Extended applicability domain with new solvent selection for the GARD platform

Jenvert RM, Larne O, Torstensdotter MattssonU. I., Johansson H, SenzaGen, Lund, Sweden

Introduction
The Genomic Allergen Rapid Detection (GARD) assay is a state of the art in vitro assay developed for the assessment of skin sensitizers. It is based on gene expression analysis of SenzaCells, a human myeloid cell line, after stimulation by the test item.
During the development of the GARD platform, two solvents were used; DMSO (0.1%) and Water. To increase the applicability domain of GARD® and the solubility of certain test items, for e.g. Medical Device extracts and UVCBs, we here show a broader range of solvents compatible with GARD.

Conclusions
Here, we show that the GARD® platform is compatible with the following solvents:
• Acetone
• Ethanol
• Glycerol
• Super refined olive oil

• DMF
• DMSO
• Water
• Isopropanol

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Exploration of the GARD® applicability domain – Sensitization assessment of UVCBs

Joint poster presented at Eurotox 2018

U. I. Torstensdotter Mattson, C. Humfrey, O. Larne, H. Johansson, L. Sweet
SenzaGen, Lund, Sweden, Lubrizol, Derbyshire, United Kingdom, Lubrizol, Ohio, United States of America

Introduction
The GARD – Genomic Allergen Rapid Detection – platform is a state of the art in vitro assay for assessment of chemical sensitizers. The GARD®skin assay is a powerful tool for assessment of chemical sensitizers, with a predictive accuracy of 94%. In this study, four UVCB test items, provided by Lubrizol and selected based on existing in vivo data (internal Lubrizol data), were evaluated. Sensitizing hazard was assessed using the GARD®skin assay, and the GARD®potency assay further subcategorized the sensitizers into strong (1A) or weak (1B) sensitizers according to GHS/CLP classification. Here we show the importance of using appropriate vehicles in order to predict a correct classification of Test items.

 

Conclusion
A UVCB Test item with poor water and DMSO solubility was assessed using a mixture of vehicles with different polarity indexes (DMF and Glycerol 1:1). This experimental vehicle mixture classified the UVCB as a skin sensitizer, being consistent with the in vivo data. This case study demonstrates the broadening of the applicability domain of the GARD -assay when assessing UVCBs.

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The Validation of GARD™skin and GARD™potency

Poster presented at Eurotox, 2018

Sandberg P, Johansson A, Agemark M, Gradin R, Larne O, Appelgren H, Forreryd A, Jerre A, Edwards A, Hoepflinger V, Burleson F, Gehrke H, Roggen E, Johansson H
SenzaGen, Lund, Sweden, Burleson Research Technologies, Morrisville, US, Eurofins, Munich, Germany

Introduction
The prevalence of allergic contact dermatitis (ACD) is estimated to >20% in the western world. Not only the individual is affected, but downstream socioeconomic effects are high. To minimize exposure, chemicals must be safety tested. Traditional testing strategies like the murine local lymph node assay (LLNA) comprise animals, but the regulatory authorities, public opinion and economic interests require animal-free models. The Genomic Allergen Rapid Detection skin (GARD®skin) is an in vitro assay addressing this need. Here, we present the results of the GARD®skin ring trial (OECD TGP 4.106) for validity of the assay. In addition, we show data for GARD®potency – a complementary assay developed to categorize identified senitizers as CLP 1A or 1B.

 

Conclusions

Transfer study
Transferability: 100%

Validation study
Reproducibility
WLR: 82 – 89%BLR: 92% (92 – 100%)
Test performance
– Accuracy: 94%
– Sensitivity: 93%
– Specificity: 96%

A blinded ring trial was performed to assess the functionality of the GARDskin assay. The data demonstrates that GARDskin is a powerful tool for assessment of chemical skin sensitizers, with a predictive accuracy of 94% and excellent reproducibility between laboratories.
In addition, we show that GARDpotency accurately assesses potency of identified sensitizers.

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Predicting skin sensitizers with confidence – Using conformal prediction to determine applicability domain

Objective
GARD – Genomic Allergen Rapid Detection – is a state of the art non-animal based technology platform for classification of skin sensitizing chemicals. The assay has proven to be reliable and highly accurate for identification of skin sensitizing chemicals, and consistently reports predictive performances > 90% across external test sets. The aim of the current project is to complement assessments of average model performance with an estimate of uncertainty involved in each individual prediction, thus allowing for classification of skin sensitizers with confidence.

Results
An Internal validation procedure was initially performed on samples in the GARD training set (n=38) using the strategy described in Fig. 3A. Results from this exercise is summarized in Fig. 4A. Conformal prediction by definition allows the user to determine a reasonable and acceptable significance level to guarantee a maximum error rate in predictions. The significance level was set to 15%, i.e. the model was allowed to make a maximum of 15% errors. Performance of the conformal predictor was measured by validity and efficiency. A model was valid if the number of prediction errors did not exceed the significance level, while efficiency corresponded to the percentage of single class predictions. Internal validation of the training data resulted in a valid and highly effective model (92% single classifications, 1 empty, 2 both), indicating that the ambitious significance level was at a reasonable level for the GARD® assay. Following internal validation, samples in a large external test set (n =70) was classified within the CP framework as described in Fig. 3B, which resulted in generation of a valid and highly efficient model (99% single classifications, 0 empty, 1 both) (Fig.4B). Additional data on model performance is illustrated in Table 1.

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