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

2025 SOT joint poster with Lundbeck on API and OHS

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Conclusion

In conclusion, the findings highlight NAMs like GARD®skin Dose-Response as useful tools for enhancing OHS and safety protocols in pharmaceutical manufacturing.

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Abstract

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

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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.

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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.

Joint poster with L’Oreal: Point of Departure for risk assessment

Improved Confidence of Quantitative Sensitizing Potency Assessment for Point of Departure Using GARD®skin Dose-Response


Presented at SOT and Eurotox 2024

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Conclusion

In conclusion, the readouts from GARDskin Dose-Response derive a quantitative continuous potency estimate of skin sensitizers that may be used directly as a PoD for a seamless integration into downstream NGRA.

​

Abstract

Identification of skin sensitization hazard and potency characterization are central aspects of risk assessment of chemicals. Current legislation advocates a transition from hazard assessment using in vivo methods to UN GHS potency subclassification and quantitative risk assessment by use of New Approach Methodologies (NAM:s) as well as Defined Approaches (DA). However, the ability of NAM assays to quantitatively estimate sensitizing potency and thereby establish a point of departure (POD) for next-generation risk assessment (NGRA) strategies is currently lacking.

To this end, the GARDskin Dose-Response (DR) method, adapted from the OECD TG 442E method GARDskin, was recently introduced. The GARDskin DR method evaluates test chemicals in a titrated range of concentrations, in order to investigate the dose-response relationship between the output from the GARDskin prediction algorithm (Decision Values; DV:s) and test chemical concentration. The combined information can be used to derive a quantitative estimation of sensitizing potency, defined as the cDV0-value, i.e, the least required dose required to elicit a positive response by the prediction model.

The current work focuses on optimizing the ability of GARDskin DR to derive a quantitative POD based on conversion to a composite Potency Value (PV; µg/cm2), taking into account both human and in vivo reference data sources. A total of 25 chemicals were used to construct predictive regression models fitted to reference PV:s. Results show that the updated models fitted to reference PV:s produced more accurate potency predictions compared with models fitted with, and aiming to predict, only LLNA EC3 and NOEL, respectively. Mean fold-change errors ranged between 2.8 and 3.2, with predicted POD:s being within or close to the range of the variation of the historical in vivo data. In addition, uncertainty in predictions was reduced, as estimated by a minimum 2-fold reduction of 95%-confidence intervals, when comparing models fitted to reference PV:s with models fitter with only LLNA EC3 and human NOEL, respectively.

In conclusion, these improvements constitute a major step forward for the ability of NAM:s to assess quantitative sensitizing potency. It demonstrates how GARDskin Dose-Response can accurately estimate a POD and be incorporated into downstream strategies for quantitative risk assessment (QRA), to ultimately contribute to the assessment of safe use levels of chemicals.

Keywords: NAM, GARDskin Dose-Response, Sensitizing potency, Quantitative risk assessment, Point of departure

Joint poster with Cargill: Case study on Hydrophobic Esters

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


Presented at Eurotox 2024

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Conclusion

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

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Abstract

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

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

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

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

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

Joint poster with Sonova: Integrating NAMs into early-stage screening of novel materials

Case studies on the use of GARD®skin Medical Device for in vitro skin sensitization assessment


Presented at SOT and Eurotox 2024

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Conclusion

In conclusion, GARDskin Medical Device can detect signals from diluted and complex extracts of solid devices, with a sensitivity superior to animal methods.

This may significantly reduce the need of animal studies, improve the safety of the final product, and avoid potential costly late-stage failures.

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Abstract

The recent advancements in New Approach Methodologies enables the use of in vitro method for skin sensitization assessment as part of the biocompatibility testing for medical devices, which is conventionally tested in vivo. GARDskin OECD TG 442E is included in ISO 10993-10:2021 as the only OECD validated in vitro assay that is compatible with both polar and non-polar extraction vehicles, in line with ISO 10993-12:2021.  GARDskin Medical Device is an adaptation of the GARDskin assay, including a pre-sample treatment procedure where solid devices are extracted using both polar and non-polar vehicles.

The aim of this study is to demonstrate the benefits of using GARD for early-stage screening of materials intended for use in medical devices for assessing their skin sensitization potential. Results from two case studies were summarized in which GARDskin Medical Device was used for skin sensitization assessment. The first case study describes the testing of an acrylic-based device with a coating consisting of a UV-cured lacquer, where chemical analysis indicated the potential for skin sensitization. The second case study describes the testing of a polymeric material consisting of Cellulose-Acetate Propionate (CAP) with a plasticizer (Triethylene glycol bis (2-ethylhexanoate), CAS# 94-28-0), with contradictive existing in vivo (negative) and in vitro (positive) data.

In the first case study, the acrylic-based device induced a positive response in both polar and non-polar vehicles in GARDskin Medical Device and was thus classified as a skin sensitizer. It was hypothesized that the positive results may be due to inadequate curing of the lacquer within cavitary structures of the devices, where UV light exposure was insufficient. To confirm the hypothesis, follow-up testing was performed on an identical device, but without cavities, which was classified as non-sensitizer. In vivo data confirmed the outcome of the in vitro assay. Consequently, a modification was made in the manufacturing process to prevent the presence of lacquer in cavitary structures of the device.

In the second case study, the CAP material was positive in the non-polar vehicle and was thus labelled as a skin sensitizer. The plasticizer was identified as a potential culprit, considering a borderline negative result in LLNA (SI=2.97) and reported positive clinical data. To support the hypothesis a follow-up study was conducted using another adaptation of the GARDskin protocol capable of providing continuous potency predictions. The results classified the plasticizer as a weak contact allergen. As a result, a different material was selected for the device.

Keywords: Biocompatiblity, ISO 10993-10, GARDskin Medical Device, Skin Sensitization, ISO 10993-12

Joint poster with RIFM and IFF: In vitro Skin Sensitization Potency Prediction Performance on the RCPL list

GARD®skin Dose-Response for skin sensitizing potency assessment: Performance on the Reference Chemical Potency List (RCPL)


Presented at BTS 2024.

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Conclusion

  • This study suggests that GARDskin Dose-Response is a potent predictor of skin sensitizing potency on a continuous scale.
  • Predicted potency values are in µg/cm2 and can be used for risk assessment and to establish a maximum allowable concentration of a sensitizer in various consumer products.

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Abstract

New Approach methods (NAMs) for assessment of skin sensitizing hazard and GHS potency have been adopted as OECD Test Guidelines. However, NAMs able to estimate skin sensitization potency on a continous scale, and thereby generating data for use as a point of departure (PoD) in next-generation risk assessment (NGRA) strategies, are currently lacking.

GARDskin Dose-Response (DR) is based on the validated protocols of GARDskin (OECD TG 442E). The readout of the assay is a continous potency prediction, or a No Expected Sensitization Induction Level (NESIL) value (ug/cm2).

The aim of this study was to evaluate the performance of the potency predictions from GARDskin DR using the chemicals in the Reference Chemical Potency List (RCPL) as a reference. The RCPL contains curated Potency Values (PVs) and was developed to serve as a resource for evaluating potency predictions from NAM-based approaches.

GARDskin data was generated (n=27 chemicals) and potency predictions were compared to PV values from the RCPL list. The continous potency predictions from GARDskin DR correlated well with PVs (p: 0.74), with an average misprediction of 3.7-fold from reference values. Furthermore, GARDskin DR provided a very similar potency ranking compared to the ranking based on PV values in the RCPL list (ρ: 0.69).

In conclusion, based on the results in this study, GARDskin DR was demonstrated to be a potent predictor of skin sensitizing potency, increasing the confidence in using the assay to conduct NGRA on new chemical entities, ultimately reducing the need for confirmatory human and/or animal studies.

Keywords: NAM, GARDskin Dose-Response, Sensitizing potency, Quantitative risk assessment, Point of departure

Joint poster with Takasago: Determining safe use level for novel fragrance ingredients

Practical application of the GARD®skin Dose-Response assay to derive a No Expected Sensitization Induction Level (NESIL) value for confirmatory human patch studies to determine safe use level for novel fragrance ingredients


Presented at ACT 2023

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Summary

  • Combining the results from the GARD®skin Dose-Response assay with other NAMs enables weight-of-evidence based approaches to determine safe use levels of novel fragrance ingredients.
  • Based on the results from the weight-of-evidence approach, confirmatory human patch test and HRIPT are conducted. HRIPT is performed at the top concentration of 11250ug/cm2. Both results are negative, confirming the predicted NESIL-value from GARD®.

 

Abstract

Skin sensitization is one of the required endpoints for the development and registration of novel fragrance ingredients. Traditionally, assessment has been performed combining in vitro and in vivo assays, but recent developments has shifted to the use of New Approach Methodologies (NAMs), without 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.

This study presents the use of the GARDskin Dose-Response assay to determine safe human user levels for one novel fragrance ingredient. The experimentally predicted NESIL value was determined to 37800µg/cm2. Using a weight-of- evidence approach mainly guided by the quantitative data from the GARDskin Dose-Response, confirmatory Human Repeated Insult Patch Testing (HRIPT) studies were conducted and at the tested concentrations, no sensitization reactions were observed. In conclusion, this study expands the toxicologist´s toolbox and illustrates the potential to use the GARDskin Dose-Response assay to derive NESIL values that are protective of human health, without having to rely on the Dermal Sensitization Threshold (DST) approach or reverting to traditional animal testing approaches.

Using in vitro data for regulatory approval of medical devices according to MDR

Regulatory approval of medical devices according to MDR using in vitro data from GARDskin Medical Device for skin sensitization assessment


Presented at Eurotox 2023

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Conclusion

  • The GARDskin Medical Device assay is a novel method for assessment of skin sensitizing properties in leachables from solid materials, according to ISO 10993 standards.
  • Here, we demonstrate how GARDskin Medical Device data, combined with endpoint data from skin irritation and cytotoxicity testing, were successfully used in a data package for biocompatibility assessment of a novel medical device.
  • Following review by a notified body, an obtained CE-mark for the medical device Tinearity® G1 highlights unique opportunities to comply with the European Medical Device Regulation 2017/745 (MDR) using only in vitro data sources.

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Abstract

Skin sensitizers in medical device extracts are conventionally assessed in vivo, primarily using the Guinea Pig Maximization Test and the Buehler Occluded Patch Test. However, there is a shift in the medical device toxicology field towards an increased use of in vitro methods for the evaluation of the biological safety of medical devices. Recently, in vitro methods for the endpoints skin irritation and skin sensitization have been included in the ISO 10993 standard, what makes it possible to perform this testing in vitro. The GARDskin assay is one of the in vitro methods for assessment of skin sensitization described in ISO 10993-10 and is the first OECD TG 442 method that has been adapted to work with oil, the non-polar extraction vehicle often used in in vivo studies for testing medical devices.

Here we share an example of how in vitro testing results, including results from the GARDskin Medical Device assay, were submitted to obtain CE-marking according to the European Medical Device Regulation 2017/745 (MDR) for Tinearity® G1, an innovative tinnitus treatment medical device. Tinearity® G1 was classified as a non-sensitizer in both polar and non-polar extracts in the GARDskin Medical Device assay. This result was used together with in vitro cytotoxicity and in vitro skin irritation results as weight of evidence together with review of chemical data in the risk assessment and biological evaluation of the medical device.

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

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


Presented at Eurotox 2023

 

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Summary

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

Abstract

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

 

Joint poster with RIFM and IFF: PoD for NGRA, a case study using isocyclocitral

The GARDskin Dose-Response assay for determination of a point-of-departure (PoD) for Next Generation Risk Assessment (NGRA) of skin sensitizers: A case study using isocyclocitral


Presented at Eurotox 2023

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Conclusion

  • 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 (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 which can be used for conducting the quantitative risk assessment for generating the IFRA standard.

Abstract

New Approach Methods (NAMs) for the assessment of skin sensitizers have been adopted as OECD Test Guidelines (TGs), supporting hazard- and GHS potency classifications. 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) of new chemical entities, which still represents a missing element in the application of NAMs for sensitization assessments.

The GARDskin assay (OECD TG 442E) provides a novel and mechanistically different method to monitor the Key Events (KE) in the Adverse Outcome Pathway (AOP) for skin sensitization and is the first harmonized test guideline based on genomics and machine leaning. A modified version of the validated protocol incorporating dose-response measurements has recently been described which uses linear models for the prediction of LLNA EC3/Human No Expected Sensitization Induction Levels (NESIL) values.

The aim of the following study, which represents a cross-sector collaboration involving industry, assay developer, and a non-profit research institute, were to perform a pre-validation exercise to evaluate the precision and reproducibility of the continous potency predictions from the GARDskin dose-response assay, and to demonstrate how the derived continous potency predictions can be implemented into available NGRA-framework to determine safe use levels in consumer products.

Predictive performance was estimated in a blinded study by evaluating a total of 17 fragrance materials, and reproducibility of the continous predictions was assessed by evaluating 11 of the materials in three replicate experiments. Results illustrate that predicted LLNA EC3/human NESIL values from the GARDskin Dose-response assay correlate well with reference data (geometric mean fold-error: 3.8 and 2.5, respectively), and that the continuous potency predictions are reproducible between experiments (geometric mean fold-change: 3.1). A case study using isocyclocitral was used to illustrate how the assay can be implemented into an NGRA-framework, which is an exposure driven risk assessment methodology. The predicted NESIL value from GARDskin Dose-response was used within a weight-of-evidence  approach to derive a PoD for use in QRA. Sensitization assessment factors were applied to the PoD to determine acceptable exposure levels at which no skin sensitization induction would be expected for different product types based on exposure.

In conclusion, the results reported from this study demonstrate that the predicted potency values from the GARDskin Dose-Response assay are reproducible between experiments and show good concordance with the published values. The case study illustrates a proof of concept and establish the assay as a relevant source of information to derive NESIL values for conducting QRA evaluations for fragrance materials without any new animal data.