The phototoxic potential of test materials after exposure to UVA/visible light is evaluated by the 3T3 Neutral Red Uptake Phototoxicity Assay using Balb/c 3T3 mouse fibroblasts (OECD TG 432). To address challenges related to testing of finished products or materials that are not completely soluble, the Reconstructed Human EpiDermis (RhE) Phototoxicity Assay (INVITTOX Protocol 121) can be used as a stand‐alone or in a tiered approach. Water and sesame oil were recommended solvents for the RhE Assay, however additional solvents may be investigated on a case‐by‐case basis to accommodate a wider variety of test materials. Alternate solvents should first be assessed to ensure that they: 1) do not cause cytotoxicity; 2) do not diminish and/or inhibit phototoxic reactions; and 3) do not interfere with the UVA exposure. We investigated 5% DMSO in Hanks’ Balanced Salt Solution (HBSS), 5% acetone in HBSS, and polyethylene glycol (PEG) and their influence on the prediction of phototoxicity of chlorpromazine, a known phototoxicant. Duplicate tissues (EpiDerm™ from MatTek Corporation, Ashland, MA) were treated with each group for 24 hours, followed by a UVA (~6 J/cm2) or dark exposure, and then a 21 hour post‐exposure period before viability assessment using MTT dye. The assay positive control, 0.02% chlorpromazine in HBSS containing 1% DMSO, was tested concurrently. A test material was considered to have
phototoxic potential if it induced a ≥30% difference in viability between tissues exposed to UVA as compared to the dark‐exposed tissues. Our data showed that the solvents performed in a comparable manner to the assay negative control (HBSS) and they did not induce significant toxicity when the viability of the UVA‐exposed and dark‐exposed tissues was compared: 92.9% and 104.8% (5% DMSO), 94.0% and 97.5% (5% acetone), and 81.3% and 90.0% (PEG), respectively. The difference between the viability of the UVA‐exposed or dark‐exposed tissues after treatment with 0.02% chlorpromazine dissolved in 1% DMSO, in 5% DMSO, and in PEG was 60.0%, 59.7%, and 72.5%, respectively. These data indicate that the new solvents we investigated were suitable for use in the detection of phototoxicity. The evaluation of 0.02% chlorpromazine in 5% acetone, as well as additional solvents (e.g. ethanol), is currently ongoing. Our future investigations will concentrate on the assessment of different solvents that can accommodate the phototoxicity testing of novel chemistries.
March 17, 2016