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

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

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

 

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
GARDskin
– Hazard identification of skin sensitizers
– Accuracy: ~90%
– Initiated ECVAM validation (OECD TGP no. 4.106)
GARDair
– Hazard identification of respiratory sensitizers
– Accuracy: ~85%

Poster:
GARD–the future of sensitization testing and safety assessment of chemicals, using a genomics-based platform

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 […]

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

Link to articel on line

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 […]

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.”

 

Press release in pdf

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, […]
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

Link to article on line

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, […]

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%.

henrik johansson senior scientist

Dr. Henrik Johansson, scientist and COO of SenzaGen AB, was awarded the LUSH Young Researcher Award 2014.

“The project for which Dr. Johansson was awarded, the Lush Prize Young Researcher Award, describes the development and use of the novel test method GARD – Genomic Allergen Rapid Detection, an assay for assessment of chemical sensitizers. …GARD utilises an in vitro model of so-called dendritic cells, a central player in the human immune system. […]

“The project for which Dr. Johansson was awarded, the Lush Prize Young Researcher Award, describes the development and use of the novel test method GARD – Genomic Allergen Rapid Detection, an assay for assessment of chemical sensitizers.

…GARD utilises an in vitro model of so-called dendritic cells, a central player in the human immune system. These cells are stimulated with any substances to be tested, and following an incubation time of 24h, the genetic material of the cells are isolated. By measurements of a certain set of genes in the cells, which could be viewed as on/off-switches of the immune response, the eventual risk of the tested substances ability to induce allergy can be predicted.” [Lush Prize Young Researcher http://www.lushprize.org/2014-prize/2014-prize-winners/]

Read more on Lush Prize Young Researcher Award web site

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 […]

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

Link to article on line

Human blood dendritic cell subsets exhibit discriminative pattern recognition receptor profiles

Immunology. 2014 Jun;142(2):279-88. doi: 10.1111/imm.12252. Lundberg K., Rydnert F, Greiff L, Lindstedt M. Abstract Dendritic cells (DCs) operate as the link between innate and adaptive immunity. Their expression of pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), enables antigen recognition and mediates appropriate immune responses. Distinct subsets of human […]

Immunology. 2014 Jun;142(2):279-88. doi: 10.1111/imm.12252.

Lundberg K., Rydnert F, Greiff L, Lindstedt M.

Abstract

Dendritic cells (DCs) operate as the link between innate and adaptive immunity. Their expression of pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), enables antigen recognition and mediates appropriate immune responses. Distinct subsets of human DCs have been identified; however their expression of PRRs is not fully clarified. Expressions of CLRs by DC subpopulations, in particular, remain elusive. This study aimed to identify and compare PRR expressions on human blood DC subsets, including CD1c(+) , CD141(+) and CD16(+) myeloid DCs and CD123(+) plasmacytoid DCs, in order to understand their capacity to recognize different antigens as well as their responsiveness to PRR-directed targeting. Whole blood was obtained from 13 allergic and six non-allergic individuals. Mononuclear cells were purified and multi-colour flow cytometry was used to assess the expression of 10 CLRs and two TLRs on distinct DC subsets. PRR expression levels were shown to differ between DC subsets for each PRR assessed. Furthermore, principal component analysis and random forest test demonstrated that the PRR profiles were discriminative between DC subsets. Interestingly, CLEC9A was expressed at lower levels by CD141(+) DCs from allergic compared with non-allergic donors. The subset-specific PRR expression profiles suggests individual responsiveness to PRR-targeting and supports functional specialization.

KEYWORDS:

C-type lectin receptors; Toll-like receptors; human dendritic cell subsets; pattern recognition receptors

Link to article on line

Evaluation of high throughput gene expression platforms using a genomic biomarker signature for prediction of skin sensitization.

BMC Genomics. 2014 May 16;15:379. doi: 10.1186/1471-2164-15-379. Forreryd A., Johansson H., Albrekt A.S., Lindstedt M Abstract BACKGROUND: Allergic contact dermatitis (ACD) develops upon exposure to certain chemical compounds termed skin sensitizers. To reduce the occurrence of skin sensitizers, chemicals are regularly screened for their capacity to induce sensitization. The recently developed Genomic Allergen Rapid Detection […]

BMC Genomics. 2014 May 16;15:379. doi: 10.1186/1471-2164-15-379.

Forreryd A., Johansson H., Albrekt A.S., Lindstedt M

Abstract

BACKGROUND:

Allergic contact dermatitis (ACD) develops upon exposure to certain chemical compounds termed skin sensitizers. To reduce the occurrence of skin sensitizers, chemicals are regularly screened for their capacity to induce sensitization. The recently developed Genomic Allergen Rapid Detection (GARD) assay is an in vitro alternative to animal testing for identification of skinsensitizers, classifying chemicals by evaluating transcriptional levels of a genomic biomarker signature. During assay development and biomarker identification, genome-wide expression analysis was applied using microarrays covering approximately 30,000 transcripts. However, the microarray platform suffers from drawbacks in terms of low sample throughput, high cost per sample and time consuming protocols and is a limiting factor for adaption of GARD into a routine assay for screening of potential sensitizers. With the purpose to simplify assay procedures, improve technical parameters and increase sample throughput, we assessed the performance of three high throughput gene expression platforms–nCounter®, BioMark HD™ and OpenArray®–and correlated their performance metrics against our previously generated microarray data. We measured the levels of 30 transcripts from the GARD biomarker signature across 48 samples. Detection sensitivity, reproducibility, correlations and overall structure of gene expression measurements were compared across platforms.

RESULTS:

Gene expression data from all of the evaluated platforms could be used to classify most of the sensitizers from non-sensitizers in the GARD assay. Results also showed high data quality and acceptable reproducibility for all platforms but only medium to poor correlations of expression measurements across platforms. In addition, evaluated platforms were superior to the microarray platform in terms of cost efficiency, simplicity of protocols and sample throughput.

CONCLUSIONS:

We evaluated the performance of three non-array based platforms using a limited set of transcripts from the GARD biomarker signature. We demonstrated that it was possible to achieve acceptable discriminatory power in terms of separation between sensitizers and non-sensitizers in the GARD assay while reducing assay costs, simplify assay procedures and increase sample throughput by using an alternative platform, providing a first step towards the goal to prepare GARD for formal validation and adaption of the assay for industrial screening of potential sensitizers.

Link to article on line

Prediction of skin sensitizers using alternative methods to animal experimentation.

Basic Clin Pharmacol Toxicol. 2014 Jul;115(1):110-7. doi: 10.1111/bcpt.12199. Epub 2014 Feb 18. Johansson H., Lindstedt M. Abstract Regulatory frameworks within the European Union demand that chemical substances are investigated for their ability to induce sensitization, an adverse health effect caused by the human immune system in response to chemical exposure. A recent ban on the […]

Basic Clin Pharmacol Toxicol. 2014 Jul;115(1):110-7. doi: 10.1111/bcpt.12199. Epub 2014 Feb 18.

Johansson H., Lindstedt M.

Abstract

Regulatory frameworks within the European Union demand that chemical substances are investigated for their ability to induce sensitization, an adverse health effect caused by the human immune system in response to chemical exposure. A recent ban on the use of animal tests within the cosmetics industry has led to an urgent need for alternative animal-free test methods that can be used for assessment of chemical sensitizers. To date, no such alternative assay has yet completed formal validation. However, a number of assays are in development and the understanding of the biological mechanisms of chemical sensitization has greatly increased during the last decade. In this MiniReview, we aim to summarize and give our view on the recent progress of method development for alternative assessment of chemical sensitizers. We propose that integrated testing strategies should comprise complementary assays, providing measurements of a wide range of mechanistic events, to perform well-educated risk assessments based on weight of evidence.