SenzaGen’s Newsletter Q4 2025
Click image to view the Newsletter in browser.
Text version:
SenzaGen Newsletter Q4, 2025
As 2025 draws to a close: |
||||||||||||||||||||||
This quarter caps a year of significant progress in advancing non-animal sensitization testing and closing critical data gaps. Combing genomics and machine leaning, GARD® continues to deliver solutions for Safe Dose Levels, Medical Devices, and Challenging Chemicals—driving the shift toward reliable, human-relevant approaches.
|
SenzaGen’s Newsletter Q3 2025
Click image to view the Newsletter in browser.
|
|
|
|
|
|
|
|
Joint poster with Urgo: Supporting hypoallergenicity claims for Medical Wound Dressings
Supporting industry decision-making and hypoallergenicity claims using an experimental setup combining GARD®skin Medical Device and Dose-Response protocols.
Presented at 2025 Eurotox
Conclusion
This case study demonstrates how NAMs like GARD® can be effectively integrated into product development for assessing skin sensitization potential in medical device extracts. It enables evidence-based decision-making and supports hypoallergenic claims, without the use of animal testing.
Abstract
Biocompatibility testing is a crucial part of medical device safety assessments both for regulatory acceptance of finished products but also within the developmental pipelines of novel devices. Along with cytotoxicity and irritation, skin sensitization is a key toxicological endpoint that must be assessed for all devices. While testing for the two former endpoints is partly conducted in vitro, traditional animal experimentations for skin sensitization testing are still routinely conducted. Thus, validation and implementation of New Approach Methods (NAMs) remains a prioritized goal of the medical device industry.
The GARDskin method (OECD 442E) is a NAM for hazard assessment of skin sensitizers based on test chemical exposure of dendritic cell-like cells followed by a genomic readout. Several method adaptations have been proposed in order to address specific needs of various industry segments. Such adaptations include the GARDskin Dose-Response (DR) method, which facilitates quantitative assessment of sensitizing potency, and the GARDskin Medical Device (MD) method, which facilitates testing of solid materials in accordance with ISO 10993 standards.
Here we report an experimental setup combining the protocols of above-mentioned GARDskin adaptations used to evaluate the relative hypoallergenicity of two sensitive skin dressings: a candidate product and a marketed comparative product. The extraction from the solid test items was performed according to ISO 10993-12, but with an increased test item to extraction vehicle ratio of 6x. The oversaturated extracts were subsequently subjected to serial dilution facilitating dose-response testing in order to evaluate, assess and compare the test items’ relative skin sensitizing potentials.
The results concluded that both test items were classified as non-sensitizers. Due to the oversaturation extraction procedure, otherwise adhering to the ISO 10993-12 standards, the results are noteworthy. Indeed, due to the manyfold increase in material to vehicle ratio, labelling the tested skin dressings as hypoallergenic, or otherwise suitable for sensitive use, may be supported by the data. Furthermore, evaluation of the relative difference between the two test items concluded that the comparative test item induced elevated response values at the highest tested concentration, whereas the candidate test item did not. As such, it could be argued that the candidate product can be claimed as hypoallergenic similarly to the comparative product.
In summary, this experimental setup combining GARDskin MD and GARDskin DR protocols can successfully be used to support claims of hypoallergenicity of medical devices, as well as comparing candidate products with respect to their relative skin sensitizing potential. These results illustrate how such strategies can be routinely integrated in the development of novel devices giving data for critical decision-making without resorting to animal experimentation for regulatory approval.
Joint poster with BIC: Case study on skin-binding dyes
In vitro skin sensitization potency assessment using GARD®skin Dose-Response: A case study on natural extracts-based skin-binding dyes and dye precursors
Presented at 2025 Eurotox
Conclusion
- GARD® skin Dose-Response provides ready-to-use NESIL values, supporting the establishment of safe dose levels for skin sensitizers.
- The assay is a non-animal alternative for skin sensitization potency testing, enabling potency ranking of candidate products and quantitative risk assessment.
Abstract
Naturally occurring skin dyes and dye precursors are widely used in temporary tattoo products, evolving from unrefined fruit extracts to more refined formulations in various ink applications. Evaluating their skin sensitization potential and establishing safe use levels for potential sensitizers are critical for product safety, which has traditionally relied on animal testing and/or human patch testing. While New Approach Methodologies (NAMs) have been developed to replace in vivo assays, a need remains for methods that can effectively and quantitatively characterize skin sensitizing potency.
GARD®skin (OECD TG 442E) is an in vitro assay that identifies chemical skin sensitizers based on the transcriptional profiling of a 196-gene biomarker signature in the dendritic-like SenzaCell® cell line. Predictions are made using a machine-learning algorithm, which classify test chemicals as sensitizers or non-sensitizers based on the assay’s readout, Decision Values (DVs). GARD®skin Dose-Response (OECD TGP 4.106) extends this approach by evaluating test chemicals across a concentration range to establish a dose-response relationship between DVs and test chemical concentration. Sensitizing potency is quantified using cDV0, the lowest dose required to elicit a positive response in GARD®skin. Depending on the need, the readout can be used to predict LLNA EC3 values, No Observable Effect Levels (NOELs)/No Expected Sensitization Induction Levels (NESILs), Human Potency (HP) Categories, and UN GHS/CLP classification (1A or 1B), all with high statistical significance.
This case study demonstrates the application of GARD®skin Dose-Response to quantitatively assess the skin sensitization potency of naturally occurring dye precursor that binds covalently to skin molecules, highlighting its use in temporary tattoo inks. Seven analogue molecules with different functional groups were investigated. While six exhibited varying degrees of sensitization potency with mean cDV0 values ranging from 3.82 µM to 34.3 µM, one functional group did not induce a positive response at any of the assayed concentrations and was subsequently classified as a non-sensitizer. Predicted LLNA EC3 values ranged from 0.60% to 4.72%, corresponding to Human Potency Categories HP2 to HP4, with NESIL estimates between 258 to 2840 µg/cm².
These findings demonstrate that GARD®skin Dose-Response provides ready-to-use NESIL values, enabling potency ranking of candidate ingredients and supporting the safety assessment of novel dyes and dye precursors. The study further highlights the assay’s value as a non-animal alternative for skin sensitization potency assessment in product development.
Joint poster with dōTERRA: Case study on Essential Oils
Comparative analysis of skin sensitization thresholds for Essential Oils: Human, murine, and GARD®skin Dose-Response
Presented at 2025 Eurotox
Conclusion
- NESIL predictions from GARD® were consistent with other skin sensitization data, particularly when compared to reference human data (HRIPT) and constituent predictions.
- The assay supports quantitative skin sensitization potency assessment of complex mixtures, offering a reliable non-animal alternative to traditional testing methods.
Abstract
Essential oils (EOs), widely used in consumer products, require robust skin sensitization hazard and potency assessment. However, dose-response thresholds for EOs remain understudied, and current classifications under CLP mixture criteria are often overly conservative. Traditional methods, such as animal testing and human patch tests, face ethical concerns, regulatory restrictions, and reliability issues. While New Approach Methodologies (NAMs) address some of these challenges, most of them are validated only for hazard identification and lack quantitative potency assessment capabilities.
GARD®skin (OECD TG 442E) is an in vitro assay that identifies chemical skin sensitizers based on the transcriptional profiling of a 196-gene biomarker signature in the dendritic-like SenzaCell® cell line. Predictions are made using a machine-learning algorithm, which classify test chemicals as sensitizers or non-sensitizers based on the assay’s readout, Decision Values (DVs). GARD®skin Dose-Response (OECD TGP 4.106) extends this approach by evaluating test chemicals across a concentration range to establish a dose-response relationship between DVs and test chemical concentration. Sensitizing potency is quantified using cDV0, the lowest dose required to elicit a positive response in GARD®skin. Depending on the need, the readout can be used to predict LLNA EC3 values, No Expected Sensitization Induction Level (NESIL), and UN GHS/CLP classification (1A or 1B), all with high statistical significance.
This study evaluates the utility of GARD®skin Dose-Response to predict NESILs for EOs, bridging the gap between hazard identification and quantitative risk assessment. Three EOs (A, B, C) were analysed using GARD®skin Dose-Response. Their skin sensitization potency threshold levels were estimated by the assay readouts (cDV0) and NESIL predictions. Results were compared to existing murine (LLNA EC3) and human (HRIPT/HMT) data through weight-of-evidence analysis.
For test item A, the predicted NESILs spanned were 5700 (GARD®), 6900 (constituent prediction), 1800 (LLNA), 2000 (HRIPT), and 3500 µg/cm² (HMT). Test item B showed NESILs of 24000 (GARD®), 29000 (constituent prediction), 6700 (LLNA), 44000 (HRIPT), and 11000 µg/cm² (HMT). Test item C was classified as a non-sensitizer by GARD®skin Dose-Response, with constituent prediction, LLNA, HRIPT, and HMT NESILs at 20000, 3900, 20000, and 690 µg/cm², respectively.
NESIL predictions from GARD®skin Dose-Response aligned with other skin sensitization test results, supporting its utility in quantitative sensitization potency assessment of complex natural extracts like EOs. While confirmatory human studies (e.g., CNIH protocols) are recommended, this assay reduces reliance on animal and human testing and advances the use of NAMs in safety assessment.
SenzaGen’s Newsletter Q2 2025
Click image to view the Newsletter in browser.
SenzaGen Newsletter 2025 Q2
SenzaGen Newsletter 2025 Q2
|
|
|
|
|
|
Published report by NICEATM: Evaluation of the GARDskin Sensitization Test Method Using Substances of Regulatory Interest
NICEATM’s report confirms the inclusion of GARDskin improved the overall performance of the Defined Approaches for Skin Sensitization (DASS)
The recent published report by NICEATM* summarizes an evaluation of the performance of the GARD®skin assay for predicting skin sensitization hazard and potency for chemicals nominated by ICCVAM member agencies.
The evaluation compared the results of the GARDskin assay alone and in applicable defined approaches for identifying skin sensitizers with reference results derived from the murine local lymph node assay, human skin sensitization reference data, and three other non-animal skin sensitization test methods.
Overall, the GARDskin assay performed well against the other assays for predicting skin sensitization hazard and potency classifications. Performance statistics for the defined approaches that included the GARDskin assay suggested that its addition improved those defined approaches.
Full report
*NICEATM (National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods) is a U.S. government center that promotes the development, validation, and adoption of alternative test methods that reduce, refine, or replace animal use in toxicology. NICEATM evaluates new in vitro, computational, and integrated approaches for regulatory safety testing and coordinates with agencies like ICCVAM to support their regulatory acceptance.
