Abstract
Several in vitro OECD test guidelines address key events 1-3 of the adverse outcome pathway for skin sensitization, but none are validated for sensitizer potency assessment. The reaction of sensitizing molecules with skin proteins is the molecular initiating event and appears to be rate-limiting, as chemical reactivity strongly correlates with sensitizer potency. The kinetic direct peptide reactivity assay (kDPRA), a modification of the DPRA (OECD TG 442C), allows derivation of rate constants of the depletion of the cysteine-containing model peptide upon reaction with the test item. Its reproducibility was demonstrated in an inter-laboratory study. Here, we present a database of rate constants, expressed as log kmax, for 180 chemicals to define the prediction threshold to identify strong sensitizers (classified as GHS 1A). A threshold of log kmax -2 offers a balanced accuracy of 85% for predicting GHS 1A sensitizers according to the local lymph node assay. The kDPRA is proposed as a stand-alone assay for identification of GHS 1A sensitizers among chemicals identified as sensitizers by other tests or defined approaches. It may also be used for the prediction of sensitizer potency on a continuous scale, ideally in combination with continuous parameters from other in vitro assays. We show how the rate constant could be combined with read-outs of other in vitro assays in a defined approach. A decision model based on log kmax alone has, however, a high predictivity and can be used as stand-alone model for identification of GHS 1A sensitizers among chemicals predicted as sensitizers.
Highlights
The field of non-animal testing for skin sensitization has rapidly advanced over the past decade, leading to the publication of the adverse outcome pathway (AOP) for skin sensitization by the OECD in 2012, in which the sensitization process has been simplified and described as a series of mechanistic key events (KE) (OECD, 2012).Three OECD test guidelines were published covering the first three KE (OECD, 2018a,b, 2020), namely KE 1 on covalent binding to proteins, KE 2 on keratinocyte activation, and KE 3 on activation of dendritic cells
Chemicals with weak peptide depletion in these previous publications were retested according to the standard operating procedure (SOP), which contains a number of rules to exclude, based on a more rigorous statistical analysis, random positive results from small fluctuations in the depletion data (DB-ALM protocol no. 217, in preparation)
The method as used in the previous publications is identical in terms of volume of the reaction, composition of the reaction, and the fluorescent read-out, and it is fully compatible with the method as described in the SOP, while the definition of the plate setup as well as the automatic evaluation and statistical analysis of the data was introduced with the SOP (DB-ALM protocol no. 217, in preparation)
Summary
The field of non-animal testing for skin sensitization has rapidly advanced over the past decade, leading to the publication of the adverse outcome pathway (AOP) for skin sensitization by the OECD in 2012, in which the sensitization process has been simplified and described as a series of mechanistic key events (KE) (OECD, 2012).Three OECD test guidelines were published covering the first three KE (OECD, 2018a,b, 2020), namely KE 1 on covalent binding to proteins (the molecular initiating event, MIE), KE 2 on keratinocyte activation, and KE 3 on activation of dendritic cells. At the time of writing, these three test guidelines cover a total of seven test methods. They were all developed for hazard identification based on a binary prediction model, while the cellular assays provide concentration-response information. Reproducibility of the concentration-response data was not evaluated in the peer-review of the validation studies, nor has a formalized way to use this quantitative information reached test guideline status. Multiple studies have investigated application of quantitative in vitro data from the validated assays for potency prediction (Hirota et al, 2018; Jaworska et al, 2013, 2015; Natsch et al, 2015; Nukada et al, 2013)
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