Reliable and Truly Animal-Free Skin Sensitization Testing – Adaption of the In vitro GARD™skin to Animal-Free Conditions

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

Andy Forreryd, Anders Jerre, Fiona Jacobs, Carol Treasure, Henrik Johansson | SenzaGen AB, 22381 Lund, Sweden; XCellR8 Ltd, Techspace One, Sci-Tech Daresbury, Cheshire WA4 4AB, UK

 

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Conclusion

  • Senzacells adapted well to routine culture in human serum with comparable cell growth, morphology, and minimal impact on cellular phenotypes.
  • GARDskin based on HS demonstrated full concordance to protocols based on animal-derived FCS for hazard identification of skin sensitizers.
  • This work represents our ambition to gradually replace all animal derived components with human equivalents to enable completely animal-free skin sensitization testing.

Abstract

A plethora of in vitro approaches for hazard assessment of skin sensitizers have recently been described and demonstrated to exhibit discriminatory properties competitive with those of accepted in vivo methods. However, the majority of these in vitro methods still use animal-derived components such as Foetal Calf Serum (FCS) within their standard protocols, imposing the question whether these methods should truly be considered as animal-free replacements.

Genomic Allergen Rapid Detection – GARD – is a genomics-based in vitro testing platform for assessment of numerous immunotoxicity endpoints. The endpoint-specific classification of skin sensitizers is a well-established application of the platform, referred to as GARDskin (OECD TGP 4.106). The assay is based on a human DC-like cell line (SenzaCells) and utilizes state-of-the-art machine learning to classify chemicals by monitoring the expression of 200 genes involved in cellular pathways associated with skin sensitization. GARDskin is progressing towards regulatory acceptance, and consistently reports accuracies > 90%.

Here, we present an adaption of the GARDskin standard protocol to enable for testing under animal-product-free conditions by replacing animal-based FCS with human derived serum. SenzaCells adapted well to routine culture in the human serum, showing comparable cell viability and growth rates to the animal-based FCS. A phenotypic analysis of common DC maturity markers showed minor changes in cell surface expression of the markers CD14 and CD1a, indicating that serum replacement did not significantly alter the phenotypic characteristics of the cells. Finally, a proficiency set of nine chemicals covering the full range from extreme sensitizers to non-sensitizers were evaluated. The protocol adapted to animal-free conditions showed full concordance to the conventional protocol, correctly classifying all chemicals.

In conclusion, this study demonstrates the potential to perform the GARDskin assay without the use of animal-derived components associated with animal welfare concerns, thus paving the way for truly animal-free and highly accurate hazard testing of skin sensitizers.

Applicability of GARD™skin for Accurate Assessment of Challenging Substances in the Context of Skin Sensitization Testing

Poster presented at ACT 2020

J. Schmidt, A. Forreryd, H. Johansson, J. Li, A. Johansson
SenzaGen, Inc., Raleigh, NC, USA, SenzaGen AB, Lund, Sweden

Link to the poster

 

Conclusion

  • GARDskin demonstrated an overall high applicability for the evaluated challenging substances with 80% predictive accuracy compared to existing human data.
  • GARDskin demonstrated excellent applicability for pre/pro-haptens and low water solubility substances, correctly classifying all such compounds in the herein investigated dataset.
  • GARDskin also showed high applicability for assessment of surfactants with 89% predictive accuracy compared to existing human data, correctly classifying 8 out of 9 internally tested surfactants, including well known challenging ones such as Sodium Dodecyl Sulphate (SDS) and Benzalkonium chloride.

Abstract

Current legislations and trends in predictive toxicology advocate a transition from in vivo methods for hazard and risk assessments to non-animal alternatives. However, certain groups of chemicals, including substances with severe membrane-damaging properties, pre- and pro-haptens, and those with high log P ratios, have been shown to be challenging to assess using cell-based assays in the context of skin sensitization testing. The aim of this study was to evaluate the applicability of GARDskin for such challenging substances, using an overlapping subset of chemicals previously tested in an integrated tested strategy (ITS) based on validated, aqueous in vitro assays, as well as in a series of Reconstructed Human Epidermis (RHE)-based assays.

The GARDskin assay (Genomic Allergen Rapid Detection) is a robust in vitro assay for identification of potential chemical skin sensitizers with over 90% prediction accuracy and broad applicability. The assay is included in the OECD Test Guideline Program (OECD TGP 4.106) and has gone through a formal validation study. The assay evaluates the gene expression of endpoint-specific genomic biomarkers in a human dendritic-like cell line following exposure to the test substance. Exposure-induced gene expression patterns are analysed using pattern recognition and machine-learning technology, providing classifications of each test item as a skin sensitizer or a non-sensitizer.

The applicability of GARDskin for a total of twelve challenging substances, including pre- and pro-haptens, low water-soluble substances, two surfactants and three additional substances known to have conflictive results when comparing in vitro and in vivo data were evaluated in this study. All twelve substances were selected from the Mehling et al. 2019 publication which reported results from three OECD validated in vitro methods, the “2 out of 3” Integrated Testing Strategy, three RHE-based models and the murine local lymph node assay (LLNA). Human potency classification was available for ten out of the twelve substances.

The GARDskin prediction results were reported from previously published studies, or from in house validation studies. Predictive accuracies were calculated by comparing skin sensitization classifications from different test methods to the available human data of each substance respectively. (N=10). To further explore and substantiate the GARDskin applicability for surfactants, additional GARDskin data for a total of nine surfactants are presented in order to complement the Mehling dataset with respect to the availability of human data.

The GARDskin assay demonstrated overall high applicability for the evaluated challenging substances, with 80% predictive accuracy compared to existing human data. GARDskin correctly classified all pre-and pro-haptens and low water-soluble substances in the data set. Furthermore, high applicability of GARDskin for severe membrane disruptive substances such as surfactants was demonstrated, with 89% predictive accuracy compared to existing human data.

 

An integrated transcriptomic- and proteomic-based approach to evaluate the human skin sensitization potential of glyphosate and its commercial agrochemical formulations

Journal of Proteomics
Available online 30 January 2020, 03647. https://doi.org/10.1016/j.jprot.2020.103647

Tim Lindberg, Renato Ivan de Ávila, Kathrin S. Zeller, Fredrik Levander, Dennis Eriksson, Aakash Chawaded, Malin Lindstedt

Highlights

  • Pure glyphosate was classified as a non-sensitizer using in vitro assessment.
  • POEA, POEA+glyphosate mixture and formulations were identified as skin sensitizers.
  • MS analysis identified protein groups related to immunologically relevant events.
  • Autophagy may be involved in the agrochemical materials-induced DC responses.

Abstract
We investigated the skin sensitization hazard of glyphosate, the surfactant polyethylated tallow amine (POEA) and two commercial glyphosate-containing formulations using different omics-technologies based on a human dendritic cell (DC)-like cell line. First, the GARD™skin assay, investigating changes in the expression of 200 transcripts upon cell exposure to xenobiotics, was used for skin sensitization prediction. POEA and the formulations were classified as skin sensitizers while glyphosate alone was classified as a non-sensitizer. Interestingly, the mixture of POEA together with glyphosate displayed a similar sensitizing prediction as POEA alone, indicating that glyphosate likely does not increase the sensitizing capacity when associated with POEA. Moreover, mass spectrometry analysis identified differentially regulated protein groups and predicted molecular pathways based on a proteomic approach in response to cell exposures with glyphosate, POEA and the glyphosate-containing formulations. Based on the protein expression data, predicted pathways were linked to immunologically relevant events and regulated proteins further to cholesterol biosynthesis and homeostasis as well as to autophagy, identifying novel aspects of DC responses after exposure to xenobiotics. In summary, we here present an integrative analysis involving advanced technologies to elucidate the molecular mechanisms behind DC activation in the skin sensitization process triggered by the investigated agrochemical materials.

Significance
The use of glyphosate has increased worldwide, and much effort has been made to improve risk assessments and to further elucidate the mechanisms behind any potential human health hazard of this chemical and its agrochemical formulations. In this context, omics-based techniques can provide a multiparametric approach, including several biomarkers, to expand the mechanistic knowledge of xenobiotics-induced toxicity. Based on this, we performed the integration of GARD™skin and proteomic data to elucidate the skin sensitization hazard of POEA, glyphosate and its two commercial mixtures, and to investigate cellular responses more in detail on protein level. The proteomic data indicate the regulation of immune response-related pathways and proteins associated with cholesterol biosynthesis and homeostasis as well as to autophagy, identifying novel aspects of DC responses after exposure to xenobiotics. Therefore, our data show the applicability of a multiparametric integrated approach for the mechanism-based hazard evaluation of xenobiotics, eventually complementing decision making in the holistic risk assessment of chemicals regarding their allergenic potential in humans.

Full article
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The GARD™Skin Assay: A New In Vitro Testing Strategy for Skin Sensitization

E. Schmidt, V. Zuckerstätter, H. Gehrke | Eurofins BioPharma Product Testing Munich GmbH

Introduction 
A skin sensitiser refers to a substance that will lead to an allergic response following skin contact as defined by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS). The potential to induce skin sensitisation is an important consideration included in procedures for the safe handling, packaging and transports of chemicals.

The assessment of skin sensitisation typically involves the use of laboratory animals. Classical methods comprise the Magnusson Kligman Guinea Pig Maximisation Test, the Buehler Test (TG 406) as well as the local lymph node assay, in its radioactive and non-radioactive form (TG 429, TG 442A/B). In order to replace in vivo experiments validation studies on alternative, mechanistically based in chemico and in vitro test methods on skin sensitisation were conducted under the auspices of ECVAM and have been considered scientifically valid for the evaluation of the skin sensitisation hazard of chemicals.

Genomic Allergen Rapid Detection (GARDTM) is an in vitro assay designed to predict the ability of chemical substances to induce skin sensitisation based on the analysis of the relative expression levels of a biomarker signature of 196 genes using a human myeloid leukaemia cell line called SenzaCells. The GARDTM assay is based on chemical stimulation of the SenzaCells, acting as an in vitro model of human Dendritic Cells (DCs). The readout of the assay is a transcriptional quantification of the genomic predictors, collectively termed the GARDTM Prediction Signature (GPS), using Nanostring nCounter technology.

Conclusion
The DPRA, KeratinoSensTM and h-CLAT are well known sensitization assays which address three different key events of the AOP. The GARDTM skin assay is a new procedure that analyses the sensitization potential based on almost 200 human genes. If a substance is a skin sensitiser with the GARDTM skin assay you have the benefit of measuring the potency on top with a different code set to make a 1A or 1B classification.
The GARDTM skin assay is especially for products that have a high log Pow (h-CLAT > 3.5, KeratinoSensTM > 7) because in those cases the classical sensitization tests are inconclusive if negative and there is no option for a replacing test method. Therefore, the GARDTM skin assay is not only an excellent alternative of the sensitization methods for these cases but it can furthermore predict the potency of a skin sensitiser, a unique feature, which makes it a testing method needed in the future.

Link to poster

Poster presented at Eurotox, Helsinki, Sep 9, 2019.

 

Identification of skin sensitizers in natural mixtures

This pilot study demonstrated the applicability of the GARDTMskin assay for identification of skin sensitizers in hair dye ingredients, delivering high prediction performance, consistent with existing human data.

The study also indicated that GARDTMskin is a promising in vitro model to identify skin sensitizers in natural mixtures.

Link to Application Note.

Extended solvent selection for in vitro sensitization testing using GARD®

Olivia Larne, Ulrika I Torstensdotter Mattson, Rikard Alm, and Gunilla Grundström.
SenzaGen, Lund, Sweden.

Introduction
The GARD®skin assay is an in vitro assay developed for the assessment of skin sensitizers. It is based on SenzaCells™, a human dendritic-like cell line, and a biomarker signature analyzed by a prediction model including pattern recognition and machine learning.

During the development of the GARD®skin platform, two solvents were used: DMSO (0.1%) and water. To increase the applicability domain of GARD®skin and the possibility to dissolve certain test items, for e.g. hard to dissolve substances and UVCBs, where show a broader range of solvents compatible with GARD®skin. Also, use of higher concentrations of the tested solvents were explored for the possibility to increase test item concentrations.

Concluding highlights
GARD®skin compatible solvents:

  • Acetone
  • DMF
  • DMF/Glycerol
  • DMSO
  • Ethanol
  • Glycerol
  • Isopropanol

Increased applicability domain.

Link to poster

Poster presented at Eurotox, Helsinki, Sep 9, 2019.

 

Validation of the GARD™skin assay for assessment of chemical skin sensitizers – ring trial results of predictive performance and reproducibility

Toxicological Sciences, kfz108, https://doi.org/10.1093/toxsci/kfz108

Henrik Johansson, Robin Gradin, Angelica Johansson, Els Adriaens, Amber Edwards, Veronika Zuckerstätter, Anders Jerre, Florence Burleson, Helge Gehrke, Erwin Roggen

Abstract
Proactive identification of chemicals with skin sensitizing properties is a key toxicological endpoint within chemical safety assessment, as required by legislation for registration of chemicals. In order to meet demands of increased animal welfare and facilitate increased testing efficiency also in non-regulatory settings, considerable efforts have been made to develop non-animal approaches to replace current animal testing.

Genomic Allergen Rapid Detection (GARD) is a state-of-the-art technology platform, the most advanced application of which is the assay for assessment of skin sensitizing chemicals, GARDskin. The methodology is based on a dendritic cell (DC)-like cell line, thus mimicking the mechanistic events leading to initiation and modulation of downstream immunological responses. Induced transcriptional changes are measured following exposure to test chemicals, providing a detailed evaluation of cell activation. These changes are associated with the immunological decision-making role of DCs in vivo and include among other phenotypic modifications, up-regulation of co-stimulatory molecules, induction of cellular and oxidative stress pathways and xenobiotic responses and provide a holistic readout of substance-induced DC activation.Here, results from an inter-laboratory ring trial of GARDskin, conducted in compliance with OECD guidance documents and comprising a blinded chemical test set of 28 chemicals, are summarized. The assay was found to be transferable to naïve laboratories, with an inter-laboratory reproducibility of 92.0%. The within-laboratory reproducibility ranged between 82.1-88.9%, while the cumulative predictive accuracy across the three laboratories was 93.8%.It was concluded that GARDskin is a robust and reliable method for the identification of skin sensitizing chemicals and suitable for stand-alone use or as a constituent of integrated testing. These data form the basis for the regulatory validation of GARDskin.
Full article
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Evaluation of in vitro testing strategies for hazard assessment of the skin sensitization potential of “real‐life” mixtures: the case of henna‐based hair coloring products containing p‐phenylenediamine

Wiley Online Library, First published: 21 April 2019,  https://doi.org/10.1111/cod.13294.

Renato I. de Ávila Danillo F. M. C. Veloso Gabriel C. Teixeira Thaisângela L. Rodrigues Tim Lindberg Malin Lindstedt Simone G. Fonseca Eliana M. Lima Marize C. Valadares

Abstract

Background
Allergic contact dermatitis reported to henna‐based hair coloring products (HPs) has been associated with adulteration of henna with p‐phenylenediamine (PPD).

Objectives
To develop a testing approach based on in vitro techniques that address key events within the skin sensitization adverse outcome pathway to evaluate allergenic potential of HPs.

Methods
The following in vitro assays were used to test the sensitizing capacity of hair dye ingredients: micro‐direct peptide reactivity assay (mDPRA); HaCaT keratinocytes‐associated IL‐18 assay; U937 cell line activation test (USENS)/IL‐8 levels; blood monocyte‐derived dendritic cell test; genomic allergen rapid detection (GARD skin). Those techniques with better human concordance were selected to evaluate the allergenic potential of ten HPs.

Results
Contrasting to the label’s information, chromatographic analyses identified PPD in all products. The main henna biomarker, lawsone, was not detected in one of 10 the products. Among the techniques evaluated by testing HDIs, mDPRA, IL‐18 assay, GARD skin and U‐SENS correlated better with human classification (concordances 91.7 to 100%) and were superior to the animal testing (concordance 78.5%). Thus, these assays were used to evaluate HPs, which were classified as skin sensitizers using different two‐out‐of‐three approaches.

Conclusions
Our findings highlight toxicological consequences and risks associated of the undisclosed use of PPD in henna‐based “natural” “real‐life” products.

This article is protected by copyright. All rights reserved.

Our view on alternative testing for product safety in Manufacturing Chemist

To replace a test animal, which is clearly a complex system with blood and organs, you need a more holistic approach to model the entire allergic response within the body. Our CEO Anki Malmborg Hager explains the benefits of using alternative testing methods to prove product safety in this week’s Manufacturing Chemist.

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Skin Sensitization Testing—What’s Next?

Int. J. Mol. Sci. 2019, 20(3), 666; https://doi.org/10.3390/ijms20030666  

Gunilla Grundström and Carl A.K. Borrebaeck

Abstract

There is an increasing demand for alternative in vitro methods to replace animal testing, and, to succeed, new methods are required to be at least as accurate as existing in vivo tests. However, skin sensitization is a complex process requiring coordinated and tightly regulated interactions between a variety of cells and molecules. Consequently, there is considerable difficulty in reproducing this level of biological complexity in vitro, and as a result the development of non-animal methods has posed a major challenge. However, with the use of a relevant biological system, the high information content of whole genome expression, and comprehensive bioinformatics, assays for most complex biological processes can be achieved. We propose that the Genomic Allergen Rapid Detection (GARD™) assay, developed to create a holistic data-driven in vitro model with high informational content, could be such an example. Based on the genomic expression of a mature human dendritic cell line and state-of-the-art machine learning techniques, GARD™ can today accurately predict skin sensitizers and correctly categorize skin sensitizing potency. Consequently, by utilizing advanced processing tools in combination with high information genomic or proteomic data, we can take the next step toward alternative methods with the same predictive accuracy as today’s in vivo methods—and beyond.

Keywords

genomics; machine learning; skin sensitization; adverse outcome pathways; next generation in vitro tests

Article online