Poster presented at ACT 2020: Applicability of GARD™skin for Accurate Assessment of Challenging Substances in the Context of Skin Sensitization Testing

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.

 

Poster presented at ACT 2020: Dose-Response Analysis in GARD™ for Assessment of Skin Sensitizer Potency

J. Schmidt, A. Forreryd, R. Gradin2, H. Johansson.
SenzaGen Inc., Raleigh, NC., SenzaGen AB, Lund, Sweden.

 

Link to the poster

 

Conclusion

  • As an adaptation from the GARDskin assay, GARDskin Dose-Response is suitable for quantitative skin sensitizing potency assessment of chemicals.
  • The experimental readout, referred to as cDV0, corresponds to the lowest dose required to elicit a positive response in GARDskin. As such, experimental protocols are analogous to the LLNA, in which the cDV0 corresponds to the EC3-value.
  • The cDV0 may be used to directly monitor sensitizing potency, or further used to extrapolate LLNA EC3-values, estimation of Human Potency categories, or CLP 1A/1B classifications.

Abstract

Several non-animal methods for identifying skin sensitizers have been developed with acceptable prediction performance. However, advancement of alternative methods for skin sensitizing potency assessment is still missing although a highly sought-after endpoint. The GARDskin assay is a genomics-based in vitro assay for hazard assessment of skin sensitizers, currently progressing towards regulatory acceptance. Here, we introduce GARDskin Dose-Response (DR), in which test chemicals are evaluated by the GARDskin assay in an extended range of concentrations, in order to investigate the dose-response relationship between GARDskin classifications and test chemical concentration.

For this work, 29 chemicals of various sensitizing potencies were used to evaluate the efficacy of applying the assay in this manner. Each chemical was analyzed at several concentrations using a slightly modified GARDskin protocol. At each concentration, a decision-value was produced and a classification prediction (sensitizing or non-sensitizing) was made by the GARDskin algorithm. Afterwards, the lowest concentration where a test item would provide a positive GARDskin prediction was found using linear interpolation. This concentration (cDV0) was then hypothesized to reflect the test items skin sensitizing potency. Furthermore, when comparing cDV0 to LLNA EC3 values, a statistically significant correlation was realized between the values (correlation coefficient =0.74, p-value=4.1*10-4).

These results suggest that modifying the GARDskin protocol to accommodate dose-response measurements can provide sensitizing potency information analogous to the gold-standard in vivo methods. This presentation will further explain the testing process, expand on results, and demonstrate how this method can be used for decision-making throughout all stages of product development, without having to use animal experimentation.

RIFM Principle Scientist Gretchen Ritacco speaks about SenzaGen and RIFM collaboration

Here is an interview with Principal Scientist Gretchen Ritacco, MS, explaining why RIFM has focused some of its research on the potential for light to activate fragrance materials resulting in reactions in the body’s largest organ, the skin.

Since June 2020, SenzaGen and the Research Institute for Fragrance Materials (RIFM) are collaborating to investigate in vitro methods and develop next-generation test for determining whether a fragrance material may cause photosensitization.

Read the RIFM interview here.

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

Full article
Article on line with open access

Watch our latest GARDskin Dose-Response Webinar

Thanks to all who attending yesterday’s GARDskin Dose-Response webinar. If you missed it, here’s a link to the recording.

CEO presentation at AGM 2020

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

2019 Annual Report – now available in English

The 2019 Annual Report in English is available.

If you want to download it click here. To order a printed version, click here.

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