A Stepwise Skin Irritation Assessment Approach Integrating in Vitro Skin Irritation Testing and Human Patch Testing

Cosmetic and quasi-drug ingredients intended for daily use require reliable skin irritation assessment. However, restrictions on animal testing, including the European Union marketing ban for cosmetics tested on animals, have prompted the adoption of reconstructed human epidermis (RhE) assays, such as Organization for Economic Co-operation and Development (OECD) Test Guideline 439 (TG439). Although TG439 identifies Globally Harmonized System of Classification and Labelling of Chemicals (GHS) Category 2 irritants and No Category (non-irritant) chemicals as an alternative to the 4-h rabbit skin irritation test, its ability to predict human responses to 24-h exposure and mild irritation remains limited. This study evaluated the reproducibility of TG439 across six laboratories using LabCyte EPI-MODEL24 and a standardized stepwise dilution design. TG439 was used to determine a "TG439 non-irritant concentration" for 17 test substances. Human 24-h closed patch tests were then performed using stepwise escalation to one-quarter and one-half of this concentration, with predefined stopping criteria to ensure subject safety. Inter-laboratory control results confirmed TG439 reproducibility. Most test substances exhibited Sugai's Irritation Index (S.I.I.) values within the "Safe" or "Acceptable" categories at both test concentrations. However, selected surfactants, cationic preservatives, and hair dye intermediates produced elevated S.I.I. values, requiring discontinuation of concentration escalation and highlighting the importance of predefined stopping criteria. These findings support TG439 as a practical starting point for human irritation testing when combined with a stepwise concentration approach and predefined safety criteria, enabling the ethical translation of in vitro findings to human testing and extending TG439-based evaluation beyond "harmless ingredients."