MEETING REQUIREMENTS OF OECD TG 404 FOR IN VITRO SKIN IRRITATION TESTING: REPRODUCIBILITY OF EPIDERM SKIN IRRITATION TEST (EPIDERM-SIT) FOLLOWING ECVAM VALIDATION AND ACCEPTANCE AS A FULL REPLACEMENT METHOD.

In autumn 2007, an international validation study was performed to evaluate the reproducibility and confirm the predictive ability of the EpiDerm Skin Irritation Test (SIT) method (1, 2). In November 2008, ECVAM endorsed the EpiDerm SIT as a full replacement method for the in vivo rabbit skin irritation test. As reflected draft OECD guidelines for skin irritation testing (TG 404), US and EU regulators have appropriately maintained that a test method must demonstrate reproducibility on an ongoing basis so that regulators and commercial end users are assured that the assay method is stable and continues to give valid test results over time. The purpose of the present study was to investigate the reproducibility of the EpiDerm SIT post-validation. Over a 7-month period in 2009, 24 independent lots of EpiDerm tissue were exposed to 3 irritants (alpha terpineol, heptanal, and butyl methacrylate), 3 non-irritants (benzyl benzoate, benzyl salicylate, and isopropanol) and the positive control (sodium dodecyl sulphate) and negative control (Dulbecco’s phosphate buffered saline) using the SIT protocol. As per the SIT method, tissue viability was determined using the robust and cost effective MTT assay following a single, 60-minute exposure and 42-hour post-exposure incubation. In all cases, the SIT method correctly identified the irritants and non-irritants. Coefficients of variation (CV) between the tests (n=24) for the tissue viability for all non-irritants were <10%, except for isopropanol (IPA) which had a CV of 11.3%. Further study of the IPA results, revealed the importance of thoroughly rinsing the tissue following the 60-minute exposure (as outlined in the SIT method). These results together with quality control results for EpiDerm over the past 14 years confirm that the EpiDerm SIT method is REACH ready and compliant with the EU 7th Amendment to the Cosmetic Directive.

EPI-200-SIT, EpiDerm-SIT, Long-term reproducibility, OECD TG 404, Performance standards, Quality control (QC), REACH, Skin irritation testing

Alpha terpineol, Benzyl benzoate, Benzyl salicytate, Butyl methacrylate, Heptanal, Isopropanol, Sodium dodecyl sulfate (5%), Triton X-100