Performance of a novel in vitro assay for skin sensitization based on activation of T lymphocytes.

Fenxia Hou

doi:10.14573/altex.2001312

Abstract

There are currently no skin sensitization assays based on T cell activation. We built a novel in vitro model to assess T cell activation and test its performance to discriminate skin sensitizers from non-sensitizers using 52 reference chemicals. Jurkat Clone E6-1 human T lymphocytes were exposed to a series of concentrations of test substances for 24 hours and CD69 expression was measured as a marker of early T cell activation with flow cytometry. Most tested sensitizers induced increased relative fluorescence intensity (RFI) of CD69 on the T cells, which was linearly correlated with the concentrations tested, indicating a statistically significant causal link between sensitizer concentration and increase of CD69 expression. CD69 RFI ≥ 1.5 was determined as the positive criterion for skin sensitizer classification. The sensitivity (79.4%), specificity (88.9%) and accuracy (82.7%) of the model for the 52 tested reference chemicals showed a good predictivity for skin sensitizers. The results were reproduced in at least two repeats; and the concurrent positive control, 2 mg/mL 2, 4-dinitrochlorobenzene, was found positive in all 25 independent runs conducted, indicating in-house reproducibility. The EC1.5 value (i.e., the concentration at which a test chemical induces a CD69 RFI of 1.5) may be used to assess skin sensitization potency of a chemical. This work may contribute to the development of an in vitro assay for skin sensitization based on the activation of T cells.

Highlights

Skin sensitization is one of the key adverse effects that many chemicals possess
The criterion to predict cluster of differentiation 69 (CD69) expression at a specific concentration of test chemical as a positive response was set as CD69 relative fluorescence intensity (RFI) ≥ 1.5 at cell viability ≥ 50%
The Jurkat Clone E6-1 human T cells were incubated directly with the test substances and CD69 expression on the T cells was determined by flow cytometry to show early T cell activation induced by sensitizers

Summary

Introduction

Skin sensitization is one of the key adverse effects that many chemicals possess. The Organization for Economic Co-operation and Development (OECD) has developed and adopted several assays for skin sensitization based on the first three of the four AOP KEs (OECD, 2015a,b, 2018). It is consistently argued that a single in vitro assay for skin sensitization is not sufficient to substitute testing in an animal model (e.g., the LLNA). The integration of information derived from assays for the first three of the four AOP KEs for the skin sensitization AOP already is the basis for several defined approaches (DA) to be used in IATA (OECD, 2016). Additional data on the fourth KE for the skin sensitization AOP may provide useful complementary information to complete the development of AOP-based DAs and IATAs and improve current predictive parameters

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Results

Conclusion