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

Alexander Edwards

doi:10.14573/altex.1710051

Abstract

Skin sensitisers are substances that can elicit allergic responses following skin contact and the process by which this occurs is described as skin sensitisation. Skin sensitisation is defined as a series of key events, that form an adverse outcome pathway (AOP). Key event three 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 (TG)442E) and can be used to assess skin sensitisation 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 in Annex II of TG442E, 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 TG442E, enabling compliance with global regulations.

Highlights

Skin sensitization is a multi-faceted process that has been described as an adverse outcome pathway (AOP)
2.1 Proficiency substances All proficiency substances were purchased from Sigma-Aldrich and selected based upon the criteria detailed in Annex II of OECD TG 442E and supporting information provided in the European Union Reference Laboratory for Alternative Methods to Animal Testing (ECVAM) Human Cell Line Activation Test (h-CLAT) Validation Study Report. 2,4-Dinitrochlorobenzene (DNCB; Sigma-Aldrich, CAS# 97-00-7) was used as the positive control for the dose finding assay
3.1 THP-1 cell culture THP-1 cells cultured in animal-product-free conditions exhibited morphology and an average population doubling time of 44 hours (n = 10 independent experiments, range 35-53 h based upon mean ±3 standard deviations) that were consistent with the ATCC THP-1 data sheet and the details given in OECD TG 442E

Summary

Introduction

Skin sensitization is a multi-faceted process that has been described as an adverse outcome pathway (AOP). Due to current legislation, including the European Cosmetics Regulation 1223/2009 and REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals), alternatives to traditionally used animal models have been sought in order to determine the skin sensitization potential of test chemicals. The h-CLAT method models KE 3 and quantifies the changes in cell surface marker expression on the THP-1 cell line, a human monocytic leukemia cell line, which mimics DCs. Surface markers CD54 and CD86 are up-regulated in the presence of skin sensitizers, indicating that DC activation has occurred (Ashikaga et al, 2006). Concurrent cytotoxicity assessment is carried out using propidium iodide (PI) to assess whether the up-regulated expression of CD54 and CD86 occurs at sub-cytotoxic test chemical concentrations (Sakaguchi et al, 2009). The data is assessed using a prediction model to discriminate between sensitizers and non-sensitizers (TG 442E; OECD, 2018b)

Methods

Results

Conclusion