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.

Poster download

The Validation of GARD™skin and GARD™potency

Presented at SOT, 2018

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 (GARDskin) is an in vitro assay addressing this need. Here, we present the results of the GARDskin ring trial (OECD TGP 4.106) for validity of the assay.

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.

Poster

The GARD™ assay for potency assessment of skin sensitizing chemicals

Kathrin S. Zeller, Andy Forreryd, Tim Lindberg, Ann-Sofie Albrekt, Aakash Chawade, Malin Lindstedt
Dept. of Immunotechnology, Lund University, Lund, Sweden; Swedish University of Agricultural Sciences, Alnarp, Sweden

Summary
The GARD assay is a cell-based transcriptional biomarker assay for the prediction of chemical  ensitizers1 targeting key event 3, dendritic cell activation, of the skin sensitization AOP. Here, we present a modified assay based on Random Forest modelling, which is capable of predicting CLP
potency classes (1A – strong sensitizers, 1B – weak sensitizers, no category – non-sensitizers) as described by the European CLP regulation with an accuracy of 75 % (no cat), 75 % (1B) and 88 % (1A) based on a test set consisting of 18 chemicals previously unseen to the model.
We further can link the activation of distinct pathways to the chemical protein reactivity, showing that our transcriptomic approach can reveal information contributing to the understanding of underlying mechanisms in sensitization.

Results and Discussion
We here present a potency prediction approach based on a Random Forest model and 18 transcripts. 18 chemicals previously unseen to the model were classified as shown in Tables 1, 4 and Fig. 1. Interestingly, diethyl maleate, misclassified as 1A instead of 1B, is a human potency class 2 according to4, and iodopropynyl butylcarbamate, wrongly predicted as 1B instead of 1A, is classified as human potency class 44. Thus, the model seems to show more agreement with human data than CLP classifications (mainly derived from animal data) based on this limited dataset. Also Fig. 1C supports the hypothesis, that both data and model contain information allowing the prediction of human potency.
Furthermore, Key Pathway Advisor analysis reveals that these data can be used to investigate the cellular response in more detail (Table 3). In conclusion, we show that the modified GARD assay is capable of providing potency information, which is imperative for quantitative risk assessment of chemical sensitizers.

The GARD assay for potency assessment of skin sensitizing chemicals_ESTIV 2016_Zeller_p