The kinetic direct peptide reactivity assay (kDPRA): Intra- and inter-laboratory reproducibility in a seven-laboratory ring trial

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Britta Wareing
Susanne N. Kolle
Barbara Birk
Nathalie Alépée
Tina Haupt
Rishil Kathawala
Petra S. Kern
Laurent Nardelli
Hans Raabe
Marián Rucki
Cindy A. Ryan
Sjoerd Verkaart
Walter M. A. Westerink
Robert Landsiedel
Andreas Natsch


While the skin sensitization hazard of substances can be identified using non-animal methods, the classification of potency into UN GHS sub-categories 1A and 1B remains challenging. The kinetic direct peptide reactivity assay (kDPRA) is a modification of the DPRA wherein the reaction kinetics of a test substance towards a synthetic cysteine-containing peptide are evaluated. For this purpose, several concentrations of the test substance are incubated with the synthetic peptide for several incubation times. The reaction is stopped by addition of monobromobimane, which forms a fluorescent complex with the free cysteine of the model peptide. The relative remaining non-depleted amount of peptide is determined. Kinetic rate constants are derived from the depletion vs concentration and time matrix and used to distinguish between UN GHS sub-category 1A sensitizers and test substances in sub-category 1B/not classified test substances. In this study, we present a ring trial of the kDPRA with 24 blind-coded test substances in seven laboratories. The intra- and inter-laboratory reproducibility were 96% and 88%, respectively (both for differentiating GHS Cat 1A sensitizers from GHS Cat 1B/not classified). Following an independent peer review, the kDPRA was considered to be acceptable for the identification of GHS Cat 1A skin sensitizers. Besides GHS Cat 1A identification, the kDPRA can be used as part of a defined approach(es) with a quantitative data integration procedure for skin sensitization potency assessment. For this aim, next to reproducibility of classification, the quantitative reproducibility and variability of the rate constants were quantified in this study.

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Wareing, B., Kolle, S. N., Birk, B., Alépée, N., Haupt, T., Kathawala, R., Kern, P. S., Nardelli, L., Raabe, H., Rucki, M., Ryan, C. A., Verkaart, S., Westerink, W. M. A., Landsiedel, R. and Natsch, A. (2020) “The kinetic direct peptide reactivity assay (kDPRA): Intra- and inter-laboratory reproducibility in a seven-laboratory ring trial”, ALTEX - Alternatives to animal experimentation, 37(4), pp. 639-651. doi: 10.14573/altex.2004291.

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