Abstract
The direct peptide reactivity assay (DPRA) is a validated method for in vitro assessment of the skin sensitization potential of chemicals. In the present work, we describe a peptide reactivity assay using 96-well plate format and systematically identified the optimal assay conditions for accurate and reproducible classification of chemicals with known sensitizing capacity. The aim of the research is to ensure that the analytical component of the peptide reactivity assay is robust, accurate, and reproducible in accordance with criteria that are used for the validation of bioanalytical methods. Analytical performance was evaluated using quality control samples (QCs; heptapeptides at low, medium, and high concentrations) and incubation of control chemicals (chemicals with known sensitization capacity, weak, moderate, strong, extreme, and non-sensitizers) with each of three synthetic heptapeptides, viz Cor1-C420 (Ac-NKKCDLF), cysteine- (Ac-RFAACAA), and lysine- (Ac-RFAAKAA) containing heptapeptides. The optimal incubation temperature for all three heptapeptides was 25°C. Apparent heptapeptide depletion was affected by vial material composition. Incubation of test chemicals with Cor1-C420, showed that peptide depletion was unchanged in polypropylene vials over 3-days storage in an autosampler but this was not the case for borosilicate glass vials. For cysteine-containing heptapeptide, the concentration was not stable by day 3 post-incubation in borosilicate glass vials. Although the lysine-containing heptapeptide concentration was unchanged in both polypropylene and borosilicate glass vials, the apparent extent of lysine-containing heptapeptide depletion by ethyl acrylate, differed between polypropylene (24.7%) and glass (47.3%) vials. Additionally, the peptide-chemical complexes for Cor1-C420-cinnamaldehyde and cysteine-containing heptapeptide-2, 4-dinitrochlorobenzene were partially reversible during 3-days of autosampler storage. These observations further highlight the difficulty in adapting in vitro methods to high-throughput format for screening the skin sensitization potential of large numbers of chemicals whilst ensuring that the data produced are both accurate and reproducible.
Highlights
Allergic contact dermatitis (ACD) is the clinically significant consequence of skin sensitization that negatively affects ∼15– 20% of the general population (Peiser et al, 2012)
Carryover was observed for Cor1-C420, such that the peak area of the heptapeptide detected in the blank was 10% of the response from the lower limit of quantitation (LLOQ)
Optimization of the assay protocol to provide favorable assay conditions for these peptides and the different chemical classes being assessed is recommended to ensure that accurate and meaningful data are obtained from the peptide reactivity assay
Summary
Allergic contact dermatitis (ACD) is the clinically significant consequence of skin sensitization that negatively affects ∼15– 20% of the general population (Peiser et al, 2012). According to the European Cosmetic Directive (EC1223/2009), the ban of cosmetic ingredients that were subjected to animal testing was enforced. On 11 March 2013, full ban on animal testing for cosmetic purposes was enforced. The EU REACH regulation (registration, evaluation, authorization, and restriction of chemicals), EC1907/2006, that came into force on 1 June 2007, provided a strong imperative for the development and implementation of rapid in vitro screening methods (EU, 2006) for assessing the skin sensitization potential of chemicals that had not been previously tested using in vivo methods. Implementation of the 3Rs, reduction, refinement and replacement of animal testing, has driven the need to adopt alternative non-animal skin sensitization screening methods. Validated in vitro methods are essential for identifying potential skin sensitizers to prevent ACD (EU, 2009)
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