ENHANCED PHOTOTOXICITY ASSAY IN RECONSTITUTED SKIN

DESCRIPTION (provided by applicant): We have conceived a high-throughput in vitro screening test for Phototoxicity designated the "Enhanced Phototoxicity Assay in Reconstituted Skin (EPARS). The phototoxic potential of chemicals, cosmetics, dietary supplements and pharmaceuticals are a growing major concern in the consumer products industry. Animal models of phototoxicity are expensive, slow, subjective, and not amendable to high throughput. The lack of rapid, economical, reliable phototoxicity screening tests inhibits the development and commercialization of many new products. Currently in the US, there are no validated regulatory agency-accepted alternatives or in vitro phototoxicity tests. The 3T3 Mouse Fibroblast Neutral Red Phototoxicity Test (3T3 NRU) is now under consideration by ICCVAM as an alternative phototoxicity method after having been pre-validated by Europe's validation agency, ECVAM. This test, however, has several weaknesses. The EPARS test overcomes many of the limitations of the 3T3 NRU 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. In the EPARS, the test substance is applied topically to the reconstructed human skin models, with and without UV irradiation. The viability of the tissues is the determined using the MU viability assay, and the irradiated and non-irradiated tissue viability is compared to determine phototoxic effects. In addition, other molecular and mechanistic endpoints relevant to phototoxicity such as PGE2 release, MHC expression, cellular proliferation and inflammatory cytokine production (IL-1a, lL1-ra, TNF-a and IFN-g) can be measured to increase the sensitivity and specificity of the test. PROPOSED COMMERCIAL APPLICATION: MB Research will offer the service of an alternative phototoxicity assay to members of the pharmaceutical, biotech, cosmetic and chemical industry. A survey of our clients indicates that there is an increasing demand for rapid and cost-effective phototoxicity tests, and a need for more mechanistic-based tests which provide both qualitative and quantitative information, In addition, we believe that flow cytometry and DNA microarrays are underutililized in the toxicity-screening field and that this technology can be used to develop other commercially viable in vivo and in vitro toxicity tests at our company.