Tag Archive for: GARDskin Dose-Response

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.

 

 

In vitro method for quantitative potency assessment of skin sensitizers during development of novel materials for intended use in medical devices

Joint poster with Sonova,
Presented at the 2022 SOT

Andy Forreryd1, Ulrika T Mattson1, David Waeckerlin2, Karla Lienau2, Robin Gradin1, Rose-Marie Jenvert1
1SenzaGen, Lund, Sweden, 2Sonova AG, Staefa, Switzerland

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Conclusion

The GARD®skin Dose-Response assay can be used as a tool for:

  • quantitative potency information of chemicals that might leach out of materials or medical devices.
  • internal decision-making during development of new materials for use in medical devices.

Abstract

New innovative materials for use in medical devices based on acrylates can bring several advantages such as super-absorbency, transparency, flexibility, toughness and hardness.

The manufacturing of acrylates typically involves using a monomer of either acrylate or methacrylate that is polymerized into the final product. The polymerization or hardening of material makes the monomers inert, however several methodologies can be used for polymerization, and they differ in the degree to which they result in a fully polymerized final product. Some products therefore contain more residual monomers than others and human exposure to these well-known skin sensitizers may increase the risk of skin sensitization and allergic contact dermatitis. To assess the risks resulting from exposure to these chemicals, potentially found in medical device material, it is necessary to accurately identify and characterize their skin sensitizing potential.

The GARDskin (OECD TGP 4.106) assay was initially developed for hazard identification of skin sensitizers. To derive potency information, a modification of the standard GARDskin protocol based on dose-response measurements has been proposed. The readout of the assay is a cDV0 value, which corresponds to the lowest concentration required to exceed a binary classification threshold in GARDskin. This concentration correlates significantly with LLNA EC3 and human NOEL values and linear regression models have been established to exploit these relationships for potency predictions. In this study, we explore the potential to use this novel assay for quantitative potency assessment of two acrylate monomers.

The GARDskin Dose-response assay classified both acrylate monomers as skin sensitizers with predicted LLNA EC3 values and human NOEL values of 0.848% and 22.4%, and 230 µg/cm2and 12200 µg/cm2, resulting in final classifications as a strong to moderate skin sensitizer (HP 2) and a moderate to weak sensitizer (HP 5), respectively. The results agreed with information in the ECHA registration dossiers and gathered human data evidence for the respective monomers, illustrating that GARDskin Dose-Response has the potential to replace the in vivo LLNA method for quantitative potency assessment of potential skin sensitizers during development of novel materials for use in medical devices.

Skin Sensitization Potency Assessments of Fragrance Materials using GARDskin Dose-Response

Joint poster with Research Institute for Fragrance Materials (RIFM),
Presented at the 2021 RIFM annual meeting & 2021 ACT annual meeting 

Mihwa Na, Ulrika Mattson, Robin Gradin, Henrik Johansson, Andy Forreryd, Anne Marie Api, Research Institute for Fragrance Materials, Inc., 50 Tice Boulevard, Woodcliff Lake, NJ, 07677, USA., SenzaGen AB, Lund, Sweden

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Conclusion

  • GARDskin Dose-Response closely approximated the potency categories of 9/12 fragrance materials tested.
  • Based on results from this dataset, GARDskin Dose-Response appears useful for potency assessment for weak sensitizers and may constitute a promising strategy for deriving a point of departure for quantitative risk assessments.

Abstract

Several New Approach Methods for hazard identification of skin sensitizers have been developed and incorporated as OECD Test Guidelines. However, the methods for potency assessment are still lacking. GARDskin (OECD TGP 4.106) was initially developed to identify skin sensitizers by monitoring transcriptional patterns of a biomarker signature in a dendritic like cell line.

The predictive capacity of GARDskin has been demonstrated previously, with 95.8% accuracy, 91.7% positive predictive value, and 100.0% negative predictive value (1 false positive, n=24) (Johansson, Gradin et al. 2019). To derive potency information, a strategy based on dose-response measurements in GARDskin, referred to as the GARDskin Dose-Response assay, has recently been proposed. The readout of the assay corresponds to the lowest concentration required to exceed the binary classification threshold in GARDskin. This concentration correlates with local lymph node assay (LLNA) EC3 and human no observed effect level (NOEL) values and linear regression models have been established to exploit these relationships for potency predictions.

In this blinded study, 12 fragrance materials (10 very weak sensitizers and 2 weak sensitizers) were evaluated in GARDskin Dose-Response. Results were evaluated by comparing predicted values to the reference potency categories. Three of the very weak sensitizers were predicted as non-sensitizers by GARDskin Dose-Response. For the remaining nine materials which were predicted as sensitizers, the predicted EC3 and NOEL values closely approximated the reference data for most materials. Based on results from this dataset, GARDskin Dose-Response appears useful for potency assessment for weak sensitizers and may constitute a promising strategy for deriving a point of departure for quantitative risk assessments.

Quantitative assessment of sensitizing potency using a dose-response adaptation of GARDskin

Nature Scientific Reports 11, 18904 (2021), https://doi.org/10.1038/s41598-021-98247-7

Robin Gradin, Andy Forreryd, Ulrika Mattson, Anders Jerre, Henrik Johansson

Abstract

Hundreds of chemicals have been identified as skin sensitizers. These are chemicals that possess the ability to induce hypersensitivity reactions in humans, giving rise to a condition termed allergic contact dermatitis. The capacity to limit hazardous exposure to such chemicals depends upon the ability to accurately identify and characterize their skin sensitizing potency. This has traditionally been accomplished using animal models, but their widespread use offers challenges from both an ethical and a scientific perspective. Comprehensive efforts have been made by the scientific community to develop new approach methodologies (NAMs) capable of replacing in vivo assays, which have successfully yielded several methods that can identify skin sensitizers. However, there is still a lack of new approaches that can effectively measure skin sensitizing potency. We present a novel methodology for quantitative assessment of skin sensitizing potency, which is founded on the already established protocols of the GARDskin assay. This approach analyses dose-response relationships in the GARDskin assay to identify chemical-specific concentrations that are sufficient to induce a positive response in the assay. We here compare results for 22 skin sensitizers analyzed using this method with both human and LLNA potency reference data and show that the results correlate strongly and significantly with both metrics (rLLNA = 0.81, p = 9.1 × 10–5; rHuman = 0.74, p = 1.5 × 10–3).

In conclusion, the results suggest that the proposed GARDskin dose-response methodology provides a novel non-animal approach for quantitative potency assessment, which could represent an important step towards reducing the need for in vivo experiments.

 

Key words: GARD, GARDskin, GARDskin Dose-Response, in vitro, sensitization, potency, chemical sensitizers, quantitative risk assessment

 

Full article
Article on line with open access

Quantitative Sensitizing Potency Assessment Using GARD™skin Dose-Response

Poster presented at the 2021 World Congress on Alternatives and Animal Use in the Life Sciences

Henrik Johansson, Robin Gradin, Andy Forreryd, Joshua Schmidt
SenzaGen AB, Lund, Sweden. SenzaGen Inc., Raleigh, NC.

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Conclusion

  • As an adaptation from the GARDskin assay, GARDskin Dose-Response is suitable for quantitative skin sensitizing potency assessment of chemicals.
  • The experimental readout, referred to as cDV0, corresponds to the lowest dose required to elicit a positive response in GARDskin. As such, experimental protocols are analogous to the LLNA, in which the cDV0 corresponds to the EC3-value.
  • The cDV0 may be used to directly monitor sensitizing potency, or further used to extrapolate LLNA EC3-values, estimation of Human potency categories, or CLP 1A/1B classifications.

Abstract

Proactive identification and characterization of sensitization hazards are central aspects of risk assessment of chemicals. Current legislations and trends in predictive toxicology advocate a transition from in vivo methods to non-animal alternatives, with a number of methods for hazard assessment of skin sensitizers currently available. However, non-animal methods capable of providing quantitative assessment of sensitizing potency are currently lacking.

The GARDskin assay is a next-generation in vitro assay for hazard assessment of skin sensitizers, currently progressing towards regulatory acceptance. Recently, the GARDskin Dose-Response (DR) testing strategy was introduced, in which test chemicals are evaluated by the GARDskin assay in a titrated range of concentrations, in order to investigate the dose-response relationship between GARDskin classifications and test chemical concentration. As such, it provides a quantitative estimation of sensitizing potency, referred to as cDV0, which corresponds to the least required dose able to generate a positive response in the GARDskin assay. The cDV0 value obtained for a test chemical may be viewed as an analogue to the LLNA EC3 value, based on which further hazard characterization and risk assessment may be performed. Statistically significant correlation between the GARDskin DR cDV0 and the LLNA EC3, as well as with human No Expected Sensitization Induction level (NESIL) estimations has been confirmed, thus enabling direct extrapolation between the different metrics.

Here, we further illustrate how these results can be used on their own to facilitate direct potency-associated ranking of test chemicals. Furthermore, we demonstrate how obtained cDV0 values can be extrapolated to LLNA EC3 values with a 95% confidence interval, thereby also facilitating potency-associated subcategorization of test chemicals according to UN GHS classification criteria. Lastly, we illustrate how results generated with GARDskin DR can be directly incorporated into existing strategies for Quantitative Risk Assessment using an entirely in vitro setup.

Quantitative Sensitizing Potency Assessment Using GARD™skin Dose-Response

Poster presented at SOT 2021

Henrik Johansson, Robin Gradin, Andy Forreryd, Joshua Schmidt
SenzaGen AB, Lund, Sweden. SenzaGen Inc., Raleigh, NC.

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Conclusion

  • As an adaptation from the GARDskin assay, GARDskin Dose-Response is suitable for quantitative skin sensitizing potency assessment of chemicals.
  • The experimental readout, referred to as cDV0, corresponds to the lowest dose required to elicit a positive response in GARDskin. As such, experimental protocols are analogous to the LLNA, in which the cDV0 corresponds to the EC3-value.
  • The cDV0 may be used to directly monitor sensitizing potency, or further used to extrapolate LLNA EC3-values, estimation of Human potency categories, or CLP 1A/1B classifications.

Abstract

Proactive identification and characterization of sensitization hazards are central aspects of risk assessment of chemicals. Current legislations and trends in predictive toxicology advocate a transition from in vivo methods to non-animal alternatives, with a number of methods for hazard assessment of skin sensitizers currently available. However, non-animal methods capable of providing quantitative assessment of sensitizing potency are currently lacking.

The GARDskin assay is a next-generation in vitro assay for hazard assessment of skin sensitizers, currently progressing towards regulatory acceptance. Recently, the GARDskin Dose-Response (DR) testing strategy was introduced, in which test chemicals are evaluated by the GARDskin assay in a titrated range of concentrations, in order to investigate the dose-response relationship between GARDskin classifications and test chemical concentration. As such, it provides a quantitative estimation of sensitizing potency, referred to as cDV0, which corresponds to the least required dose able to generate a positive response in the GARDskin assay. The cDV0 value obtained for a test chemical may be viewed as an analogue to the LLNA EC3 value, based on which further hazard characterization and risk assessment may be performed. Statistically significant correlation between the GARDskin DR cDV0 and the LLNA EC3, as well as with human No Expected Sensitization Induction level (NESIL) estimations has been confirmed, thus enabling direct extrapolation between the different metrics.

Here, we further introduce the GARDskin DR protocols, as proposed in a standardized testing strategy. By studying a concentration range of 6 concentration points titrated from the experimentally derived GARD input concentration in biological duplicates, a test chemical-specific cDV0 is established by linear interpolation. We illustrate how these results can be used on their own to facilitate direct potency-associated ranking of test chemicals. Furthermore, we demonstrate how obtained cDV0 values can be extrapolated to LLNA EC3 values with a 95% confidence interval, thereby also facilitating potency-associated subcategorization of test chemicals according to UN GHS classification criteria. Lastly, we illustrate how results generated with GARDskin DR can be directly incorporated into existing strategies for Quantitative Risk Assessment using an entirely in vitro setup.

 

Dose-Response Analysis in GARD™ for Assessment of Skin Sensitizer Potency

Poster presented at ACT 2020

J. Schmidt, A. Forreryd, R. Gradin, H. Johansson.
SenzaGen Inc., Raleigh, NC., SenzaGen AB, Lund, Sweden.

Link to the poster

 

Conclusion

  • As an adaptation from the GARDskin assay, GARDskin Dose-Response is suitable for quantitative skin sensitizing potency assessment of chemicals.
  • The experimental readout, referred to as cDV0, corresponds to the lowest dose required to elicit a positive response in GARDskin. As such, experimental protocols are analogous to the LLNA, in which the cDV0 corresponds to the EC3-value.
  • The cDV0 may be used to directly monitor sensitizing potency, or further used to extrapolate LLNA EC3-values, estimation of Human Potency categories, or CLP 1A/1B classifications.

Abstract

Several non-animal methods for identifying skin sensitizers have been developed with acceptable prediction performance. However, advancement of alternative methods for skin sensitizing potency assessment is still missing although a highly sought-after endpoint. The GARDskin assay is a genomics-based in vitro assay for hazard assessment of skin sensitizers, currently progressing towards regulatory acceptance. Here, we introduce GARDskin Dose-Response (DR), in which test chemicals are evaluated by the GARDskin assay in an extended range of concentrations, in order to investigate the dose-response relationship between GARDskin classifications and test chemical concentration.

For this work, 29 chemicals of various sensitizing potencies were used to evaluate the efficacy of applying the assay in this manner. Each chemical was analyzed at several concentrations using a slightly modified GARDskin protocol. At each concentration, a decision-value was produced and a classification prediction (sensitizing or non-sensitizing) was made by the GARDskin algorithm. Afterwards, the lowest concentration where a test item would provide a positive GARDskin prediction was found using linear interpolation. This concentration (cDV0) was then hypothesized to reflect the test items skin sensitizing potency. Furthermore, when comparing cDV0 to LLNA EC3 values, a statistically significant correlation was realized between the values (correlation coefficient =0.74, p-value=4.1*10-4).

These results suggest that modifying the GARDskin protocol to accommodate dose-response measurements can provide sensitizing potency information analogous to the gold-standard in vivo methods. This presentation will further explain the testing process, expand on results, and demonstrate how this method can be used for decision-making throughout all stages of product development, without having to use animal experimentation.

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