Reliable and Truly Animal-Free Skin Sensitization Testing – Adaption of the In vitro GARD™skin to Animal-Free Conditions

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

Andy Forreryd, Anders Jerre, Fiona Jacobs, Carol Treasure, Henrik Johansson | SenzaGen AB, 22381 Lund, Sweden; XCellR8 Ltd, Techspace One, Sci-Tech Daresbury, Cheshire WA4 4AB, UK

 

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Conclusion

  • Senzacells adapted well to routine culture in human serum with comparable cell growth, morphology, and minimal impact on cellular phenotypes.
  • GARDskin based on HS demonstrated full concordance to protocols based on animal-derived FCS for hazard identification of skin sensitizers.
  • This work represents our ambition to gradually replace all animal derived components with human equivalents to enable completely animal-free skin sensitization testing.

Abstract

A plethora of in vitro approaches for hazard assessment of skin sensitizers have recently been described and demonstrated to exhibit discriminatory properties competitive with those of accepted in vivo methods. However, the majority of these in vitro methods still use animal-derived components such as Foetal Calf Serum (FCS) within their standard protocols, imposing the question whether these methods should truly be considered as animal-free replacements.

Genomic Allergen Rapid Detection – GARD – is a genomics-based in vitro testing platform for assessment of numerous immunotoxicity endpoints. The endpoint-specific classification of skin sensitizers is a well-established application of the platform, referred to as GARDskin (OECD TGP 4.106). The assay is based on a human DC-like cell line (SenzaCells) and utilizes state-of-the-art machine learning to classify chemicals by monitoring the expression of 200 genes involved in cellular pathways associated with skin sensitization. GARDskin is progressing towards regulatory acceptance, and consistently reports accuracies > 90%.

Here, we present an adaption of the GARDskin standard protocol to enable for testing under animal-product-free conditions by replacing animal-based FCS with human derived serum. SenzaCells adapted well to routine culture in the human serum, showing comparable cell viability and growth rates to the animal-based FCS. A phenotypic analysis of common DC maturity markers showed minor changes in cell surface expression of the markers CD14 and CD1a, indicating that serum replacement did not significantly alter the phenotypic characteristics of the cells. Finally, a proficiency set of nine chemicals covering the full range from extreme sensitizers to non-sensitizers were evaluated. The protocol adapted to animal-free conditions showed full concordance to the conventional protocol, correctly classifying all chemicals.

In conclusion, this study demonstrates the potential to perform the GARDskin assay without the use of animal-derived components associated with animal welfare concerns, thus paving the way for truly animal-free and highly accurate hazard testing of skin sensitizers.

Hazard Assessment of Photoallergens Using GARD™skin

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

Andy Forreryd, Angelica Johansson, Gretchen Ritacco, Anne Marie Api, Henrik Johansson | SenzaGen AB, 22381 Lund, Sweden; Research Institute for Fragrance Materials, Inc., 50 Tice Boulevard, Woodcliff Lake, NJ, 07677, USA.

 

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Conclusion

  • The GARDskin assay for assessment of chemical skin sensitizers can be used to predict photoallergens.
  • By implementing a UV-light exposure step into the conventional GARDskin protocols, photoactivation was achieved. Non-irradiated photoallergens remain accurately classified as non-sensitizers.
  • UV-light exposure did not have an impact on classifications on true non-sensitizing and conventional sensitizing controls.

Abstract

Chemicals of different categories, such as cosmetics and drugs, have the potential to become photoactivated when exposed to UV light, giving rise to otherwise dormant adverse effects such as sensitization. Such chemicals, referred to as photoallergens, causes a Type IV delayed hypersensitivity, typically manifested as allergic contact dermatitis. While in vitro assays for prediction of a chemical’s potential to provoke phototoxicity (photoirritation) have been proposed, there is no recognized assay that specifically predicts photoallergens. Therefore, development of accurate in vitro assays that can detect photoallergens remains a high priority.

The Genomic Allergen Rapid Detection – GARD – platform constitutes a unique framework for classification of numerous immunotoxicity endpoints. The endpoint-specific classification of skin sensitizing chemicals is a well-established application of the platform, referred to as the GARDskin assay. GARDskin utilizes the readout of a genomic biomarker signature of 200 genes, which allows for machine learning-assisted classification of skin sensitizers. The assay is progressing towards regulatory acceptance, and demonstrates high predictive performance.

Here, we present an adaptation of GARD protocols, allowing for assessment of chemical photoallergens. By incorporating UVA exposure during sample preparation, photoactivation of latent photoallergens has been demonstrated. In a first step, protocols were optimized using the photoallergens 6-methylcoumarin and Ketoprofen, exposed to UVA light, both prior to and in association with cellular exposure, along with appropriate radiated/non-radiated controls. Photoallergenicity was accurately predicted in both test chemicals exposed to UVA light, while non-radiated counterparts were accurately classified as non-sensitizers.

In summary, our initial data demonstrates a potential of GARDskin to assess photoallergenicity of chemicals. Further evaluation and optimization of the method are currently in progress, in which an extended panel of fragrances are being studied in a collaboration with the Research Institute for Fragrance Materials (RIFM).

Applicability domain of the GARD™skin Medical Device test for in vitro skin sensitization testing of medical devices

Poster presented at SOT 2021

Joshua Schmidt, Ron Brown and Rose-Marie Jenvert
SenzaGen Inc., Raleigh, NC, USA, Risk Science Consortium LLC, Arnold, MD, USA, SenzaGen AB, Lund, Sweden.

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Conclusion

  • The chemical space of compounds tested in GARD closely approximates the chemical space of compounds known to be released from medical device materials.
  • GARDskin is able to predict the skin sensitization potential of compounds released from medical device materials with a high degree of sensitivity and specificity, including: metals, lipophilic compounds and pre/pro haptens.

Abstract

Medical device toxicology is undergoing an exciting evolution; transitioning from a process that largely relied on the results of animal testing to evaluate the biological safety of devices in patients to one which is increasingly focused on the use of in vitro methods for the safety assessment of device materials.

Recently, in vitro methods to assess endpoints such as skin irritation and pyrogenicity have been validated and proposed for medical device testing, but a method to assess the potential for device-related skin sensitization to occur has not been sufficiently qualified. A number of in vitro skin sensitization test methods have been shown to have acceptable predictive ability for known skin sensitizers with structures that span a broad range of chemical classes, but the predictive ability of these methods has not been specifically evaluated using compounds typically found in materials used to manufacture medical devices. As a result, the need exists to qualify in vitro methods to assess the skin sensitization of compounds that may be released from medical devices, taking into account the applicability domain of known or potential skin sensitizers, including metals.

To address this challenge, the predictive ability of the GARD assay has been evaluated using a dataset of compounds known to be released from device materials.  Against these data, the assay correctly predicted 19 out of 21 lipophilic and pre-/pro-hapten compounds (90.5% accuracy), with one false positive (95.2% sensitivity) and one false negative (95.2% specificity) being predicted, thus increasing the confidence in use of this in vitro assay to assess the skin sensitization potential of medical devices.  Furthermore, we have also demonstrated that the GARD assay correctly predicts the skin sensitization response of nickel and cobalt salts (sensitizers) and a zinc salt (non-sensitizer). Overall, our data support the use of the GARDskin Medical Device assay as an in vitro alternative for the in vivo methods (e.g., GPMT, LLNA) that are typically used to assess skin sensitization as part of the biological safety assessment of medical devices.

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.

Applicability of GARD™skin for Accurate Assessment of Challenging Substances in the Context of Skin Sensitization Testing

Poster presented at ACT 2020

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.

 

The GARD™Skin Assay: A New In Vitro Testing Strategy for Skin Sensitization

E. Schmidt, V. Zuckerstätter, H. Gehrke | Eurofins BioPharma Product Testing Munich GmbH

Introduction 
A skin sensitiser refers to a substance that will lead to an allergic response following skin contact as defined by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS). The potential to induce skin sensitisation is an important consideration included in procedures for the safe handling, packaging and transports of chemicals.

The assessment of skin sensitisation typically involves the use of laboratory animals. Classical methods comprise the Magnusson Kligman Guinea Pig Maximisation Test, the Buehler Test (TG 406) as well as the local lymph node assay, in its radioactive and non-radioactive form (TG 429, TG 442A/B). In order to replace in vivo experiments validation studies on alternative, mechanistically based in chemico and in vitro test methods on skin sensitisation were conducted under the auspices of ECVAM and have been considered scientifically valid for the evaluation of the skin sensitisation hazard of chemicals.

Genomic Allergen Rapid Detection (GARDTM) is an in vitro assay designed to predict the ability of chemical substances to induce skin sensitisation based on the analysis of the relative expression levels of a biomarker signature of 196 genes using a human myeloid leukaemia cell line called SenzaCells. The GARDTM assay is based on chemical stimulation of the SenzaCells, acting as an in vitro model of human Dendritic Cells (DCs). The readout of the assay is a transcriptional quantification of the genomic predictors, collectively termed the GARDTM Prediction Signature (GPS), using Nanostring nCounter technology.

Conclusion
The DPRA, KeratinoSensTM and h-CLAT are well known sensitization assays which address three different key events of the AOP. The GARDTM skin assay is a new procedure that analyses the sensitization potential based on almost 200 human genes. If a substance is a skin sensitiser with the GARDTM skin assay you have the benefit of measuring the potency on top with a different code set to make a 1A or 1B classification.
The GARDTM skin assay is especially for products that have a high log Pow (h-CLAT > 3.5, KeratinoSensTM > 7) because in those cases the classical sensitization tests are inconclusive if negative and there is no option for a replacing test method. Therefore, the GARDTM skin assay is not only an excellent alternative of the sensitization methods for these cases but it can furthermore predict the potency of a skin sensitiser, a unique feature, which makes it a testing method needed in the future.

Link to poster

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

 

Identification of skin sensitizers in natural mixtures

This pilot study demonstrated the applicability of the GARDTMskin assay for identification of skin sensitizers in hair dye ingredients, delivering high prediction performance, consistent with existing human data.

The study also indicated that GARDTMskin is a promising in vitro model to identify skin sensitizers in natural mixtures.

Link to Application Note.

In vitro skin sensitization testing of Medical Devices using GARD™

Rose-Marie Jenvert, Angelica Johansson, Olivia Larne, Emelie Danefur, Emil Altonen, Anders Jerre, Robin Gradin, Gunilla Grundström.
SenzaGen, Lund, Sweden.

Introduction
All medical devices need to be evaluated for the end point skin sensitization according to the Biological Evaluation of Medical Devices (ISO 10993-1:2018), today commonly involving in vivo assays. Here, we show that the in vitro assay GARDskin Medical Devices can classify leachables as either skin sensitizers or non-sensitizers in polar and non-polar extraction of Medical Devices.

Conclusion
GARDskin Medical Device

  • is an in vitro alternative for assessment of skin sensitization of Medical Devices
  • is compatible with the extraction vehicles salin, olive oil and sesame oil.

Link to poster

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

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.

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 presented at Eurotox, Helsinki, Sep 9, 2019.

 

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 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