Tag Archive for: Skin Sensitization

GARDskin Dose-Response assay for PoD determination of fragrance materials and its application in conducting Quantitative Risk Assessment (QRA)

Presented at SOT 2023

GARDskin Dose-Response assay for PoD determination of fragrance materials and its application in conducting Quantitative Risk Assessment (QRA)

Shashi Donthamsetty1 , Andy Forreryd2, Paul Sterchele1, Xiao Huang1, Robin Gradin2, Henrik Johansson2, Ulrika Mattsson2, Isabelle Lee3, Anne Marie Api3, Gregory Ladics1

1 International Flavors & Fragrances USA ,2 SenzaGen AB Sweden, 3Research Institute for Fragrance Materials USA

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Conclusion

  • The continous readout from the assay is reproductible and the assay predicts LLNA EC3 and human NESIL values with high correlation to reference benchmark data (geometric mean fold-misprediction factors of 3.8 and 2.5 respectively).
  • The assay provides a nice tool for the fragrance industry to predict the NESIL value to be used for conducting the quantitative risk assesment for generating the IFRA standard.

Abstract

The global fragrance industry applies Quantitative risk assessment (QRA) to develop risk management practices (IFRA Standards) for ingredients that are identified as potential dermal sensitizers. An important step in QRA is determination of a ”No Expected Sensitization Induction Level” (NESIL), which has historically been determined using human data with the support of animal data (e.g., murine local lymph node assay (LLNA). The EC3 value determined in the LLNA is used as the guidance for selection of the dose level in HRIPTs (Human Repteated Insult Patch Test) to confirm a NESIL value. The fragrance industry has adopted new approach methodologies (NAM) to address skin sensitization. Although several NAMs for identifying skin sensitizers have been accepted as Test Guidelines by OECD, these methods have thus far been validated only for hazard identification. Since a NESIL value is a key requirement to evaluate sensitizing potency for conducting QRA evaluations, development of a NAM-based strategy capable of providing potency data in the form of NESIL remains a high priority for the fragrance industry. The in vitro GARDskin assay was recently adopted by the OECD (TG 442E) for the hazard identification of skin sensitizers. Continuous potency predictions are derived using a modified protocol that incorporates dose-response measurements. Linear regression models have further been developed to predict LLNA EC3 and human NESIL values. The aim of the study was to evaluate the precision and reproducibility of the continuous potency predictions from the GARDskin Dose-Response assay. A total of 17 test materials were evaluated, 11 of which were evaluated in three blinded studies separated in time. Preliminary results indicated that the GARDskin Dose-Response model predicted LLNA EC3 values and human NESIL values with geometric mean fold-misprediction factors of 3.8 and 2.5, respectively. For comparative reasons, the LLNA EC3 predicted the human NESIL values with a fold-misprediction factor of 3.7 in the same dataset. Results from the repeated assessment of the test materials were reproducible, with an estimated geometric mean range of fold-changes between replicates of 2.9. Using isocyclocitral (CAS 1335-66-6) as an example, a QRA was conducted to determine its safe use levels in different consumer product types. The results demonstrate that the LLNA EC3 values and the human NESIL values predicted from the GARDskin Dose-Response assay are reproducible between experiments and show good concordance with the published NESIL and EC3 values. Together with the reported performance data, this represents a major step towards the establishment of the assay as a relevant source of information to derive NESIL values for conducting QRA evaluations for fragrance materials to ensure product safety while avoiding the generation of new animal data.

 

 

 

 

In vitro assays for assessment of the skin sensitization hazard and potency of isobornyl acrylate

Presented at SOT 2023

In vitro assays for assessment of the skin sensitization hazard and potency of isobornyl acrylate

​Rose-Marie Jenvert1, Olivia Larne1, Ulrika Mattson1, Robin Gradin1, Kelly P Coleman2 and Andy Forreryd1  |  SenzaGen, Lund, Sweden1, Medtronic, Minneapolis, USA2

 

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Conclusion

The GARD®skin Medical Device assay can be used​

  • with the extraction vehicles saline and oil as described ​
    in ISO 10993. ​
  • to detect low concentrations of skin sensitizers in a mixture and have the potential to replace in vivo tests ​
    for risk assessment of skin sensitizers in medical devices. ​
  • as risk assessment tool in combination with the ​
    GARD®skin Dose-Reponse assay during development of medical devices containing acrylates. ​

​

Abstract

In Vitroassays for Assessment of the Skin SensitizationHazard and Potency of Isobornyl AcrylateRose-Marie Jenvert1, Olivia Larne1, Ulrika Mattsson1, Robin Gradin1, Kelly P Coleman2and Andy Forreryd1SenzaGen, Lund, Sweden1, Medtronic, Minneapolis, USA2Acrylateshave a wide range of applications for medical devicesas they can bring several advantages such as transparency, super-absorbency and hardness in combination with flexibility. The manufacturing of acrylic materials typically involves using at least one monomer of either acrylate or methacrylate which react to form a polymer. Severalmethodologies can be used for polymerizationand the degree of polymerization of the final material can vary, hence some products can contain more residual monomers than others,and human exposure to these well-known skin sensitizers may increase the risk of developing the adverse immunologicalresponse, allergic contact dermatitis.In 2020,theacrylate monomer,isobornyl acrylate(IBOA; CAS 5888-33-5),was named allergen of the year by American Contact Dermatitis Societydue to the increased number of patients that weresensitized to IBOA found in glucose sensors andglucose pumps. IBOA is alsopresentin othermedical devices as plastic materials, coatings, sealants, glues, adhesivesand inks.As a result, it is important to find alternatives to the animal methods used today to assess skin sensitization to properly identify the skin sensitizing potentialof IBOAin medical devicesand avoid the risk of sensitizing more individualsto this chemical. The GARDskin (OECD TG 442E) assay,initially developed for hazard identification of a wide range of skin sensitizers,hasbeenadapted for use withpolar and non-polar solvents as described in ISO 10993-12:2021and can be used toassesstheskin sensitization of medical devices. Further development of the GARDskin protocol hasalso enabledthe prediction ofskin sensitization potency byusing a dose-response measurements. ThecDV0valuederived from the assaycorresponds to the lowest concentration required to exceed abinary classification threshold in GARDskin, and this concentration correlatessignificantly with LLNA EC3 and human NOEL values. Linear regression models have been established to exploit these relationships for potency predictions.In this study,we exploredthe ability of GARDskin Medical Device assaytodetect the skin sensitizing potentialof IBOA in a mixture of chemicals extracted from a siliconematerial (Nunsil MED-2000) in both saline and oil extract.Additionally, the skin sensitizing potency of IBOA was also investigated using the novel GARDskin Dose-Response assay.The acrylate monomer,IBOA,was correctly classified as a skin sensitizerin extracts of thesilicon materialspiked with IBOAusing the GARDskin Medical Device assay. The correct classification of IBOA as a skin sensitizer providesevidencethat the GARDskin Medical Device assay is sensitive enough to detect low concentrations of device-related skin sensitizers in a mixture of extracted chemicals and hence hasthe potential to replace in vivotestsfor risk assessment of medical devices.Furthermore, IBOA was classified as a strong to moderate skin sensitizer (HP 2) with a predicted LLNA EC3 value of 0.848% and human NOEL value of 230μg/cm2using the GARDskin Dose-Response assay. These predictions agreewith existing human data and information from the ECHA registration dossier, illustrating the potential of theGARDskin Dose-Response assay to replace in vivotests forquantitative potency assessment of potential skin sensitizers.

GARD®skin Dose-Response for Photosensitization: ​Assessment of Reference Photoirritants and Photoallergens

Presented at SOT 2023

GARD®skin Dose-Response for Photosensitization: ​Assessment of Reference Photoirritants and Photoallergens

Tim Lindberg1, Gretchen Ritacco2, Anders Jerre1, Robin Gradin1, Andy Forreryd1, Henrik Johansson1, Anne Marie Api2  | 1SenzaGen, Lund, Sweden, 2Research Institute for Fragrance Materials. Woodcliff lake, NJ, USA

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Conclusion

  • Increase in cytotoxicity after UV exposure strongly linked with predominantly photo irritating properties.​
  • Decrease in GARDskin Dose-Response cDV0-value after UV exposure indicative of photoallergenic properties​.

Abstract

Dermal exposure to certain chemical compounds, so-called sensitizers, can give rise to adverse outcomes induced by an immunological response towards the specific compound. One such class of compounds, photosensitizers, needs to be activated by UV rays to elicit an immune response. Although rare in occurrence, it is a critical human health endpoint in need of investigation to limit potential exposures. Other phototoxic skin reactions include photoirritation, which is manifested as a one-time occurrence at the site of exposure that goes away over time. While testing schemes for photoirritation are clear, testing for photosensitization remains a challenge and no established in vitro model to evaluate this endpoint currently exists. For risk management purposes, distinguishing between phototoxic properties is important, as concentration limits can be set for photoirritants whereas fragrance photoallergens have historically been banned. 

The GARDskin assay is a next-generation in vitro method for hazard classification of conventional skin sensitizers, included in OECD TG 442E. 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. The GARDskin Dose-Response assay is based on the validated GARDskin protocols but instead of giving a binary classification it provides quantitative information about the lowest concentration needed to induce a positive classification in the assay, termed the cDV0 concentration. To investigate phototoxicity, an extra UV-exposure step was added to the original protocol, with photosensitization determined by a drop in cDV0 concentration after UV-exposure, i.e., the cDV0 concentration of the specific compound is lower after UV-exposure than in the non-exposed counterpart.  

The study presented here aimed at investigating the applicability of the GARDskin Photo Dose-Response assay to correctly assess photoallergens and distinguish them from photoirritant effects. Previous studies have indicated that a shift in cytotoxic profile after UV-exposure may indicate a predominantly photoirritant activity rather than photosensitizing and this was also investigated in the present study. Six reference photoirritants and six reference photoallergens were investigated using the GARD®skin Dose-Response assay in combination with a UV irradiation protocol. Cytotoxic profiles and cDV0-values were established for each compound in the presence and absence of UV exposure. 5 out of 6 photoirritants were correctly predicted based on their cytotoxic profile while 3 out of 6 photoallergens where correctly predicted based on the decrease in cDV0-value after UV-exposure. In conclusion, functionality of combining GARDskin Dose-Response protocols with UV irradiation to investigate phototoxicity was shown. Further, photoirritant effects were strongly correlated to a shift in cytotoxic profile after UV-exposure and a decrease in cDV0 values after UV-exposure may indicate on photosensitizing effects. However, further work may be warranted to establish a final prediction model for photosensitization.  

Keywords: Predictive Toxicology, GARDskin, Phototoxicity, Quantitative Risk Assesment

 

 

 

 

The GARDskin assay: Investigation of the applicability domain for metals

Joint publication with Johnson Matthey

ALTEX – Alternatives to animal experimentation, published Nov 03, 2022, accepted manuscript

DOI: https://doi.org/10.14573/altex.2203021

Forreryd, A., Gradin, R., Larne, O., Rajapakse, N., Deag, E. and Johansson, H.


Abstract

New approach methods (NAMs) for hazard identification of skin sensitizing chemicals have been adopted as test guidelines by the OECD during the last decade as alternatives to animal models. These models align to individual key events (KE) in the adverse outcome pathway (AOP) for skin sensitization for which the molecular initiating event (MIE) is covalent binding to proteins. As it currently stands, the AOP does not include mechanistic events of sensitization by metals, and limited information is available on whether NAMs accurately the predict sensitization potential of such molecules, which have been proposed to act via alternative mechanisms to organic chemicals.

Methods for assessing the sensitization potential of metals would comprise valuable tools to support risk management within e.g., occupational settings during production of new metal salts or within the medical device industry to evaluate leachables from metal alloys.

This paper describes a systematic evaluation of the applicability domain of the GARD™skin assay for assessment of metals. Hazard classifications were supplemented with an extended analysis of gene expression profiles induced by metal sensitizers to compare the induction of toxicity pathways between metals and organic sensitizers. Based on the results of this study, the accuracy, sensitivity, and specificity of GARD™skin for prediction of skin sensitizing hazard were 92% (12/13), 100% (7/7) and 83% (5/6), respectively.

Thus, the performance of GARD™skin for assessment of metals was found to be similar to what is observed on conventional organic substances, providing support for inclusion of metals within the applicability domain of the test method.

Keywords

skin sensitization, metals, regulatory testing, medical devices

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Next Generation Risk Assessment (NGRA) using NAMs for skin sensitization: Reproducibility and precision of the GARDskin Dose-Response assay for PoD determination of fragrance chemicals.

Presented at ASCCT 2022

Next Generation Risk Assessment (NGRA) using NAMs for skin sensitization: Reproducibility and precision of the GARDskin Dose-Response assay for PoD determination of fragrance chemicals.

Andy Forreryd1, Shashi Donthamsetty2, Paul Sterchele2, Xiao Huang2, Gregory Ladics2, Mihwa Na3, Isabelle Lee3, Anne Marie Api3, Robin Gradin1, Henrik Johansson1
1SenzaGen, Lund, Sweden , 2International Flavors & Fragrances, Hazlet, NJ, USA, 3Research Institute for Fragrance Materials. Woodcliff lake, NJ, USA

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Conclusion

  • GARD®skin Dose-Response can be used for continous predictions of skin sensitizing potency.
  • The continous readout from the assay is reproducible and the assay predicts LLNA EC3 and human NESIL values with high correlation to reference benchmark data.
  • The assay provides a nice tool for the fragrance industry to predict the NESIL value which can be used for conducting the quantitative risk assessment for generating the IFRA standard.

Abstract

New Approach Methods (NAMs) for assessment of skin sensitizers have been adopted as Test Guidelines (TGs) by OECD. When combined into Integrated Approaches to Testing and Assessment (IATA) or defined approaches (DA), they provide data supporting hazard classifications and GHS potency subcategorization. However, more granular potency information, preferably on a continuous scale, is needed to derive a point-of-departure (PoD) for Next Generation Risk Assessment (NGRA).

GARDskin was recently adopted into OECD TG 442E to support discrimination of skin sensitizers and non-sensitizers. Continous potency predictions are derived using a modified protocol that incorporates dose-response measurements. Linear regression models have further been developed to predict LLNA EC3 and human NOEL values. The aim of the following study, which represents a cross-sector collaboration was to evaluate precision and reproducibility of the potency predictions from GARDskin Dose-Response in blinded studies.

Preliminary results from estimate of precision (n=36 materials) indicated that GARDskin Dose-Response predicted LLNA EC3/ human NOEL values with median fold-misprediction factors < 3.0 and < 2.0, respectively. Interestingly, LLNA predicted human NOEL with a fold-change > 2 in the same dataset. For reproducibility assessment, test materials (n=11) were evaluated in separate experiments (n=3), which generated highly reproducible results, with an average median range of fold-changes between replicates of 2.5.

Results from this study demonstrate that continous potency predictions from GARDskin Dose-Response are reproducible. Together with performance data, this represents a major step towards establishment of the assay as a relevant source of information to derive a PoD for NGRA, avoiding generation of new animal data.

 

 

 

The use of the GARD®skin Dose-Response assay to assess skin sensitizing potency in developing novel fragrance ingredients

Presented at ASCCT 2022

The use of the GARD®skin Dose-Response assay to assess skin sensitizing potency in developing novel fragrance ingredients

Tim Lindberg1, Christopher Choi2, Ulrika Mattson1 and Satoshi Sasaki3
1SenzaGen, Lund, Sweden , 2Takasago International Corp, Rockleigh NJ, USA ,3Takasago International Corp, Hiratsuka city, Kanagawa, Japan

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Conclusion

The present study aimed at investigating the skin sensitizing potency of two novel fragrances, Fragrance 1 and 2, using three NAMs, the kDPRA, KeratinoSens and GARD®skin Dose-Response assays.

To move away from traditional safety testing, which includes animal studies, there is a paradigm shift towards the use of multiple NAMs in a weight-of-evidence approach when risk assessment of novel fragrance ingredients are conducted. However, the lack of established potency assays puts the alternative methods at a disadvantage as compared to the in vivo counterparts.

  • Fragrance 1 showed similar results across the three NAMs, with the GARD®skin Dose-Response assay predicting the cDV0–value to 18.4 µM, which in turn was used to predict a NESIL-value of 659 µg/cm2.
  • For Fragrance 2, equivocal results were seen, where the kDPRA assay predicting the ingredient not to be a category 1A skin sensitizer while KeratinoSens predicted it as a non-skin sensitizer. GARD®skin Dose-Response predicted the cDV0–value to 296 µM, which was used to predict a NESIL-value of 16600 µg/cm2. Combining the results from all three NAM assays, a confirmatory HRIPT testing concentration was determined for both ingredients, 562.5 µg/cm2 and 15000 µg/cm2 for Fragrance 1 and Fragrance 2, respectively.

In conclusion the data presented here show how the use of the GARD®skin Dose-Response assay in combination with other NAMs can be used as a replacement of animal studies for quantitative risk assessment of novel fragrance materials.

Abstract

Skin sensitization is one of the required endpoints for the development and registration of novel fragrance ingredients. Traditionally, testing has been performed using a combination of in vitro and in vivo assays, but recent developments has shifted the paradigm towards the use of New Approach Methodologies (NAMs), without the need for in vivo methods. However, none of the proposed NAMs are currently validated for continuous potency predictions, which is required for quantitative risk assessments of novel fragrance ingredients.

The GARD®skin assay (OECD TG 442E) is a genomics-based assay for hazard identification of sensitizers. To meet the need for quantitative potency information, GARD®skin Dose-Response has been developed based on the validated protocols of GARD®skin and generates a dose-response curve to identify the lowest concentration of a test compound required to elicit a positive classification (cDV0 value). These values correlate significantly to LLNA EC3 and human NESIL values.

The aim of this study was to investigate the sensitizing potency of two novel fragrance ingredients and to identify predicted non-sensitizing levels. Testing was performed in GARD®skin Dose-Response, with predicted EC3 and NESIL values of 1.93% and 27.8%, and 659µg/cm2 and 16600µg/cm2, for fragrance ingredients 1 and 2, respectively. These results in combination with data from kDPRA, KeratinoSens and in silico read- across, established the concentrations for confirmatory HRIPT testing (562.5µg/cm2 and 15000µg/cm2).

In conclusion, this study demonstrates how GARD®skin Dose-Response combined with other NAMs can be used for risk assessments and to establish a concentration for confirmatory HRIPT testing of novel fragrance ingredients.

 

 

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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