Using novel in vitro methods to predict of the skin sensitization potential of botanical mixtures

Determination of skin sensitization is a critical toxicological endpoint in the safety evaluation of botanical ingredients. With the European Union regulatory ban of animal testing for cosmetic ingredients, and many companies proactively choosing to eliminate animal testing due to ethical considerations, non-animal methods are increasingly being used for safety assessments. The KeratinoSens assay is a cell-based reporter gene assay which may be used to assess the potential of chemicals to induce skin sensitization in humans. A feature all chemical allergens have in common is their intrinsic electrophilicity, or their potential to be transformed to electrophilic chemicals. The Nrf-2-electrophile sensing pathway comprised of the repressor protein Keap1, the transcription factor Nrf2 and the antioxidant response element (ARE), is capable of detecting skin sensitizers. In the KeratinoSens assay, the induction of a luciferase gene under the control of the antioxidant response element derived from the human gene AKR1C2 gene is quantified. Botanical mixtures were tested in the KeratinoSens assay and the resulting data compared to available historical animal data or clinical data. Since the botanical extract portion may only be a small fraction of the complete mixture, as a proof of concept low levels of known sensitizers were spiked into botanical mixtures with different excipient solvent systems and luciferase induction and cytotoxicity measured. Even at levels as low as 1%, the assay was capable of detecting the spiked sensitizers. These data suggest that the KeratinoSens assay has the potential to identify electrophile allergens within a botanical extract mixture.