Next Generation Risk Assessment (NGRA) using NAMs for skin sensitization: Reproducibility and precision of the GARDskin Dose-Response assay for PoD determination of fragrance chemicals.

Presented at ASCCT 2022

Next Generation Risk Assessment (NGRA) using NAMs for skin sensitization: Reproducibility and precision of the GARDskin Dose-Response assay for PoD determination of fragrance chemicals.

Andy Forreryd1, Shashi Donthamsetty2, Paul Sterchele2, Xiao Huang2, Gregory Ladics2, Mihwa Na3, Isabelle Lee3, Anne Marie Api3, Robin Gradin1, Henrik Johansson1
1SenzaGen, Lund, Sweden , 2International Flavors & Fragrances, Hazlet, NJ, USA, 3Research Institute for Fragrance Materials. Woodcliff lake, NJ, USA

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Conclusion

  • GARD®skin Dose-Response can be used for continous predictions of skin sensitizing potency.
  • The continous readout from the assay is reproducible and the assay predicts LLNA EC3 and human NESIL values with high correlation to reference benchmark data.
  • The assay provides a nice tool for the fragrance industry to predict the NESIL value which can be used for conducting the quantitative risk assessment for generating the IFRA standard.

Abstract

New Approach Methods (NAMs) for assessment of skin sensitizers have been adopted as Test Guidelines (TGs) by OECD. When combined into Integrated Approaches to Testing and Assessment (IATA) or defined approaches (DA), they provide data supporting hazard classifications and GHS potency subcategorization. However, more granular potency information, preferably on a continuous scale, is needed to derive a point-of-departure (PoD) for Next Generation Risk Assessment (NGRA).

GARDskin was recently adopted into OECD TG 442E to support discrimination of skin sensitizers and non-sensitizers. Continous potency predictions are derived using a modified protocol that incorporates dose-response measurements. Linear regression models have further been developed to predict LLNA EC3 and human NOEL values. The aim of the following study, which represents a cross-sector collaboration was to evaluate precision and reproducibility of the potency predictions from GARDskin Dose-Response in blinded studies.

Preliminary results from estimate of precision (n=36 materials) indicated that GARDskin Dose-Response predicted LLNA EC3/ human NOEL values with median fold-misprediction factors < 3.0 and < 2.0, respectively. Interestingly, LLNA predicted human NOEL with a fold-change > 2 in the same dataset. For reproducibility assessment, test materials (n=11) were evaluated in separate experiments (n=3), which generated highly reproducible results, with an average median range of fold-changes between replicates of 2.5.

Results from this study demonstrate that continous potency predictions from GARDskin Dose-Response are reproducible. Together with performance data, this represents a major step towards establishment of the assay as a relevant source of information to derive a PoD for NGRA, avoiding generation of new animal data.

 

 

 

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

Poster presented at the 2021 World Congress on Alternatives and Animal Use in the Life Sciences

Robin Gradin, Angelica Johansson, Andy Forreryd, Amber Edwards, Veronika Hoepflinge, Florence Burleson, Helge Gehrke, Erwin Roggen, Henrik Johansson| SenzaGen AB, 22381 Lund, Sweden; Burleson Research Technologies, Morrisville, NC 27560, USA; Eurofins BioPharma Product Testing Munich Gmbh, 82152 Planegg, Germany; 43RsMC Aps, 2800 Kongens Lyngby, Denmark

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Conclusion

  • GARDpotency is an assay for sub-categorization of strong sensitizers (CLP sub-category 1A), allowing for discrimination from weak sensitizers (CLP sub-category 1B) and non-sensitizers. The method is based on the GARD platform, combining human immune cells, a genomic biomarker readout and machine learning-assisted classifications.
  • Sequential combination of GARDskin and GARDpotency forms the GARD Defined Approach, for complete hazard and risk assessment of skin sensitizers into three categories (CLP 1A, CLP 1B, non-sensitizers).
  • A blinded ring trial, comprising 28 chemicals, demonstrated that GARDpotency is functional and reproducible, with an accumulated predictive accuracy of 91% across three laboratories. In the same dataset, the GARD Defined Approach classifies chemicals into three categories with 86% accuracy.

Abstract

The advancement of non-animal approaches for hazard assessment of skin sensitizers have generated a variety of alternative assays with discriminatory properties comparable with those of accepted in vivo methods. However, hazard identification is rarely sufficient and information permitting the relative ranking of chemicals’ skin sensitization potency is desired. For example, the globally harmonized system of classification and labelling of chemicals (GHS/CLP) extends the binary hazard assessment with a qualitative subcategorization to distinguish between weak and strong skin sensitizers.

Though substantial efforts have been made towards developing alternative methods for potency assessment, none have gained regulatory acceptance, emphasizing that continued development of improved alternative assays remains a high priority.

The genomic allergen rapid detection (GARD) is an in vitro testing platform for assessment and characterization of chemical sensitizers, based on evaluation of transcriptional patterns of endpoint-specific genomic biomarker signatures in a human dendritic-like cell line following chemical exposure, in order to provide machine learning-assisted classifications of tested substances. The GARDskin assay was recently subjected to a formal validation procedure (OECD TGP 4.106) and reported a reproducibility between laboratories of 92%, as well as a predictive accuracy of 94%, for sensitization hazard assessment.

Here, we present the implementation of the related GARD application GARDpotency, for potency-associated subcategorization of chemical sensitizers. Following prediction model establishment, the functionality of the assay was validated in a blinded ring-trial, in accordance with OECD-guidance documents, by assessing predictive performance and reproducibility. It was found that the assay is functional and predictive, with an estimated cumulative accuracy of 88% across three laboratories and nine independent experiments. The within-laboratory reproducibility measures ranged between 63-89%, and the between-laboratory reproducibility was estimated to 61%. In conclusion, the in vitro GARDpotency assay constitute a standardized, functional assay, which could be a valuable tool for hazard characterization of skin sensitizer potency.

GARDskin and GARDpotency: a proof of concept study to investigate the applicability domain for agrochemical formulations

Joint poster with Corteva,
Presented at the 2021 SOT Virtual Conference

M. Corvaro, J. Henriquez, R. Settivari, U.T. Mattson, S. Gehen | Corteva Agriscience Italia, Rome, ITA; Corteva Agriscience, Indianapolis, IN, USA;  Corteva Agriscience, Newark, DE, USA; SenzaGen AB, Lund, SWE

 

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Conclusion

  • GARDskin and GARDpotency, showed a satisfactory performance in this initial proof of concept.
  • The accuracy, sensitivity, and specificity for prediction of hazard were 77.8% (14/18), 87.5% (7/8) and 70.0% (7/10), when using available LLNA results as classification reference.
  • Where the GARDskin correctly predicted hazard category, the GARDpotency of GHS potency was correct in 6/7 cases, with 1 underpredicted formulation

Abstract

In vitro methods for detection of delayed dermal sensitization have been formally validated for regulatory use in the last two decades as an alternative to the animal use. Some methods have reached regulatory acceptance as OECD test guidelines. The Genomic Allergen Rapid Detection (GARD™) is a genomic based assay platform which is currently being assessed for inclusion in the OECD test guideline program. GARD is available in the two variants, GARDskin and GARDpotency, addresses Key Event 3 (dendritic cell activation) of the skin sensitization Adverse Outcome Pathway (AOP), and provides reliably potency information for several chemical classes.

Understanding of the applicability domain of test methods is pivotal in providing confidence in assay outcomes, facilitating regulatory uptake in specific industry sectors. The purpose of this work is to verify the applicability domain of GARDskin and GARDpotency, for the product class of agrochemical formulations.

For this proof of concept, 20 agrochemical formulations were tested using GARDskin. When GARDskin was positive, GARDpotency assay was used to determine the severity of sensitization potential. Tests were conducted according to the assay developer Standard Operating Procedures. The selected agrochemical formulations were liquid (11 water based; and 9 organic solvent based) with a balanced distribution (11 not classified; 7 GHS cat 1B; 2 GHS cat 1A, which is rare for agrochemical formulations). GARD results (available for 18 formulations at this time) were compared with in vivo data (mouse LLNA) already available for registration purpose, in order to verify concordance (GHS hazard and potency categories). For hazard, GARDskin was able to correctly identify 7/10 not classified (true negatives) and 7/8 GHS1B/1A (true positives), with 1 false negative and 3 false positives. The accuracy, sensitivity, and specificity for prediction of hazard were 77.8% (14/18), 87.5% (7/8) and 70.0% (7/10), when using available LLNA results as classification reference. Additionally, GARDpotency was able to correctly identify 5 GHS cat 1B and 1 GHS cat 1A out of 7 correctly predicted sensitizer (underprediction from 1A to 1B occurred in 1 case).

In conclusion, GARDskin and GARDpotency, showed a satisfactory performance in this initial proof of concept.

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

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.

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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 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

The use of Genomic Allergen Rapid Detection (GARD) assays to predict the respiratory and skin sensitising potential of e-liquids

Regulatory Toxicology and Pharmacology. Volume 103, April 2019, Pages 158-165 https://doi.org/10.1016/j.yrtph.2019.01.001

Matthew Stevenson, Lukasz Czekala, Liam Simms, Nicole Tschierske, Olivia Larne, Tanvir Walele

Abstract
Electronic cigarettes (e-cigarettes) are an increasingly popular alternative to combustible tobacco cigarettes among smokers worldwide. A growing body of research indicates that flavours play a critical role in attracting and retaining smokers into the e-cigarette category, directly contributing to declining smoking rates and tobacco harm reduction. The responsible selection and inclusion levels of flavourings in e-liquids must be guided by toxicological principles. Some flavour ingredients, whether natural extracts or synthetic, are known allergens. In this study, we used the Genomic Allergen Rapid Detection (GARD) testing strategy to predict and compare the respiratory and skin sensitising potential of three experimental and two commercial e-liquids. These novel, myeloid cell-based assays use changes in the transcriptional profiles of genomic biomarkers that are collectively relevant for respiratory and skin sensitisation. Our initial results indicate that the GARD assays were able to differentiate and broadly classify e-liquids based on their sensitisation potential, which are defined mixtures. Further studies need to be conducted to assess whether and how these assays could be used for the screening and toxicological assessment of e-liquids to support product development and commercialisation.

Keywords
Electronic cigarettesSkin sensitisationRespiratory sensitisationIn vitroAlternative methods

Article on line

The Validation of GARD™skin and GARD™potency

Poster presented at Eurotox, 2018

Sandberg P, Johansson A, Agemark M, Gradin R, Larne O, Appelgren H, Forreryd A, Jerre A, Edwards A, Hoepflinger V, Burleson F, Gehrke H, Roggen E, Johansson H
SenzaGen, Lund, Sweden, Burleson Research Technologies, Morrisville, US, Eurofins, Munich, Germany

Introduction
The prevalence of allergic contact dermatitis (ACD) is estimated to >20% in the western world. Not only the individual is affected, but downstream socioeconomic effects are high. To minimize exposure, chemicals must be safety tested. Traditional testing strategies like the murine local lymph node assay (LLNA) comprise animals, but the regulatory authorities, public opinion and economic interests require animal-free models. The Genomic Allergen Rapid Detection skin (GARD®skin) is an in vitro assay addressing this need. Here, we present the results of the GARD®skin ring trial (OECD TGP 4.106) for validity of the assay. In addition, we show data for GARD®potency – a complementary assay developed to categorize identified senitizers as CLP 1A or 1B.

 

Conclusions

Transfer study
Transferability: 100%

Validation study
Reproducibility
WLR: 82 – 89%BLR: 92% (92 – 100%)
Test performance
– Accuracy: 94%
– Sensitivity: 93%
– Specificity: 96%

A blinded ring trial was performed to assess the functionality of the GARDskin assay. The data demonstrates that GARDskin is a powerful tool for assessment of chemical skin sensitizers, with a predictive accuracy of 94% and excellent reproducibility between laboratories.
In addition, we show that GARDpotency accurately assesses potency of identified sensitizers.

Poster download

SenzaGen receives patent protection for GARDskin™ in China

The recently granted Chinese patent covers the analytical methods and genetic signatures that form the basis of SenzaGen’s GARDskin™ technology. The patent is valid until October 2031.

“China is a potentially important future market for us, and patent protection in China is another confirmation of our technology’s merits and a new milestone in establishing our unique GARDskin™ test method as a global industry standard. This complements European patent protection and further strengthens our potential in the global market of in vitro tests,” says SenzaGen CEO, Anki Malmborg Hager.

A vital part of SenzaGen’s business development is relations with contract laboratories, industry, government agencies and NGOs concerned with alternative testing methods. The market for in vitro methods is growing rapidly, although experiments on animals are still permitted for allergy tests in some parts of the world. Asia is a huge market and is growing fast, and it is therefore essential for SenzaGen to prepare the way for a future introduction of its tests there.

The announcement from the Chinese Patent Office relates to Chinese Patent No. CN 103429756 B entitled: “Analytical methods and arrays for use in the identification of agents inducing sensitization in human skin”. Corresponding patent applications are currently being processed by the patent authorities in Brazil, Canada, Hong Kong, India, South Korea and the United States. 

For more information, please contact:
Anki Malmborg Hager, CEO, SenzaGen AB
Email: anki.malmborg.hager@senzagen.com
Telephone: +46 768 284822

About GARD™
GARD™ is a group of tests for assessing chemical skin sensitizers. The tests make use of genetic biomarkers for more than 200 genes which cover the entire immune reaction and are relevant to predicting the risk of hypersensitivity. The tests have over 90 percent reliability. This compares with the current predominant test method, experiments on mice, which has an accuracy of 70-75 percent. SenzaGen’s tests are also capable of measuring the potency of a substance’s allergenic properties. Consequently, GARD tests provide a much more comprehensive basis for determining whether a substance should be classified as an allergen than current testing methods.

About SenzaGen
SenzaGen makes it possible to replace animal experiments with in vitro genetic testing to determine the allergenicity of the chemicals we come into contact with in our daily lives, such as for example in cosmetics, pharmaceuticals, food products and dyes. The company’s patented tests are the most reliable on the market and provide more information than traditional evaluation methods. We ourselves sell the tests in Sweden and the USA, and we sell through partners in several other countries. Over the next few years the company will expand geographically, make alliances with more distribution partners and launch further unique tests. SenzaGen has its headquarters in Lund in Sweden and a subsidiary in San Francisco, USA. For more information visit http://www.senzagen.com.

The information was submitted for publication, through the contact person set out above on July 11th 2018 at 08:50

SenzaGen AB is listed on Nasdaq First North in Stockholm and FNCA is the company’s Certified Adviser. For more information, please visit http://www.senzagen.com.

Attached file

SenzaGen’s final validation report for the animal-free allergy test GARDpotency™ has been submitted to the regulatory authorities

GARDpotency™ is based on the same biological platform as GARDskin™ and both tests are expected to be approved and recommended at the same time. Together with GARDpotency™, which complements GARDskin™, SenzaGen’s allergy test will be unique on the market, as it is the only test that offers animal-free tests of chemicals in line with the EU’s CLP classification. The CLP classification follows the Global Harmonized System, GHS, developed by the UN to create common criteria for the classification and labelling of chemicals.

The EU Chemicals Agency ECHA, which regulates chemical use based on the REACH Regulation, requires that chemicals that may induce sensitivity (sensitization) must be potency-classified according to CLP. This has so far only been possible with a so-called LLNA (Local Lymph Node Assay) evaluation, which is an in vivo test using animals. SenzaGen’s GARD™ test platform is based on human cells in vitro and genomic biomarkers, constantly delivering better accuracy than animal tests.

“Potency is extremely difficult to measure. We are therefore very happy to be able to report these results. In view of the impressive validation results for GARDpotency™, we look forward to receiving the authorities’ response regarding GARDskin™ and GARDpotency™ in 2019. As a consequence of these good results, we are planning to communicate sales targets for the coming years during the second half of this year. A positive response would make our allergy test the first animal-free test that can be used for classification in accordance with CLP, the EU’s standard,” says Anki Malmborg Hager, CEO of Senzagen.

The results from the validation report will be presented at forthcoming scientific conferences.

For more information, please contact:
Anki Malmborg Hager, CEO, SenzaGen AB
Email: anki.malmborg.hager@senzagen.com
Telephone: +46 768 284822

About GARD™
GARD™ is a group of tests for assessing chemical skin sensitizers. The tests make use of genetic biomarkers for more than 200 genes which cover the entire immune reaction and are relevant to predicting the risk of hypersensitivity. The tests have over 90 percent reliability. This compares with the current predominant test method, experiments on mice, which has an accuracy of 70-75 percent. SenzaGen’s tests are also capable of measuring the potency of a substance’s allergenic properties. Consequently, GARD tests provide a much more comprehensive basis for determining whether a substance should be classified as an allergen than current testing methods.

About SenzaGen
SenzaGen makes it possible to replace animal experiments with in vitro genetic testing to determine the allergenicity of the chemicals we come into contact with in our daily lives, such as for example in cosmetics, pharmaceuticals, food products and dyes. The company’s patented tests are the most reliable on the market and provide more information than traditional evaluation methods. We ourselves sell the tests in Sweden and the USA, and we sell through partners in several other countries. Over the next few years the company will expand geographically, make alliances with more distribution partners and launch further unique tests. SenzaGen has its headquarters in Lund in Sweden and a subsidiary in San Francisco, USA. For more information visit http://www.senzagen.com.

The information was submitted for publication, through the contact person set out above on the 6 July 2018 at 08:50

SenzaGen AB is listed on Nasdaq First North in Stockholm and FNCA is the company’s Certified Adviser. For more information, please visit http://www.senzagen.com.

Attached file