"Enhanced Phototoxicity screening Assay in Reconstituted Skin" (EPARS)

The phototoxic potential of chemicals, cosmetics, dietary supplements and pharmaceuticals is a major and growing concern in the consumer products industry. The lack of rapid and reliable screening tests and the cost of animal-based phototoxicity tests inhibits the development and commercialization of many new products. To date, the U.S. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) has not validated an alternative non-animal phototoxicity test. In contrast, the European Commission’s Validation Agency (ECVAM) has validated and accepted the 3T3 Mouse Fibroblast Neutral Red Uptake Assay (3T3 NRU) as an alternative phototoxicity assay.

EPARS employs 3-dimensional keratinocyte-based reconstituted skin “tissue equivalents”, specifically MatTek Corporation’s EpiDerm™ and EpiOcular™ tissue models. The ability of these models to predict irritancy has been highly characterized (Koschier et al., 1997; Faller et al., 2002). If developed to its full potential, this assay would be able to rapidly and objectively identify agents that have phototoxic potential in humans. The EPARS test overcomes many of the limitations of the 3T3 NRU phototoxicity test in that: 1) EPARS employs multi-layer tissues that closely parallel human skin morphology, instead of a fibroblast monolayer; 2) non-aqueous soluble formulations can be tested, in contrast to the 3T3 NRU Assay, in which test substances need to dosed via the culture media; 3) the human primary keratinocyte-based tissues are a more relevant model than a mouse tumor cell line.

Using a validated MTT viability assay, the cytotoxicity of the agent is determined. Cytokine release can be used to evaluate the irritant properties of test chemicals in these tissues (Bernhofer et al.,1999). Furthermore, molecular and mechanistic endpoints relevant to phototoxicity such as PGE2 release, and inflammatory cytokine production (IL-1α, IL-1ra, IL-8 and TNFα) can also be evaluated as endpoints which increase the sensitivity and specificity of the test.

Methods and Materials

Test Substances and Dosing
MatTek EpiDerm PHO tissues are equilibrated according to standard protocol and dosed with test substances falling into one of three groups: 1) known phototoxins, 2) irritants (non-phototoxic), and 3) non-irritants (non-phototoxic). Tissues suspended in 0.9 ml assay media are dosed with vehicle control or 100 ul test substance applied directly to tissue surface, and then incubated for 18-24 hours.

UV Irradiation
Following treatment, a Honle SOL 500 solar simulator (Honle UV America, Malboro, MA) is used to deliver UVA light doses of 6 J/cm2 (approximately 60 min. at 1.7 mW/cm2) to the tissue equivalents. Unirradiated control tissues were incubated at room temperature in the dark. After UVA treatment, tissues are rinsed with PBS and returned to the incubator.

MTT Viability Assay Endpoint
At 18-24 hours, post-irradiation, tissue viability is measured using the MatTek MTT Viability Assay protocol. Briefly, tissues are incubated with 1 mg/ml MTT in unsupplemented DMEM for 3 hours. Following a wash step, the reduced formazan product is extracted overnight at room temperature. Absorbance is measured on a microplate reader. The percent viability is calculated as follows:

% Tissue Viability = 100*(OD sample/OD vehicle control).

PGE2 and Cytokine ELISA Endpoint Analysis
At the end of the post-irradiation period, and before addition of MTT solution, aliquots of cell culture media are collected for PGE2 and cytokine ELISA analysis. The media samples are diluted within range of the ELISAs according to ELISA kit instructions.