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Skin Sensitization Potency Assessments of Fragrance Materials using GARDskin Dose-Response

Joint poster with Research Institute for Fragrance Materials (RIFM),
Presented at the 2021 RIFM annual meeting & 2021 ACT annual meeting 

Mihwa Na, Ulrika Mattson, Robin Gradin, Henrik Johansson, Andy Forreryd, Anne Marie Api, Research Institute for Fragrance Materials, Inc., 50 Tice Boulevard, Woodcliff Lake, NJ, 07677, USA., SenzaGen AB, Lund, Sweden

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Conclusion

  • GARDskin Dose-Response closely approximated the potency categories of 9/12 fragrance materials tested.
  • Based on results from this dataset, GARDskin Dose-Response appears useful for potency assessment for weak sensitizers and may constitute a promising strategy for deriving a point of departure for quantitative risk assessments.

Abstract

Several New Approach Methods for hazard identification of skin sensitizers have been developed and incorporated as OECD Test Guidelines. However, the methods for potency assessment are still lacking. GARDskin (OECD TGP 4.106) was initially developed to identify skin sensitizers by monitoring transcriptional patterns of a biomarker signature in a dendritic like cell line.

The predictive capacity of GARDskin has been demonstrated previously, with 95.8% accuracy, 91.7% positive predictive value, and 100.0% negative predictive value (1 false positive, n=24) (Johansson, Gradin et al. 2019). To derive potency information, a strategy based on dose-response measurements in GARDskin, referred to as the GARDskin Dose-Response assay, has recently been proposed. The readout of the assay corresponds to the lowest concentration required to exceed the binary classification threshold in GARDskin. This concentration correlates with local lymph node assay (LLNA) EC3 and human no observed effect level (NOEL) values and linear regression models have been established to exploit these relationships for potency predictions.

In this blinded study, 12 fragrance materials (10 very weak sensitizers and 2 weak sensitizers) were evaluated in GARDskin Dose-Response. Results were evaluated by comparing predicted values to the reference potency categories. Three of the very weak sensitizers were predicted as non-sensitizers by GARDskin Dose-Response. For the remaining nine materials which were predicted as sensitizers, the predicted EC3 and NOEL values closely approximated the reference data for most materials. Based on results from this dataset, GARDskin Dose-Response appears useful for potency assessment for weak sensitizers and may constitute a promising strategy for deriving a point of departure for quantitative risk assessments.

Assessment of the skin sensitizing potential of pandemic-associated medical devices using the GARDskin Medical Device assay

Joint poster with Essity Hygiene & Health AB,
Presented at the 2021 Eurotox annual meeting

P. Mohlin, A. Forreryd, O. Larne, R.-M. Jenvert, H. Johansson | Essity Hygiene & Health AB, Product Safety, Clinical & Regulatory Affairs, Mölndal, Sweden; SenzaGen AB, 22381 Lund, Sweden

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Conclusion

  • GARDskin is well adapted to risk assess the skin sensitizing potential of medical devices in accordance with ISO 10993.
  • GARDskin Medical Device classified the tested commercially available face masks as non-sensitizers and the nitrile glove as sensitizers.
  • New in vitro technologies, like GARD, is well suited as a routine tool to increase the speed of decision making in extraordinary situations as a pandemic.

Abstract

The current SARS-CoV-2 pandemic have led to an increased use of medical devices such as face masks and nitrile gloves, within occupational groups of medical care as well as within the general population. Consequently, the incidence reports of adverse effects associated with use of such medical devices have increased manyfold, including reports of allergic skin reactions.

The cause of allergic skin reactions, referred to as Allergic Contact Dermatitis (ACD) is the immunological process known as skin sensitization, which is induced by so-called chemical sensitizers. Assessment of skin sensitizing potential of leachables from medical devices is a part of biocompatibility testing of medical devices and is typically performed by use of the Guinea Pig Maximization Test (GPMT), in accordance with the ISO 10993 series of standards. However, recent developments of in vitro assays for assessment of chemical sensitizers motivates the exploration of such methods in the context of rapid pandemic-associated testing.

The GARDskin assay [1] is a next-generation in vitro assay for hazard assessment of skin sensitizers, currently progressing towards regulatory acceptance. The method evaluates the transcriptional patterns of a genomic biomarker signature in a human dendritic-like cell line following exposure, in order to provide hazard assessments of tested substances. The method has been adapted to testing of solid materials from e.g. medical device products, by application of extraction protocols using polar- and non-polar extraction vehicles, in accordance with ISO 10993-12.

Here, we present results from testing of a commonly used face mask and a nitrile glove in the GARDskin Medical Device assay. Results indicate that the face mask does not leach any compounds with skin sensitizing potential (among four different batches tested), while the nitrile glove was classified as a skin sensitizer. These results harmonize with preexisting experience of similar models of nitril gloves, which are known to be associated with adverse skin reactions, potentially induced by skin sensitization. Furthermore, these results may have implications on the continued use of similar medical devices throughout the pandemic and beyond, as the appropriate application and removal of face masks may indeed benefit from the discontinued simultaneous use of nitrile gloves.

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.

 

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.