Abstract
DPRA (direct peptide reactivity assay) and ADRA (amino acid derivative reactivity assay), which are based on the biological events of skin sensitization, were developed as alternatives to the controversial animal experiments. These assays are described in the OECD (Organization for Economic Co-operation and Development) guideline, Test No. 442C. Although these assays have been endorsed by the industries and internationally accepted as promising and effective tests for in vitro skin sensitization, they suffer from several drawbacks, such as incompatibility with hydrophobic chemicals and complicated sample processing. Here, we demonstrated a chromophore-based solid phase peptide reaction assay in vitro using peptides immobilized on magnetic beads (C-SPRA-MB). We successfully synthesized lysine (Lys) and cysteine (Cys) immobilized on magnetic microbeads. However, Cys immobilized magnetic microbeads showed gradual decomposition of the magnetic beads due to SH oxidation. Using Lys immobilized magnetic microbeads, we demonstrated the capacity of C-SPRA-MB to predict skin sensitization by measuring free amino groups of the Lys after reaction with test chemicals. First, the free amines on the microbeads were reacted with bromophenol blue (BB). Then, by treatment with a saturated solution of Lys, the bound BBs were released and quantified. C-SPRA-MB provides high-throughput and accurate assays for assessments of chemicals, including with low-potency as skin sensitizers and poor water solubility. C-SPRA-MB may be useful for effective prediction of their skin sensitization potential in the process of compound screening, especially in the case of misclassified by DPRA and ADRA. Thus, C-SPRA-MB can be applied to assessing the sensitization potential of medical, pharmaceutical, cosmetics, and industrial compounds.
Highlights
IntroductionAnimal experiments for assessing such risk potential, especially in cosmetics, were prohibited in the EU
The potential to trigger skin sensitization must be effectively assessed for various chemical compounds present in industrial, cosmetics and pharmaceutical products to minimize the risk of developing skin diseases
After optimizing the synthesis protocol [15], we assessed the purity of the beads loading the amino acids
Summary
Animal experiments for assessing such risk potential, especially in cosmetics, were prohibited in the EU. The potential to trigger skin sensitization must be effectively assessed for various chemical compounds present in industrial, cosmetics and pharmaceutical products to minimize the risk of developing skin diseases. Animal experiments, including local lymph node assay (LLNA) [1], were widely used to assess the sensitization potential of chemical compounds. Processes 2020, 8, 1257 experiments for assessing such risk potential, especially in cosmetics, were prohibited in the EU in. The in 2011 [2]
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