SenzaGen’s Newsletter April 2023
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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
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.
In vitro assays for assessment of the skin sensitization hazard and potency of isobornyl acrylate
Presented at SOT 2023
In vitro assays for assessment of the skin sensitization hazard and potency of isobornyl acrylate
Rose-Marie Jenvert1, Olivia Larne1, Ulrika Mattson1, Robin Gradin1, Kelly P Coleman2 and Andy Forreryd1 | 1SenzaGen, Lund, Sweden, 2Medtronic, Minneapolis, USA
Conclusion
The GARDskin Medical Device assay can be used
- with the extraction vehicles saline and oil as described
in ISO 10993. - to detect low concentrations of skin sensitizers in a mixture and have the potential to replace in vivo tests
for risk assessment of skin sensitizers in medical devices. - as risk assessment tool in combination with the
GARDskin Dose-Reponse assay during development of medical devices containing acrylates.
Abstract
Acrylates have a wide range of applications for medical devices as they can bring several advantages such as transparency, super-absorbency and hardness in combination with flexibility. The manufacturing of acrylic materials typically involves using at least one monomer of either acrylate or methacrylate which react to form a polymer. Several methodologies can be used for polymerization and the degree of polymerization of the final material can vary, hence some products can contain more residual monomers than others, and human exposure to these well-known skin sensitizers may increase the risk of developing the adverse immunological response, allergic contact dermatitis.
In 2020, the acrylate monomer, isobornyl acrylate (IBOA; CAS 5888-33-5), was named allergen of the year by American Contact Dermatitis Society due to the increased number of patients that were sensitized to IBOA found in glucose sensors and glucose pumps. IBOA is also present in other medical devices as plastic materials, coatings, sealants, glues, adhesives and inks. As a result, it is important to find alternatives to the animal methods used today to assess skin sensitization to properly identify the skin sensitizing potential of IBOA in medical devices and avoid the risk of sensitizing more individuals to this chemical.
The GARDskin (OECD TG 442E) assay, initially developed for hazard identification of a wide range of skin sensitizers, has been adapted for use with polar and non-polar solvents as described in ISO 10993-12:2021 and can be applied to assess the skin sensitization of medical devices. Further development of the GARDskin protocol has also enabled the prediction of skin sensitization potency by using a dose-response measurements. The cDV0 value derived from the assay corresponds to the lowest concentration required to exceed a binary classification threshold in GARDskin, and this concentration correlates significantly with LLNA EC3 and human NOEL values. Linear regression models have been established to exploit these relationships for potency predictions.
In this study, we explored the ability of GARDskin Medical Device assay to detect the skin sensitizing potential of IBOA in a mixture of chemicals extracted from a silicone material (Nunsil MED-2000) in both saline and oil extract. Additionally, the skin sensitizing potency of IBOA was also investigated using the novel GARDskin Dose-Response assay. The acrylate monomer, IBOA, was correctly classified as a skin sensitizer in extracts of the silicon material spiked with IBOA using the GARDskin Medical Device assay. The correct classification of IBOA as a skin sensitizer provides evidence that the GARDskin Medical Device assay is sensitive enough to detect low concentrations of device-related skin sensitizers in a mixture of extracted chemicals and hence has the potential to replace in vivo tests for risk assessment of medical devices. Furthermore, IBOA was classified as a strong to moderate skin sensitizer (HP 2) with a predicted LLNA EC3 value of 0.848% and human NOEL value of 230 µg/cm2 using the GARDskin Does-Response assay. These predictions agree with existing human data and information from the ECHA registration dossier, illustrating the potential of the GARDskin Dose-Response assay to replace in vivo tests for quantitative potency assessment of potential skin sensitizers.
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
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
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 SOT 2023
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.
SenzaGen has acquired ToxHub to strengthen toxicology offering
ToxHub has joined the SenzaGen Group as independent regulatory toxicology experts. Our customer-centric in vitro testing services and cutting-edge GARD® technologyies have been complemented by ToxHub’s tailored toxicology advisory services. Based in Rome, Italy, ToxHub specializes in toxicological risk assessment and regulatory strategy consulting, with specific expertise in medical devices and pharmacology.
About ToxHub
Founded by toxicologists with 25-years of experience in the pharmaceutical industry, ToxHub provides tailored and cost-effective toxicology advisory services to customers across industries. ToxHub’s risk assessment and regulatory compliance services include the following key areas:
- Hazard identification, hazard characterization including dose-response analysis to define a Point of Departure, exposure evaluation and risk characterization for pharmaceutical and non-pharmaceutical ingredients, chemicals, contaminants and impurities.
- Regulatory strategy advisory and compilation of regulatory documentation (IB, IMPDs, INDs, etc).
- Regulatory support when interacting with authorities, including responses to deficiency letters.
- Biological evaluation of medical devices (MDR).
https://toxhub-consulting.com
Joint poster with Coty: Case study on Cosmetic Fragrance Formulations
EFFECT OF A MODULATOR ON THE SKIN SENSITIZATION POTENCY OF COSMETIC FRAGRANCE FORMULATIONS
Presented at 2022 ESTIV
Conclusion
The GARDskin Dose-Response assay, thanks to its ability to obtain continuous potency predictions, in our case on complex mixtures, allowed to identify a reduction of the skin sensitization potency following the spike of a modulator into fragrance formulations.
Analyzing different fragrance modulators at varying doses, it can be concluded that modulator 1 allowed a reduction of the sensitizing potential of Formula 1 at its two highest tested doses. The observed effect was not dose dependent. At its highest concentration, the same effect was observed in Formula 2 for the two samples that contain the same amount of fragrance oil than Formula 1. The effect of modulator 1 was observed on the two fragrance designs studied. In contrast, modulators 2 and 3 were not efficient in impacting the sensitizing effect of Formula 1 at the tested concentrations. Only modulator 1 was of interest regarding sensitizing potency reduction in a fragrance formula.
The addition of the modulator 1 on the 2 lowest concentrations of perfume oil in Formula 2 generated, as previously, a similar reduction of the sensitization, contrary to the highest concentration for which no effect was observed. By varying the proportion of fragrance oil in the Formula 2, we were able to demonstrate the existence of a threshold from which the effect of modulator 1 on the sensitizing potential of this formula was no longer detected.
In conclusion, modulator 1 appeared to be of interest showing an ability to reduce the sensitizing potential of the tested fragrance formulations. It could be interesting to evaluate its impact on other cosmetic or fragrance formulas containing different fragrance oils and perfume raw materials. Another next step should be to define the lowest concentration of modulator 1 allowing a significant reduction of the skin sensitizing potential of perfume formulas.
Abstract
Perfume long lasting is an important concern that is widely addressed in fragrance research and innovation. To this end, there is a need for new technologies to prolong the perception and intensity of fragrances over time. The intended function of fragrance modulators is to slow down the evaporation rate of perfume raw materials. But while they may improve fragrance properties, they may also impact the toxicological profile of the final formulation. This work evaluated the impact of a modulator on the skin sensitizing potency of a fragrance formulation using the GARDskin Dose-response assay.
GARDskin Dose-Response is a modification of the validated protocols of GARDskin (OECD TGP 4.106) that incorporates dose-response analysis. The readout is a cDV0 value, describing the lowest concentration required to generate a positive classification. This value correlates with potency and can be used to rank test items by their relative sensitizing potency. The assay was used due to its capacity to evaluate complex mixtures and because it delivers continuous potency predictions, which was crucial for effectively comparing the modulator’s effect on the formulation’s skin sensitizing potency.
This study examined the effect on a formulation’s skin sensitizing potency when a modulator was added by assaying two otherwise identical formulations. Testing was performed using GARDskin Dose-Response, and the derived cDV0 values were compared using 95% confidence intervals (CI).
Fragrance formulations gave rise to monotonically increasing dose-response curves and cDV0 values were estimated. The cDV0 value for the fragrance formulation containing the modulator was significantly higher (458ppm, 95%CI: 332-626) compared with the cDV0 value for the naïve fragrance formulation (268ppm, 95%CI: 248-292), indicating a potential for the modulator to reduce the sensitization potency of the evaluated fragrance formulation.
Based on the encouraging data reported in this study, the modulator appears to reduce the sensitization potency of the evaluated fragrance mixture.
Joint poster with DSM: Case study on process-related impurities in polymeric materials
In vitro assessment of skin sensitizing potential of process-related impurities in polymeric materials during product development.
Presented at 2022 ESTIV
Abstract
Andy Forreryd1, Stefan Kaiser2, Roman Goy2, Florian Glaus2, Ulrika Mattson1, Robin Gradin1, Henrik Johansson1
1 SenzaGen AB, 22381 Lund, Sweden
2 DSM Nutritional Products Ltd, 4303 Kaiseraugst, Switzerland
Skin sensitization testing represents a key toxicological endpoint during safety evaluation of ingredients intended for consumer products with topical exposure, both in a regulatory context and during product development, to early understand the toxicological profile of the end-product and potential impurities.
The inclusion of New Approach Methods (NAMs) for skin sensitization testing into OECD TGs have resulted in a broader acceptance of such methods as replacements to animal models. However, these methods are not universally applicable, and compounds with certain properties, such as lipophilicity, or of complex composition, are frequently considered outside the applicability domain.
GARDskin is a genomic-based next-generation in vitro assay for assessment of skin sensitizers progressing towards regulatory acceptance. The assay is highly sensitive, is compatible with a variety of solvents and has a demonstrated applicability for testing of lipophilic materials.
The aim of this study was to evaluate the skin sensitization potential of a lipophilic polymeric material (Mw > 2000g/mol) containing approximately 2% impurities, during product development. The polymeric material was initially flagged as a skin sensitizer. A preparative procedure was applied to purify the polymer from impurities, while generating enriched by-product fractions (oligomeric fraction, Mw: 500-1000 g/mol and small molecule fraction, Mw: <500 g/mol). The fractions were evaluated in GARDskin, using acetone or DMSO as solvents, and classified as skin sensitizers (by-products) and non-sensitizers (purified polymers, n=2), respectively, indicating that the impurities were responsible for the positive classification of the initial non-purified polymeric material.
In conclusion, the GARDskin protocol enabled for testing of the lipophilic materials, using a selection of solvents to increase solubility. Results from this study informed that actions to reduce concentration of impurities may be a useful strategy to prevent skin sensitization properties of the final end-product, highlighting the importance of skin sensitization testing during the production development.
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