Joint publication with IFF and RIFM: GARDskin dose-response assay and its application in conducting Quantitative Risk Assessment (QRA) for fragrance materials using a Next Generation Risk Assessment (NGRA) framework

New joint publication with International Flavors & Fragrances Inc (IFF) and Research Institute for Fragrance Materials (RIFM).

SenzaGen scientists, alongside the scientific teams at International Flavors & Fragrances Inc and Research Institute for Fragrance Materials, have jointly published an article in Regulatory Toxicology and Pharmacology, presenting new peer-reviewed evidence on the performance of the GARD®skin Dose-Response assay for Quantitative Risk Assessment of fragrance materials.

The study results confirm the ability of GARD®skin Dose-Response to predict human NESIL values with good predictive performance, showing good concordance with published reference Human data and demonstrating good reproducibility.

Furthermore, the paper also presents a case study to illustrate how the predicted NESIL value from GARDskin Dose-Response can be used in practice within a NGRA framework to establish a maximum allowable concentration of a sensitizer in different consumer products.

The study represents a major step towards the establishment of the assay to derive NESIL values for conducting QRA evaluations for fragrance materials using an NGRA framework.

Shashikiran Donthamsetty, Andy Forreryd, Paul Sterchele, Xiao Huang, Robin Gradin, Henrik Johansson, Ulrika Mattson, Isabelle Lee, Anne Marie Api, Gregory Ladics,
Regulatory Toxicology and Pharmacology, Volume 149, 2024, 105597, ISSN 0273-2300,
https://doi.org/10.1016/j.yrtph.2024.105597

Keywords

QRA (Quantitative Risk Assesment); Dermal sensitization; Fragrance materials; Next Generation Risk Assesment (NGRA); GARD assay; No Expected Sensitization Induction Level (NESIL); New Approach Methodologies (NAMs); OECD 442E

Highlights

  • Developed a Next Generation Risk Assessment (NGRA) framework for conducting QRA2 for fragrance materials.
  • The GARDskin Dose Response (DR) assay is a reliable and reproducible method for predicting NESIL for fragrance materials.
  • NGRA for QRA2 was validated using isocyclocitral as a case study.


Abstract

Development of New Approach Methodologies (NAMs) capable of providing a No Expected Sensitization Induction Level (NESIL) value remains a high priority for the fragrance industry for conducting a Quantitative Risk Assesment (QRA) to evaluate dermal sensitization. The in vitro GARDskin assay was recently adopted by the OECD (TG 442E) for the hazard identification of skin sensitizers. Continuous potency predictions are derived using a modified protocol that incorporates dose-response measurements. Linear regression models have been developed to predict human NESIL values. The aim of the study was to evaluate the precision and reproducibility of the continuous potency predictions from the GARDskin Dose-Response (DR) assay and its application in conducting QRA for fragrance materials using a Next Generation Risk Assessment (NGRA) framework. Results indicated that the GARDskin Dose-Response model predicted human NESIL values with a good degree of concordance with published NESIL values, which were also reproducible in 3 separate experiments. Using Isocyclocitral as an example, a QRA was conducted to determine its safe use levels in different consumer product types using a NGRA framework. This study represents a major step towards the establishment of the assay to derive NESIL values for conducting QRA evaluations for fragrance materials using a NGRA framework.

Evaluation of the applicability of GARDskin to predict skin sensitizers in extracts from medical device materials

Peer-reviewed article in Frontiers in Toxicology.

The application of GARD®skin to predict potential skin sensitizers in extracts from Medical Device materials has recently been published in a peer-reviewed article in Frontiers in Toxicology.

The study results provide evidence recognizing the GARD®skin Medical Device assay as a scientifically sound and ethical alternative to conventional animal methods, compatible with both polar and non-polar extraction vehicles, in line with the ISO 10993-12:2021 standard.

We are proud to provide the only OECD-validated in vitro assay for sensitization that is fully compatible with testing requirements specified in ISO 10993-12.

Jenvert Rose-Marie, Larne Olivia, Johansson Angelica, Berglin Mattias, Pedersen Emma, Johansson Henrik
Frontiers in Toxicology, Volume 6, 2024,ISSN 2673-3080
DOI=10.3389/ftox.2024.1320367

Keywords

GARD™; In vitro; Skin sensitisation; NAMs; Medical Device, ISO 10993, Biocompatibility

Abstract

Biocompatibility testing of medical devices is governed by the ISO 10993 series of standards and includes evaluation of skin sensitization potential of the final product. A majority of all medical devices are tested using in vivo methods, largely due to the lack of in vitro methods validated within the applicability domain of solid materials. The GARDskin method for assessment of chemical skin sensitizers is a validated method included in the OECD Test Guideline 442E, based on evaluation of transcriptional patterns of an endpoint-specific genomic biomarker signature in a dendritic cell-like cell, following test chemical exposure. The current study aimed to evaluate the applicability of GARDskin for the purpose of testing solid materials by incorporation of extraction procedures described in ISO 10993-12:2021, as well as to demonstrate the functionality of the proposed protocols, by testing of custom-made materials spiked with sensitizing agents. It was shown that GARDskin is compatible with both polar and non-polar extraction vehicles frequently used for the purpose of medical device biological testing. Further, exploring three different material types spiked with up to four different sensitizing agents, as well as three unspiked control materials and commercial reference products, it was shown that the method correctly classified all evaluated test materials. Taken together, the data presented suggest that GARDskin may constitute a valid alternative to in vivo experimentation for the purpose of skin sensitization assessment of medical devices.

 

Joint publication with Corteva Agriscience: GARD™skin and GARD™potency: A proof-of-concept study investigating applicability domain for agrochemical formulations

New joint publication with Corteva Agriscience.

SenzaGen scientists, alongside the toxicology team at Corteva Agriscience, have recently published a joint study in Regulatory Toxicology and Pharmacology, presenting new peer-reviewed evidence on the applicability of GARD® for agrochemical formulations.

The study demonstrates a satisfactory performance of GARD®skin and GARD®potency for skin sensitization hazard and GHS potency categorization of tested agrochemical formulations.

Marco Corvaro, Joseph Henriquez, Raja Settivari, Ulrika Mattson, Andy Forreryd, Robin Gradin, Henrik Johansson, Sean Gehen,
Regulatory Toxicology and Pharmacology, Volume 148, 2024, 105595, ISSN 0273-2300,
https://doi.org/10.1016/j.yrtph.2024.105595.

Keywords

GARD™; In vitro; Skin sensitisation; NAMs; Agrochemical formulations

Highlights

  • Tested 42 agrochemical formulations to expand applicability domain of GARD.

  • GARDskin showed good accuracy (76.2%), sensitivity (85.0%) and specificity (68.2%).
  • GARDpotency correctly subcategorized 14/17, correctly predicted sensitisers.
  • GARD satisfactory for Key Event 3 characterisation of agrochemical formulations.


Abstract

Several New Approach Methodologies (NAMs) for hazard assessment of skin sensitisers have been formally validated. However, data regarding their applicability on certain product classes are limited. The purpose of this project was to provide initial evidence on the applicability domain of GARD™skin and GARD™potency for the product class of agrochemical formulations.

For this proof of concept, 30 liquid and 12 solid agrochemical formulations were tested in GARDskin for hazard predictions. Formulations predicted as sensitisers were further evaluated in the GARDpotency assay to determine GHS skin sensitisation category. The selected formulations were of product types, efficacy groups and sensitisation hazard classes representative of the industry’s products.

The performance of GARDskin was estimated by comparing results to existing in vivo animal data. The overall accuracy, sensitivity, and specificity were 76.2% (32/42), 85.0% (17/20), and 68.2% (15/22), respectively, with the predictivity for liquid formulations being slightly higher compared to the solid formulations. GARDpotency correctly subcategorized 14 out of the 17 correctly predicted sensitisers. Lack of concordance was justifiable by compositional or borderline response analysis. In conclusion, GARDskin and GARDpotency showed satisfactory performance in this initial proof-of-concept study, which supports consideration of agrochemical formulations being within the applicability domain of the test methods.

 

SenzaGen’s Newsletter Year-end 2023

Click image to view the newsletter in browser.

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

Download a copy

 

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.

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.

Greminger A, Frasca J, Goyak K, North C. 
Challenges integrating skin sensitization data for assessment of difficult to test substances. 
ALTEX - Alternatives to animal experimentation, published Oct 12, 2023
doi: 10.14573/altex.2201122. Epub ahead of print. PMID: 37843019.

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

SenzaGen is on a growth path. Watch CEO Peter Nählstedt present the company at BioStock Life Science Summit 2023.

#Summit #BioStockSWE

SenzaGen’s Newsletter October 2023

Click image to view the newsletter in browser.

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

Download a copy

 

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.

​

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

 

Download a copy

 

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.