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The intra- and inter-laboratory reproducibility and predictivity of the KeratinoSens assay to predict skin sensitizers in vitro: Results of a ring-study in five laboratories |
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| Authors: | Natsch Andreas Bauch CarolineFoertsch Leslie Gerberick FrankNorman Kimberly Hilberer AllisonInglis Heather Landsiedel RobertOnken Stefan Reuter HendrikSchepky Andreas Emter Roger |
| Affiliation: | a Givaudan Schweiz AG, Ueberlandstrasse 138, CH-8600 Duebendorf, Switzerland b BASF SE, GV/TB - Z570, D-67056 Ludwigshafen, Germany c Procter and Gamble Inc., Miami Valley Laboratories, Cincinnati, USA d The Institute for In Vitro Sciences, Inc. (IIVS), 30 W. Watkins Mill Rd., Ste. 100, Gaithersburg, MD 20878, USA e Beiersdorf AG, Unnastrasse 48, D-20253 Hamburg, Germany |
| Abstract: | Due to regulatory constraints and ethical considerations, research on alternatives to animal testing to predict the skin sensitization potential of novel chemicals has gained a high priority. Accordingly, different in vitro, in silico and in chemico approaches have been described in the scientific literature to achieve this goal. To replace regulatory approved animal tests, these alternatives need to be transferable to other labs, their within and between laboratory reproducibility must be assured, and their predictivity should be high. The KeratinoSens assay is a cell-based reporter gene assay to screen substances with a full dose-response assessment. It is based on a stable transgenic keratinocyte cell line. The induction of a luciferase gene under the control of the antioxidant response element (ARE) derived from the human AKR1C2 gene is determined. Here we report on the results of a ring-study with five laboratories performing the KeratinoSens assay on a set of 28 test substances. The assay was found to be easily transferable to all laboratories. Overall both the qualitative (sensitizer/non-sensitizer categorization) and the quantitative (concentration for significant gene induction) results were reproducible between laboratories. A detailed analysis of the transferability, the within- and between laboratory reproducibility and the predictivity is presented. |
| Keywords: | LLNA, local lymph node assay Nrf2, nuclear factor-erythroid 2-related factor 2 Keap1, Kelch-like ECH-associated protein 1 ARE, antioxidant response element SOP, standard operating procedure WLR, within laboratory reproducibility BLR, between laboratory reproducibility PC, predictive capacity ECVAM, European centre for the validation of alternative methods to animal testing ICCVAM, interagency coordinating committee on the validation of alternative methods RT-PCR, reverse transcriptase polymerase chain reaction DMSO, dimethylsulfoxide DNCB, 2,4-dinitrochlorobenzene SLS, sodium lauryl sulphate MCI, (5-Chloro)-methyl-isothiazolinone IC50, inhibitory concentration for 50% reduction in viability as determined with the MTT assay EC 1.5, extrapolated concentration for 1.5-fold luciferase induction above threshold REACH, registration, evaluation, authorization and restriction of chemicals h-CLAT, human cell line activation test MUSST, myeloid U937 skin sensitization test |
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