Next Generation Risk Assessment (NGRA) using NAMs for skin sensitization: Reproducibility and precision of the GARDskin Dose-Response assay for PoD determination of fragrance chemicals.

Presented at ASCCT 2022

Next Generation Risk Assessment (NGRA) using NAMs for skin sensitization: Reproducibility and precision of the GARDskin Dose-Response assay for PoD determination of fragrance chemicals.

Andy Forreryd1, Shashi Donthamsetty2, Paul Sterchele2, Xiao Huang2, Gregory Ladics2, Mihwa Na3, Isabelle Lee3, Anne Marie Api3, Robin Gradin1, Henrik Johansson1
1SenzaGen, Lund, Sweden , 2International Flavors & Fragrances, Hazlet, NJ, USA, 3Research Institute for Fragrance Materials. Woodcliff lake, NJ, USA

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Conclusion

  • GARD®skin Dose-Response can be used for continous predictions of skin sensitizing potency.
  • 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.
  • 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 assessment of skin sensitizers have been adopted as Test Guidelines (TGs) by OECD. When combined into Integrated Approaches to Testing and Assessment (IATA) or defined approaches (DA), they provide data supporting hazard classifications and GHS potency subcategorization. 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).

GARDskin was recently adopted into OECD TG 442E to support discrimination of skin sensitizers and non-sensitizers. Continous 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 NOEL values. The aim of the following study, which represents a cross-sector collaboration was to evaluate precision and reproducibility of the potency predictions from GARDskin Dose-Response in blinded studies.

Preliminary results from estimate of precision (n=36 materials) indicated that GARDskin Dose-Response predicted LLNA EC3/ human NOEL values with median fold-misprediction factors < 3.0 and < 2.0, respectively. Interestingly, LLNA predicted human NOEL with a fold-change > 2 in the same dataset. For reproducibility assessment, test materials (n=11) were evaluated in separate experiments (n=3), which generated highly reproducible results, with an average median range of fold-changes between replicates of 2.5.

Results from this study demonstrate that continous potency predictions from GARDskin Dose-Response are reproducible. Together with performance data, this represents a major step towards establishment of the assay as a relevant source of information to derive a PoD for NGRA, avoiding generation of new animal data.

 

 

 

The use of the GARD®skin Dose-Response assay to assess skin sensitizing potency in developing novel fragrance ingredients

Presented at ASCCT 2022

The use of the GARD®skin Dose-Response assay to assess skin sensitizing potency in developing novel fragrance ingredients

Tim Lindberg1, Christopher Choi2, Ulrika Mattson1 and Satoshi Sasaki3
1SenzaGen, Lund, Sweden , 2Takasago International Corp, Rockleigh NJ, USA ,3Takasago International Corp, Hiratsuka city, Kanagawa, Japan

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Conclusion

The present study aimed at investigating the skin sensitizing potency of two novel fragrances, Fragrance 1 and 2, using three NAMs, the kDPRA, KeratinoSens and GARD®skin Dose-Response assays.

To move away from traditional safety testing, which includes animal studies, there is a paradigm shift towards the use of multiple NAMs in a weight-of-evidence approach when risk assessment of novel fragrance ingredients are conducted. However, the lack of established potency assays puts the alternative methods at a disadvantage as compared to the in vivo counterparts.

  • Fragrance 1 showed similar results across the three NAMs, with the GARD®skin Dose-Response assay predicting the cDV0–value to 18.4 µM, which in turn was used to predict a NESIL-value of 659 µg/cm2.
  • For Fragrance 2, equivocal results were seen, where the kDPRA assay predicting the ingredient not to be a category 1A skin sensitizer while KeratinoSens predicted it as a non-skin sensitizer. GARD®skin Dose-Response predicted the cDV0–value to 296 µM, which was used to predict a NESIL-value of 16600 µg/cm2. Combining the results from all three NAM assays, a confirmatory HRIPT testing concentration was determined for both ingredients, 562.5 µg/cm2 and 15000 µg/cm2 for Fragrance 1 and Fragrance 2, respectively.

In conclusion the data presented here show how the use of the GARD®skin Dose-Response assay in combination with other NAMs can be used as a replacement of animal studies for quantitative risk assessment of novel fragrance materials.

Abstract

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

The aim of this study was to investigate the sensitizing potency of two novel fragrance ingredients and to identify predicted non-sensitizing levels. Testing was performed in GARD®skin Dose-Response, with predicted EC3 and NESIL values of 1.93% and 27.8%, and 659µg/cm2 and 16600µg/cm2, for fragrance ingredients 1 and 2, respectively. These results in combination with data from kDPRA, KeratinoSens and in silico read- across, established the concentrations for confirmatory HRIPT testing (562.5µg/cm2 and 15000µg/cm2).

In conclusion, this study demonstrates how GARD®skin Dose-Response combined with other NAMs can be used for risk assessments and to establish a concentration for confirmatory HRIPT testing of novel fragrance ingredients.

 

 

SenzaGen’s Newsletter Oct 2022

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