Adaptation of the human Cell Line Activation Test (h-CLAT) to Animal-Product-Free Conditions

Skin sensitizers are substances that can elicit allergic responses following skin contact. The process by which this occurs, i.e., skin sensitization, is a series of key events that form an adverse outcome pathway (AOP). Key event 3 in the AOP is dendritic cell activation that can be modelled by the human Cell Line Activation Test (h-CLAT) and is typified by changes in cell surface markers CD54 and CD86 in dendritic cells. The h-CLAT is accepted at a regulatory level (OECD Test Guideline 442E) and can be used to assess skin sensitization potential as part of an integrated approach to testing and assessment (IATA). Stakeholders in the cosmetics and chemical industries have scientific and ethical concerns relating to use of animal-derived material and have communicated a strong preference for fully human-based in vitro methods. Therefore, we adapted the h-CLAT to animal-product-free conditions and validated the adapted method with the proficiency panel substances listed in Annex II of TG 442E using 3 independent batches of pooled human serum. The modified method showed equivalence to the validated reference method (VRM), as all proficiency substances were correctly classified. Comparable values for CV75 (concentration yielding 75% cell viability), EC150 and EC200 (concentration yielding RFI of >= 150 for CD86 and >= 200 for CD54) were obtained. Data generated using the adapted method may be used in European REACH submissions, provided the proficiency data is included. We are seeking formal inclusion of the adaptation into TG 442E, enabling compliance with global regulations.