Tag Archive for: Fragrance materials

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.

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.

 

 

 

 

Joint poster with RIFM and IFF: PoD determination of fragrance materials

GARDskin Dose-Response assay for PoD determination of fragrance materials and its application in conducting Quantitative Risk Assessment (QRA)


Presented at SOT 2023


Download a copy

 

 

Conclusion

  • The continous readout from the assay is reproductible 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 to be used for conducting the quantitative risk assesment for generating the IFRA standard.

Abstract

The global fragrance industry applies Quantitative risk assessment (QRA) to develop risk management practices (IFRA Standards) for ingredients that are identified as potential dermal sensitizers. An important step in QRA is determination of a ”No Expected Sensitization Induction Level” (NESIL), which has historically been determined using human data with the support of animal data (e.g., murine local lymph node assay (LLNA). The EC3 value determined in the LLNA is used as the guidance for selection of the dose level in HRIPTs (Human Repteated Insult Patch Test) to confirm a NESIL value. The fragrance industry has adopted new approach methodologies (NAM) to address skin sensitization. Although several NAMs for identifying skin sensitizers have been accepted as Test Guidelines by OECD, these methods have thus far been validated only for hazard identification. Since a NESIL value is a key requirement to evaluate sensitizing potency for conducting QRA evaluations, development of a NAM-based strategy capable of providing potency data in the form of NESIL remains a high priority for the fragrance industry. 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 further been developed to predict LLNA EC3 and 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 assay. A total of 17 test materials were evaluated, 11 of which were evaluated in three blinded studies separated in time. Preliminary results indicated that the GARDskin Dose-Response model predicted LLNA EC3 values and human NESIL values with geometric mean fold-misprediction factors of 3.8 and 2.5, respectively. For comparative reasons, the LLNA EC3 predicted the human NESIL values with a fold-misprediction factor of 3.7 in the same dataset. Results from the repeated assessment of the test materials were reproducible, with an estimated geometric mean range of fold-changes between replicates of 2.9. Using isocyclocitral (CAS 1335-66-6) as an example, a QRA was conducted to determine its safe use levels in different consumer product types. The results demonstrate that the LLNA EC3 values and the human NESIL values predicted from the GARDskin Dose-Response assay are reproducible between experiments and show good concordance with the published NESIL and EC3 values. Together with the reported performance data, this represents a major step towards the establishment of the assay as a relevant source of information to derive NESIL values for conducting QRA evaluations for fragrance materials to ensure product safety while avoiding the generation of new animal data.

 

 

 

 

Joint poster with RIFM: ​Assessment of Reference Photoirritants and Photoallergens using GARD

GARD®skin Dose-Response for Photosensitization: ​Assessment of Reference Photoirritants and Photoallergens


Presented at SOT 2023

 

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Conclusion

  • Increase in cytotoxicity after UV exposure strongly linked with predominantly photo irritating properties.​
  • Decrease in GARDskin Dose-Response cDV0-value after UV exposure indicative of photoallergenic properties​.

Abstract

Dermal exposure to certain chemical compounds, so-called sensitizers, can give rise to adverse outcomes induced by an immunological response towards the specific compound. One such class of compounds, photosensitizers, needs to be activated by UV rays to elicit an immune response. Although rare in occurrence, it is a critical human health endpoint in need of investigation to limit potential exposures. Other phototoxic skin reactions include photoirritation, which is manifested as a one-time occurrence at the site of exposure that goes away over time. While testing schemes for photoirritation are clear, testing for photosensitization remains a challenge and no established in vitro model to evaluate this endpoint currently exists. For risk management purposes, distinguishing between phototoxic properties is important, as concentration limits can be set for photoirritants whereas fragrance photoallergens have historically been banned. 

The GARDskin assay is a next-generation in vitro method for hazard classification of conventional skin sensitizers, included in OECD TG 442E. The assay is based on a human dendritic -like cell line and combines genomics and machine learning to achieve a high predictive performance with a large applicability domain. The GARDskin Dose-Response assay is based on the validated GARDskin protocols but instead of giving a binary classification it provides quantitative information about the lowest concentration needed to induce a positive classification in the assay, termed the cDV0 concentration. To investigate phototoxicity, an extra UV-exposure step was added to the original protocol, with photosensitization determined by a drop in cDV0 concentration after UV-exposure, i.e., the cDV0 concentration of the specific compound is lower after UV-exposure than in the non-exposed counterpart.  

The study presented here aimed at investigating the applicability of the GARDskin Photo Dose-Response assay to correctly assess photoallergens and distinguish them from photoirritant effects. Previous studies have indicated that a shift in cytotoxic profile after UV-exposure may indicate a predominantly photoirritant activity rather than photosensitizing and this was also investigated in the present study. Six reference photoirritants and six reference photoallergens were investigated using the GARD®skin Dose-Response assay in combination with a UV irradiation protocol. Cytotoxic profiles and cDV0-values were established for each compound in the presence and absence of UV exposure. 5 out of 6 photoirritants were correctly predicted based on their cytotoxic profile while 3 out of 6 photoallergens where correctly predicted based on the decrease in cDV0-value after UV-exposure. In conclusion, functionality of combining GARDskin Dose-Response protocols with UV irradiation to investigate phototoxicity was shown. Further, photoirritant effects were strongly correlated to a shift in cytotoxic profile after UV-exposure and a decrease in cDV0 values after UV-exposure may indicate on photosensitizing effects. However, further work may be warranted to establish a final prediction model for photosensitization.  

Keywords: Predictive Toxicology, GARDskin, Phototoxicity, Quantitative Risk Assesment