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

•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.

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%.