Subcategorization of skin sensitizers into UN GHS categories using GARDskin Dose-Response
Presented at 2025 SOT
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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.
Identify workplace hazards by testing skin and respiratory sensitizers
SenzaGen’s Newsletter Q3 2025
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Safe dose levels for sensitizers, NESIL values for HRIPT, and PoD-based QRA
Webinar: In vitro skin sensitization potency assessment
OECD TG 497 regulatory update at Eurotox
Connect with our specialists to discuss what this means for your regulatory testing strategy
GARD® enters final ISO validation
– 100% reproducibility and 97% concordance
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Upcoming events
🌎 ASCCT 2025 | Gaithersburg, USA | Oct 21-23
🌎 3rd Annual Medical Device Biocompatibility Conference | Berlin | Nov 6-7
🎧 Webinar: How GARDskin fits into OECD TG 497 | Nov 20
Joint poster with Urgo: Supporting hypoallergenicity claims for Medical Wound Dressings
Supporting industry decision-making and hypoallergenicity claims using an experimental setup combining GARD®skin Medical Device and Dose-Response protocols.
Presented at 2025 Eurotox
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This case study demonstrates how NAMs like GARD® can be effectively integrated into product development for assessing skin sensitization potential in medical device extracts. It enables evidence-based decision-making and supports hypoallergenic claims, without the use of animal testing.
Biocompatibility testing is a crucial part of medical device safety assessments both for regulatory acceptance of finished products but also within the developmental pipelines of novel devices. Along with cytotoxicity and irritation, skin sensitization is a key toxicological endpoint that must be assessed for all devices. While testing for the two former endpoints is partly conducted in vitro, traditional animal experimentations for skin sensitization testing are still routinely conducted. Thus, validation and implementation of New Approach Methods (NAMs) remains a prioritized goal of the medical device industry.
The GARDskin method (OECD 442E) is a NAM for hazard assessment of skin sensitizers based on test chemical exposure of dendritic cell-like cells followed by a genomic readout. Several method adaptations have been proposed in order to address specific needs of various industry segments. Such adaptations include the GARDskin Dose-Response (DR) method, which facilitates quantitative assessment of sensitizing potency, and the GARDskin Medical Device (MD) method, which facilitates testing of solid materials in accordance with ISO 10993 standards.
Here we report an experimental setup combining the protocols of above-mentioned GARDskin adaptations used to evaluate the relative hypoallergenicity of two sensitive skin dressings: a candidate product and a marketed comparative product. The extraction from the solid test items was performed according to ISO 10993-12, but with an increased test item to extraction vehicle ratio of 6x. The oversaturated extracts were subsequently subjected to serial dilution facilitating dose-response testing in order to evaluate, assess and compare the test items’ relative skin sensitizing potentials.
The results concluded that both test items were classified as non-sensitizers. Due to the oversaturation extraction procedure, otherwise adhering to the ISO 10993-12 standards, the results are noteworthy. Indeed, due to the manyfold increase in material to vehicle ratio, labelling the tested skin dressings as hypoallergenic, or otherwise suitable for sensitive use, may be supported by the data. Furthermore, evaluation of the relative difference between the two test items concluded that the comparative test item induced elevated response values at the highest tested concentration, whereas the candidate test item did not. As such, it could be argued that the candidate product can be claimed as hypoallergenic similarly to the comparative product.
In summary, this experimental setup combining GARDskin MD and GARDskin DR protocols can successfully be used to support claims of hypoallergenicity of medical devices, as well as comparing candidate products with respect to their relative skin sensitizing potential. These results illustrate how such strategies can be routinely integrated in the development of novel devices giving data for critical decision-making without resorting to animal experimentation for regulatory approval.
Joint poster with BIC: Case study on skin-binding dyes
In vitro skin sensitization potency assessment using GARD®skin Dose-Response: A case study on natural extracts-based skin-binding dyes and dye precursors
Presented at 2025 Eurotox
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Naturally occurring skin dyes and dye precursors are widely used in temporary tattoo products, evolving from unrefined fruit extracts to more refined formulations in various ink applications. Evaluating their skin sensitization potential and establishing safe use levels for potential sensitizers are critical for product safety, which has traditionally relied on animal testing and/or human patch testing. While New Approach Methodologies (NAMs) have been developed to replace in vivo assays, a need remains for methods that can effectively and quantitatively characterize skin sensitizing potency.
GARD®skin (OECD TG 442E) is an in vitro assay that identifies chemical skin sensitizers based on the transcriptional profiling of a 196-gene biomarker signature in the dendritic-like SenzaCell® cell line. Predictions are made using a machine-learning algorithm, which classify test chemicals as sensitizers or non-sensitizers based on the assay’s readout, Decision Values (DVs). GARD®skin Dose-Response (OECD TGP 4.106) extends this approach by evaluating test chemicals across a concentration range to establish a dose-response relationship between DVs and test chemical concentration. Sensitizing potency is quantified using cDV0, the lowest dose required to elicit a positive response in GARD®skin. Depending on the need, the readout can be used to predict LLNA EC3 values, No Observable Effect Levels (NOELs)/No Expected Sensitization Induction Levels (NESILs), Human Potency (HP) Categories, and UN GHS/CLP classification (1A or 1B), all with high statistical significance.
This case study demonstrates the application of GARD®skin Dose-Response to quantitatively assess the skin sensitization potency of naturally occurring dye precursor that binds covalently to skin molecules, highlighting its use in temporary tattoo inks. Seven analogue molecules with different functional groups were investigated. While six exhibited varying degrees of sensitization potency with mean cDV0 values ranging from 3.82 µM to 34.3 µM, one functional group did not induce a positive response at any of the assayed concentrations and was subsequently classified as a non-sensitizer. Predicted LLNA EC3 values ranged from 0.60% to 4.72%, corresponding to Human Potency Categories HP2 to HP4, with NESIL estimates between 258 to 2840 µg/cm².
These findings demonstrate that GARD®skin Dose-Response provides ready-to-use NESIL values, enabling potency ranking of candidate ingredients and supporting the safety assessment of novel dyes and dye precursors. The study further highlights the assay’s value as a non-animal alternative for skin sensitization potency assessment in product development.
Joint poster with dōTERRA: Case study on Essential Oils
Comparative analysis of skin sensitization thresholds for Essential Oils: Human, murine, and GARD®skin Dose-Response
Presented at 2025 Eurotox
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Essential oils (EOs), widely used in consumer products, require robust skin sensitization hazard and potency assessment. However, dose-response thresholds for EOs remain understudied, and current classifications under CLP mixture criteria are often overly conservative. Traditional methods, such as animal testing and human patch tests, face ethical concerns, regulatory restrictions, and reliability issues. While New Approach Methodologies (NAMs) address some of these challenges, most of them are validated only for hazard identification and lack quantitative potency assessment capabilities.
GARD®skin (OECD TG 442E) is an in vitro assay that identifies chemical skin sensitizers based on the transcriptional profiling of a 196-gene biomarker signature in the dendritic-like SenzaCell® cell line. Predictions are made using a machine-learning algorithm, which classify test chemicals as sensitizers or non-sensitizers based on the assay’s readout, Decision Values (DVs). GARD®skin Dose-Response (OECD TGP 4.106) extends this approach by evaluating test chemicals across a concentration range to establish a dose-response relationship between DVs and test chemical concentration. Sensitizing potency is quantified using cDV0, the lowest dose required to elicit a positive response in GARD®skin. Depending on the need, the readout can be used to predict LLNA EC3 values, No Expected Sensitization Induction Level (NESIL), and UN GHS/CLP classification (1A or 1B), all with high statistical significance.
This study evaluates the utility of GARD®skin Dose-Response to predict NESILs for EOs, bridging the gap between hazard identification and quantitative risk assessment. Three EOs (A, B, C) were analysed using GARD®skin Dose-Response. Their skin sensitization potency threshold levels were estimated by the assay readouts (cDV0) and NESIL predictions. Results were compared to existing murine (LLNA EC3) and human (HRIPT/HMT) data through weight-of-evidence analysis.
For test item A, the predicted NESILs spanned were 5700 (GARD®), 6900 (constituent prediction), 1800 (LLNA), 2000 (HRIPT), and 3500 µg/cm² (HMT). Test item B showed NESILs of 24000 (GARD®), 29000 (constituent prediction), 6700 (LLNA), 44000 (HRIPT), and 11000 µg/cm² (HMT). Test item C was classified as a non-sensitizer by GARD®skin Dose-Response, with constituent prediction, LLNA, HRIPT, and HMT NESILs at 20000, 3900, 20000, and 690 µg/cm², respectively.
NESIL predictions from GARD®skin Dose-Response aligned with other skin sensitization test results, supporting its utility in quantitative sensitization potency assessment of complex natural extracts like EOs. While confirmatory human studies (e.g., CNIH protocols) are recommended, this assay reduces reliance on animal and human testing and advances the use of NAMs in safety assessment.
Biocompatibility Testing of Medical Devices
SenzaGen’s Newsletter Q2 2025
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SenzaGen Newsletter 2025 Q2
SenzaGen Newsletter 2025 Q2
GARD®skin is now included in OECD TG 497
Learn more about how GARDskin fits into TG 497.
Webcast: How GARDskin enhances Defined Approaches for Skin Sensitization
FDA shift highlights need for innovative, human-relevant test methods
Read more in the full FDA article
Expanded collaboration with RIFM to advance fragrance safety
Learn more about our work with RIFM
Upcoming events
🌎 Eurotox 2025 | Athens, Greece | Sep 14-17
🌎 ICT 2025 | Beijing, China | Oct 15-18
🌎 ASCCT 2025 | Gaithersburg, USA | Oct 21-23
Skin Sensitization Potency Assessment
SenzaGen’s Newsletter Q1 2025
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Text-only version:
Scientific highlights from SOT 2025
SOT 2025 in Orlando was a fantastic opportunity to connect with industry leaders and present the latest advancements in GARD® for Skin Sensitization. The strong interest in GARD® was further reinforced by our presentations on its use for Natural Extracts and Medical Devices, as well as its ability to provide quantitative data for safe dose levels and potency GHS/CLP classification. It was great to meet so many of you at SOT, and we look forward to continuing the conversation!
A special thank you to our research partners for their invaluable contributions: dōTERRA | Unilever | Ron Brown | Clarins | Lundbeck | SafeBridge
Advancing NAMs for fragrance safety
A new peer-reviewed study published in ALTEX, conducted in collaboration with RIFM and IFF, confirms the predictive ability of GARD®skin Dose-Response in assessing the skin sensitization potency of fragrance ingredients. The study supports the assay’s application in Quantitative Risk Assessment (QRA) and Next Generation Risk Assessment (NGRA).
With data on 100 fragrance ingredients spanning diverse chemical structures and potency levels, this research represents another step toward more reliable, non-animal testing methods.
Read the new scientific publication
Broadened regulatory testing services
The SenzaGen Group is pleased to announce the implementation of EpiSensA (OECD TG 442D) – making us the first European service provider to offer the new method based on a Reconstructed Human Epidermis (RhE) model.
Talk to one of our experts to learn more
Upcoming events
🌎 Conference: World Congress 13 | Rio de Janeiro, Brazil | Aug 31- Sep 4
🌎 Conference: Eurotox 2025 | Athens, Greece | Sep 14 – 17
Joint poster with Lundbeck: Case study on Occupational Health and Safety
Assessing Skin Sensitizing Hazard of Drug Products and Active Pharmaceutical Ingredients using GARD®: New Approach Methodology in Occupational Health and Safety
Presented at 2025 SOT
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In conclusion, the findings highlight NAMs like GARD®skin Dose-Response as useful tools for enhancing OHS and safety protocols in pharmaceutical manufacturing.
Background and Purpose:
Skin sensitizers are chemicals capable of inducing hypersensitivity reactions, such as Allergic Contact Dermatitis (ACD). Identifying and characterizing these chemicals’ skin sensitizing potential is essential for limiting hazardous exposure. Significant efforts have been made in the scientific community to develop New Approach Methodologies (NAMs) to replace animal testing for skin sensitization. Internationally recognized test guidelines as well as integrated strategies have been developed, combining in vitro, in chemico, and in silico approaches for predicting skin sensitization potential.
While widely accepted in cosmetics and personal care, NAMs’ application in the pharmaceutical sector for product development and Occupational Health and Safety (OHS) is less prominent. The aim of this study is to demonstrate how NAMs, including the in vitro GARD® assay, combined with in silico data, can be used by the pharmaceutical industry to assess the skin sensitization potential of drug products and Active Pharmaceutical Ingredients (APIs) to take protective measures and improve occupational safety in production environments.
In this study, the drug product is an oral medicine used to treat mental health problems, and the two APIs are compounds that affects how dopamine works in the brain, helping with symptoms like hallucinations or delusions, and helps boost certain chemicals in the brain to improve mood and reduce feelings of depression, respectively.
Methods:
Following reports of allergic reactions in production and quality control, a drug product and two APIs A and B were assessed. The drug product contained approximately 0.5% of API A and 9.4% of API B. In silico predictions using Nexus Derek software identified all compounds as potential sensitizers. The in vitro assay GARDskin Dose-Response (GARDskin DR) was conducted to confirm these predictions.
The GARDskin method (OECD 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.
GARDskin DR is an adaptation of the conventional GARDskin method, in which test chemicals are evaluated by the GARDskin prediction algorithm in an extended range of concentrations, to investigate the dose-response relationship between GARDskin DVs and test chemical concentration. 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. The readouts can predict correlating LLNA EC3 values, which are traditionally used to measure the skin sensitizing potency of chemicals. Furthermore, it can predict human skin sensitizing potency NOEL and GHS/CLP classification (1A or 1B), all with high statistical significance.
Results:
All test items were identified as sensitizers by GARDskin DR with the following results:
• Drug product: cDV0 = 12.8 µg/ml. Predictions: LLNA EC3 = 7.63%, NOEL = 3108 µg/cm², GHS/CLP 1B.
• API A: cDV0 = 1.51 µg/ml. Predictions: LLNA EC3 = 1.08%, NOEL = 254 µg/cm², GHS/CLP 1A.
• API B: cDV0 = 1.77 µg/ml. Predictions: LLNA EC3 = 1.19%, NOEL = 315 µg/cm², GHS/CLP 1A.
Conclusions:
Both APIs were classified as strong sensitizers, while the drug product was classified as a weak sensitizer, reflecting the dilution effect of excipients. The GARDskin DR assay demonstrated a clear dose-dependent increase in DVs for all three test items, confirming their classification as skin sensitizers, with varying potencies.
Based on the test outcomes, recommendations were made to improve occupational safety in handling these sensitizers:
• Avoiding dust inhalation and skin/eye contact.
• Minimizing prolonged and/or repeated exposure.
• Using appropriate personal protective equipment (PPE).
• Removing contaminated clothing and washing it before reuse.
• Ensuring thorough hand washing after handling, during breaks, and at the end of each shift.
• Handling substances in closed systems with proper ventilation.
These findings highlight NAMs like GARDskin DR as useful tools for enhancing OHS and safety protocols in pharmaceutical manufacturing.
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.