Adaptation of the KeratinoSens™ skin sensitization test to animal-product-free cell culture

Nathalie Belot

doi:10.14573/altex.1701311

Abstract

Skin sensitization is the process by which a substance induces an allergic reaction following skin contact. The process has been described as an adverse outcome pathway (AOP), including several key events, from skin penetration and covalent protein binding, to keratinocyte activation, dendritic cell activation and T-lymphocyte proliferation. The in vitro assay KeratinoSens™ measures the activation of keratinocytes. It is fully accepted at a regulatory level (OECD TG 442d) and complies with a range of legislation including the EU Cosmetics Regulation, REACH, and the CLP Regulation. Currently, many in vitro methods use animal-derived components in their cell culture systems. Many stakeholders in the cosmetics industry have both scientific and ethical concerns relating to this issue and have stated a strong preference for fully human in vitro test systems. We have adapted the KeratinoSens™ method to animal product-free conditions, and carried out an in-house validation with 21 reference substances, including those listed in the Performance Standards associated with OECD TG442d. The modified method was shown to be equivalent to the Validated Reference Method (VRM), with comparable values for accuracy (85.7%), sensitivity (84.6%) and specificity (87.5%), and all acceptance criteria being met. In Europe, data generated by the adapted method may be used in REACH submissions, and we are now seeking approval to list the adaptation in OECD TG 442d, enabling formal compliance with a range of global regulations.

Highlights

Skin sensitization is a multi-step process that has been described as an Adverse Outcome Pathway (AOP)
KeratinoSensTM cells are derived from the human keratinocyte cell line HaCat, containing a luciferase gene that is under the control of a constitutive promoter fused with the antioxidant response element (ARE) from a gene that is known to be up-regulated by contact sensitizers
As per the VRM (Validated Reference Method), the following parameters were calculated: the Imax value; the EC1.5 value, representing the lowest concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e., 50% enhanced luciferase activity); and the IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability, respectively

Summary

Introduction

Skin sensitization is a multi-step process that has been described as an Adverse Outcome Pathway (AOP). Dendritic cells migrate to the lymph nodes and induce the proliferation of T-lymphocytes (key event 4). Alternatives for key events 1-3 have recently been adopted as OECD guidelines: DPRA (OECD TG 442c), KeratinoSensTM (OECD TG 442d) and h-CLAT (OECD TG 442e). The KeratinoSensTM method addresses the second key event of skin sensitization, i.e., the activation of keratinocytes. The majority of skin sensitizers induce this pathway and, the luciferase signal reflects the activation by sensitizers of endogenous Nrf dependent genes. This method was fully validated and gained regulatory acceptance in 2015 (Natsch et al, 2010, 2011, 2013)

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Conclusion