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

Abstract

Proactive identification and characterization of hazards attributable to chemicals are central aspects of risk assessments. Current legislations and trends in predictive toxicology advocate a transition from in vivo methods to nonanimal alternatives. For skin sensitization assessment, several OECD validated alternatives exist for hazard identification, but nonanimal methods capable of accurately characterizing the risks associated with sensitizing potency are still lacking. The GARD (Genomic Allergen Rapid Detection) platform utilizes exposure-induced gene expression profiles of a dendritic-like cell line in combination with machine learning to provide hazard classifications for different immunotoxicity endpoints. Recently, a novel genomic biomarker signature displaying promising potency-associated discrimination between weak and strong skin sensitizers was proposed. Here, we present the adaptation of the defined biomarker signature on a gene expression analysis platform suited for routine acquisition, confirm the validity of the proposed biomarkers, and define the GARDpotency assay for prediction of skin sensitizer potency. The performance of GARDpotency was validated in a blinded ring trial, in accordance with OECD guidance documents. The cumulative accuracy was estimated to 88.0% across 3 laboratories and 9 independent experiments. The within-laboratory reproducibility measures ranged between 62.5% and 88.9%, and the between-laboratory reproducibility was estimated to 61.1%. Currently, no direct or systematic cause for the observed inconsistencies between the laboratories has been identified. Further investigations into the sources of introduced variability will potentially allow for increased reproducibility. In conclusion, the in vitro GARDpotency assay constitutes a step forward for development of nonanimal alternatives for hazard characterization of skin sensitizers.

Adjuvants in fungicide formulations can be skin sensitizers and cause different types of cell stress responses

“…Test materials were classified as skin sensitizers when the mean of the support vector machine decision values obtained for triplicate samples was > 0. Some of the skin sensitizers were further predicted into 1A and 1B potency classes [26] . The potency predictions are based on the same physical samples and processing as for the GARD®skin based assay, with the difference that RNA expression was analyzed for a different biomarker signature consisting of 52 transcripts and processed with another algorithm that also takes the input concentration into account [23] , [26] .…”

“…Some of the skin sensitizers were further predicted into 1A and 1B potency classes [26] . The potency predictions are based on the same physical samples and processing as for the GARD®skin based assay, with the difference that RNA expression was analyzed for a different biomarker signature consisting of 52 transcripts and processed with another algorithm that also takes the input concentration into account [23] , [26] . Senzagen AB did not know about the identity of the substances used to expose the cell model at the time of analysis.…”

Potential of concentration-response data to broaden regulatory application of in vitro test guidelines

“…Cloud-based machine learning approaches are now also being integrated into draft TGs. The Genomic Allergen Rapid Detection GARD TM assay uses a novel genomic biomarker signature that discriminates between weak and strong skin sensitizers (Gradin et al, 2020). The prediction model is constantly optimized as new data is generated, and the EURL ECVAM Scientific Advisory committee (ESAC) peer review 3 was able to support progression of GARDskin towards a TG, but a need for further work for GARDpotency was identified.…”

Impact of mono-culture vs. Co-culture of keratinocytes and monocytes on cytokine responses induced by important skin sensitizers

“…Besides the already fullyvalidated tests, there are several other methods at different stages of development and acceptance (Bergal et al 2020). Several of these are based on gene expression analysis either using a dendritic-like cell model (MUTZ-3) as in the GARD V R (Genomic Allergen Rapid Detection) assay (Gradin et al 2020) or using Reconstituted human Epidermis (RhE) models as in the SENS-IS assay (Cottrez et al 2015), the EpiSensA (Saito et al 2013), and RhE IL-18 potency test (Gibbs et al 2013).…”