3.16, 1.00 and 0.316 μg/mL. The final concentration of DMSO in the culture medium was 1.0% (v/v). After removal of culture medium and DPBS washing: (i) aliquots (200 μL) of EBSS medium containing concentrations of the test item, positive control and negative control were applied to duplicate plates. For all compounds, 8 test concentrations were tested in 8-fold. Columns 9-12 contained incubation medium. (ii) Plates were incubated in the dark at 37°C in a humidified atmosphere (80-100%) of 5% (v/v) CO2 in air for 60 minutes. (iii) One plate for the test item, negative control, and positive control (designated “+Irr” plates) was then to be irradiated using the light source (with lid). Plates were irradiated for 18 min receiving a dose of 5 J/cm2 UV-A. The remaining plates (designated “-Irr” plates) were kept in the dark at room temperature for the same period of time. The cytotoxicity was expressed as a concentration-dependent reduction of the uptake of the Neutral Red approximately 20-24 h after treatment with the test item, in the presence and absence of irradiation. Immediately following the visual assessment, medium was removed from the cells and 100 μL of Neutral Red dye (50 μg/mL) was added. The plates were then incubated at 37 ± 1°C in a humidified atmosphere (80-100%) of 5% (v/v) CO2 in air for approximately 3.5 hours. Following the incubation, the Neutral Red solution was removed and the cells were washed at least once with 150 μL of DPBS which was removed before adding 150 μL of Neutral Red de-stain solution (ethanol : acetic acid : distilled water, 50:1:49 ratio). Plates were shaken on a plate shaker for approximately 20-40 minutes until all Neutral Red was extracted from the cells. Analysis was performed using a TECAN Infinite M200 Pro Plate Reader at a wavelength setting of 540 nm. Neutral Red absorbances were expressed in terms of absolute optical density (OD540). The OD540 was measured for each concentration of the test item, negative control, and positive control on the “+Irr” and “-Irr” plates and were compared to the OD540 of the vehicle control(s), as appropriate. The test assay passed the acceptance criteria. Specifically, the mean viability of the vehicle treated irradiated cells compared to non-irradiated cells was with 92% higher than the acceptance criteria of 80%. The OD540 value of non-irradiated vehicle control cells was with 0.63 higher than 0.40. The negative control SDS showed a PIF value of 1.19 and is therefore classified as non-phototoxic. The positive control Anthracene showed a PIF value of > 921 and is therefore classified as phototoxic. For the test item, both the IC50(+Irr) and IC50(-Irr) could not be determined since no clear dose response could be observed and no sigmoidal curve was obtained. Therefore, no Photo Irritation Factor (PIF) could be calculated. Furthermore, the curves of the presence (+Irr) and absence (-Irr) of irradiation showed similar progressions indicating that the test item has no phototoxic potential in the assay leading to the classification non-phototoxic. Under the conditions of this study, the substance is considered non-phototoxic.