GENOTOXICITY TESTING USING THE NORMAL HUMAN CELL BASED 3D EPITHELIAL MODELS.

Safety assessment of new products for human use requires genotoxicity testing to ensure their non-carcinogenicity. Current in vitro genotoxicity assays have low specificity resulting in unacceptably high rates of false positives. Moreover, starting in 2009, in vivo genotoxicity testing was banned by the 7th Amendment to the Cosmetics Directive. Since epithelial tissues have the highest exposure to many household, cosmetic, and personal care products, 3D epithelial models can better reflect human exposure and metabolism. The Reconstructed Skin Micronucleus (RSMN) assay utilizes MatTek’s highly differentiated EpiDerm™ tissue model. The RSMN assay is a well established procedure that is currently part of a COLIPA sponsored pre-validation study. Test materials are topically applied (similar to actual human contact) in the presence of Cytochalasin-B. Damage to the DNA is measured by counting dividing cells that contain micronuclei (MNC). We have shown dose-related, statistically significant increases in MNC for 9 direct genotoxins and 4 genotoxins that require metabolic activation; 4 non-genotoxins were negative. When the same protocol was applied to other MatTek tissue models (e.g. EpiOral™, EpiAirway™, EpiVaginal™, and EpiOcular™), the % dividing cells varied significantly between models (11.6-53.6%) but statistically significant increases in MNC (0.3 to 1.2%) after treatment were observed while background levels of MNC remained low. Hence, the RSMN assay that utilizes EpiDerm is very useful for predicting genotoxicity and the assay can be applied to other human epithelial tissues to predict genotoxic effects following real life exposure. Preliminary results indicate that other epithelial tissues are also amenable to the comet assay. Note: Subsequent to submission of this abstract, additional data were obtained which have been included in this presentation.

AIR-100, Acridine orange, Binucleated cells, Comet assay, Cytochrome P450 drug metabolizing enzymes, DNA damage, EFT-300, EPI-200, EpiAirway, EpiDerm, EpiDermFT, EpiOcular, EpiOral, EpiVaginal, EpiVaginalFT, Genotoxicity tests, Micronuclei, Mutagenic hazard, OCL-200, ORL-200, Reconstructed skin micronucleus, Trypan Blue Dye exclusion, VEC-100, VEC-100-FT

1,2 Epoxydodecane , 4-Nitrophenol (p-Nitrophenol) , 4-Nitroquinoline 1-Oxide , Acetone, Apigenin , Benzo[a]pyrene , Cyclophosphamide , Cyctochalisin-B, Dibenz[a,h]anthracene , Dimethylbenz[a]-Anthracene , Dimethylcarbamoyl Chloride , Dimethylnitrosamine , Ethohexadiol (2-ethyl-1,3-hexandediol) , Methyl Methanesulfonate , Mitomycin C , Trichloroethylene , Tris[2,3-dibromopropyl] phosphate , Vinblastine Sulfate , n-Ethyl-nitrosourea , n-Methyl-N-Nitrosourea , â-Butyrolactone