Characterization of respiratory sensitizing properties of the protein Subtilisin, using GARD®air
Presented at the 12th World Congress on Alternatives and Animal Use in the Life Sciences 2023
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Conclusion
GARDair is an experimental in vitro assay for assessment of respiratory sensitizers, based on evaluation of exposure-induced gene expression changes of genomic biomarkers in a Dendritic Cell-like cell line and machine learning-assisted classifications.
GARDair has previously been shown to be functional, having been subjected to a validating ring trial, demonstrating its capability to predict respiratory sensitization properties of low-molecular weight test chemicals.
Here, we investigated the hypothesis that protein allergens engage similar toxicity pathways as low-molecular weight chemicals and demonstrated that GARDair can be successfully used to predict the respiratory sensitizing properties of the model allergen Subtilisin.
Abstract
Sensitization is a condition induced by an immune system response to a variety of molecules, including proteins or chemicals, referred to as sensitizers. Proactive identification of sensitizers is central in hazard and risk assessment of both biologics and chemicals, for regulatory registration or to ensure occupational safety. While large investments in New Approach Methodologies for assessment of dermal sensitizers have been made, the ability to accurately predict respiratory sensitizers in vitro, including both low-molecular weight chemicals and biologics such as proteins and peptides, remains unfulfilled.
The Genomic Allergen Rapid Detection assay for hazard assessment of respiratory sensitizers (GARDair) is an experimental method, originally developed to provide binary hazard identification of chemical respiratory sensitizers. Based on the same technological framework as the OECD validated GARDskin assay (OECD TG 442E), the method evaluates the transcriptional patterns of disease-associated genes in the dendritic cell-like cell line SenzaCell™, following test item exposure. Here, we hypothesized similar toxicity pathway engagement by protein sensitizers as for low-molecular weight chemicals. Using the model protein allergen Subtilisin, a well-known inducer of occupational asthma, we here demonstrate the ability of GARDair to assess the respiratory sensitization potential of proteins.
The GARDair test results of the Subtilisin and control samples are presented in Figure 3. Subtilisin gives rise to clearly positive response signals, along with the low-molecular weight positive control (Reactive black 5), while the negative (vehicle) control does not. Evidently, the protein allergen Subtilisin triggers, at least to a limited albeit sufficient extent, similar toxicity pathways as the low-molecular weight chemical space used to define the method. While these findings are solely originating from an in vitro setting, we hypothesize that such toxicity pathways would be similarly engaged also in an in vivo setting, thereby, at least partly, explaining the allergenic effects known to be associated with Subtilisin exposure. Further, we hypothesize that such toxicity pathways would, at least partly, be associated with the GARDair GPS, which is proposed to monitor transcriptional changes induced specifically by respiratory sensitizers, related to the bridging of innate and adaptive immune functions and skewing towards Th2 type immune responses (including TSLP and IL-7R-alpha chain).
Based on the findings reported in this study, GARDair may constitute a valuable tool for assessment of respiratory sensitization properties of chemicals and proteins. Given the limitation of this study with respect to sample size, next steps include the generation of more data to support the predictive capacity, including the assessment of proteins with low (or lack of ) sensitization potential.
SenzaGen’s Newsletter Year-end 2023
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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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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.
New scientific publication by ExxonMobil: Challenges integrating skin sensitization data for assessment of difficult to test substances
New scientific publication by ExxonMobil.
Check out this newly published article by ExxonMobil focusing on the challenges of determining skin sensitization hazard in the case of difficult-to-test substances with conflicting or low-confidence data, where GARDskin data on UVCBs and hydrophobic substances provides valuable input for the integrated hazard assessment.
The article provides new peer-reviewed evidence for the applicability domain of GARDskin on UVCBs, hydrophobic and highly complex substances.
Keywords
LLNA; ToxPi; new approach methodologies; skin sensitization; weight of evidence.
Abstract
Difficult to test substances including poorly soluble, mildly irritating, or Unknown or Variable Composition Complex reaction products or Biological Materials (UVCBs), producing weak or borderline in vivo results, face additional challenges in in vitro assays that often necessitates data integration in a weight of evidence (WOE) approach to inform skin sensitization potential. Here we present several case studies on difficult to test substances and highlight the utility of Toxicological Prioritization Index (ToxPi) as a data visualization tool to compare skin sensitization biological activity. The case study test substances represent two poorly soluble substances, tetrakis (2-ethylbutyl) orthosilicate and decyl palmitate, and two UVCB substances, alkylated anisole and hydrazinecarboximidamide, 2-[(2-hydroxyphenyl)methylene]-, reaction products with 2 undecanone. Data from key events within the skin sensitization adverse outcome pathway were gathered from publicly available sources or specifically generated. Incorporating the data for these case study test substances as well as on chemicals of a known sensitization class (sensitizer, irritating non-sensitizer, and non-sensitizer) into ToxPi produced biological activity profiles which were grouped using unsupervised hierarchical clustering. Three of the case study test substances concluded to lack skin sensitization potential by traditional WOE produced biological activity profiles most consistent with non-sensitizing substances, whereas the prediction was less definitive for a substance considered positive by traditional WOE. Visualizing the data using bioactivity profiles can provide further support for WOE conclusions in certain circumstances but is unlikely to replace WOE as a stand-alone prediction due to limitations of the method including the impact of missing data points.
SenzaGen presented at the BioStock Life Science Fall Summit in Lund on October 26
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SenzaGen’s Newsletter October 2023
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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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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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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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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.
Joint poster with Johnson Matthey: Case study on Metals
Expanding the applicability domain of NAMs for skin sensitization testing: A case study using GARDskin for assessment of metals
Presented at the 12th World Congress on Alternatives and Animal Use in the Life Sciences, 2023
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New Approach Methods (NAMs) for detection of sensitization have been validated and adopted as OECD TGs during the last decade. These assays target different Key Events (KE) in the AOP for skin sensitization and are increasingly being applied to replace animal models within different chemical sectors. However, further characterization of the applicability domain (AD) of these assays is critical to understand limitations and to facilitate regulatory uptake in other industrial sectors. Of particular interest from a scientific and regulatory perspective is the potential to use NAMs for assessment of metals, which have been proposed to act via alternative mechanisms to organic chemicals. The current study describes a joint effort by industry and assay developers to evaluate the AD of the GARDskin assay for metal compounds. GARDskin is the first harmonised method utilizing a combination of genomics and machine learning for a regulatory endpoint and was recently adopted into OECD TG442E.
A selection of metal salts (n=13) was evaluated and the accuracy, sensitivity, and specificity for prediction of skin sensitizing hazard of metals were estimated to 92% (12/13), 100% (7/7) and 83% (5/6), respectively. Interestingly, transcriptomic analysis revealed almost identical response patterns in dendritic cells for metals and organic compounds, indicating a high similarity in the toxicity pathways driving classifications. In conclusion, the result from this study supports the inclusion of metals into the AD of GARDskin, which is an important step to ensure scientific/regulatory confidence to reduce the need for animal testing within the metal production and medical device sector.
Joint poster with Merck: case study on protein allergy
Characterization of respiratory sensitizing properties of the protein Subtilisin, using GARD®air
Presented at the 12th World Congress on Alternatives and Animal Use in the Life Sciences 2023
Download a copy in PDF
GARDair is an experimental in vitro assay for assessment of respiratory sensitizers, based on evaluation of exposure-induced gene expression changes of genomic biomarkers in a Dendritic Cell-like cell line and machine learning-assisted classifications.
GARDair has previously been shown to be functional, having been subjected to a validating ring trial, demonstrating its capability to predict respiratory sensitization properties of low-molecular weight test chemicals.
Here, we investigated the hypothesis that protein allergens engage similar toxicity pathways as low-molecular weight chemicals and demonstrated that GARDair can be successfully used to predict the respiratory sensitizing properties of the model allergen Subtilisin.
Sensitization is a condition induced by an immune system response to a variety of molecules, including proteins or chemicals, referred to as sensitizers. Proactive identification of sensitizers is central in hazard and risk assessment of both biologics and chemicals, for regulatory registration or to ensure occupational safety. While large investments in New Approach Methodologies for assessment of dermal sensitizers have been made, the ability to accurately predict respiratory sensitizers in vitro, including both low-molecular weight chemicals and biologics such as proteins and peptides, remains unfulfilled.
The Genomic Allergen Rapid Detection assay for hazard assessment of respiratory sensitizers (GARDair) is an experimental method, originally developed to provide binary hazard identification of chemical respiratory sensitizers. Based on the same technological framework as the OECD validated GARDskin assay (OECD TG 442E), the method evaluates the transcriptional patterns of disease-associated genes in the dendritic cell-like cell line SenzaCell™, following test item exposure. Here, we hypothesized similar toxicity pathway engagement by protein sensitizers as for low-molecular weight chemicals. Using the model protein allergen Subtilisin, a well-known inducer of occupational asthma, we here demonstrate the ability of GARDair to assess the respiratory sensitization potential of proteins.
The GARDair test results of the Subtilisin and control samples are presented in Figure 3. Subtilisin gives rise to clearly positive response signals, along with the low-molecular weight positive control (Reactive black 5), while the negative (vehicle) control does not. Evidently, the protein allergen Subtilisin triggers, at least to a limited albeit sufficient extent, similar toxicity pathways as the low-molecular weight chemical space used to define the method. While these findings are solely originating from an in vitro setting, we hypothesize that such toxicity pathways would be similarly engaged also in an in vivo setting, thereby, at least partly, explaining the allergenic effects known to be associated with Subtilisin exposure. Further, we hypothesize that such toxicity pathways would, at least partly, be associated with the GARDair GPS, which is proposed to monitor transcriptional changes induced specifically by respiratory sensitizers, related to the bridging of innate and adaptive immune functions and skewing towards Th2 type immune responses (including TSLP and IL-7R-alpha chain).
Based on the findings reported in this study, GARDair may constitute a valuable tool for assessment of respiratory sensitization properties of chemicals and proteins. Given the limitation of this study with respect to sample size, next steps include the generation of more data to support the predictive capacity, including the assessment of proteins with low (or lack of ) sensitization potential.