Catch-up validation study of an in vitro skin irritation test method based on an open source reconstructed epidermis (phase I). (October 2016)

• Record Type: Journal Article
• Authors: Mewes, K.R.; Fischer, A.; Zöller, N.N.; Laubach, V.; Bernd, A.; Jacobs, A.; van Rompay, A.; Liebsch, M.; Pirow, R.; Petersohn, D.
• Is Part Of: Toxicology in vitro. Volume 36(2016)
• Page Start: 238
• Page End: 253
• Publication Date: 2016-10
• Subjects: 1-BH 1-bromohexane -- ALI Air-Liquid Interface -- BLR Between-laboratory-reproducibility -- CyA Cyclamen aldehyde -- D-PBS Dulbecco's phosphate-buffered saline -- ECETOC European Centre for Ecotoxicology and Toxicology -- EURL-ECVAM European Union Reference Laboratory for the Validation of Alternative Methods -- ET50 effective time at 50% viability -- EU European Union -- GC/MS gas chromatography/mass spectroscopy -- MTT 3-(4, 5-dimethylthiazo-2-yl)-2, 5-diphenyltetrazoliumbromide -- NC negative control -- OD optical density -- OECD Organization for Economic Co-operation and Development -- OS-REp open source reconstructed epidermis -- PC positive control -- PS performance standards -- REACH Registration, Evaluation, Authorisation and Restriction of Chemical Substances -- RC reference chemical -- RHE reconstructed human epidermis -- SD standard deviation -- SDS sodium dodecyl sulfate -- SOP standard operating procedure -- SIT skin irritation testing -- SIVT skin irritation validation test -- TG testing guideline -- VRM validated reference method -- WLR within-laboratory-reproducibility -- ZEBET Center for Documentation and Evaluation of Alternative Methods to Animal Experiments at the BfR (Federal Institute for Risk Assessment); Skin irritation test -- Reconstructed human epidermis -- Open source concept -- Catch-up validation study -- Cross-contamination -- OECD performance standards; Toxicity testing -- In vitro -- Periodicals; Toxicology -- Periodicals; 615.9
• DOI: 10.1016/j.tiv.2016.07.007
• Languages: English
• ISSNs: 0887-2333

Abstract

Abstract: We have developed a new in vitro skin irritation test based on an open source reconstructed epidermis (OS-REp) with openly accessible protocols for tissue production and test performance. Due to structural, mechanistic and procedural similarity, a blinded catch-up validation study for skin irritation according to OECD Performance Standards (PS) was conducted in three laboratories to promote regulatory acceptance, with OS-REp models produced at a single production site only. While overall sensitivity and predictive capacity met the PS requirements, overall specificity was only 57%. A thorough analysis of the test results led to the assumption that some of the false-positive classifications could have been evoked by volatile skin-irritating chemicals tested in the same culture plate as the non-irritants falsely predicted as irritants. With GC/MS and biological approaches the cross-contamination effect was confirmed and the experimental set-up adapted accordingly. Retesting of the affected chemicals with the improved experimental set-up and otherwise identical protocol resulted in correct classifications as non-irritants. Taking these re-test results into account, 93% overall sensitivity, 70% specificity and 82% accuracy was achieved, which is in accordance with the OECD PS. A sufficient reliability of the method was indicated by a within-laboratory-reproducibility of 85–95% and a between-laboratory-reproducibility of 90%. Highlights: An in vitro skin irritation test based on the open source reconstructed epidermis (OS-REp) was developed. Open source means freely available protocols for tissue construction and test performance without legal restrictions. A catch-up validation study with 20 reference chemicals was conducted in 3 laboratories. Cross-contamination of OS-REp tissues with volatile irritants was identified as reason for initial low specificity. After re-testing with improved experimental set-up 93% overall sensitivity, 70% specificity and 82% accuracy were achieved.