Case studies on the use of GARD®skin Medical Device for in vitro skin sensitization assessment
Presented at SOT and Eurotox 2024
Download a copy in PDF
Conclusion
In conclusion, GARDskin Medical Device can detect signals from diluted and complex extracts of solid devices, with a sensitivity superior to animal methods.
This may significantly reduce the need of animal studies, improve the safety of the final product, and avoid potential costly late-stage failures.
Abstract
The recent advancements in New Approach Methodologies enables the use of in vitro method for skin sensitization assessment as part of the biocompatibility testing for medical devices, which is conventionally tested in vivo. GARDskin OECD TG 442E is included in ISO 10993-10:2021 as the only OECD validated in vitro assay that is compatible with both polar and non-polar extraction vehicles, in line with ISO 10993-12:2021. GARDskin Medical Device is an adaptation of the GARDskin assay, including a pre-sample treatment procedure where solid devices are extracted using both polar and non-polar vehicles.
The aim of this study is to demonstrate the benefits of using GARD for early-stage screening of materials intended for use in medical devices for assessing their skin sensitization potential. Results from two case studies were summarized in which GARDskin Medical Device was used for skin sensitization assessment. The first case study describes the testing of an acrylic-based device with a coating consisting of a UV-cured lacquer, where chemical analysis indicated the potential for skin sensitization. The second case study describes the testing of a polymeric material consisting of Cellulose-Acetate Propionate (CAP) with a plasticizer (Triethylene glycol bis (2-ethylhexanoate), CAS# 94-28-0), with contradictive existing in vivo (negative) and in vitro (positive) data.
In the first case study, the acrylic-based device induced a positive response in both polar and non-polar vehicles in GARDskin Medical Device and was thus classified as a skin sensitizer. It was hypothesized that the positive results may be due to inadequate curing of the lacquer within cavitary structures of the devices, where UV light exposure was insufficient. To confirm the hypothesis, follow-up testing was performed on an identical device, but without cavities, which was classified as non-sensitizer. In vivo data confirmed the outcome of the in vitro assay. Consequently, a modification was made in the manufacturing process to prevent the presence of lacquer in cavitary structures of the device.
In the second case study, the CAP material was positive in the non-polar vehicle and was thus labelled as a skin sensitizer. The plasticizer was identified as a potential culprit, considering a borderline negative result in LLNA (SI=2.97) and reported positive clinical data. To support the hypothesis a follow-up study was conducted using another adaptation of the GARDskin protocol capable of providing continuous potency predictions. The results classified the plasticizer as a weak contact allergen. As a result, a different material was selected for the device.
Keywords: Biocompatiblity, ISO 10993-10, GARDskin Medical Device, Skin Sensitization, ISO 10993-12
Identify workplace hazards by testing skin and respiratory sensitizers
SenzaGen’s Newsletter Oct 2024
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Joint poster with L’Oreal: Point of Departure for risk assessment
Improved Confidence of Quantitative Sensitizing Potency Assessment for Point of Departure Using GARD®skin Dose-Response
Presented at SOT and Eurotox 2024
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In conclusion, the readouts from GARDskin Dose-Response derive a quantitative continuous potency estimate of skin sensitizers that may be used directly as a PoD for a seamless integration into downstream NGRA.
Identification of skin sensitization hazard and potency characterization are central aspects of risk assessment of chemicals. Current legislation advocates a transition from hazard assessment using in vivo methods to UN GHS potency subclassification and quantitative risk assessment by use of New Approach Methodologies (NAM:s) as well as Defined Approaches (DA). However, the ability of NAM assays to quantitatively estimate sensitizing potency and thereby establish a point of departure (POD) for next-generation risk assessment (NGRA) strategies is currently lacking.
To this end, the GARDskin Dose-Response (DR) method, adapted from the OECD TG 442E method GARDskin, was recently introduced. The GARDskin DR method evaluates test chemicals in a titrated range of concentrations, in order to investigate the dose-response relationship between the output from the GARDskin prediction algorithm (Decision Values; DV:s) and test chemical concentration. The combined information can be used to derive a quantitative estimation of sensitizing potency, defined as the cDV0-value, i.e, the least required dose required to elicit a positive response by the prediction model.
The current work focuses on optimizing the ability of GARDskin DR to derive a quantitative POD based on conversion to a composite Potency Value (PV; µg/cm2), taking into account both human and in vivo reference data sources. A total of 25 chemicals were used to construct predictive regression models fitted to reference PV:s. Results show that the updated models fitted to reference PV:s produced more accurate potency predictions compared with models fitted with, and aiming to predict, only LLNA EC3 and NOEL, respectively. Mean fold-change errors ranged between 2.8 and 3.2, with predicted POD:s being within or close to the range of the variation of the historical in vivo data. In addition, uncertainty in predictions was reduced, as estimated by a minimum 2-fold reduction of 95%-confidence intervals, when comparing models fitted to reference PV:s with models fitter with only LLNA EC3 and human NOEL, respectively.
In conclusion, these improvements constitute a major step forward for the ability of NAM:s to assess quantitative sensitizing potency. It demonstrates how GARDskin Dose-Response can accurately estimate a POD and be incorporated into downstream strategies for quantitative risk assessment (QRA), to ultimately contribute to the assessment of safe use levels of chemicals.
Keywords: NAM, GARDskin Dose-Response, Sensitizing potency, Quantitative risk assessment, Point of departure
Joint poster with Cargill: Case study on Hydrophobic Esters
The applicability of GARD®skin for assessing skin sensitization potential of hydrophobic esters during product development
Presented at Eurotox 2024
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In conclusion, the inclusion of GARD®skin in the OECD test guidelines has expanded the applicability of NAMs for skin sensitization assessment.
This study demonstrates the applicability of the GARDskin assay to assess skin-sensitizing hazard of hydrophobic esters, which provides an ethical alternative to animal methods for safety assessment during product development.
The field of skin sensitization assessment is rapidly evolving and the recent advancements in New Approach Methodologies (NAMs) has made it possible for the industry to perform in vitro skin ssensitization testing with good predictivity across a large chemical space. However, challenges remain for “difficult-to-test” chemicals, those with challenging physical/chemical properties or of Unknown or Variable composition, Complex reaction products or Biological materials (UVCBs), which are often outside the applicability domain of conventional cell-based assays. GARDskin (OECD TG 442E) is a genomic-based assay with demonstrated applicability to “difficult-to-test” substances.
The aim of this study is to assess the skin sensitization potential of two ester substances of biological origin, substance A and B, using the GARDskin assay. These substances are very hydrophobic and fall outside of the applicability domain of the conventional in vitro assays.
Both substances were successfully solubilised in cell media by utilizing a combination of heating, sonication, and selection of appropriate solvent vehicles (ethanol or acetone). No cytotoxicity was observed for either substance, thus 500 μM was chosen as the input concentration for cellular stimulations. GARDskin combines a genomic readout with machine learning to predict skin sensitizing hazard, where values above the threshold (DV=0) is predicted as skin sensitizing and below as non-sensitizing. Both substances resulted in negative mean Decisions Values and thus were classified as non-sensitizers in GARDskin (A: -1.54, B: -0.339).
In conclusion, with the inclusion of GARDskin into the OECD test guidelines, the range to where NAMs are appropriate has been increased. This study demonstrates the applicability of the GARDskin assay to assess skin sensitizing hazard of hydrophobic ethyl esters, which provides an ethical alternative to animal methods for safety assessment during product development.
Keywords: NAM, skin sensitization, difficult-to-test, UVCB, Applicability domain
Joint poster with Sonova: Integrating NAMs into early-stage screening of novel materials
Case studies on the use of GARD®skin Medical Device for in vitro skin sensitization assessment
Presented at SOT and Eurotox 2024
Download a copy in PDF
In conclusion, GARDskin Medical Device can detect signals from diluted and complex extracts of solid devices, with a sensitivity superior to animal methods.
This may significantly reduce the need of animal studies, improve the safety of the final product, and avoid potential costly late-stage failures.
The recent advancements in New Approach Methodologies enables the use of in vitro method for skin sensitization assessment as part of the biocompatibility testing for medical devices, which is conventionally tested in vivo. GARDskin OECD TG 442E is included in ISO 10993-10:2021 as the only OECD validated in vitro assay that is compatible with both polar and non-polar extraction vehicles, in line with ISO 10993-12:2021. GARDskin Medical Device is an adaptation of the GARDskin assay, including a pre-sample treatment procedure where solid devices are extracted using both polar and non-polar vehicles.
The aim of this study is to demonstrate the benefits of using GARD for early-stage screening of materials intended for use in medical devices for assessing their skin sensitization potential. Results from two case studies were summarized in which GARDskin Medical Device was used for skin sensitization assessment. The first case study describes the testing of an acrylic-based device with a coating consisting of a UV-cured lacquer, where chemical analysis indicated the potential for skin sensitization. The second case study describes the testing of a polymeric material consisting of Cellulose-Acetate Propionate (CAP) with a plasticizer (Triethylene glycol bis (2-ethylhexanoate), CAS# 94-28-0), with contradictive existing in vivo (negative) and in vitro (positive) data.
In the first case study, the acrylic-based device induced a positive response in both polar and non-polar vehicles in GARDskin Medical Device and was thus classified as a skin sensitizer. It was hypothesized that the positive results may be due to inadequate curing of the lacquer within cavitary structures of the devices, where UV light exposure was insufficient. To confirm the hypothesis, follow-up testing was performed on an identical device, but without cavities, which was classified as non-sensitizer. In vivo data confirmed the outcome of the in vitro assay. Consequently, a modification was made in the manufacturing process to prevent the presence of lacquer in cavitary structures of the device.
In the second case study, the CAP material was positive in the non-polar vehicle and was thus labelled as a skin sensitizer. The plasticizer was identified as a potential culprit, considering a borderline negative result in LLNA (SI=2.97) and reported positive clinical data. To support the hypothesis a follow-up study was conducted using another adaptation of the GARDskin protocol capable of providing continuous potency predictions. The results classified the plasticizer as a weak contact allergen. As a result, a different material was selected for the device.
Keywords: Biocompatiblity, ISO 10993-10, GARDskin Medical Device, Skin Sensitization, ISO 10993-12
Joint publication with L’Oréal: In vitro prediction of skin sensitizing potency using the GARDskin Dose-Response assay: A simple regression approach
New joint publication with L’Oréal.
We are excited to announce the recent publication of a collaborative scientific paper by the expert teams at L’Oréal and SenzaGen, in Toxics MDPI. This peer-reviewed article presents new evidence on the performance of the GARD®skin Dose-Response for quantitative potency assessment of skin sensitizers.
With an extended set of 30 chemicals and a composite potency model for the prediction of sensitizing potency, the study demonstrates the ability of GARD®skin Dose-Response to predict a Point-of-Departure (PoD) for potential skin sensitizers, showing concordance with NESIL values derived from LLNA and Human data.
This research represents a significant advancement in deriving PoD values for chemicals which can be used directly in improving downstream risk assessment strategies.
Keywords
NAM; GARDskin Dose-Response; Sensitizing potency; Quantitative risk assessment; Point of departure
Abstract
Toxicological assessments of skin sensitizers have progressed towards a higher reliance on non-animal methods. Current technological trends aim to extend the utility of non-animal methods to accurately characterize skin sensitizer potency.
The GARDskin Dose-Response assay was previously described where it was shown that its main readout, the cDV0 concentration, was associated with skin sensitizing potency. The ability to predict potency in the form of NESILs derived from LLNA or human NOEL, from cDV0, was evaluated. The assessment of a dataset of 30 chemicals showed that the cDV0 values still correlated strongly and significantly with both LLNA EC3 and human NOEL values (ρ = 0.645-0.787 [p < 1×10-3]).
A composite potency value that combined LLNA and human potency data was defined, which aided the performance of the proposed model for the prediction of NESIL. The potency model accurately predicted sensitizing potency, with cross-validation errors of 2.75 and 3.22 fold changes compared with NESILs from LLNA and human, respectively.
In conclusion, the results suggest that the GARDskin Dose-Response assay may be used to derive an accurate quantitative continuous potency estimate of skin sensitizers.
CEO comments on successful financing round to accelerate portfolio expansion
We are excited to announce that SenzaGen has secured financing to accelerate portfolio expansion within non-animal testing.With this, we are poised for an even brighter growth path.
The funding enables us to better meet customer needs within in vitro testing, fostering innovation and advancing non-animal testing solutions.
CEO Peter Nählstedt comments: “This achievement underscores our commitment to non-animal testing, innovation, and excellence. We are proud of the progress we have made and look forward to continuing to drive the transition from animal testing to methods better suited to reflect human biology. This will contribute to creating safer and more efficacious products for society.”
CEO interview by Redeye analyst Gustaf Meyer (ENG)
SenzaGen’s Newsletter April 2024
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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.
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
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.
Evaluation of the applicability of GARDskin to predict skin sensitizers in extracts from medical device materials
Peer-reviewed article in Frontiers in Toxicology.
The application of GARD®skin to predict potential skin sensitizers in extracts from Medical Device materials has recently been published in a peer-reviewed article in Frontiers in Toxicology.
The study results provide evidence recognizing the GARD®skin Medical Device assay as a scientifically sound and ethical alternative to conventional animal methods, compatible with both polar and non-polar extraction vehicles, in line with the ISO 10993-12:2021 standard.
We are proud to provide the only OECD-validated in vitro assay for sensitization that is fully compatible with testing requirements specified in ISO 10993-12.
Keywords
GARD™; In vitro; Skin sensitisation; NAMs; Medical Device, ISO 10993, Biocompatibility
Abstract
Biocompatibility testing of medical devices is governed by the ISO 10993 series of standards and includes evaluation of skin sensitization potential of the final product. A majority of all medical devices are tested using in vivo methods, largely due to the lack of in vitro methods validated within the applicability domain of solid materials. The GARDskin method for assessment of chemical skin sensitizers is a validated method included in the OECD Test Guideline 442E, based on evaluation of transcriptional patterns of an endpoint-specific genomic biomarker signature in a dendritic cell-like cell, following test chemical exposure. The current study aimed to evaluate the applicability of GARDskin for the purpose of testing solid materials by incorporation of extraction procedures described in ISO 10993-12:2021, as well as to demonstrate the functionality of the proposed protocols, by testing of custom-made materials spiked with sensitizing agents. It was shown that GARDskin is compatible with both polar and non-polar extraction vehicles frequently used for the purpose of medical device biological testing. Further, exploring three different material types spiked with up to four different sensitizing agents, as well as three unspiked control materials and commercial reference products, it was shown that the method correctly classified all evaluated test materials. Taken together, the data presented suggest that GARDskin may constitute a valid alternative to in vivo experimentation for the purpose of skin sensitization assessment of medical devices.
Joint publication with Corteva Agriscience: GARD™skin and GARD™potency: A proof-of-concept study investigating applicability domain for agrochemical formulations
New joint publication with Corteva Agriscience.
SenzaGen scientists, alongside the toxicology team at Corteva Agriscience, have recently published a joint study in Regulatory Toxicology and Pharmacology, presenting new peer-reviewed evidence on the applicability of GARD® for agrochemical formulations.
The study demonstrates a satisfactory performance of GARD®skin and GARD®potency for skin sensitization hazard and GHS potency categorization of tested agrochemical formulations.
Keywords
GARD™; In vitro; Skin sensitisation; NAMs; Agrochemical formulations
Highlights
Tested 42 agrochemical formulations to expand applicability domain of GARD.
Abstract
Several New Approach Methodologies (NAMs) for hazard assessment of skin sensitisers have been formally validated. However, data regarding their applicability on certain product classes are limited. The purpose of this project was to provide initial evidence on the applicability domain of GARD™skin and GARD™potency for the product class of agrochemical formulations.
For this proof of concept, 30 liquid and 12 solid agrochemical formulations were tested in GARDskin for hazard predictions. Formulations predicted as sensitisers were further evaluated in the GARDpotency assay to determine GHS skin sensitisation category. The selected formulations were of product types, efficacy groups and sensitisation hazard classes representative of the industry’s products.
The performance of GARDskin was estimated by comparing results to existing in vivo animal data. The overall accuracy, sensitivity, and specificity were 76.2% (32/42), 85.0% (17/20), and 68.2% (15/22), respectively, with the predictivity for liquid formulations being slightly higher compared to the solid formulations. GARDpotency correctly subcategorized 14 out of the 17 correctly predicted sensitisers. Lack of concordance was justifiable by compositional or borderline response analysis. In conclusion, GARDskin and GARDpotency showed satisfactory performance in this initial proof-of-concept study, which supports consideration of agrochemical formulations being within the applicability domain of the test methods.