Abstract
<jats:p>Reconstructed human epidermis (RhE) irritation testing under OECD Test Guideline 439 is among the most successfully validated replacements for animal testing in regulatory toxicology. Independent validation exercises report within-laboratory reproducibility of 87 to 100% and between-laboratory reproducibility of 80 to 100%, and a seventeen-laboratory transfer of the medical device protocol achieved 98% agreement of predictions. Against the roughly 58% agreement obtained when the same materials are circulated for ISO 10993-5 cytotoxicity assessment, this is a demonstration that reproducible in vitro testing is achievable when performance standards, reference chemical panels and defined acceptance criteria are specified. This review examines a gap in that achievement. The viability endpoint of TG 439 and of ISO 10993-23 is reduction of a tetrazolium salt, and the applicability domain of both was established using neat chemicals and their mixtures. Nanomaterials were not part of that domain, yet RhE models are now routinely applied to them. A separate and substantial literature establishes that nanomaterials interfere directly with tetrazolium chemistry: six commonly used tetrazolium salts are affected by carbon nanotubes, with absorbance driven below the 70% classification cut-off at cell-free concentrations, and zinc ions released from degrading alloys shift WST-8 signal in the direction of apparent viability against direct microscopic observation. These two literatures barely intersect. Of eight recent RhE studies of nanomaterials examined here, one reported an explicit interference control. That study found no interference for its material class and concluded, correctly for that class, that the guideline was applicable. A single negative result does not establish an applicability domain, and the organosilicon case shows what happens when domain limits are assumed rather than tested: both validated RhE methods failed to predict rabbit outcomes and agreed with each other in only 60% of cases. Recommendations are made for a mandatory cell-free interference control at every tested concentration, routine use of the orthogonal endpoints the guideline already permits, and a formal applicability-domain extension exercise for nanomaterials.</jats:p>