Quantification of Chemical Peptide Reactivity for Screening Contact Allergens: A Classification Tree Model Approach

Abstract

In the interest of reducing animal use, in vitro alternatives for skin sensitization testing are under development. One unifying characteristic of chemical allergens is the requirement that they react with proteins for the effective induction of skin sensitization. The majority of chemical allergens are electrophilic and react with nucleophilic amino acids. To determine whether and to what extent reactivity correlates with skin sensitization potential, 82 chemicals comprising allergens of different potencies and nonallergenic chemicals were evaluated for their ability to react with reduced glutathione (GSH) or with two synthetic peptides containing either a single cysteine or lysine. Following a 15-min reaction time with GSH, or a 24-h reaction time with the two synthetic peptides, the samples were analyzed by high-performance liquid chromatography. UV detection was used to monitor the depletion of GSH or the peptides. The peptide reactivity data were compared with existing local lymph node assay data using recursive partitioning methodology to build a classification tree that allowed a ranking of reactivity as minimal, low, moderate, and high. Generally, nonallergens and weak allergens demonstrated minimal to low peptide reactivity, whereas moderate to extremely potent allergens displayed moderate to high peptide reactivity. Classifying minimal reactivity as nonsensitizers and low, moderate, and high reactivity as sensitizers, it was determined that a model based on cysteine and lysine gave a prediction accuracy of 89%. The results of these investigations reveal that measurement of peptide reactivity has considerable potential utility as a screening approach for skin sensitization testing, and thereby for reducing reliance on animal-based test methods.

Patch testing – Revisited

“…An in vitro test for cell-mediated immunity is possible and could be accomplished experimentally, perhaps by peptide reactivity measurements [20]. This will enable patients to give a small amount of blood for an accurate diagnosis.…”

Alternative Methods to Animal Testing for the Safety Evaluation of Cosmetic Ingredients: An Overview

“…Other were accepted in 2015, including Test 442C, the In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA) [29], which was developed some years ago [30,31] and Test 442D: In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method [32]. This last method was proposed to demonstrate the second event that takes place in the sensitisation mechanism occurring in the keratinocytes and includes inflammatory responses as well as gene expression associated with specific cell signaling pathways such as the antioxidant/electrophile response element [33].…”

T-cell recognition of chemicals, protein allergens and drugs: towards the development of in vitro assays

“…The latter point is not addressed, for example, when haptenated proteins are used as T-cell antigens. However, chemical reactivity seems to correlate with allergenic potency [40,41]. Therefore, it is absolutely crucial to know the questions that can be addressed by the type of T-cell assay used ( Table 1).…”

“…It is clear that knowledge of the chemistry is required to perform and refine T-cell-based assays, and that this involves close collaborations with chemists possessing this expertise. Historical data are available and more experience is gained with the development of assays that are based on the chemical reactivity to peptides [40,41]. It remains an open and difficult question whether and how T-cell-based assays can incorporate pre-and pro-haptens [86,87].…”