Validation of the GARD™skin Assay for Assessment of Chemical Skin Sensitizers: Ring Trial Results of Predictive Performance and Reproducibility

Abstract

Proactive identification of chemicals with skin sensitizing properties is a key toxicological endpoint within chemical safety assessment, as required by legislation for registration of chemicals. In order to meet demands of increased animal welfare and facilitate increased testing efficiency also in nonregulatory settings, considerable efforts have been made to develop nonanimal approaches to replace current animal testing. Genomic Allergen Rapid Detection (GARD™) is a state-of-the-art technology platform, the most advanced application of which is the assay for assessment of skin sensitizing chemicals, GARD™skin. The methodology is based on a dendritic cell (DC)-like cell line, thus mimicking the mechanistic events leading to initiation and modulation of downstream immunological responses. Induced transcriptional changes are measured following exposure to test chemicals, providing a detailed evaluation of cell activation. These changes are associated with the immunological decision-making role of DCs in vivo and include among other phenotypic modifications, up-regulation of co-stimulatory molecules, induction of cellular and oxidative stress pathways and xenobiotic responses, and provide a holistic readout of substance-induced DC activation. Here, results from an inter-laboratory ring trial of GARD™skin, conducted in compliance with OECD guidance documents and comprising a blinded chemical test set of 28 chemicals, are summarized. The assay was found to be transferable to naïve laboratories, with an inter-laboratory reproducibility of 92.0%. The within-laboratory reproducibility ranged between 82.1% and 88.9%, whereas the cumulative predictive accuracy across the 3 laboratories was 93.8%. It was concluded that GARD™skin is a robust and reliable method for the identification of skin sensitizing chemicals and suitable for stand-alone use or as a constituent of integrated testing. These data form the basis for the regulatory validation of GARD™skin.

The GARDpotency Assay for Potency-Associated Subclassification of Chemical Skin Sensitizers—Rationale, Method Development, and Ring Trial Results of Predictive Performance and Reproducibility

“…All cellular protocols associated with GARDpotency are identical to those of GARDskin, which has been previously described ( Forreryd et al , 2016 ; Johansson et al , 2019 ). For further details, the GARD SOP is available and attached to this publication as Supplementary material 1, including attachments amending the data analysis section for potency subclassification using GARDpotency.…”

“…The design of the interlaboratory ring trial was to a great extent based on the structure of a previously described interlaboratory ring study performed with the purpose of validating the GARDskin assay ( Johansson et al , 2019 ), which was conducted in accordance with OECD guidance documents ( OECD, 2005 ). Briefly, SenzaGen AB (SenzaGen, Lund, Sweden) initiated the study and acted as lead laboratory throughout the experiments.…”

“…Due to the identical experimental protocols of the GARDskin and the GARDpotency assays ( Supplementary material 1), and because both participating laboratories had already shown proficiency in running the GARDskin assay ( Johansson et al , 2019 ), neither transfer nor training phases were performed.…”

“…For assessment of GARDpotency’s predictive performance and reproducibility, the VMG determined that the 28 chemicals that were assayed for the purpose of validating GARDskin ( Johansson et al , 2019 ) would be used, see Table 2 . Only chemicals that were classified as skin sensitizers by the GARDskin assay (by respective laboratory in each experiment) were assessed in the GARDpotency assay, as defined by the GARD tiered approach.…”

“…GARDskin was the first GARD platform application described and is consequently the most advanced in terms of regulatory acceptance. An interlaboratory ring trial was conducted in adherence with OECD guidance documents for acceptance of novel alternative methods ( OECD, 2005 , 2009 ), demonstrating that GARDskin is a powerful tool for assessment of chemical skin sensitizers, with a predictive accuracy of 93.8% and high reproducibility between laboratories ( Johansson et al , 2019 ).…”