Abstract
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.
Highlights
The mechanism underlying skin sensitization is complex, but it is well understood and described as an adverse outcome pathway by Organisation for Economic Co-operation and Development (OECD) (OECD, 2014)
The results reported were very similar to those obtained by the two lead labs (Fig. 2) with the exception of formaldehyde, which was much less reactive in all three repetitions performed at Lab E
The inter-laboratory reproducibility was high for most test substances with an average standard deviation (SD) for inter-laboratory comparison of 0.244, which was slightly higher than in the intra-laboratory comparisons and which corresponds to a variation around the geometric mean of 1.75 fold
Summary
The mechanism underlying skin sensitization is complex, but it is well understood and described as an adverse outcome pathway by OECD (OECD, 2014). Significant progress has been made in the field of non-animal tests, and several test methods to address skin sensitization have been adopted as OECD test guidelines (TGs). The molecular initiating event in the adverse outcome pathway for skin sensitization is the covalent binding of the sensitizer to skin proteins, and a first method to address this key event was adopted as OECD TG 442C in 2015. This method, the direct peptide reactivity assay (DPRA), uses two synthetic peptides (one containing a cysteine, one containing a lysine residue) that are incubated with a single concentration of the test substance. After 24 hours incubation, the concentrations of remaining, non-depleted peptide are determined using HPLC
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