GARDair – A novel assay for assessment of chemical respiratory sensitizers based on a genomic platform

Henrik Johansson, Andy Forreryd, Robin Gradin, Angelica Johansson, Olivia Larne, Emil Aaltonen, Anders Jerre, Carl A.K. Borrebaeck and Malin Lindstedt SenzaGen AB, Lund, Sweden. Department of Immunotechnology, Lund, Sweden. Introduction Exposure to chemicals may induce allergic hypersensitivity reactions in skin or respiratory tract. To minimize exposure, chemicals are routinely screened for their sensitizing potential. Proactive […]
Henrik Johansson, Andy Forreryd, Robin Gradin, Angelica Johansson, Olivia Larne, Emil Aaltonen, Anders Jerre, Carl A.K. Borrebaeck and Malin Lindstedt
SenzaGen AB, Lund, Sweden. Department of Immunotechnology, Lund, Sweden.

Introduction
Exposure to chemicals may induce allergic hypersensitivity reactions in skin or respiratory tract. To minimize exposure, chemicals are routinely screened for their sensitizing potential. Proactive identification has historically been performed using animal models, but the use of animals for safety assessment of cosmetics was recently banned within EU. Today, similar trends are spreading both globally and across industry and market segments. Methods for specific identification of respiratory sensitizers are greatly underdeveloped, with no validated, or even widely used assay readily available. Thus, there is an urgent need for development of non-animal-based methods for hazard classification of respiratory sensitizing chemicals.

GARD– Genomic Allergen Rapid Detection – is a state of the art technology platform for assessment of chemical sensitizers (Figure 1). It is based on a dendritic cell (DC)-like cell line, thus mimicking the cell type involved in the initiation of the response leading to sensitization. Following test chemical exposure, induced transcriptional changes are measured to study the activation state of the cells. These changes are associated with the immunological decision-making role of DCs in vivo and constitutes of e.g. up-regulation of co-stimulatory molecules, induction of cellular and oxidative stress pathways and an altered phenotype associated with recognition of xenobiotic matter. By using state-of-the-art gene expression technologies, high informational content data is generated, that allows the user to get a holistic view of the cellular response induced by the test substance.

Conclusion
GARDair is a novel assay for assessment of respiratory sensitizers. It is an adaptation of the GARD platform, utilizing gene expression analysis of predictive biomarker signatures and state-of-the-art data analysis methodology. GARDair has been proven functional and is currently progressing towards industrial implementation and  regulatory acceptance with financial support from the EU programme Horizon 2020. This progress includes scientific verification of results, assay optimization, assay
transfer and formal validation by a blinded ring trial.

Link to poster

A mechanistic reinterpretation of the AOP for skin sensitisation

David W Roberts, Liverpool John Moores University, Liverpool Introduction – Non-Animal Prediction: the 21st Century Consensus Because of the biological complexity of the skin sensitisation process no single in chemico or in vitro assay will be an appropriate replacement for an animal-based assay such as LLNA or GPMT… …to ensure a mechanistic basis and cover the […]

David W Roberts, Liverpool John Moores University, Liverpool

Introduction – Non-Animal Prediction: the 21st Century Consensus
Because of the biological complexity of the skin sensitisation process no single in chemico or in vitro assay will be an appropriate replacement for an animal-based assay such as LLNA or GPMT…
…to ensure a mechanistic basis and cover the complexity, multiple methods should be integrated into a testing strategy, in accordance with the adverse outcome pathway that describes all key events in skin sensitisation.

We need an ITS based on the KEs of the AOP…but
Is that what we really need?

Conclusion

A single assay, GARD™, predicts sensitisation potential and absence of sensitisation potential better than any of, or combinations of, the OECD guideline assays DPRA, KeratinosensTM (ARE-Nrf2 ) and h-CLAT.

We do not really need an ITS covering all KE’s of the AOP.

Link to poster

The Validation of GARD®skin

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. […]

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

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 […]

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.

Poster download

Prediction of chemical respiratory sensitizers using GARD

a novel in vitro assay based on a genomic biomarker signature Henrik Johansson, Andy Forreryd, Robin Gradin, Angelica Johansson, Olivia Larne, Emil Aaltonen, Anders Jerre, Carl A.K. Borrebaeck and Malin Lindstedt SenzaGen AB, Lund, Sweden. Department of Immunotechnology, Lund, Sweden.   Introduction Exposure to chemicals may induce allergic hypersensitivity reactions in skin or respiratory tract. […]

a novel in vitro assay based on a genomic biomarker signature

Henrik Johansson, Andy Forreryd, Robin Gradin, Angelica Johansson, Olivia Larne, Emil Aaltonen, Anders Jerre, Carl A.K. Borrebaeck and Malin Lindstedt
SenzaGen AB, Lund, Sweden. Department of Immunotechnology, Lund, Sweden.

 

Introduction

Exposure to chemicals may induce allergic hypersensitivity reactions in skin or respiratory tract. To minimize exposure, chemicals are routinely screened for their sensitizing potential. Proactive identification has historically been performed using animal models, but the use of animals for safety assessment of cosmetics was recently banned within EU. Today, similar trends are spreading both globally and across industry and market segments. Methods for specific identification of respiratory sensitizers are greatly underdeveloped, with no validated, or even widely used assay readily available. Thus, there is an urgent need for development of non-animal based methods for hazard classification of respiratory sensitizing chemicals.
GARD – Genomic Allergen Rapid Detection – is a state of the art technology platform for assessment of chemical sensitizers (Figure 1). It is based on a dendritic cell (DC)-like cell line, thus mimicking the cell type involved in the initiation of the response leading to sensitization. Following test chemical exposure, induced transcriptional changes are measured to study the activation state of the cells. These changes are associated with the immunological decision-making role of DCs in vivo and constitutes of e.g. up-regulation of co-stimulatory molecules, induction of cellular and oxidative stress pathways and an altered phenotype associated with recognition of xenobiotic matter. By using state-of-the-art gene expression technologies, high informational content data is generated, that allows the user to get a holistic view of the cellular response induced by the test substance.

Conclusion

GARDair is a novel assay for assessment of respiratory sensitizers. It is an adaptation of the GARD platform, utilizing gene expression analysis of predictive biomarker signatures and state-of-the-art data analysis methodology. GARDair has been proven functional and is currently progressing towards industrial implemetation with financial support from the EU programme Horizon 2020. This progress will include scientific verification of results, assay optimization, transfer and formal validation.

Poster:

Prediction of chemical respiratory sensitizers using GARD_LIVe2018

The Validation of GARDskin

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 […]

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

Integrated hazard identification of chemical sensitizers using in vitro and in silico readouts – A comparative evaluation of predictive performance

By Lhasa Donna Macmillan, Henrik Johansson, Olivia Larne, Malin Lindstedt 1. Granary Wharf House, 2 Canal Wharf, Leeds, LS11 5PS 2. SenzaGen, Lund, Sweden 3. Lund University, Lund, Sweden   Introduction There has been a significant drive to reduce, refine and replace animal models for the prediction of skin sensitization. This is in part due […]

By Lhasa

Donna Macmillan, Henrik Johansson, Olivia Larne, Malin Lindstedt
1. Granary Wharf House, 2 Canal Wharf, Leeds, LS11 5PS
2. SenzaGen, Lund, Sweden
3. Lund University, Lund, Sweden

 

Introduction

There has been a significant drive to reduce, refine and replace animal models for the prediction of skin sensitization. This is in part due to the implementation of EU regulation 1223/20091 which prohibits the sale and marketing of any cosmetics and cosmetic ingredients which have been tested on animals, alongside REACH2 and CLP3 regulations which state that non-animal methods must be exhausted prior to considering the use of animal tests. The use and availability of non-animal methods is ever-increasing and 3 assays have been validated by the OECD thus far; the in chemico DPRA, the in vitro KeratinoSens™ and the in vitro h-CLAT. A number of other assays are undergoing OECD validation, including the GARDskin assay (Genomic Allergen Rapid Detection), a dendritic cell-based assay which identifies skin sensitizers from 200 genomic biomarkers4. However, it is generally accepted that no single non-animal method can be used as a standalone approach to replace animal models such as the murine local lymph node assay (LLNA). The focus has instead turned to combining multiple in chemico/in vitro/in silico assays and/or molecular descriptors to derive a more accurate assessment of hazard or risk, known as integrated testing strategies (ITS)5. The GARDskin assay has demonstrated high predictivity and has been reported as ready to use in an ITS6, therefore, it was decided to investigate the effect on performance when GARD was used in combination with Derek Nexus – and to compare these results against Derek with the DPRA, KeratinoSens™ and h-CLAT.

 

Conclusion

Using Derek skin sensitization predictions in combination with in chemico/in vitro assay results has a beneficial effect when predicting the LLNA outcome. GARDskin in particular performs extremely well when used with Derek in a conservative call approach. Human sensitization is more challenging to predict and GARDskin performs less well for this compared to predicting the LLNA – attributed to the small number of chemicals with both GARDskin and human data (n = 57), in addition to the positive bias in the GARD dataset (70%). However, the addition of Derek predictions clearly improve assay performance. Future work will focus on repeating this analysis on a larger, more balanced dataset.

Poster at Lhasa website
https://www.lhasalimited.org/publications/integrated-hazard-identification-of-chemical-sensitizers-using-in-vitro-and-in-silico-readouts-a-comparative-evaluation-of-predictive-performance/4428

 

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. […]

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

GARD – The story

Olivia Larne, Andy Forreryd, Ann Sofie Albrekt, Carl Arne Krister Borrebaeck, Henrik Johansson, Malin Lindstedt SenzaGen, Sweden, Lund, Department of Immunotechnology, Lund University, Sweden, Lund   Background To prevent the general population for unnecessary exposure to sensitizing substances, the substances have to be safety tested. Regulatory authorities and economic interests request animal free methodology. Genomic […]

Olivia Larne, Andy Forreryd, Ann Sofie Albrekt, Carl Arne Krister Borrebaeck, Henrik Johansson, Malin Lindstedt
SenzaGen, Sweden, Lund, Department of Immunotechnology, Lund University, Sweden, Lund

 

Background

To prevent the general population for unnecessary exposure to sensitizing substances, the substances have to be safety tested. Regulatory authorities and economic interests request animal free methodology. Genomic Allergen Rapid Detection, GARD, is an in vitro test developed for the prediction of sensitizing chemicals. It is based on differential expression of disease-associated genomic biomarkers in a human myeloid dendritic cell line.
Here, we describe the development of the GARD platform and its downstream innovations.

Poster:
GARD – The Story_ESTIV 2016

GARD – the future of sensitization testing and safety assessment of chemicals using a genomic-based platform

BACKGROUND Genomic Allergen Rapid Detection, GARD, is an in vitro test developed for the prediction of sensitizing chemicals. It is based on differential expression of disease-associated genomic biomarkers in a human myeloid dendritic cell line. Here, we describe the development, scientific validation, applications and the current state of the GARD platform. The scientific rationale behind […]

BACKGROUND
Genomic Allergen Rapid Detection, GARD, is an in vitro test developed for the prediction of sensitizing chemicals. It is based on differential expression of disease-associated genomic biomarkers in a human myeloid dendritic cell line.
Here, we describe the development, scientific validation, applications and the current state of the GARD platform. The scientific rationale behind the use of genomic biomarker signatures are detailed, linked to the AOP in a biological context, and to advantages realized through multivariate computational prediction models in a technological context.

RESUME
The GARD assay is elastic, it can be used for several applications. Today, two are developed:

GARDskin
– Hazard identification of skin sensitizers
– Accuracy: ~90%
– Initiated ECVAM validation (OECD TGP no. 4.106)

GARDair
– Hazard identification of respiratory sensitizers
– Accuracy: ~85%

GARD–the future of sensitization testing and safety assessment of chemicals, using a genomics-based platform_Eurotox 2016_p