Subcategorization of skin sensitizers into UN GHS categories using GARDskin Dose-Response
Presented at 2025 SOT
Download a copy in PDF
Conclusion
GARD®skin Dose-Response provides accurate and robust UN GHS subclassifications, with performance and reproducibility levels comparable to, or surpassing, those of current in vitro counterparts.
Abstract
Background and Purpose:
Proactive identification and characterization of sensitization hazards and risks are central aspects of risk assessment of chemicals. Current legislations and trends in predictive toxicology advocate a transition from in vivo methods to new approach methodologies (NAM:s). For the purpose of hazard identification, numerous successful innovations have led to both the development and validation of several NAM:s for assessment of chemical skin sensitizers. Similarly, subcategorization of skin sensitizers according to requirements defined by United Nations Globally Harmonized System (UN GHS) has recently been demonstrated to be achievable by so-called Defined Approaches (DA:s), purely constituting NAM data sources. However, the ability of NAM:s to perform UN GHS-associated subcategorization may be considered subjects of optimization, both in terms of performance, applicability and the number of data sources required to obtain robust and accurate results.
The GARDskin Dose-Response (DR) method, adapted from the conventional OECD TG 442E method GARDskin, provides quantitative potency assessment of skin sensitizers. The method has been demonstrated to be useful for various potency-associated problem situations, including for definition of a point of departure (PoD) for downstream risk assessment, prediction of LLNA potency categories and weight of evidence (WoE)-based categorization of test chemicals. However, the ability of the method to contribute to UN GHS subcategorization, i.e., categorizing skin sensitizers into category 1A (strong) and category 1B (weak) sensitizers, has as of to date not been described. Here, a meta-analysis of all available and peer-reviewed GARDskin DR data is presented, aiming to describe the performance and reproducibility with which the GARDskin DR method can be used for UN GHS subcategorization.
Methods:
The GARDskin method (OECD TG 442E) is an in vitro assay for assessment of chemical skin sensitizers. The method provides binary hazard identification of skin sensitizers by evaluation of transcriptional patterns of an endpoint-specific genomic biomarker signature, comprising 196 genes, referred to as the GARDskin Genomic Prediction Signature (GPS), in the SenzaCell cell line. Final classifications are provided by a machine-learning prediction algorithm in the form of decision values (DV), the sign of which is evaluated by the prediction model; Any test chemical with a positive mean DV is classified as a skin sensitizer. Conversely, any test chemical with a negative mean DV is classified as non-skin sensitizer.
The GARDskin DR is an expanded adaptation of the conventional GARDskin method, in which test chemicals are evaluated by the GARDskin prediction algorithm in an extended range of concentrations, in order to investigate the dose-response relationship between GARDskin DVs and test chemical concentration. As such, it provides a quantitative estimation of sensitizing potency, referred to as cDV0, which corresponds to the lowest required dose able to generate a positive mean DV.
As cDV0 is typically reported as experimentally derived concentrations in the units of molar concentration or in the unit of μg/ml, improved interpretation may be facilitated by conversion to a predicted potency value (PPV) in the unit of µg/cm2, as recently described. As such, a PPV can readily be used to predict a human NESIL-value, derived from e.g. LLNA EC3 or human NOEL-values. Lastly, a PPV can be evaluated with respect to the UN GHS classification threshold of 500 µg/cm2 (corresponding to an LLNA EC3 of 2%). As such, a test chemical with a PPV smaller than 500 µg/cm2 is classified as a cat. 1A sensitizer. Conversely, a test chemical with a PPV greater than 500 µg/cm2 is classified as a non-cat. 1A sensitizer, which in the presence of positive GARDskin results is indicative of a cat. 1B sensitizer.
In the present study, a meta-analysis was conducted considering all available data from published GARDskin DR resources, which comprises > 150 unique test chemicals all of which have been assayed blindly in partner-controlled studies. The dataset was cross-referenced for UN GHS categories according to both human and LLNA data, as extracted from the Annex 2 of the Supporting document to the Guideline (GL) on Defined Approaches (DAs) for Skin Sensitization (GL 497). Taken together, the union of available data and GL 497 references included 87 test chemicals, with 69 and 38 test chemicals having available LLNA and human subcategory references, respectively. In addition, 42 chemicals had multiple results from repeated testing in 2-6 separate experiments, allowing also for an estimation of reproducibility.
Results:
The ability of the GARDskin DR method to perform UN GHS-associated subcategorization of skin sensitizers was estimated by evaluating published data with respect to the classification threshold of 500 µg/cm2 (corresponding to an LLNA EC3 of 2%). Considering all available published data with corresponding reference values (human and/or LLNA) available in the Annex 2 of the Supporting document to GL 497, results indicated that GARDskin DR was able to subcategorize test chemicals as cat. 1A or non-cat. 1A with a predictive accuracy of 88.6% and 91.3%, when considering human (N = 38) and LLNA (N = 69) references, respectively. Furthermore, from repeated testing of N = 42 chemicals, results indicate that the method was 92.9% reproducible, with concordant subcategorizations for 39 of the 42 chemicals. For this subset of data, test chemicals had been repeatedly tested in 2 (N = 22), 3 (N = 15), 4 (N = 4) or 6 (N = 1) independent experiments.
When put into context, these estimates indicate that GARDskin DR performs at levels comparable to, or surpassing, those of the DA:s of GL 497, when studying the same datasets, in terms of predictive accuracy and reproducibility.
Conclusions:
Taken together, this herein presented study indicates the usefulness of the GARDskin DR method to contribute to the prediction of skin sensitizing potency and associated UN GHS subcategorizations. Of particular note, the method is estimated to be comparable to existing DA:s, as described in GL 497, while being based on a single method. Should the regulatory context approve of classification and labelling based on individual data sources to the same extent as for DA:s, it is foreseen that such a testing strategy could potentially allow for resource-effective testing, with maintained high robustness and accuracy.
Poster: Skin sensitization potency classification according to GHS/CLP
Subcategorization of skin sensitizers into UN GHS categories using GARDskin Dose-Response
Presented at 2025 SOT
Download a copy in PDF
GARD®skin Dose-Response provides accurate and robust UN GHS subclassifications, with performance and reproducibility levels comparable to, or surpassing, those of current in vitro counterparts.
Background and Purpose:
Proactive identification and characterization of sensitization hazards and risks are central aspects of risk assessment of chemicals. Current legislations and trends in predictive toxicology advocate a transition from in vivo methods to new approach methodologies (NAM:s). For the purpose of hazard identification, numerous successful innovations have led to both the development and validation of several NAM:s for assessment of chemical skin sensitizers. Similarly, subcategorization of skin sensitizers according to requirements defined by United Nations Globally Harmonized System (UN GHS) has recently been demonstrated to be achievable by so-called Defined Approaches (DA:s), purely constituting NAM data sources. However, the ability of NAM:s to perform UN GHS-associated subcategorization may be considered subjects of optimization, both in terms of performance, applicability and the number of data sources required to obtain robust and accurate results.
The GARDskin Dose-Response (DR) method, adapted from the conventional OECD TG 442E method GARDskin, provides quantitative potency assessment of skin sensitizers. The method has been demonstrated to be useful for various potency-associated problem situations, including for definition of a point of departure (PoD) for downstream risk assessment, prediction of LLNA potency categories and weight of evidence (WoE)-based categorization of test chemicals. However, the ability of the method to contribute to UN GHS subcategorization, i.e., categorizing skin sensitizers into category 1A (strong) and category 1B (weak) sensitizers, has as of to date not been described. Here, a meta-analysis of all available and peer-reviewed GARDskin DR data is presented, aiming to describe the performance and reproducibility with which the GARDskin DR method can be used for UN GHS subcategorization.
Methods:
The GARDskin method (OECD TG 442E) is an in vitro assay for assessment of chemical skin sensitizers. The method provides binary hazard identification of skin sensitizers by evaluation of transcriptional patterns of an endpoint-specific genomic biomarker signature, comprising 196 genes, referred to as the GARDskin Genomic Prediction Signature (GPS), in the SenzaCell cell line. Final classifications are provided by a machine-learning prediction algorithm in the form of decision values (DV), the sign of which is evaluated by the prediction model; Any test chemical with a positive mean DV is classified as a skin sensitizer. Conversely, any test chemical with a negative mean DV is classified as non-skin sensitizer.
The GARDskin DR is an expanded adaptation of the conventional GARDskin method, in which test chemicals are evaluated by the GARDskin prediction algorithm in an extended range of concentrations, in order to investigate the dose-response relationship between GARDskin DVs and test chemical concentration. As such, it provides a quantitative estimation of sensitizing potency, referred to as cDV0, which corresponds to the lowest required dose able to generate a positive mean DV.
As cDV0 is typically reported as experimentally derived concentrations in the units of molar concentration or in the unit of μg/ml, improved interpretation may be facilitated by conversion to a predicted potency value (PPV) in the unit of µg/cm2, as recently described. As such, a PPV can readily be used to predict a human NESIL-value, derived from e.g. LLNA EC3 or human NOEL-values. Lastly, a PPV can be evaluated with respect to the UN GHS classification threshold of 500 µg/cm2 (corresponding to an LLNA EC3 of 2%). As such, a test chemical with a PPV smaller than 500 µg/cm2 is classified as a cat. 1A sensitizer. Conversely, a test chemical with a PPV greater than 500 µg/cm2 is classified as a non-cat. 1A sensitizer, which in the presence of positive GARDskin results is indicative of a cat. 1B sensitizer.
In the present study, a meta-analysis was conducted considering all available data from published GARDskin DR resources, which comprises > 150 unique test chemicals all of which have been assayed blindly in partner-controlled studies. The dataset was cross-referenced for UN GHS categories according to both human and LLNA data, as extracted from the Annex 2 of the Supporting document to the Guideline (GL) on Defined Approaches (DAs) for Skin Sensitization (GL 497). Taken together, the union of available data and GL 497 references included 87 test chemicals, with 69 and 38 test chemicals having available LLNA and human subcategory references, respectively. In addition, 42 chemicals had multiple results from repeated testing in 2-6 separate experiments, allowing also for an estimation of reproducibility.
Results:
The ability of the GARDskin DR method to perform UN GHS-associated subcategorization of skin sensitizers was estimated by evaluating published data with respect to the classification threshold of 500 µg/cm2 (corresponding to an LLNA EC3 of 2%). Considering all available published data with corresponding reference values (human and/or LLNA) available in the Annex 2 of the Supporting document to GL 497, results indicated that GARDskin DR was able to subcategorize test chemicals as cat. 1A or non-cat. 1A with a predictive accuracy of 88.6% and 91.3%, when considering human (N = 38) and LLNA (N = 69) references, respectively. Furthermore, from repeated testing of N = 42 chemicals, results indicate that the method was 92.9% reproducible, with concordant subcategorizations for 39 of the 42 chemicals. For this subset of data, test chemicals had been repeatedly tested in 2 (N = 22), 3 (N = 15), 4 (N = 4) or 6 (N = 1) independent experiments.
When put into context, these estimates indicate that GARDskin DR performs at levels comparable to, or surpassing, those of the DA:s of GL 497, when studying the same datasets, in terms of predictive accuracy and reproducibility.
Conclusions:
Taken together, this herein presented study indicates the usefulness of the GARDskin DR method to contribute to the prediction of skin sensitizing potency and associated UN GHS subcategorizations. Of particular note, the method is estimated to be comparable to existing DA:s, as described in GL 497, while being based on a single method. Should the regulatory context approve of classification and labelling based on individual data sources to the same extent as for DA:s, it is foreseen that such a testing strategy could potentially allow for resource-effective testing, with maintained high robustness and accuracy.
SenzaGen at 2025 SOT: Advancing NAMs for Skin Sensitization Testing
The SenzaGen team had a fantastic time at SOT this year in Orlando, connecting with peers and presenting the latest GARD® data from our industry collaborations.
A special thank you to everyone who joined our scientific sessions and to our guest speakers, Ron Brown and Joe Dawson, for their insightful talks on emerging trends and New Approach Methodologies (NAMs) in skin sensitization assessment of chemicals and Medical Devices.
Refresh your mind with science and Swedish fika at our hosted session at 2025 SOT!
Join us for an interactive session that combines regulatory updates, case studies, and a live Q&A. You’ll gain exclusive insights on the latest GARD® data from SenzaGen’s research and industry collaborations.
📅 Tuesday, March 18
Exhibitor Hosted Session: Advancing NAMS for skin sensitization testing
10:45 am – 11.45 am | Room W208B
Key Topics
Speakers
Don’t miss these thought leadership presentations and networking opportunities. Our experts will be available to discuss how GARD® innovations can address your needs and advance your work in skin sensitization testing.
Advancing in vitro skin sensitization testing with GARD® and EpiSensA
Exciting news from the SenzaGen Group! Our Group company VitroScreen has recently validated and implemented the in vitro skin sensitization assay EpiSensA (OECD TG 442D).
With this new achievement, we are proud to be the first service provider in Europe offering both GARDskin (OECD TG 442E) and EpiSensA (OECD TG 442D) for in vitro skin sensitization testing!
By integrating GARD®skin’s genomic biomarker approach with EpiSensA’s reconstructed human epidermis (RhE) model, we provide a complementary platform to address KE2 and KE3 in the AOP and strengthen your skin sensitization testing strategy. This synergy enhances predictivity and supports regulatory compliance, reinforcing our commitment to advancing NAMs for in vitro skin sensitization testing.
Want to learn how this can support your safety assessments?
Get in touch!
Bring your questions and testing challenges to our booth and scientific sessions at 2025 SOT!
The SenzaGen team is ready to connect at this year’s SOT in Orlando, USA! Join us for an interactive program featuring a hosted session and multiple poster presentations showcasing advancements in skin sensitization testing.
Our experts will be on-site to discuss how GARD® innovations can support your testing needs and accelerate your research:
A Quick Guide to Skin Sensitization Testing
Joint publication with RIFM and IFF: Determining a Point of Departure for Skin Sensitization Potency and Quantitative Risk Assessment of Fragrance Ingredients Using the GARD®skin Dose-Response Assay
Advancing NAMs for Fragrance Safety: Collaborative Research with RIFM and IFF.
As innovators in in vitro skin sensitization testing, addressing key data gaps, we are pleased to share the publication of a new peer-reviewed article in ALTEX, which underscores the performance of GARD®skin Dose-Response in predicting the skin sensitization potency of fragrance ingredients. This study, conducted in collaboration with the expert teams at the Research Institute for Fragrance Materials, Inc. (RIFM) and IFF, contributes valuable insights to the growing body of research supporting more reliable, non-animal testing methods.
The article presents a comprehensive dataset of 100 fragrance ingredients, covering a broad chemical domain with diverse structural reactivity domains and potency levels. The results confirm GARD®skin Dose-Response‘s robust predictivity for skin sensitization potency across this wide chemical spectrum, strengthening its application for deriving No Expected Sensitization Induction Level (NESIL) values in Quantitative Risk Assessment (QRA) within frameworks like Next Generation Risk Assessment (NGRA). These advancements are pivotal in further reducing reliance on animal testing while enhancing risk assessment capabilities.
We would like to extend our gratitude to the exceptional teams who contributed to this collaboration: Isabelle Lee, Andy Forreryd, Mihwa Na, Isabella Schember, Maura Lavelle, Robin Gradin, Ulrika Mattson, Henrik Johansson, Shashi Donthamsetty, Gregory Ladics, and Anne Marie Api.
Keywords
skin sensitization, fragrance materials, point of departure, OECD TG 442E, GARDskin, new approach methodology
Abstract
Potency and quantitative risk assessment are essential for determining safe concentrations for the formulation of potential skin sensitizers into consumer products. Several new approach methodologies (NAMs) for skin sensitization hazard assessment have been developed, validated, and adopted in OECD test guidelines. However, work is ongoing to develop NAMs for predicting skin sensitization potency on a quantitative scale for use as a point of departure (POD) in next-generation risk assessment (NGRA). GARDskin Dose-Response (DR) is an adaptation of the validated GARDskin assay (OECD TG 442E), and the readout of the assay is a quantitative potency prediction similar to the No Expected Sensitization Induction Level (NESIL) value (µg/cm2). The goal of this study was to evaluate the performance of the GARDskin DR assay for potency prediction of fragrance ingredients. One hundred (100) fragrance ingredients from a reference database covering varied structural reactivity domains and potency were tested in GARDskin DR. Materials tested had varied protein-binding reactivity alerts, including Schiff base, Michael addition, SN2, and acylation. Potency categories were predicted with a total accuracy of 37% and an approximate accuracy (exact match or off by 1 category) of 81%. Combining predicted weak and very weak categories increased total accuracy to 53% and approximate accuracy to 98%. The mean prediction error for the NESIL and local lymph node assay (LLNA) EC3 was 3.15- and 3.36-fold, respectively. Based on the results of this study, GARDskin DR is a promising predictor of skin sensitization potency with an applicability domain covering a wide range of fragrance ingredient reaction mechanisms, increasing the confidence in using the assay to conduct NGRA, ultimately reducing the need for animal testing.
Plain language summary
This study focused on testing a new in vitro method, GARDskin Dose-Response (DR), to predict the quantitative potency of fragrance ingredients in causing skin sensitization. This potency is important for setting safe levels of chemicals in consumer products. The GARDskin DR assay, based on an existing skin sensitization test (OECD TG 442E), provides a quantitative measure of potency similar to the No Expected Sensitization Induction Level (NESIL). One hundred (100) fragrance ingredients with different chemical structures and reactivity patterns were tested. The assay accurately distinguished between sensitizers and non-sensitizers for 81% of the materials, and also correctly predicted their approximate potency categories. The results show that GARDskin DR is a promising tool for predicting quantitative potency for skin sensitization risk, helping to reduce animal testing and support safer product development.
SenzaGen’s Newsletter Dec 2024
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SenzaGen’s Newsletter Dec 2024
As 2024 ends:
We’re proud to share how our innovative GARD® method is addressing key challenges in skin sensitization testing. In Q4, we’ve focused on filling data gaps in areas like Medical Devices, Safe Dose Levels, and Complex Mixtures—driving the shift to reliable, human-relevant solutions.
Building confidence in NAMs for Biological safety testing of Medical Devices
Progress is underway to integrate New Approach Methodologies (NAMs) into skin sensitization testing standards for Medical Devices within ISO. At the recent ISO TC 194/WG 8 meeting in Paris, SenzaGen’s Andy Forreryd presented data from the GARD®skin Medical Device pre-validation study—an important step toward its inclusion in the normative section of ISO 10993-10.
Since 2021, GARD®skin has been recognized in Annex C of the standard, and its further integration underscores the advancement of NAMs in improving biological safety assessments while reducing reliance on animal testing.
Explore how you can use in vitro methods for Biocompatibility testing
Discussing safe dose levels at ASCCT
At the recent ASCCT conference, SenzaGen presented exciting new data on assessing skin sensitizing potency, including a poster that highlighted the GARD®skin Dose-Response assay’s performance on the Reference Chemical Potency List (RCPL).
Further confirmation of the assay’s effectiveness comes from two recent publications, developed in collaboration with industry leaders L’Oréal, RIFM, and IFF. These studies demonstrate the assay’s ability to:
Access our latest publications for more insights
Conclusive testing results for Natural Extracts
In our latest webinar, we explored advancements in skin sensitization testing, focusing on challenging samples—specifically, substances that fall outside the applicability domains of conventional in vitro assays outlined in OECD Test Guidelines.
GARD®skin (OECD TG 442E) stands out as a reliable method for testing complex mixtures like natural extracts, backed by scientific data and successful industry collaborations.
Contact us for a case study on Natural Extracts
Discovery our advisory service
Toxicology, Pharmacology and Regulatory Compliance
Area of Expertise
Redeye: CEO interview Q3 2024
Redeye interviewed the CEO of SenzaGen, Peter Nählstedt, where they discussed the company’s recently released Q3 2024 report.
View CEO interview
Penser Access by Carnegie: CEO interview (in Swedish)
Penser Access by Carnegie har intervjuat SenzaGens VD, Peter Nählstedt, i samband med bolagets Q3-rapport.
Intervjun behandlar utvecklingen under kvartalet, bolagets syn på det aktuella marknadsläget samt framtidsutsikter och strategiska prioriteringar framöver.
VD-intervju – Stark tillväxt i Q3
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