Reconstructed human epidermis‐based testing strategy of skin sensitization potential and potency classification using epidermal sensitization assay and in silico data

Abstract

The hazards and potency of skin sensitizers are traditionally determined using animal tests such as the local lymph node assay (LLNA); however, significant progress has been made in the development of non‐animal test methods addressing the first three mechanistic key events of adverse outcome pathway in skin sensitization. We developed the epidermal sensitization assay (EpiSensA), which is a reconstructed human epidermis‐based assay, by measuring four genes related to critical keratinocyte responses during skin sensitization. Four in vitro skin sensitization test methods (EpiSensA, direct peptide reactivity assay [DPRA], KeratinoSens™, and human cell line activation test [h‐CLAT]) were systematically evaluated using 136 chemicals including lipophilic chemicals and pre/pro‐haptens, which may be related to assay‐specific limitations. The constructed database included existing and newly generated data. The EpiSensA showed a broader applicability domain and predicted the hazards with 82.4% and 78.8% accuracy than LLNA and human data. The EpiSensA could detect 76 out of 88 sensitizers at lower concentrations than the LLNA, indicating that the EpiSensA has higher sensitivity for the detection of minor sensitizing constituents. These results confirmed the potential use of the EpiSensA in evaluating a mixture of unknown compositions that can be evaluated by animal tests. To combine different information sources, the reconstructed human epidermis‐based testing strategy (RTS) was developed based on weighted multiple information from the EpiSensA and TImes MEtabolism Simulator platform for predicting Skin Sensitization (TIMES‐SS; RTSv1) or Organization for Economic Cooperation and Development (OECD) QSAR Toolbox automated workflow (RTSv2). The predictivities of the hazards and Globally Harmonized System (GHS) subcategories were equal to or better than the defined approaches (2 out of 3, integrated testing strategy [ITS]v1, and ITSv2) adopted as OECD Guideline 497.

Adhesive Antibacterial Moisturizing Nanostructured Skin Patch for Sustainable Development of Atopic Dermatitis Treatment in Humans

“…Following the MTT assay to assess tissue metabolism, the next step involved determining the activity of lactate dehydrogenase (LDH) in culture media (Figure c). This sequence of tests enables a comprehensive analysis of metabolic activity, where the MTT assay provides information on the tissue’s ability to reduce substrates, whereas LDH confirms enzymatic activity indicative of intermediate metabolism. , To validate the method’s reliability, a positive control, PC (SDS treatment), yielded negative results in all parameters tested, confirming the method’s accuracy. It is important to underscore the significance of the fact that the optimal exposure time-point for the Epiderm tissue model was determined to be 18 h, as per the extensive optimization carried out in the literature for the Epiderm skin irritation test (EPI-200-SIT-MD) .…”

Proof of concept testing of a positive reference material for in vivo and in vitro sensitization testing of medical devices

“…EpiSensA was performed as previously reported. 9 The LabCyte EPI-MODEL24 (Japan Tissue Engineering Co. Ltd., Aichi, Japan) was used for the in vitro skin sensitization test. This RhE model is included in OECD TG 439.…”

“…The epidermal sensitization assay (EpiSensA) is an alternative method that uses reconstructed human epidermis in the same way as animal tests to evaluate oil-soluble raw materials. [7][8][9] KE3 (as determined by the expression of specific cell surface markers and the production of chemokines and cytokines) can be evaluated using the human cell line activation test (h-CLAT), U-SENS™, and IL-8 Luc assay, as described in OECD TG 442E. 10 Annex C of ISO 10993-10 describes various nonanimal methods for the sensitization testing of medical devices.…”