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.
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
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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.
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
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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The GARDskin assay: Investigation of the applicability domain for metals
Joint publication with Johnson Matthey
ALTEX – Alternatives to animal experimentation, published Nov 03, 2022, accepted manuscript
DOI: https://doi.org/10.14573/altex.2203021
Forreryd, A., Gradin, R., Larne, O., Rajapakse, N., Deag, E. and Johansson, H.
Abstract
New approach methods (NAMs) for hazard identification of skin sensitizing chemicals have been adopted as test guidelines by the OECD during the last decade as alternatives to animal models. These models align to individual key events (KE) in the adverse outcome pathway (AOP) for skin sensitization for which the molecular initiating event (MIE) is covalent binding to proteins. As it currently stands, the AOP does not include mechanistic events of sensitization by metals, and limited information is available on whether NAMs accurately the predict sensitization potential of such molecules, which have been proposed to act via alternative mechanisms to organic chemicals.
Methods for assessing the sensitization potential of metals would comprise valuable tools to support risk management within e.g., occupational settings during production of new metal salts or within the medical device industry to evaluate leachables from metal alloys.
This paper describes a systematic evaluation of the applicability domain of the GARD™skin assay for assessment of metals. Hazard classifications were supplemented with an extended analysis of gene expression profiles induced by metal sensitizers to compare the induction of toxicity pathways between metals and organic sensitizers. Based on the results of this study, the accuracy, sensitivity, and specificity of GARD™skin for prediction of skin sensitizing hazard were 92% (12/13), 100% (7/7) and 83% (5/6), respectively.
Thus, the performance of GARD™skin for assessment of metals was found to be similar to what is observed on conventional organic substances, providing support for inclusion of metals within the applicability domain of the test method.
Keywords
skin sensitization, metals, regulatory testing, medical devices
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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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
Download a copy
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.
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.
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.
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Laboratory Technicians till SenzaGen i Lund
Vår verksamhet i Lund växer starkt och vi söker fler drivna medarbetare till vårt labb
SenzaGen avser att bli en ledande aktör inom djurfri toxikologisk testning och driva omställningen från djurtester till metoder som bättre överensstämmer med hur människor biologiskt fungerar. Som innovativt bolag har SenzaGen utvecklat en teknologiplattform som ersätter djurförsök vid bedömningar om kemikalier kan orsaka allergiska reaktioner på huden eller i luftvägarna. GARD-plattformen kombinerar genomiska data från mänskliga celler med maskininlärning vilket gör metoden både effektivare och mer träffsäker än traditionella djurbaserade metoder. Nyligen godkändes testmetoden GARDskin av OECD och bolaget står nu inför sitt kommersiella genombrott.
Älskar du att labba och vill vara med på en spännande tillväxtresa?
Vi söker två Laboratory Technicians som är nyexaminerade eller med något års erfarenhet, som vill arbeta med den senaste tekniken för att bedöma om kemikalier kan påverka människors hälsa. Hos oss får du arbeta med engagerade och drivna kollegor, utvecklas inom ditt arbetsområde samt arbeta med ett meningsfullt uppdrag. Organisationen präglas av en entreprenörsdriven kultur där alla medarbetare arbetar prestigelöst mot tydliga och ambitiösa mål och där varje medarbetares bidrag gör stor skillnad för bolagets väg framåt.
Dina arbetsuppgifter
Din utbildning, erfarenhet och personliga egenskaper
Du är utbildad till Biomedicinsk analytiker (BMA) eller har en annan likvärdig universitetsutbildning. Du talar och skriver flytande svenska samt behärskar engelska på en hög kommunikativ nivå. Det är meriterande om du har erfarenhet av laboratoriearbete inom cell- eller molekylärbiologi och erfarenhet av att arbeta i ett kommersiellt ackrediterat laboratorium.
Som person är du är ansvarstagande, noggrann och förstår värdet av dokumentation och kvalitetsarbete i laboratoriet. Samtidigt är du bra på att hitta lösningar, är flexibel och kan hantera förändringsarbete i en kommersiellt driven verksamhet. Du gillar att arbeta i team men trivs också med att ta eget ansvar och leverera resultat med hög kvalitet och enligt tidsplan.
Vi erbjuder
Heltidsarbete, 40h/vecka, i bolagets lokaler i Lund. I tjänsten kan det framöver komma att ingå även schematjänstgöring på kvällar och helger.
Ansökan
Vi är inne i en semesterperiod vilket innebär att vår tillgänglighet för frågor är något begränsad. Intervjuerna beräknas börja i augusti.
Skicka din ansökan med CV och personligt brev till Åsa Nyhlén, VP Operations senast den 31 juli 2022: asa.nyhlen@senzagen.com.
The GARDskin Assay: Investigation of the Applicability Domain of Indirectly Acting Haptens
Presented at the 2022 SOT
Tim Lindberg1, Andy Forreryd1, Robin Gradin1 and Henrik Johansson1
1SenzaGen, Lund, Sweden
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Hypersensitivity reactions in the skin, clinically manifested as Allergic Contact Dermatitits (ACD), are caused by the ensuing immunological response to low-molecular weight compounds termed skin sensitizers. Such substances, often referred to as haptens, have the inherent property to react with skin proteins and form immune inducing complexes. However, indirectly acting haptens need to be transformed to protein-reactive intermediates either through biotic (pro-hapten) or abiotic (pre-hapten) conversion in order to elicit an immune response.
Conventionally, safety tests of skin sensitizers have been done using animal experiments, but New Approach Methodologies (NAMs) have been developed over the past decades to replace the use of animals in such testing. However, one potential problem faced with the in vitro and in chemico alternatives is the lack of metabolic and chemical activity as compared to an in vivo system, which in turn may lead to false predictions for pre- and pro-haptens.
The GARDskin assay is a next-generation NAM for hazard classification of skin sensitizers. 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. Currently, the method is approaching regulatory acceptance as an OECD test guideline.
The study presented here aimed to explore the applicability domain of the GARDskin assay, specifically the capability to predict indirectly acting haptens. Available data obtained from GARDskin testing of indirectly acting haptens were compiled, resulting in a set of 28 substances. Further subcategorization identified 5 pro-haptens and 11 pre-haptens, while 12 substances were unable to be unambiguously assigned as either exclusively a pro- or a pre-hapten, due to the dual nature of the protein-reactive activity. Skin sensitizing hazard sensitivity of indirectly acting haptens (n=28) was 89% (25/28) while pro-haptens (n=5) and pre-haptens (n=11) were 80% and 100%, respectively. These data support GARDskin applicability in the domain of indirectly acting haptens, demonstrating that the method has the capacity to accurately assess both pre- and pro-haptens.