The GARD™potency assay for potency-associated subclassification of chemical skin sensitizers – Rationale, method development and ring trial results of predictive performance and reproducibility

Poster presented at the 2021 World Congress on Alternatives and Animal Use in the Life Sciences

Robin Gradin, Angelica Johansson, Andy Forreryd, Amber Edwards, Veronika Hoepflinge, Florence Burleson, Helge Gehrke, Erwin Roggen, Henrik Johansson| SenzaGen AB, 22381 Lund, Sweden; Burleson Research Technologies, Morrisville, NC 27560, USA; Eurofins BioPharma Product Testing Munich Gmbh, 82152 Planegg, Germany; 43RsMC Aps, 2800 Kongens Lyngby, Denmark

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Conclusion

  • GARDpotency is an assay for sub-categorization of strong sensitizers (CLP sub-category 1A), allowing for discrimination from weak sensitizers (CLP sub-category 1B) and non-sensitizers. The method is based on the GARD platform, combining human immune cells, a genomic biomarker readout and machine learning-assisted classifications.
  • Sequential combination of GARDskin and GARDpotency forms the GARD Defined Approach, for complete hazard and risk assessment of skin sensitizers into three categories (CLP 1A, CLP 1B, non-sensitizers).
  • A blinded ring trial, comprising 28 chemicals, demonstrated that GARDpotency is functional and reproducible, with an accumulated predictive accuracy of 91% across three laboratories. In the same dataset, the GARD Defined Approach classifies chemicals into three categories with 86% accuracy.

Abstract

The advancement of non-animal approaches for hazard assessment of skin sensitizers have generated a variety of alternative assays with discriminatory properties comparable with those of accepted in vivo methods. However, hazard identification is rarely sufficient and information permitting the relative ranking of chemicals’ skin sensitization potency is desired. For example, the globally harmonized system of classification and labelling of chemicals (GHS/CLP) extends the binary hazard assessment with a qualitative subcategorization to distinguish between weak and strong skin sensitizers.

Though substantial efforts have been made towards developing alternative methods for potency assessment, none have gained regulatory acceptance, emphasizing that continued development of improved alternative assays remains a high priority.

The genomic allergen rapid detection (GARD) is an in vitro testing platform for assessment and characterization of chemical sensitizers, based on evaluation of transcriptional patterns of endpoint-specific genomic biomarker signatures in a human dendritic-like cell line following chemical exposure, in order to provide machine learning-assisted classifications of tested substances. The GARDskin assay was recently subjected to a formal validation procedure (OECD TGP 4.106) and reported a reproducibility between laboratories of 92%, as well as a predictive accuracy of 94%, for sensitization hazard assessment.

Here, we present the implementation of the related GARD application GARDpotency, for potency-associated subcategorization of chemical sensitizers. Following prediction model establishment, the functionality of the assay was validated in a blinded ring-trial, in accordance with OECD-guidance documents, by assessing predictive performance and reproducibility. It was found that the assay is functional and predictive, with an estimated cumulative accuracy of 88% across three laboratories and nine independent experiments. The within-laboratory reproducibility measures ranged between 63-89%, and the between-laboratory reproducibility was estimated to 61%. In conclusion, the in vitro GARDpotency assay constitute a standardized, functional assay, which could be a valuable tool for hazard characterization of skin sensitizer potency.

Quantitative Sensitizing Potency Assessment Using GARD™skin Dose-Response

Poster presented at the 2021 World Congress on Alternatives and Animal Use in the Life Sciences

Henrik Johansson, Robin Gradin, Andy Forreryd, Joshua Schmidt
SenzaGen AB, Lund, Sweden. SenzaGen Inc., Raleigh, NC.

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Conclusion

  • As an adaptation from the GARDskin assay, GARDskin Dose-Response is suitable for quantitative skin sensitizing potency assessment of chemicals.
  • The experimental readout, referred to as cDV0, corresponds to the lowest dose required to elicit a positive response in GARDskin. As such, experimental protocols are analogous to the LLNA, in which the cDV0 corresponds to the EC3-value.
  • The cDV0 may be used to directly monitor sensitizing potency, or further used to extrapolate LLNA EC3-values, estimation of Human potency categories, or CLP 1A/1B classifications.

Abstract

Proactive identification and characterization of sensitization hazards are central aspects of risk assessment of chemicals. Current legislations and trends in predictive toxicology advocate a transition from in vivo methods to non-animal alternatives, with a number of methods for hazard assessment of skin sensitizers currently available. However, non-animal methods capable of providing quantitative assessment of sensitizing potency are currently lacking.

The GARDskin assay is a next-generation in vitro assay for hazard assessment of skin sensitizers, currently progressing towards regulatory acceptance. Recently, the GARDskin Dose-Response (DR) testing strategy was introduced, in which test chemicals are evaluated by the GARDskin assay in a titrated range of concentrations, in order to investigate the dose-response relationship between GARDskin classifications and test chemical concentration. As such, it provides a quantitative estimation of sensitizing potency, referred to as cDV0, which corresponds to the least required dose able to generate a positive response in the GARDskin assay. The cDV0 value obtained for a test chemical may be viewed as an analogue to the LLNA EC3 value, based on which further hazard characterization and risk assessment may be performed. Statistically significant correlation between the GARDskin DR cDV0 and the LLNA EC3, as well as with human No Expected Sensitization Induction level (NESIL) estimations has been confirmed, thus enabling direct extrapolation between the different metrics.

Here, we further illustrate how these results can be used on their own to facilitate direct potency-associated ranking of test chemicals. Furthermore, we demonstrate how obtained cDV0 values can be extrapolated to LLNA EC3 values with a 95% confidence interval, thereby also facilitating potency-associated subcategorization of test chemicals according to UN GHS classification criteria. Lastly, we illustrate how results generated with GARDskin DR can be directly incorporated into existing strategies for Quantitative Risk Assessment using an entirely in vitro setup.

Quantitative Sensitizing Potency Assessment Using GARD™skin Dose-Response

Poster presented at SOT 2021

Henrik Johansson, Robin Gradin, Andy Forreryd, Joshua Schmidt
SenzaGen AB, Lund, Sweden. SenzaGen Inc., Raleigh, NC.

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Conclusion

  • As an adaptation from the GARDskin assay, GARDskin Dose-Response is suitable for quantitative skin sensitizing potency assessment of chemicals.
  • The experimental readout, referred to as cDV0, corresponds to the lowest dose required to elicit a positive response in GARDskin. As such, experimental protocols are analogous to the LLNA, in which the cDV0 corresponds to the EC3-value.
  • The cDV0 may be used to directly monitor sensitizing potency, or further used to extrapolate LLNA EC3-values, estimation of Human potency categories, or CLP 1A/1B classifications.

Abstract

Proactive identification and characterization of sensitization hazards are central aspects of risk assessment of chemicals. Current legislations and trends in predictive toxicology advocate a transition from in vivo methods to non-animal alternatives, with a number of methods for hazard assessment of skin sensitizers currently available. However, non-animal methods capable of providing quantitative assessment of sensitizing potency are currently lacking.

The GARDskin assay is a next-generation in vitro assay for hazard assessment of skin sensitizers, currently progressing towards regulatory acceptance. Recently, the GARDskin Dose-Response (DR) testing strategy was introduced, in which test chemicals are evaluated by the GARDskin assay in a titrated range of concentrations, in order to investigate the dose-response relationship between GARDskin classifications and test chemical concentration. As such, it provides a quantitative estimation of sensitizing potency, referred to as cDV0, which corresponds to the least required dose able to generate a positive response in the GARDskin assay. The cDV0 value obtained for a test chemical may be viewed as an analogue to the LLNA EC3 value, based on which further hazard characterization and risk assessment may be performed. Statistically significant correlation between the GARDskin DR cDV0 and the LLNA EC3, as well as with human No Expected Sensitization Induction level (NESIL) estimations has been confirmed, thus enabling direct extrapolation between the different metrics.

Here, we further introduce the GARDskin DR protocols, as proposed in a standardized testing strategy. By studying a concentration range of 6 concentration points titrated from the experimentally derived GARD input concentration in biological duplicates, a test chemical-specific cDV0 is established by linear interpolation. We illustrate how these results can be used on their own to facilitate direct potency-associated ranking of test chemicals. Furthermore, we demonstrate how obtained cDV0 values can be extrapolated to LLNA EC3 values with a 95% confidence interval, thereby also facilitating potency-associated subcategorization of test chemicals according to UN GHS classification criteria. Lastly, we illustrate how results generated with GARDskin DR can be directly incorporated into existing strategies for Quantitative Risk Assessment using an entirely in vitro setup.

 

Dose-Response Analysis in GARD™ for Assessment of Skin Sensitizer Potency

Poster presented at ACT 2020

J. Schmidt, A. Forreryd, R. Gradin, H. Johansson.
SenzaGen Inc., Raleigh, NC., SenzaGen AB, Lund, Sweden.

Link to the poster

 

Conclusion

  • As an adaptation from the GARDskin assay, GARDskin Dose-Response is suitable for quantitative skin sensitizing potency assessment of chemicals.
  • The experimental readout, referred to as cDV0, corresponds to the lowest dose required to elicit a positive response in GARDskin. As such, experimental protocols are analogous to the LLNA, in which the cDV0 corresponds to the EC3-value.
  • The cDV0 may be used to directly monitor sensitizing potency, or further used to extrapolate LLNA EC3-values, estimation of Human Potency categories, or CLP 1A/1B classifications.

Abstract

Several non-animal methods for identifying skin sensitizers have been developed with acceptable prediction performance. However, advancement of alternative methods for skin sensitizing potency assessment is still missing although a highly sought-after endpoint. The GARDskin assay is a genomics-based in vitro assay for hazard assessment of skin sensitizers, currently progressing towards regulatory acceptance. Here, we introduce GARDskin Dose-Response (DR), in which test chemicals are evaluated by the GARDskin assay in an extended range of concentrations, in order to investigate the dose-response relationship between GARDskin classifications and test chemical concentration.

For this work, 29 chemicals of various sensitizing potencies were used to evaluate the efficacy of applying the assay in this manner. Each chemical was analyzed at several concentrations using a slightly modified GARDskin protocol. At each concentration, a decision-value was produced and a classification prediction (sensitizing or non-sensitizing) was made by the GARDskin algorithm. Afterwards, the lowest concentration where a test item would provide a positive GARDskin prediction was found using linear interpolation. This concentration (cDV0) was then hypothesized to reflect the test items skin sensitizing potency. Furthermore, when comparing cDV0 to LLNA EC3 values, a statistically significant correlation was realized between the values (correlation coefficient =0.74, p-value=4.1*10-4).

These results suggest that modifying the GARDskin protocol to accommodate dose-response measurements can provide sensitizing potency information analogous to the gold-standard in vivo methods. This presentation will further explain the testing process, expand on results, and demonstrate how this method can be used for decision-making throughout all stages of product development, without having to use animal experimentation.

Exploration of the GARD applicability domain – Skin sensitization assessment of UVCBs

Poster presented at Eurotox 2018 in collaboration with Lubrizol

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U. I.Torstensdotter Mattson1, C. Humfrey2, O. Larne1, H. Johansson1, L. Sweet3 1SenzaGen, Lund, Sweden, 2Lubrizol, Derbyshire, United Kingdom, 3Lubrizol, Ohio, United States of America

Conclusion

This case study demonstrates the broadening of the applicability domain of the GARD assay when assessing UVCBs.

Abstract

In this study, four test items were evaluated. All the test items were “Unknown or Variable composition, Complex reaction products and Biological materials” materials (UVCBs), which were provided by Lubrizol and selected based on existing in vivo data (internal Lubrizol data). Skin sensitizing hazard was assessed using the GARDskin assay, and the GARDpotency assay for further subcategorized the sensitizers into strong 1 A) or weak 1 B) sensitizers according to GHS/CLP classification. The GARDskin predictions for test items 1, 2, 3 and the GARDpotency classifications for test item 2 and 3 were consistent with the in vivo data, whereas test item 4 showed inconsistency between the in vitro and in vivo methods. These results indicate the importance of screening a panel of different vehicles or mixtures thereof, in order to choose the appropriate solvent For one of the Test items, the DMSO extraction procedure generates a negative prediction while the experimental vehicle mixture, Glycerol and DMF, classifies the chemical as a skin sensitizer This case study demonstrates the broadening in applicability domain of the GARDassays when assessing UVCBs.

 

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