The GARD platform for potency assessment of skin sensitizing chemicals

ALTEX Online first published April 12, 2017, version 2 https://doi.org/10.14573/altex.1701101

Kathrin S. Zeller, Andy Forreryd, Tim Lindberg, Robin Gradin, Aakash Chawade and Malin Lindstedt,

Summary

Contact allergy induced by certain chemicals is a common health concern, and several alternative methods have been developed to fulfill the requirements of European legislation with regard to hazard assessment of potential skin sensitizers. However, validated methods, which provide information about the potency of skin sensitizers, are still lacking. The cell-based assay Genomic Allergen Rapid Detection (GARD), targeting key event 3, dendritic cell activation, of the skin sensitizer AOP, uses gene expression profiling and a machine learning approach for the prediction of chemicals as sensitizers or non-sensitizers. Based on the GARD platform, we here expanded the assay to predict three sensitizer potency classes according to the European Classification, Labelling and Packaging (CLP) Regulation, targeting categories 1A (strong), 1B (weak) and no cat (non-sensitizer). Using a random forest approach and 70 training samples, a potential biomarker signature of 52 transcripts was identified. The resulting model could predict an independent test set consisting of 18 chemicals, six from each CLP category and all previously unseen to the model, with an overall accuracy of 78%. Importantly, the model was shown to be conservative and only underestimated the class label of one chemical. Furthermore, an association of defined chemical protein reactivity with distinct biological pathways illustrates that our transcriptional approach can reveal information contributing to the understanding of underlying mechanisms in sensitization.

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Evaluation of the GARD assay in a blind Cosmetics Europe study

ALTEX Online first published February 17, 2017 https://doi.org/10.14573/altex.1701121

Johansson H., Gradin R., Forreryd A., Agemark M., Zeller K., Johansson A., Larne O., van Vliet E.,  Borrebaeck C., Lindstedt M.,

Summary

Chemical hypersensitivity is an immunological response towards foreign substances, commonly referred to as sensitizers, which gives rise primarily to the clinical symptoms known as allergic contact dermatitis. For the purpose of mitigating risks associated with consumer products, chemicals are screened for sensitizing effects. Historically, such predictive screenings have been performed using animal models. However, due to industrial and regulatory demand, animal models for the purpose of sensitization assessment are being replaced by animalfree testing methods, a global trend that is spreading across industries and market segments. To meet this demand, the Genomic Allergen Rapid Detection (GARD) assay was developed. GARD is a novel, cell-based assay that utilizes the innate recognition of xenobiotic substances by dendritic cells, as measured by a multivariate readout of genomic biomarkers. Following cellular stimulation, chemicals are classified as sensitizers or non-sensitizers based on induced transcriptional profiles. Recently, a number of animal-free methods were comparatively evaluated by Cosmetic Europe, using a coherent and blinded test panel of reference chemicals with human and local lymph node assay data, comprising a wide range of sensitizers and non-sensitizers. In this paper, the outcome of the GARD assay is presented. It was demonstrated that GARD is a highly functional assay with a predictive performance of 83% in this Cosmetics Europe dataset. The average accumulated predictive accuracy of GARD across independent datasets was 86%, for skin sensitization hazard. Keywords: GARD, sensitization, in vitro, predictive accuracy, alternative methods

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Testing Human Skin and Respiratory Sensitizers—What Is Good Enough?

Int. J. Mol. Sci. 2017, 18(2), 241; doi:10.3390/ijms18020241

Malmborg A., Borrebaeck C. A.K.

Abstract

Alternative methods for accurate in vitro assessment of skin and respiratory sensitizers are urgently needed. Sensitization is a complex biological process that cannot be evaluated accurately using single events or biomarkers, since the information content is too restricted in these measurements. On the contrary, if the tremendous information content harbored in DNA/mRNA could be mined, most complex biological processes could be elucidated. Genomic technologies available today, including transcriptional profiling and next generation sequencing, have the power to decipher sensitization, when used in the right context. Thus, a genomic test platform has been developed, denoted the Genomic Allergen Rapid Detection (GARD) assay. Due to the high informational content of the GARD test, accurate predictions of both the skin and respiratory sensitizing capacity of chemicals, have been demonstrated. Based on a matured dendritic cell line, acting as a human-like reporter system, information about potency has also been acquired. Consequently, multiparametric diagnostic technologies are disruptive test principles that can change the way in which the next generation of alternative methods are designed.
Keywords:

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

Next generation skin sensitisation testing.

Personal Care Magazine Europe, sept 2016

Malmborg Hager A., Johansson H., Lindstedt M., Borrebaeck CA.

 

DNA is the software that predicts human phenotypes. Genomics utilises DNA as such a piece of software, which in several recent applications has been demonstrated to be able to predict a number of biological features, enabling anything from face recognition to vaccine development. The tremendous information content, harboured in the DNA, should be harnessed in tests of cosmetic ingredients and formulations, to get a complete insight into what is happening in the body when these types of products are applied to the human skin. This type of complex information gives a holistic view of a human condition that many of us are familiar with, such as e.g. allergic contact dermatitis, and has the potential not only to classify chemicals used in cosmetics but also to determine the magnitude to which a
chemical affects the human body (the potency).

This distinguishes genomics from most other test principles, where in many cases only one or two markers are being
monitored, such as DC activation markers (CD86, CD54), genes involved in cytoprotective responses to oxidative stress or electrophilic compounds (Nrf2, Keap1), or proinflammatory cytokines, (e.g. IL18).

Next generation skin sensitisation testing. PC Magazine, Sept 2016. Malmborg Hager A., Johansson H., Lindstedt M., Borrebaeck CA.

From genome-wide arrays to tailor-made biomarker readout – Progress towards routine analysis of skin sensitizing chemicals with GARD.

Toxicol In Vitro. 2016 Dec;37:178-188. doi: 10.1016/j.tiv.2016.09.013. Epub 2016 Sep 13.

Forreryd A., Zeller K., Lindberg T., Johansson H., Lindstedt M

Abstract

Allergic contact dermatitis (ACD) initiated by chemical sensitizers is an important public health concern. To prevent ACD, it is important to identify chemical allergens to limit the use of such compounds in various products. EU legislations, as well as increased mechanistic knowledge of skin sensitization have promoted development of non-animal based approaches for hazard classification of chemicals. GARD is an in vitro testing strategy based on measurements of a genomic biomarker signature. However, current GARD protocols are optimized for identification of predictive biomarker signatures, and not suitable for standardized screening. This study describes improvements to GARD to progress from biomarker discovery into a reliable and cost-effective assay for routine testing. Gene expression measurements were transferred to NanoString nCounter platform, normalization strategy was adjusted to fit serial arrival of testing substances, and a novel strategy to correct batch variations was presented. When challenging GARD with 29 compounds, sensitivity, specificity and accuracy could be estimated to 94%, 83% and 90%, respectively. In conclusion, we present a GARD workflow with improved sample capacity, retained predictive performance, and in a format adapted to standardized screening. We propose that GARD is ready to be considered as part of an integrated testing strategy for skin sensitization.

KEYWORDS:

GARD; In vitro assay; Predictive genomic biomarker signature; Skin sensitization

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Next generation skin sensitisation testing

Malmborg Hager A., Johansson H., Lindstedt M., Borrebaeck CA. Next generation skin sensitisation testing. PC Magazine, Sept 2016

senzagen member of the board

Ian Kimber joins the board of SenzaGen AB

SenzaGen AB, a diagnostic spin-off company from Lund University, is proud to announce that Prof. Ian Kimber will join the Board of Directors.

SenzaGen AB is a diagnostic company working to replace animal testing for sensitization predictions.

Prof Ian Kimber is currently Professor of Toxicology and Associate Dean for Business Development in the Faculty of Life Sciences at the University of Manchester. He has broad research interests at the interface between toxicology and immunology, with a particular focus on allergy and inflammation.  Professor Kimber holds, and has held, a variety of positions on national and international expert and scientific advisory committees. Currently these include the following:  Member UK Medicines and Healthcare products Regulatory Agency (MHRA) Committee for Safety of Devices, Programme Advisor Food Standards Agency Food Allergy and Intolerance Research Programme, and member MRC Translational Research Group. Professor Kimber was previously President of the British Toxicology Society (BTS) (2012-2014), and Chairman of the Board of the UK National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) (2008-2013).  

“I am delighted to be joining the Board of SenzaGen at such an exciting period of growth. Since our initial studies that led to the development and ultimate validation of the Local Lymph Node Assay I have maintained a keen interest in chemical allergy and in the development of novel approaches for improved hazard identification and risk assessment. In this respect harnessing fully the opportunities afforded by developments in the biomedical sciences is critical, and the work of SenzaGen in exploiting a genomic approach to pathway analysis and risk assessment is at the cutting edge of new developments”, says Prof Kimber.

Ian Kimber has published over 600 research papers, review articles and book chapters, and serves currently on the editorial boards of toxicology, immunology, dermatology and pathology journals.

“We are very happy to have Prof Kimber on board in SenzaGen”, since he is one of the forerunners in sensitization testing, says Prof Carl Borrebaeck, “his knowledge and interest in the field of allergy and replacement of animal testing with new technologies is very important to us. We value his participation in developing the next generation tests.”

Systematic evaluation of non-animal test methods for skin sensitisation safety assessment.

Toxicology in Vitro   Volume 29, Issue 1, February 2015, Pages 259–270

Kerstin Reisinger, Sebastian Hoffmann, Nathalie Alépée, Takao Ashikaga, Joao Barroso, Cliff Elcombe, Nicola Gellatly, Valentina Galbiati, Susan Gibbs, Hervé Groux, Jalila Hibatallah, Donald Keller, Petra Kern, Martina Klaric, Susanne Kolle, Jochen Kuehnl, Nathalie Lambrechts, Malin Lindstedt, Marion Millet, Silvia Martinozzi-Teissier, Andreas Natsch, Dirk Petersohn, Ian Pike, Hitoshi Sakaguchi, Andreas Schepky, Magalie Tailhardat, Marie Templier, Erwin van Vliet, Gavin Maxwell

 

Abstract

The need for non-animal data to assess skin sensitisation properties of substances, especially cosmetics ingredients, has spawned the development of many in vitro methods. As it is widely believed that no single method can provide a solution, the Cosmetics Europe Skin Tolerance Task Force has defined a three-phase framework for the development of a non-animal testing strategy for skin sensitisation potency prediction. The results of the first phase – systematic evaluation of 16 test methods – are presented here. This evaluation involved generation of data on a common set of ten substances in all methods and systematic collation of information including the level of standardisation, existing test data, potential for throughput, transferability and accessibility in cooperation with the test method developers. A workshop was held with the test method developers to review the outcome of this evaluation and to discuss the results. The evaluation informed the prioritisation of test methods for the next phase of the non-animal testing strategy development framework. Ultimately, the testing strategy – combined with bioavailability and skin metabolism data and exposure consideration – is envisaged to allow establishment of a data integration approach for skin sensitisation safety assessment of cosmetic ingredients.

Keywords

Skin sensitisationTesting strategySafety assessmentNon-animal test methodsAdverse Outcome Pathways

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SenzaGen AB completes successful collaboration with Beiersdorf AG.

SenzaGen AB, a diagnostic spin-off company from Lund University, reports the successful completion of a collaboration with Beiersdorf.

SenzaGen AB, a diagnostic company working to replace animal testing, has been collaborating with Beiersdorf to evaluate its skin sensitization test for chemicals.

SenzaGen AB is based on research at the Department of Immunotechnology at Lund University, providing animal-free, in vitro sensitization tests for the cosmetic, chemical, pharmaceutical and food industries.

Beiersdorf has been working in the development and recognition of alternative methods for more than 20 years and is one of the main players in the cosmetic industry in Europe. An industry that is highly interested in recommendations in this field – especially after the ban in March 2013 on animal testing in the cosmetic industry.

“It has been very valuable for SenzaGen to be able to challenge its skin sensitization test in an industrial setting.” says Prof. Carl Borrebaeck, chairman of the board of SenzaGen.

In the collaboration, blind testing of two industrial test sets of chemicals were performed using the SenzaGen proprietary GARD assay, resulting in an accuracy of 89%.

Genomic allergen rapid detection in-house validation-a proof of concept.

Toxicol Sci. 2014 Jun;139(2):362-70. doi: 10.1093/toxsci/kfu046. Epub 2014 Mar 27.

Johansson H., Rydnert F., Kuehnl J., Schepky A., Borrebaeck C.A.K., Lindstedt M.

Abstract

Chemical sensitization is an adverse immunologic response to chemical substances, inducing hypersensitivity in exposed individuals. Identifying chemical sensitizers is of great importance for chemical, pharmaceutical, and cosmetic industries, in order to prevent the use of sensitizers in consumer products. Historically, chemical sensitizers have been assessed mainly by in vivo methods, however, recently enforced European legislations urge and promote the development of animal-free test methods able to predict chemical sensitizers. Recently, we presented a predictive biomarker signature in the myeloid cell line MUTZ-3, for assessment of skinsensitizers. The identified genomic biomarkers were found to be involved in immunologically relevant pathways, induced by recognition of foreign substances and regulating dendritic cell maturation and cytoprotective mechanisms. We have developed the usage of this biomarker signature into a novel in vitro assay for assessment of chemical sensitizers, called Genomic Allergen Rapid Detection (GARD). The assay is based on chemical stimulation of MUTZ-3 cultures, using the compounds to be assayed as stimulatory agents. The readout of the assay is a transcriptional quantification of the genomic predictors, collectively termed the GARD Prediction Signature (GPS), using a complete genome expression array. Compounds are predicted as either sensitizers or nonsensitizers by a Support Vector Machine model. In this report, we provide a proof of concept for the functionality of the GARD assay by describing the classification of 26 blinded and 11 nonblinded chemicals as sensitizers or nonsensitizers. Based on these classifications, the accuracy, sensitivity, and specificity of the assay were estimated to 89, 89, and 88%, respectively.

KEYWORDS:

GARD; allergic contact dermatitis; chemical sensitizers; in vitro assay; predictive assay; skin sensitization

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