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.

Poster dowload

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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The use of GARDskin for sensitization evaluation of cosmetic ingredients and ‘real-life’ mixtures

Renato Ivan de Ávila, Tim Lindbergh, Malin Lindstedt and Marize Campos Valadares
Lab. of Education and Research in Pharmacology and Cellular Toxicology, Faculty of Pharmacy, Federal University of Goiás, Goiânia, Goiás, Brazil.
Department of Immunotechnology, Medicon Village, Lund University, Lund, Sweden.

 

Introduction
Genomic fingerprints in dendritic cells after chemical exposure is a recent strategy in in vitro techniques for skin sensitization hazard. Within this perspective, Genomic Allergen Rapid Detection (GARDskinTM), an assay based on a support vector machine (SVM) model, was developed for identifying contact allergens using a myeloid cell line as a surrogate for dendritic cells. Predictive system behind the GARDskin™ consists on the transcriptional quantitative analysis of 200 genes, referred as the GARDskin™ prediction signature. Mechanistically, GARDskin™ is linked to key event 3 “Activation of DCs”, as defined by the Adverse Outcome Pathways for skin sensitization published in 2012 by OECD (https://read.oecd-ilibrary.org/environment/the-adverse-outcome-pathway-for-skin-sensitisation-initiated-by-covalent-binding-to-proteins_9789264221444-en#page1)

 

Resluts
Information declared on the label and lawsone and PDD levels found in ten commercial henna-based hair coloring cosmetics are show in Table 1. Since all products analyzed were declared as henna cosmetics by the manufactures, the presence of LAW, the main active phytochemical of henna, was then expected in all samples. However, HPLC analysis showed no LAW level in the product nº 2, suggesting falsification. Furthermore, the presence of PPD was declared on the products nº 2 and 8 only by the manufactures. However, this substance was detected in all products, suggesting undisclosed adulteration.

Poster:
The use of GARDskin for sensitization evaluation of cosmetic ingredients and ‘real-life’ mixtures

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. 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 In Vitro assessment of Respiratory Sensitisation Potential of Electronic Cigarette Liquids

Matthew Stevenson, Lukasz Czekala, Liam Simms, Nicole Tschierske, Henrik Johansson, Tanvir Walele
Imperial Tobacco Ltd, Reemtsma Cigarettenfabriken GmbH, an Imperial Brands PLC Company, SenzaGen AB, Fontem Ventures B.V., an Imperial Brands PLC Company,

 

Introduction and Objectives

There is a general consensus amongst the scientific and public health community that e-cigarettes constitute a less harmful source of nicotine than combustible cigarettes, and that flavours play a critical role in attracting and retaining smokers into the vaping category. Due to the dynamic nature of innovation with e-cigarettes new assays are required to quickly determine the subtle biological response of these products for product stewardship activities. The size of this task is considerable as recent estimates state that more than 8,000 e-liquid flavours are on the market (Hartung, 2016). One particular toxicological endpoint which is of interest for the Stewardship of e-liquids, is Respiratory Sensitisation.
Respiratory sensitization (RS) is an allergic type I hypersensitivity reaction of the upper and lower respiratory tract caused by an immune response triggered by low molecular weight compounds or other environmental proteins. Clinical symptoms of RS include asthmatic attacks, bronchoconstriction and wheezing upon repeated exposure to the same compound. However, respiratory sensitisers are rare, with around 100 well characterised substances described in the literature.
It is Fontem Ventures policy to screen all novel e-liquid ingredients for Respiratory sensitising activities using published literature and in silico techniques. However, there is a need for alternative techniques to fill data gaps and add to a weight of evidence. Several in vitro assays have been described and validated to assess skin sensitisation, however for respiratory sensitization there are no validated predictive assays. It is of note that not all skin sensitizers are also respiratory sensitizers. In 2015, Basketter and Kimber concluded that “…airborne fragrance materials, including skin sensitising fragrance materials, do not pose a risk of the induction or elicitation of allergic reactions consequent upon exposure via the respiratory tract”. Therefore, it is critical that any assays developed to determine the sensitising properties of a chemical can distinguish between dermal and respiratory activity.
The objective of this study was to assess experimental and commercial e-liquids in GARDair™; an assay which claims to detect respiratory sensitisers.
GARDair measures the genomic biomarker signature of a human myeloid leukemia-derived cell line exposed to test substances; making this technology in keeping with the 3Rs (Reduce, Replace and Refine) and Toxicity Testing in the 21st Century principles. Gene expression analysis is performed using Affymetrix microarray technology and a prediction model is used to classify each sample according to its respiratory sensitizing potential.

 

Conclusions

• From the Benchmark Control data it was estimated that GARDair™ had a sensitivity and specificity of 71% and 100% respectively; with an overall predictive accuracy estimated as 89%.
• Extensive validation of this assay is ongoing, however, the lack of well characterised Chemical Respiratory Sensitisers may limit this.
• None of the experimental or commercial samples were classified as respiratory sensitisers.
• Further exploration of this assay is required, particularly its ability to detect low concentrations of sensitiser in complex mixtures and to ensure that the e-liquid matrix does not interfere with the detection of activity.

Poster:
The In Vitro assessment of Respiratory Sensitisation Potential of Electronic Cigarette Liquids_British ToxSocCong 2018 Imperial

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

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 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

 

Performance of the GARD assay in a blind Cosmetics Europe study

Johansson H, Gradin R, Forreryd A, Agemark M, Zeller K, Malmborg-Hager A1, Johansson A, Larne O, Van Vliet E3, Borrebaeck C, Lindstedt M,
SenzaGen AB, Lund, Sweden, Department of Immunotechnology, Lund University, Lund, Sweden, Cosmetics Europe –The Personal Care Association, Brussels, Belgium

Introduction

Chemical hypersensitivity is an immunological response to foreign substances. Primarily, these give rise to the clinical symptoms known as allergic contact dermatitis. To mitigate risks associated with consumer products, chemicals are screened for sensitizing effects. Historically, such predictive screenings have been performed using animal models, but industrial and regulatory authorities now demand animal-free methods for the assessment of sensitization. This is a global development spreading across industries and markets. To meet this demand, the Genomic Allergen Rapid Detection (GARD) assay has been developed. Here, we present novel data reconfirming the performance and accuracy of GARD.

Resume

The performance of GARD is highly accurate
83% (72 chemicals)
86% (127 chemicals)
83% (72 chemicals)
Correlated GARD predictions and potency classifications

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