The molecular initiating event in skin sensitization is thought to be the covalent binding of electrophilic chemicals to nucleophilic centres in epidermal proteins. This binding is the target of a number of experimental and clinical strategies designed to prevent or delay skin sensitization.
The Direct Peptide Reactivity Assay (DPRA) is an OECD-adopted method for evaluating whether a chemical can react with epidermal proteins to form a protein-complex that triggers skin sensitization. In the DPRA, an aqueous solution of a test chemical is incubated with an excess of either cysteine or lysine peptide and the extent of peptide depletion is evaluated by HPLC. This allows the chemical to be classified into four reactivity categories and discriminated between sensitizers and non-sensitizers.
A spectrophotometric method, Spectro-DPRA, has been developed to obviate some limitations of the DPRA by assessing the reaction of the chemical with two model peptides in a high throughput manner. Pre-validation studies at three laboratories confirmed high transferability and proficiency for Spectro-DPRA.
In a typical assay, the peptides are Fmoc-protected and incubated in an aqueous solution of the test chemical at a concentration of 1 to 10 g/mL. The samples are then subjected to a series of chromatographic conditions (scan times, UV wavelength, retention time, elution volume, and pH). A reference sample is also run to ensure that the results can be interpreted correctly. A cinnamic aldehyde is used as an elution control to assess whether the direct peptides are being absorbed or removed by the solvent system.
A number of chemicals have been found to give erratic DPRA results. These are generally chemicals that are not reactive with peptides but which can be sensitizing as a result of oxidation to a reactive species under exposure conditions. This can occur for example when the phenolic group of PPD is converted to a quinoline and is reacted with the peptide’s -SH group in an oxygen-dependent reaction.
The kDPRA protocol cannot directly determine the true rate constant for the reaction of the peptide with the test chemical under the assay conditions and this is why this approach is not able to predict sensitization. For the seventeen chemicals that have a clear trend, the calculated k value decreases with increasing t. However, for seven chemicals the k values do not show a clear correlation with t and therefore their logk value alone can not be reliably taken as an indication of a chemical’s ability to interact with other nucleophiles. In these cases a more detailed analysis is required to identify the reason for their erratic DPRA results.