DEVELOPMENT OF A NEW RECONSTRUCTED HUMAN EPIDERMIS (RHE)-BASED SCREENING ASSAY FOR CONTACT ALLERGENS.

Recent changes in regulatory restrictions and social views against animal testing called for the development of reliable in vitro tests for predicting skin sensitizing potential of a broad range of cosmetic raw materials. Also, many of these raw materials include lipophilic compounds and complex mixtures, which could be out of the applicability domain for known cell-based assays and peptide reactivity assays. To address both the political and technical issues, we employed commercially available reconstructed human epidermis (EpiDerm), on which test samples can be applied at high concentrations in the same manner as animal testings. We first determined gene expression profiles in EpiDerm after application of known allergens, DNFB and oxazolone (OXA). A unique set of genes encoding redox regulatory enzymes was upregulated significantly by DNFB and/or OXA, but not by a non-allergen, benzalkonium chloride. Real-time PCR analysis demonstrated that expression of the selected target gene(s) was robustly (>3-fold) induced by hexyl cinnamic aldehyde, abietic acid, and ethyleneglycol dimethacrylate, which are categorized as weak allergen in LLNA, but not by non-allergens, methylsalicylate and lactic acid. Furthermore, lipophilic sensitizing compounds, such as b-propiolactone and palmitoyl chloride, induced marked expression of target genes without affecting cell viability, but not a lipophilic non-allergen, hexane. Not only do our results demonstrate the predictivity of our new assay platform, which we called Epidermal Sensitization Assay (EpiSensA), they also highlight the much broader applicability domain against raw materials including highly-lipophilic compounds and complex mixtures.

ATF3 gcol, Complex mixtures, Contact allergen, DNAJA4 gcol, DNAJB4 gcol, DPRA, Epidermal sensitization assay (EpiSensA), GCLM gcol, GeneChip,h-CLAT, HSPA1A gcol, HSPA6 gcol, HSPH1 gcol, Lipophilic compounds, Real-time PCR, Sensitizers, Stress related genes, Weak allergen, ZFAND2A gcol

2,4-dinitroflurobenzene, abietic acid, benzalkonium chloride, beta-propiolactone, ethyleneglycol dimethacrylate, hexane, hexyl cinnamic aldehyde, lactic acid, methyl salicylate, oxazolone, palmitoyl chloride, phenyl acetaldehyde