Extended solvent selection for in vitro sensitization testing using GARD®

Olivia Larne, Ulrika I Torstensdotter Mattson, Rikard Alm, and Gunilla Grundström. SenzaGen, Lund, Sweden. Introduction The GARD®skin assay is an in vitro assay developed for the assessment of skin sensitizers. It is based on SenzaCells™, a human dendritic-like cell line, and a biomarker signature analyzed by a prediction model including pattern recognition and machine learning. […]
Olivia Larne, Ulrika I Torstensdotter Mattson, Rikard Alm, and Gunilla Grundström.
SenzaGen, Lund, Sweden.

Introduction
The GARD®skin assay is an in vitro assay developed for the assessment of skin sensitizers. It is based on SenzaCells™, a human dendritic-like cell line, and a biomarker signature analyzed by a prediction model including pattern recognition and machine learning.

During the development of the GARD®skin platform, two solvents were used: DMSO (0.1%) and water. To increase the applicability domain of GARD®skin and the possibility to dissolve certain test items, for e.g. hard to dissolve substances and UVCBs, where show a broader range of solvents compatible with GARD®skin. Also, use of higher concentrations of the tested solvents were explored for the possibility to increase test item concentrations.

Concluding highlights
GARD®skin compatible solvents:

  • Acetone
  • DMF
  • DMF/Glycerol
  • DMSO
  • Ethanol
  • Glycerol
  • Isopropanol

Increased applicability domain.

Link to poster

Poster publication at Eurotox, Helsinki, Sep 9, 2019.

 

Validation of the GARD™skin assay for assessment of chemical skin sensitizers – ring trial results of predictive performance and reproducibility

Toxicological Sciences, kfz108, https://doi.org/10.1093/toxsci/kfz108 Henrik Johansson, Robin Gradin, Angelica Johansson, Els Adriaens, Amber Edwards, Veronika Zuckerstätter, Anders Jerre, Florence Burleson, Helge Gehrke, Erwin Roggen Abstract Proactive identification of chemicals with skin sensitizing properties is a key toxicological endpoint within chemical safety assessment, as required by legislation for registration of chemicals. In order to meet demands of increased animal welfare and facilitate increased testing efficiency […]

Toxicological Sciences, kfz108, https://doi.org/10.1093/toxsci/kfz108

Henrik Johansson, Robin Gradin, Angelica Johansson, Els Adriaens, Amber Edwards, Veronika Zuckerstätter, Anders Jerre, Florence Burleson, Helge Gehrke, Erwin Roggen

Abstract
Proactive identification of chemicals with skin sensitizing properties is a key toxicological endpoint within chemical safety assessment, as required by legislation for registration of chemicals. In order to meet demands of increased animal welfare and facilitate increased testing efficiency also in non-regulatory settings, considerable efforts have been made to develop non-animal approaches to replace current animal testing.

Genomic Allergen Rapid Detection (GARD) is a state-of-the-art technology platform, the most advanced application of which is the assay for assessment of skin sensitizing chemicals, GARDskin. The methodology is based on a dendritic cell (DC)-like cell line, thus mimicking the mechanistic events leading to initiation and modulation of downstream immunological responses. Induced transcriptional changes are measured following exposure to test chemicals, providing a detailed evaluation of cell activation. These changes are associated with the immunological decision-making role of DCs in vivo and include among other phenotypic modifications, up-regulation of co-stimulatory molecules, induction of cellular and oxidative stress pathways and xenobiotic responses and provide a holistic readout of substance-induced DC activation.Here, results from an inter-laboratory ring trial of GARDskin, conducted in compliance with OECD guidance documents and comprising a blinded chemical test set of 28 chemicals, are summarized. The assay was found to be transferable to naïve laboratories, with an inter-laboratory reproducibility of 92.0%. The within-laboratory reproducibility ranged between 82.1-88.9%, while the cumulative predictive accuracy across the three laboratories was 93.8%.It was concluded that GARDskin is a robust and reliable method for the identification of skin sensitizing chemicals and suitable for stand-alone use or as a constituent of integrated testing. These data form the basis for the regulatory validation of GARDskin.Keywords
GARD™, GARD™skin, in vitro, sensitization, chemical sensitizers

Full article
Article on line ahead of print with open access 

Evaluation of in vitro testing strategies for hazard assessment of the skin sensitization potential of “real‐life” mixtures: the case of henna‐based hair coloring products containing p‐phenylenediamine

Wiley Online Library, First published: 21 April 2019,  https://doi.org/10.1111/cod.13294. Renato I. de Ávila Danillo F. M. C. Veloso Gabriel C. Teixeira Thaisângela L. Rodrigues Tim Lindberg Malin Lindstedt Simone G. Fonseca Eliana M. Lima Marize C. Valadares Abstract Background Allergic contact dermatitis reported to henna‐based hair coloring products (HPs) has been associated with adulteration of […]

Wiley Online Library, First published: 21 April 2019,  https://doi.org/10.1111/cod.13294.

Renato I. de Ávila Danillo F. M. C. Veloso Gabriel C. Teixeira Thaisângela L. Rodrigues Tim Lindberg Malin Lindstedt Simone G. Fonseca Eliana M. Lima Marize C. Valadares

Abstract

Background
Allergic contact dermatitis reported to henna‐based hair coloring products (HPs) has been associated with adulteration of henna with p‐phenylenediamine (PPD).

Objectives
To develop a testing approach based on in vitro techniques that address key events within the skin sensitization adverse outcome pathway to evaluate allergenic potential of HPs.

Methods
The following in vitro assays were used to test the sensitizing capacity of hair dye ingredients: micro‐direct peptide reactivity assay (mDPRA); HaCaT keratinocytes‐associated IL‐18 assay; U937 cell line activation test (USENS)/IL‐8 levels; blood monocyte‐derived dendritic cell test; genomic allergen rapid detection (GARD skin). Those techniques with better human concordance were selected to evaluate the allergenic potential of ten HPs.

Results
Contrasting to the label’s information, chromatographic analyses identified PPD in all products. The main henna biomarker, lawsone, was not detected in one of 10 the products. Among the techniques evaluated by testing HDIs, mDPRA, IL‐18 assay, GARD skin and U‐SENS correlated better with human classification (concordances 91.7 to 100%) and were superior to the animal testing (concordance 78.5%). Thus, these assays were used to evaluate HPs, which were classified as skin sensitizers using different two‐out‐of‐three approaches.

Conclusions
Our findings highlight toxicological consequences and risks associated of the undisclosed use of PPD in henna‐based “natural” “real‐life” products.

This article is protected by copyright. All rights reserved.

Our view on alternative testing for product safety in Manufacturing Chemist

To replace a test animal, which is clearly a complex system with blood and organs, you need a more holistic approach to model the entire allergic response within the body. Our CEO Anki Malmborg Hager explains the benefits of using alternative testing methods to prove product safety in this week’s Manufacturing Chemist. Read the article […]

To replace a test animal, which is clearly a complex system with blood and organs, you need a more holistic approach to model the entire allergic response within the body. Our CEO Anki Malmborg Hager explains the benefits of using alternative testing methods to prove product safety in this week’s Manufacturing Chemist.

Read the article here

Skin Sensitization Testing—What’s Next?

Int. J. Mol. Sci. 2019, 20(3), 666; https://doi.org/10.3390/ijms20030666   Gunilla Grundström and Carl A.K. Borrebaeck Abstract There is an increasing demand for alternative in vitro methods to replace animal testing, and, to succeed, new methods are required to be at least as accurate as existing in vivo tests. However, skin sensitization is a complex process […]

Int. J. Mol. Sci. 2019, 20(3), 666; https://doi.org/10.3390/ijms20030666  

Gunilla Grundström and Carl A.K. Borrebaeck

Abstract

There is an increasing demand for alternative in vitro methods to replace animal testing, and, to succeed, new methods are required to be at least as accurate as existing in vivo tests. However, skin sensitization is a complex process requiring coordinated and tightly regulated interactions between a variety of cells and molecules. Consequently, there is considerable difficulty in reproducing this level of biological complexity in vitro, and as a result the development of non-animal methods has posed a major challenge. However, with the use of a relevant biological system, the high information content of whole genome expression, and comprehensive bioinformatics, assays for most complex biological processes can be achieved. We propose that the Genomic Allergen Rapid Detection (GARD™) assay, developed to create a holistic data-driven in vitro model with high informational content, could be such an example. Based on the genomic expression of a mature human dendritic cell line and state-of-the-art machine learning techniques, GARD™ can today accurately predict skin sensitizers and correctly categorize skin sensitizing potency. Consequently, by utilizing advanced processing tools in combination with high information genomic or proteomic data, we can take the next step toward alternative methods with the same predictive accuracy as today’s in vivo methods—and beyond.

Keywords

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

Article online

A mechanistic reinterpretation of the AOP for skin sensitisation

David W Roberts, Liverpool John Moores University, Liverpool Introduction – Non-Animal Prediction: the 21st Century Consensus Because of the biological complexity of the skin sensitisation process no single in chemico or in vitro assay will be an appropriate replacement for an animal-based assay such as LLNA or GPMT… …to ensure a mechanistic basis and cover the […]

David W Roberts, Liverpool John Moores University, Liverpool

Introduction – Non-Animal Prediction: the 21st Century Consensus
Because of the biological complexity of the skin sensitisation process no single in chemico or in vitro assay will be an appropriate replacement for an animal-based assay such as LLNA or GPMT…
…to ensure a mechanistic basis and cover the complexity, multiple methods should be integrated into a testing strategy, in accordance with the adverse outcome pathway that describes all key events in skin sensitisation.

We need an ITS based on the KEs of the AOP…but
Is that what we really need?

Conclusion

A single assay, GARD™, predicts sensitisation potential and absence of sensitisation potential better than any of, or combinations of, the OECD guideline assays DPRA, KeratinosensTM (ARE-Nrf2 ) and h-CLAT.

We do not really need an ITS covering all KE’s of the AOP.

Link to poster

Is a combination of assays really needed for non-animal prediction of skin sensitization potential? Performance of the GARD™ (Genomic Allergen Rapid Detection) assay in comparison with OECD guideline assays alone and in combination

Regulatory Toxicology and Pharmacology, Volume 98, October 2018, Pages 155-160, https://doi.org/10.1016/j.yrtph.2018.07.014. David W.Roberts Highlights Prediction of skin sensitization potential does not need multiple assays representing Key Events of the AOP. This has been argued on theoretical grounds and is now tested against published data. A single assay, GARD™, can outperform combinations of OECD test guideline […]

Regulatory Toxicology and Pharmacology, Volume 98, October 2018, Pages 155-160, https://doi.org/10.1016/j.yrtph.2018.07.014.

David W.Roberts

Highlights

  • Prediction of skin sensitization potential does not need multiple assays representing Key Events of the AOP.
  • This has been argued on theoretical grounds and is now tested against published data.
  • A single assay, GARD™, can outperform combinations of OECD test guideline assays.

Abstract
To meet regulatory requirements, and avoid or minimize animal testing, there is a need for non-animal methods to assess the potential of chemicals to cause skin sensitization. It is widely assumed that no one test will be sufficient and that combined data from several assays spanning key events from the adverse outcome pathway will be required. This paper challenges that assumption. The predictive performance of a single assay, the Genomic Allergen Rapid Detection (GARD™) assay, was compared with the performance, singly and in combination, of three formally validated non-animal approaches that appear as OECD test guidelines: the direct peptide reactivity assay (DPRA), the ARE-Nrf2 luciferase test method, and the human cell line activation test (h-CLAT).

It is shown here that GARD™ alone outperforms each of DPRA, ARE-Nrf2 luciferase or h-CLAT, alone or in any combination as a 2 out of 3 strategy, in terms of sensitivity, specificity and accuracy.

Based on the datasets analysed here, the sensitivity and specificity of GARD™ alone are 90–92% and 79–84% (“2 out of 3”, 86% and 76%). Thus, in any situation where the 2 out of 3 strategy is considered adequate, GARD™ alone could be used with equal or better performance.

Keywords
Skin sensitization potential, Non-animal assays, Integrated testing strategies, GARD™

Full article
Article on line

GARD® and SenzaGen mentioned in Chemical Risk Manager, September 18, 2018

Don’t miss the interesting article in Chemical Risk Manager in Chemical Watch, written by Dr. Emma Davies, with the title; Skin sensitization integrated testing strategies need rethink, expert suggests. Emma Davies is interviewing the UK academic Dr. David Roberts after his publication of an article in Regulatory Toxicology and Pharmacology in July where he is discussing […]

Don’t miss the interesting article in Chemical Risk Manager in Chemical Watch, written by Dr. Emma Davies, with the title; Skin sensitization integrated testing strategies need rethink, expert suggests.

Emma Davies is interviewing the UK academic Dr. David Roberts after his publication of an article in Regulatory Toxicology and Pharmacology in July where he is discussing the relevance of Integrated Testing Strategy (ITS) for skin sensitization testing as well as he is presenting data on the performance of the GARDskin test method compared to the OECD -tests.  

In short, David Roberts from Liverpool John Moores University questions the science behind integrated testing strategies (ITS) for combining in vitro test methods to predict skin sensitisation, as required by REACH. Instead, Roberts suggests, ”… that the genomic allergen rapid detection (GARD) assay for skin sensitisation may outperform other OECD validated test methods, either alone or in combination.”

Dr. David Roberts will present his data and the research behind the article at the EUSAAT meeting 23-26 September in Linz, and at the ESTIV conference 15-18 October in Berlin

Read the article here (requires subscription to Chemical Watch):
https://chemicalwatch.com/crmhub/70359/skin-sensitisation-integrated-testing-strategies-need-rethink-expert-suggests

 

 

Extended applicability domain with new solvent selection for the GARD platform

Jenvert RM, Larne O, TorstensdotterMattssonU. I., Johansson H, SenzaGen, Lund, Sweden Introduction The Genomic Allergen Rapid Detection (GARD) assay is a state of the art in vitro assay developed for the assessment of skin sensitizers. It is based on gene expression analysis of SenzaCells, a human myeloid cell line, after stimulation by the test item. During […]

Jenvert RM, Larne O, TorstensdotterMattssonU. I., Johansson H, SenzaGen, Lund, Sweden

Introduction
The Genomic Allergen Rapid Detection (GARD) assay is a state of the art in vitro assay developed for the assessment of skin sensitizers. It is based on gene expression analysis of SenzaCells, a human myeloid cell line, after stimulation by the test item.
During the development of the GARD platform, two solvents were used; DMSO (0.1%) and Water. To increase the applicability domain of GARD® and the solubility of certain test items, for e.g. Medical Device extracts and UVCBs, we here show a broader range of solvents compatible with GARD.

Conclusions
Here, we show that the GARD® platform is compatible with the following solvents:
• Acetone
• Ethanol
• Glycerol
• Super refined olive oil

• DMF
• DMSO
• Water
• Isopropanol

Poster download