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.

In vitro method for quantitative potency assessment of skin sensitizers during development of novel materials for intended use in medical devices

Joint poster with Sonova,
Presented at the 2022 SOT

Andy Forreryd1, Ulrika T Mattson1, David Waeckerlin2, Karla Lienau2, Robin Gradin1, Rose-Marie Jenvert1
1SenzaGen, Lund, Sweden, 2Sonova AG, Staefa, Switzerland

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Conclusion

The GARD®skin Dose-Response assay can be used as a tool for:

  • quantitative potency information of chemicals that might leach out of materials or medical devices.
  • internal decision-making during development of new materials for use in medical devices.

Abstract

New innovative materials for use in medical devices based on acrylates can bring several advantages such as super-absorbency, transparency, flexibility, toughness and hardness.

The manufacturing of acrylates typically involves using a monomer of either acrylate or methacrylate that is polymerized into the final product. The polymerization or hardening of material makes the monomers inert, however several methodologies can be used for polymerization, and they differ in the degree to which they result in a fully polymerized final product. Some products therefore contain more residual monomers than others and human exposure to these well-known skin sensitizers may increase the risk of skin sensitization and allergic contact dermatitis. To assess the risks resulting from exposure to these chemicals, potentially found in medical device material, it is necessary to accurately identify and characterize their skin sensitizing potential.

The GARDskin (OECD TGP 4.106) assay was initially developed for hazard identification of skin sensitizers. To derive potency information, a modification of the standard GARDskin protocol based on dose-response measurements has been proposed. The readout of the assay is a cDV0 value, which corresponds to the lowest concentration required to exceed a binary classification threshold in GARDskin. This concentration correlates significantly with LLNA EC3 and human NOEL values and linear regression models have been established to exploit these relationships for potency predictions. In this study, we explore the potential to use this novel assay for quantitative potency assessment of two acrylate monomers.

The GARDskin Dose-response assay classified both acrylate monomers as skin sensitizers with predicted LLNA EC3 values and human NOEL values of 0.848% and 22.4%, and 230 µg/cm2and 12200 µg/cm2, resulting in final classifications as a strong to moderate skin sensitizer (HP 2) and a moderate to weak sensitizer (HP 5), respectively. The results agreed with information in the ECHA registration dossiers and gathered human data evidence for the respective monomers, illustrating that GARDskin Dose-Response has the potential to replace the in vivo LLNA method for quantitative potency assessment of potential skin sensitizers during development of novel materials for use in medical devices.

Exploration of the GARDskin applicability domain: Indirectly acting haptens, hydrophobic substances and UVCBs

Joint publication with the Lubrizol Corporation

ALTEX – Alternatives to animal experimentation, published April 21, 2022, accepted manuscript, https://doi.org/10.14573/altex.2201281

Forreryd, A., Gradin, R., Humfrey, C., Sweet, L. and Johansson, H.

Abstract

Hazard assessments of skin sensitizers are increasingly being performed using new approach methodologies (NAMs), with several in chemico, in vitro and most recently also defined approaches (DAs) being accepted for regulatory use. However, keeping track of potential limitations of each method in order to define applicability domains remains a crucial component to ensure adequate predictivity as well as facilitating the appropriate selection of method(s) for each hazard assessment task. The objective of this report is to share test results generated with the GARD™skin assay on chemicals that have traditionally been considered as difficult to test in some of the conventional in vitro and in chemico OECD Test Guidelines for skin sensitization. Such compounds may include, for example, indirectly acting haptens, hydrophobic substances, and substances of unknown variable composition or biological substances (UVCBs). Based on the results of this study, the sensitivity for prediction of skin sensitizing hazard of indirectly acting haptenswas92.4%and 87.5%, when compared with LLNA(n=25)and human data(n=8), respectively. Similarly, the sensitivity for prediction of skin sensitizing hazard of hydrophobic substances was 85.1% and 100%, when compared with LLNA(n=24)and human data(n=9), respectively. Lastly, a case study involving the assessment of a set of hydrophobic UVCBs(n=7) resulted in a sensitivity of 100, as compared to available reference data. Thus, it was concluded that these data provide support for the inclusion of such chemistries in the GARD™skin applicability domain, without an increased risk of false negative classifications.

Key words: GARD, GARDskin, skin sensitization, applicability domain, difficult to test substances, Indirectly acting haptens, hydrophobic substances, UVCBs

 

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