Sensitivity of the Kinetic Direct Peptide Reactivity Assay (kDPRA)

The kinetic direct peptides peptide reactivity assay (kDPRA) is a non-animal test method that assesses the reaction kinetics of a chemical with a synthetic cysteine-containing model peptide. The reaction is stopped by addition of monobromobimane, which forms a fluorescent complex with the free cysteine in the model peptide. The relative amount of depleted peptide is determined by the absorbance at 405 nm. In combination with the results of two other in vivo cell-based assays, the kDPRA provides a skin sensitisation hazard prediction. This article investigates the sensitivity of this prediction when it is used to distinguish chemicals in binary mixtures, which differ significantly in their DP value with respect to the model peptide.

The ability of a chemical to form a covalent bond with the thiol group of the cysteine residue in the peptidyl-cysteine protein (peptidyl-cysteine) is an important property that determines its permeability and toxicity, but it also reflects the extent to which the molecule can be converted to transient redox-active disulfide bonds. This redox activity can cause oxidative damage to proteins and other molecules, but it is also responsible for the labile nature of the bonds formed in the context of a reactivity assay with peptides.

The kDPRA performs well when used to assign chemicals to the UN GHS potency categories 1A and 1B/NS on the basis of peptide depletion, but there are groups of chemicals for which it is not a good predictor of sensitisation. The most prominent examples are the aldehydes chlorpromazine, formaldehyde and glutaraldehyde, which need to be activated by chemical oxidation to release the reactive thiol group. A small number of other chemicals, such as phthalic and trimellitic anhydrides, which are both classed as acyl transfer agents, are outside the chemistry-potency applicability domain of the kDPRA because they react with the peptidyl-cysteine in a similar way but have different hydrophobicity and therefore exhibit very similar logkmax values.

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