Influence of acidic extraction conditions on formazan assay: Assessment of test substances using in vitro EpiSkin skin irritation test method by HPLC and colorimetry

The skin irritation potential of a test substance is typically determined by measuring cell viability in treated tissues by means of the colorimetric MTT reduction assay after topical application of a test substance. In the EpiSkin validated skin irritation test method, tetrazolium salt-based formazan assay is performed by using acidific isopropanol extraction conditions (ESAC 2007, OECD TG439 2010). The current work evaluated the data obtained with the EpiSkin model for the 20 reference chemicals (listed in the OECD TG439) following formazan extraction with acidified (IPA) or non acidified (IP) isopropanol solutions to evaluate the influence of acidic extraction conditions. Therefore the objectives of this work was to establish the use of HPLC measurements as a complementary assay to standard photometry assay for detection of reduced MTT, a known limitation for test substances that are highly coloured.

The cell viability in EpiSkin quantitatively measured after acidified (IPA) or non acidified (IP) isopropanol extractions from tissues were equivalent ranging from 6.4 to 104% by colorimetry (reference method). Following HPLC measurement, formazan quantitative analyses were comprised between 7.9 to 99.2%. The standard deviation between those 2 conditions was +/- 18%. Therefore, within-laboratory variability assessed in 3 runs showed similar concordance of classification for each condition. The sensitivity (based on 10 GHS Cat.2 substances) was 90% and the specificity (based on 10 No-category substances) was 70%. The HPLC provides data comparable to those using standard photometry assays for 20 test substances indicative of the use of HPLC as a complementary assay to photometry in EpiSkin skin irritation test method. Therefore, those results suggest that acidific isopropanol extraction conditions did not affect the EpiSkin skin irritation outcomes.