Skin Sensitization: Challenging the Conventional Thinking – A Case Against 2 Out of 3 as Integrated Testing Strategy

Toxicological Sciences, Volume 159, Issue 1, 1 September 2017, Pages 3–5, https://doi.org/10.1093/toxsci/kfx115
Published: 27 June 2017

Henrik Johansson,  Robin Gradin

Background

Recent years have seen a surge in development of alternative methods for assessment of skin sensitizers. This has been in order to meet the regulatory and industrial demand for accurate safety assessment without the use of animal experimentation, and to address societal expectations. Today, 3 such assays are formally validated and have achieved OECD Guideline status (EC, 201320142015). However, partly due to insufficient predictive capacity, none of the validated assays have received a recommendation for use as a stand-alone method. In addition, there is a view that accurate hazard identification must be based upon an assessment of several key events in the relevant Adverse Outcome Pathway (AOP).

For these reasons it is proposed that tests are used in combinations, forming so-called Integrated Testing Strategies (ITSs), to address deficiencies in the performance of individual methods and to incorporate end points that reflect several key events in the AOP (Hartung etal., 2013Jaworska and Hoffmann, 2010Rovida etal., 2015). Although such ITSs may be configured in a number of ways, one heavily advocated approach relies on the majority vote of individual assays, often referred to as a 2 out of 3 ITS. That is, the identification of hazard is dependent upon the chemical eliciting a positive response in at least 2 of 3 test methods. This approach is based on the view that this will provide an increased weight of evidence and thereby greater confidence in hazard characterization and risk assessment.

Here, we present arguments opposing the claimed benefits of 2 out of 3 ITSs by examining the theoretical probability propagation of combined outcomes, simulated predictions generated by hypothetical assays, and actual data obtained from recently published literature. We argue that the added value of combining the top-performing assay with other assays with less predictive accuracy is overestimated, if present, and may actually be detrimental.

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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

Prediction of chemical respiratory sensitizers using GARD, a novel in vitro assay based on a genomic biomarker signature.

PLoS One. 2015 Mar 11;10(3):e0118808. doi: 10.1371/journal.pone.0118808. eCollection 2015.

Forreryd A., Johansson H., Albrekt AS, Borrebaeck CA, Lindstedt M.

Abstract

BACKGROUND:

Repeated exposure to certain low molecular weight (LMW) chemical compounds may result in development of allergic reactions in the skin or in the respiratory tract. In most cases, a certain LMW compound selectively sensitize the skin, giving rise to allergic contact dermatitis (ACD), or the respiratory tract, giving rise to occupational asthma (OA). To limit occurrence of allergic diseases, efforts are currently being made to develop predictive assays that accurately identify chemicals capable of inducing such reactions. However, while a few promising methods for prediction of skin sensitization have been described, to date no validated method, in vitro or in vivo, exists that is able to accurately classify chemicals as respiratory sensitizers.

RESULTS:

Recently, we presented the in vitro based Genomic Allergen Rapid Detection (GARD) assay as a novel testing strategy for classification of skin sensitizing chemicals based on measurement of a genomic biomarker signature. We have expanded the applicability domain of the GARD assay to classify also respiratory sensitizers by identifying a separate biomarker signature containing 389 differentially regulated genes for respiratory sensitizers in comparison to non-respiratory sensitizers. By using an independent data set in combination with supervised machine learning, we validated the assay, showing that the identified genomic biomarker is able to accurately classify respiratory sensitizers.

CONCLUSIONS:

We have identified a genomic biomarker signature for classification of respiratory sensitizers. Combining this newly identified biomarker signature with our previously identified biomarker signature for classification of skin sensitizers, we have developed a novel in vitro testing strategy with a potent ability to predict both skin and respiratory sensitization in the same sample.

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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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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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