Joint poster with Cargill: Case study on Hydrophobic Esters

The applicability of GARD®skin for assessing skin sensitization potential of hydrophobic esters during product development


Presented at Eurotox 2024

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Conclusion

In conclusion, the inclusion of GARD®skin in the OECD test guidelines has expanded the applicability of NAMs for skin sensitization assessment.
This study demonstrates the applicability of the GARDskin assay to assess skin-sensitizing hazard of hydrophobic esters, which provides an ethical alternative to animal methods for safety assessment during product development.

Abstract

The field of skin sensitization assessment is rapidly evolving and the recent advancements in New Approach Methodologies (NAMs) has made it possible for the industry to perform in vitro skin ssensitization testing with good predictivity across a large chemical space. However, challenges remain for “difficult-to-test” chemicals, those with challenging physical/chemical properties or of Unknown or Variable composition, Complex reaction products or Biological materials (UVCBs), which are often outside the applicability domain of conventional cell-based assays. GARDskin (OECD TG 442E) is a genomic-based assay with demonstrated applicability to “difficult-to-test” substances.

The aim of this study is to assess the skin sensitization potential of two ester substances of biological origin, substance A and B, using the GARDskin assay. These substances are very hydrophobic and fall outside of the applicability domain of the conventional in vitro assays.

Both substances were successfully solubilised in cell media by utilizing a combination of heating, sonication, and selection of appropriate solvent vehicles (ethanol or acetone). No cytotoxicity was observed for either substance, thus 500 μM was chosen as the input concentration for cellular stimulations. GARDskin combines a genomic readout with machine learning to predict skin sensitizing hazard, where values above the threshold (DV=0) is predicted as skin sensitizing and below as non-sensitizing. Both substances resulted in negative mean Decisions Values and thus were classified as non-sensitizers in GARDskin (A: -1.54, B: -0.339).

In conclusion, with the inclusion of GARDskin into the OECD test guidelines, the range to where NAMs are appropriate has been increased. This study demonstrates the applicability of the GARDskin assay to assess skin sensitizing hazard of hydrophobic ethyl esters, which provides an ethical alternative to animal methods for safety assessment during product development.

Keywords: NAM, skin sensitization, difficult-to-test, UVCB, Applicability domain

Joint poster with ExxonMobil: Case study on UVCBs and Formulated Lubricant Products

Assessing the Utility of the Genomic Allergen Rapid Detection (GARDskin) Assay to Detect Dermal Sensitization Potential in UVCBs and Formulated Lubricant Products


Presented at Eurotox 2023

 

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Summary

  • GARD®skin is considered to provide useful information in an overall weight of evidence assessment for difficult to test materials (mixtures, UVCBs) with challenging physical chemical properties.
  • The accuracy for prediction of skin sensitization hazard ranged from 66% for formulated lubricants/greases to 100% for synthetic base oils compared to expected outcomes based on reference data.

Abstract

Advances in new approach methods and their combinations into defined approaches can provide clarity and confidence in concluding on skin sensitization potential. However, challenges remain in utilizing these approaches for difficult to test materials such as those with challenging physical chemical properties (low water solubility, hydrophobic substances) or complex compositions like Unknown or Variable Composition Complex reaction products or Biological Materials (UVCBs) and formulated mixtures. The previously developed available non-animal test methods for skin sensitization based on key-events of the adverse outcome pathway (AOP) have clearly defined requirements for test material properties that impact feasibility or confound reliance on negative results particularly for difficult to test materials and impedes the application of defined approaches to conclude on skin sensitization hazard. A set of difficult to test materials were evaluated in the recently validated GARDskin assay since it offered advantages such as a broader applicability domain, availability of additional validated test solvents for poorly soluble materials and provides mechanistically relevant information on key events from across the skin sensitization AOP. The aim of the study was to evaluate the accuracy of the GARDskin assay for a set of synthetic base oils (UVCBs), lubricant additives (UVCBs/poorly soluble substances) and fully formulated lubricants/greases (mixtures) as well as to provide additional information to assist in a weight of evidence determination given that several of the test materials had borderline or conflicting data from other key events within the skin sensitization AOP. All test items were adequately solubilized in one of the following solvents, Ethanol (0.1% final), DMSO (0.25% or 0.1% final), or Xylenes (0.1% final). SenzaCells were incubated in triplicate under standard conditions with the test items at a max concentration of 500uM for those with a known molecular weight or 100 ppm (w/v) for those without a known molecular weight. Following cell stimulations, RNA was isolated and endpoint measurements were performed using the GARDskin genomic profile signature. Based on the results of this study, the accuracy for prediction of skin sensitization hazard was 100% for synthetic base oils (n=4), 83% for lubricant additives (n=6), and 66% for formulated lubricants/greases (n=6) compared to expected outcomes based on available reference data. In some cases, the available reference data was borderline or considered to have low confidence due to confounding factors such as irritation, and nonmonotonic dose responses impacting the accuracy determination when compared one to one with either animal or human data. However, the GARDskin assay is considered to provide useful insight into the overall weight of evidence for difficult to test materials with conflicting datasets as it provides an additional profile of bioactivity across the skin sensitization adverse outcome pathway. 

 

Joint poster with Johnson Matthey: Case study on Metals

Expanding the applicability domain of NAMs for skin sensitization testing: A case study using GARDskin for assessment of metals


Presented at the 12th World Congress on Alternatives and Animal Use in the Life Sciences, 2023

 

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Summary

  • Limited data are currently available to support the inclusion of metals into the applicability domain of the OECD TG 442 series of assays.
  • GARD®skin (OECD TG 442 E) correctly predicted 11/12 metals in this study, including nickel which is a false negative in LLNA.
  • GARD®skin has the potential to reduce the need for animal testing for the endpoint of skin sensitization within the metal production and medical device sectors.

 

Abstract

New Approach Methods (NAMs) for detection of sensitization have been validated and adopted as OECD TGs during the last decade. These assays target different Key Events (KE) in the AOP for skin sensitization and are increasingly being applied to replace animal models within different chemical sectors. However, further characterization of the applicability domain (AD) of these assays is critical to understand limitations and to facilitate regulatory uptake in other industrial sectors. Of particular interest from a scientific and regulatory perspective is the potential to use NAMs for assessment of metals, which have been proposed to act via alternative mechanisms to organic chemicals. The current study describes a joint effort by industry and assay developers to evaluate the AD of the GARDskin assay for metal compounds.  GARDskin is the first harmonised method utilizing a combination of genomics and machine learning for a regulatory endpoint and was recently adopted into OECD TG442E.

A selection of metal salts (n=13) was evaluated and the accuracy, sensitivity, and specificity for prediction of skin sensitizing hazard of metals were estimated to 92% (12/13), 100% (7/7) and 83% (5/6), respectively. Interestingly, transcriptomic analysis revealed almost identical response patterns in dendritic cells for metals and organic compounds, indicating a high similarity in the toxicity pathways driving classifications. In conclusion, the result from this study supports the inclusion of metals into the AD of GARDskin, which is an important step to ensure scientific/regulatory confidence to reduce the need for animal testing within the metal production and medical device sector.

Joint poster with ExxonMobil: Case study on UVCBs and Formulated Lubricant Products

Assessing the Utility of the Genomic Allergen Rapid Detection (GARDskin) Assay to Detect Dermal Sensitization Potential in UVCBs and Formulated Lubricant Products


Presented at SOT 2023

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Summary

  • GARD®skin is considered to provide useful information in an overall weight of evidence assessment for difficult to test materials (mixtures, UVCBs) with challenging physical chemical properties.
  • The accuracy for prediction of skin sensitization hazard ranged from 66% for formulated lubricants/greases to 100% for synthetic base oils compared to expected outcomes based on reference data.

Abstract

Advances in new approach methods and their combinations into defined approaches can provide clarity and confidence in concluding on skin sensitization potential. However, challenges remain in utilizing these approaches for difficult to test materials such as those with challenging physical chemical properties (low water solubility, hydrophobic substances) or complex compositions like Unknown or Variable Composition Complex reaction products or Biological Materials (UVCBs) and formulated mixtures. The previously developed available non-animal test methods for skin sensitization based on key-events of the adverse outcome pathway (AOP) have clearly defined requirements for test material properties that impact feasibility or confound reliance on negative results particularly for difficult to test materials and impedes the application of defined approaches to conclude on skin sensitization hazard. A set of difficult to test materials were evaluated in the recently validated GARDskin assay since it offered advantages such as a broader applicability domain, availability of additional validated test solvents for poorly soluble materials and provides mechanistically relevant information on key events from across the skin sensitization AOP. The aim of the study was to evaluate the accuracy of the GARDskin assay for a set of synthetic base oils (UVCBs), lubricant additives (UVCBs/poorly soluble substances) and fully formulated lubricants/greases (mixtures) as well as to provide additional information to assist in a weight of evidence determination given that several of the test materials had borderline or conflicting data from other key events within the skin sensitization AOP. All test items were adequately solubilized in one of the following solvents, Ethanol (0.1% final), DMSO (0.25% or 0.1% final), or Xylenes (0.1% final). SenzaCells were incubated in triplicate under standard conditions with the test items at a max concentration of 500uM for those with a known molecular weight or 100 ppm (w/v) for those without a known molecular weight. Following cell stimulations, RNA was isolated and endpoint measurements were performed using the GARDskin genomic profile signature. Based on the results of this study, the accuracy for prediction of skin sensitization hazard was 100% for synthetic base oils (n=4), 83% for lubricant additives (n=6), and 66% for formulated lubricants/greases (n=6) compared to expected outcomes based on available reference data. In some cases, the available reference data was borderline or considered to have low confidence due to confounding factors such as irritation, and nonmonotonic dose responses impacting the accuracy determination when compared one to one with either animal or human data. However, the GARDskin assay is considered to provide useful insight into the overall weight of evidence for difficult to test materials with conflicting datasets as it provides an additional profile of bioactivity across the skin sensitization adverse outcome pathway. 

 

The GARDskin Assay: Investigation of the Applicability Domain of Indirectly Acting Haptens

Presented at the 2022 SOT

Tim Lindberg1, Andy Forreryd1, Robin Gradin1 and Henrik Johansson1
1SenzaGen, Lund, Sweden

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Conclusion

  • The GARD®skin assay can accurately predict indirectly acting haptens and has the capacity to assess both pre- and pro-haptens as skin sensitizers.
  • No increased risk of false negative classifications due to possible limitations in metabolic capacity of the cell system.

Abstract

Hypersensitivity reactions in the skin, clinically manifested as Allergic Contact Dermatitits (ACD), are caused by the ensuing immunological response to low-molecular weight compounds termed skin sensitizers. Such substances, often referred to as haptens, have the inherent property to react with skin proteins and form immune inducing complexes. However, indirectly acting haptens need to be transformed to protein-reactive intermediates either through biotic (pro-hapten) or abiotic (pre-hapten) conversion in order to elicit an immune response.

Conventionally, safety tests of skin sensitizers have been done using animal experiments, but New Approach Methodologies (NAMs) have been developed over the past decades to replace the use of animals in such testing. However, one potential problem faced with the in vitro and in chemico alternatives is the lack of metabolic and chemical activity as compared to an in vivo system, which in turn may lead to false predictions for pre- and pro-haptens.

The GARDskin assay is a next-generation NAM for hazard classification of skin sensitizers. The assay is based on a human dendritic -like cell line and combines genomics and machine learning to achieve a high predictive performance with a large applicability domain. Currently, the method is approaching regulatory acceptance as an OECD test guideline.

The study presented here aimed to explore the applicability domain of the GARDskin assay, specifically the capability to predict indirectly acting haptens. Available data obtained from GARDskin testing of indirectly acting haptens were compiled, resulting in a set of 28 substances. Further subcategorization identified 5 pro-haptens and 11 pre-haptens, while 12 substances were unable to be unambiguously assigned as either exclusively a pro- or a pre-hapten, due to the dual nature of the protein-reactive activity. Skin sensitizing hazard sensitivity of indirectly acting haptens (n=28) was 89% (25/28) while pro-haptens (n=5) and pre-haptens (n=11) were 80% and 100%, respectively. These data support GARDskin applicability in the domain of indirectly acting haptens, demonstrating that the method has the capacity to accurately assess both pre- and pro-haptens.

Ability of the GARDskin assay to Predict Skin Sensitization Response in the Guinea Pig Maximization Test

Joint poster with Risk Science Consortium,
Presented at the 2022 SOT

Rose-Marie Jenvert1, Alexandra Zambriczki Lee2, Ronald P Brown2
1SenzaGen, Lund, Sweden, 2Risk Science Consortium, LLC, Arnold, MD USA

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Conclusion

  • The GARD®skin assay is able to predict skin sensitization potential in humans with a level of accuracy that is equal to or exceeds that of GPMT and the LLNA.
  • As a result, the GARDskin assay serves as a promising alternative to assess the skin sensitization potential of medical devices.

Abstract

The preclinical safety assessment of medical devices typically involves an evaluation of the skin sensitization potential of the device. The GARDskin assay is being proposed as an in vitro alternative to the animal-based tests, Local Lymph Node Assay (LLNA) and Guinea Pig Maximization Test (GPMT), that are typically used to assess the skin sensitization potential of medical devices. The ability of the GARDskin assay to replace LLNA for prediction of skin sensitization response has been evaluated (e.g., Johansson et al., 2019) but since GARDskin has also been proposed as an alternative to the GPMT, it is important to compare the concordance of the prediction of the GARDskin assay with the in vivo response obtained in both of the animal-based tests.  Based on the results of the GARDskin assay for 122 compounds, this in vitro assay shows a high concordance with the predicted results of the LLNA (87.5%); however, the concordance for results obtained in the GPMT is much lower (71.2%). The concordance of the GARDskin assay and the GPMT is impacted by the relatively high number of false positive results (15 out of 73) compared to the false positives seen in the comparison between GARDskin and LLNA (2 out of 80).   The high number of false positives found when comparing the results from GARDskin and the GPMT results from the inaccurate characterization of the human skin sensitization potential of these compounds by the GPMT. Therefore, the low concordance between the GARDskin assay and the GPMT is due largely to inaccurate predictions of human skin sensitization potential by the GPMT and not by shortcomings of the GARDskin assay. Notably, the GARDskin assay (88.7% accuracy) outperforms the GPMT (83.0% accuracy) in the ability to predict the human sensitization response of compounds in this dataset. The results of this project show that the GARDskin assay is able to predict skin sensitization potential with a level of accuracy that is equal to or exceeds that of the currently accepted animal-based tests, suggesting that the GARDskin assay can serve as a promising alternative to the GPMT and the LLNA, and provide a more human relevant result for assessment of the skin sensitization potential of medical devices.

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.