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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).

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.

Quantitative Sensitizing Potency Assessment Using GARD™skin Dose-Response

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

Henrik Johansson, Robin Gradin, Andy Forreryd, Joshua Schmidt
SenzaGen AB, Lund, Sweden. SenzaGen Inc., Raleigh, NC.

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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

Proactive identification and characterization of sensitization hazards are central aspects of risk assessment of chemicals. Current legislations and trends in predictive toxicology advocate a transition from in vivo methods to non-animal alternatives, with a number of methods for hazard assessment of skin sensitizers currently available. However, non-animal methods capable of providing quantitative assessment of sensitizing potency are currently lacking.

The GARDskin assay is a next-generation in vitro assay for hazard assessment of skin sensitizers, currently progressing towards regulatory acceptance. Recently, the GARDskin Dose-Response (DR) testing strategy was introduced, in which test chemicals are evaluated by the GARDskin assay in a titrated range of concentrations, in order to investigate the dose-response relationship between GARDskin classifications and test chemical concentration. As such, it provides a quantitative estimation of sensitizing potency, referred to as cDV0, which corresponds to the least required dose able to generate a positive response in the GARDskin assay. The cDV0 value obtained for a test chemical may be viewed as an analogue to the LLNA EC3 value, based on which further hazard characterization and risk assessment may be performed. Statistically significant correlation between the GARDskin DR cDV0 and the LLNA EC3, as well as with human No Expected Sensitization Induction level (NESIL) estimations has been confirmed, thus enabling direct extrapolation between the different metrics.

Here, we further illustrate how these results can be used on their own to facilitate direct potency-associated ranking of test chemicals. Furthermore, we demonstrate how obtained cDV0 values can be extrapolated to LLNA EC3 values with a 95% confidence interval, thereby also facilitating potency-associated subcategorization of test chemicals according to UN GHS classification criteria. Lastly, we illustrate how results generated with GARDskin DR can be directly incorporated into existing strategies for Quantitative Risk Assessment using an entirely in vitro setup.

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.

Quantitative Sensitizing Potency Assessment Using GARD™skin Dose-Response

Poster presented at SOT 2021

Henrik Johansson, Robin Gradin, Andy Forreryd, Joshua Schmidt
SenzaGen AB, Lund, Sweden. SenzaGen Inc., Raleigh, NC.

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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

Proactive identification and characterization of sensitization hazards are central aspects of risk assessment of chemicals. Current legislations and trends in predictive toxicology advocate a transition from in vivo methods to non-animal alternatives, with a number of methods for hazard assessment of skin sensitizers currently available. However, non-animal methods capable of providing quantitative assessment of sensitizing potency are currently lacking.

The GARDskin assay is a next-generation in vitro assay for hazard assessment of skin sensitizers, currently progressing towards regulatory acceptance. Recently, the GARDskin Dose-Response (DR) testing strategy was introduced, in which test chemicals are evaluated by the GARDskin assay in a titrated range of concentrations, in order to investigate the dose-response relationship between GARDskin classifications and test chemical concentration. As such, it provides a quantitative estimation of sensitizing potency, referred to as cDV0, which corresponds to the least required dose able to generate a positive response in the GARDskin assay. The cDV0 value obtained for a test chemical may be viewed as an analogue to the LLNA EC3 value, based on which further hazard characterization and risk assessment may be performed. Statistically significant correlation between the GARDskin DR cDV0 and the LLNA EC3, as well as with human No Expected Sensitization Induction level (NESIL) estimations has been confirmed, thus enabling direct extrapolation between the different metrics.

Here, we further introduce the GARDskin DR protocols, as proposed in a standardized testing strategy. By studying a concentration range of 6 concentration points titrated from the experimentally derived GARD input concentration in biological duplicates, a test chemical-specific cDV0 is established by linear interpolation. We illustrate how these results can be used on their own to facilitate direct potency-associated ranking of test chemicals. Furthermore, we demonstrate how obtained cDV0 values can be extrapolated to LLNA EC3 values with a 95% confidence interval, thereby also facilitating potency-associated subcategorization of test chemicals according to UN GHS classification criteria. Lastly, we illustrate how results generated with GARDskin DR can be directly incorporated into existing strategies for Quantitative Risk Assessment using an entirely in vitro setup.

 

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.

 

Exploration of the GARD applicability domain – Skin sensitization assessment of UVCBs

Poster presented at Eurotox 2018 in collaboration with Lubrizol

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U. I.Torstensdotter Mattson1, C. Humfrey2, O. Larne1, H. Johansson1, L. Sweet3 1SenzaGen, Lund, Sweden, 2Lubrizol, Derbyshire, United Kingdom, 3Lubrizol, Ohio, United States of America

Conclusion

This case study demonstrates the broadening of the applicability domain of the GARD assay when assessing UVCBs.

Abstract

In this study, four test items were evaluated. All the test items were “Unknown or Variable composition, Complex reaction products and Biological materials” materials (UVCBs), which were provided by Lubrizol and selected based on existing in vivo data (internal Lubrizol data). Skin sensitizing hazard was assessed using the GARDskin assay, and the GARDpotency assay for further subcategorized the sensitizers into strong 1 A) or weak 1 B) sensitizers according to GHS/CLP classification. The GARDskin predictions for test items 1, 2, 3 and the GARDpotency classifications for test item 2 and 3 were consistent with the in vivo data, whereas test item 4 showed inconsistency between the in vitro and in vivo methods. These results indicate the importance of screening a panel of different vehicles or mixtures thereof, in order to choose the appropriate solvent For one of the Test items, the DMSO extraction procedure generates a negative prediction while the experimental vehicle mixture, Glycerol and DMF, classifies the chemical as a skin sensitizer This case study demonstrates the broadening in applicability domain of the GARDassays when assessing UVCBs.