The GARDpotency assay for potency-associated subclassification of chemical skin sensitizers – Rationale, method development and ring trial results of predictive performance and reproducibility

Toxicological Sciences, kfaa068, https://doi.org/10.1093/toxsci/kfaa068

Robin Gradin, Angelica Johansson, Andy Forreryd, Emil Aaltonen, Anders Jerre, Olivia Larne, Ulrika Mattson, Henrik Johansson

Abstract
Proactive identification and characterization of hazards attributable to chemicals are central aspects of risk assessments. Current legislations and trends in predictive toxicology advocate a transition from in vivo methods to non-animal alternatives. For skin sensitization assessment, several OECD validated alternatives exist for hazard identification, but non-animal methods capable of accurately characterizing the risks associated with sensitizing potency are still lacking.

The GARDTM platform utilizes exposure-induced gene expression profiles of a dendritic -like cell line in combination with machine learning to provide hazard classifications for different immunotoxicity endpoints. Recently, a novel genomic biomarker signature displaying promising potency-associated discrimination between weak and strong skin sensitizers was proposed. Here, we present the adaptation of the defined biomarker signature on a gene expression analysis platform suited for routine acquisition, confirm the validity of the proposed biomarkers, and define the GARDTMpotency assay for prediction of skin sensitizer potency. The performance of GARDTMpotency was validated in a blinded ring-trial, in accordance with OECD-guidance documents. The cumulative accuracy was estimated to 88.0% across three laboratories and nine independent experiments. The within-laboratory reproducibility measures ranged between 62.5% and 88.9%, and the between-laboratory reproducibility was estimated to 61.1%. Currently, no direct or systematic cause for the observed inconsistencies between the laboratories have been identified. Further investigations into the sources of introduced variability will potentially allow for increased reproducibility.

In conclusion, the in vitro GARDTMpotency assay constitute a step forward for development of non-animal alternatives for hazard characterization of skin sensitizers.

Key words: GARD, GARDpotency, in vitro, sensitization, potency, chemical sensitizers

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CEO presentation at AGM 2020

Comments to the 2019 closing and 2020 update by CEO Axel Sjöblad. 

2019 Annual Report – now available in English

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Our lab is open – rely on us for prompt and accurate results

SenzaGen has received questions from customers and partners about how COVID-19 has affected our daily operations. We continue to monitor the development of the COVID-19 epidemic closely and recognize that this situation represents an unprecedented challenge to the daily lives of our communities across the globe.

To date, we continue to offer our full range of testing services

SenzaGen acts in adherence with the authorities’ guidelines in order to protect the health of its employees and minimize the spread of the virus. To ensure that SenzaGen remains in operation, we have implemented a number of safety steps to adapt our way of working in order to limit the exposure of our laboratory personnel. Furthermore, inventory of consumables critical to our operations have been secured to facilitate the uninterrupted laboratory work necessary to supporting customers’ in vitro safety testing needs.

 

Contact us about your challenging substances – we are here to support your testing needs.

 

Tell us about your challenging substances

SenzaGen provides a highly accurate method for both skin and respiratory sensitization assessment of chemicals, mixtures, and materials, including so-called “difficult-to-test” substances. Customers across the industry use the GARD assays for a variety of reasons, including:

  • High accuracy – low number of false positives/negatives
  • Ability to handle complex mixtures, UVCBs, surfactants and solid materials
  • Fast results – test time 2 weeks
  • Low sample requirement
  • Scientific support and flexibility

Web-based conference on “difficult-to-test” substances and regulatory testing

Join SenzaGen’s web-based conference on “Difficult-to-test” substances and regulatory testing in place of our exhibitor hosted session at SOT 2020

Take this chance and participate in our web-based conference on the latest news about GARD™ and how customers use the assays for skin and respiratory sensitization testing.

Key takeaways

  • Insights on sensitization assessment of “difficult-to-test” substances
  • Experiences on how to use GARD for regulatory testing
  • Results from industry partners from several applications and case studies
  • Updates on the GARD assays by SenzaGen experts

Speakers

  • Andy Forreryd, SenzaGen AB
  • Charles Humfrey/Yafan Li/Len Sweet: Collaborators at the Lubrizol Corporation.
  • Helge Gehrke, Eurofins BioPharma Product Testing
  • George DeGeorge, MB Research Labs

Available on two occasions
EU Time Zone | Mar 26, 11:00 CET | Register here
US Time Zone | Mar 26, 11:00 EDT |Register here

SenzaGen’s operation during COVID-19

SenzaGen has received questions from customers and partners about how COVID-19 has affected our daily operations. We continue to monitor the development of the COVID-19 epidemic closely and recognize that this situation represents an unprecedented challenge to the daily lives of our communities across the globe.

SenzaGen acts in adherence with the authorities’ guidelines in order to protect the health of its employees and to minimize the spread of the virus. Currently, measures have been taken to adapt our way of working in order to limit the exposure of our laboratory personnel. Furthermore, inventories of critical consumables have been secured to enable the uninterrupted continuation of laboratory work necessary to support customers’ in vitro safety testing needs.

We hope that everybody stays safe in these challenging times and SenzaGen remains ready to serve where we can.

Web-based SOT 2020 poster presentation session

Join SenzaGen’s free web-based SOT 2020 poster presentation session

Don’t miss the opportunity to participate in our web-based conference on the latest news about the GARD™ assays for skin and respiratory sensitization testing.

Key takeaways

Comparative study: GARDskin for challenging substances
How to test UVCBs and Mixtures
Formal validation of GARDpotency for subclassification of skin sensitizers
GARDair technology update and case study

Available on two occasions

EU Time Zone | Mar 19, 11:00 CET | Register here
US Time Zone | Mar 19, 11:00 EDT | Register here

Do you have challenging samples?
Complex mixtures, natural extracts or other low solubility substances – what are your difficult-to-test samples? GARD is a genomic-based in vitro test with high performance and broad applicability specializing in difficult-to-test samples that can help you through the hurdles in your product life cycle. Hundreds of samples have been tested and successfully reported. 

Free Webinar: In vitro sensitization testing based on genomics and machine learning

Did you miss our popular webinar ‘GARD technology: Genomics and Machine Learning’ last year? Here is a new opportunity to participate in a live webinar presented by GARD experts at SenzaGen followed by a Q&A session.

Join us and you’ll learn the principles of the technology platform and get an updated overview of its broad applicability. This webinar will give you in-depth knowledge about GARD.

Key takeaways

  • The story behind the development of the technology platform
  • How the platform works and why it is highly accurate – the prediction model, genomics and machine learning technology
  • Applicability of the GARD assays – what chemicals have been tested? How about complex mixtures, natural extracts and low solubility substances?

Available on two occasions

EU Time Zone | Feb 25, 11:00 CET | Register here
US Time Zone | Feb 25, 11:00 EST| Register here

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.

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