As the cosmetics and personal care industry moves away from animal testing, researchers are turning to innovative in vitro methods to assess ingredient safety. One promising approach is the GARDskin Dose-Response (DR) assay, which quantitatively evaluates the skin sensitization potential of fragrance ingredients.
In this Q&A, Dr. Isabelle Lee, PhD, from the Research Institute for Fragrance Materials (RIFM), discussed why her team explored this method, how it compares to traditional testing, and what it could mean for formulators and regulators.
As lead author on RIFM’s latest study, “Determining a Point of Departure for Skin Sensitization Potency and Quantitative Risk Assessment of Fragrance Ingredients Using the GARDskin Dose-Response Assay,” she also shared the research’s key findings, including insights on accuracy, regulatory acceptance, and future research directions.
CDU: What motivated your team to explore using the GARDskin dose-response (DR) assay to assess the potency of fragrance ingredients?
Isabelle Lee, PhD: As part of our ongoing safety assessment of fragrance ingredients, the Research Institute for Fragrance Materials (RIFM) gauges the potential of these ingredients to cause skin sensitization, a critical safety concern. RIFM’s goal is to prevent the induction of skin sensitization.
RIFM does not use animal testing for any of its human health endpoints, and the GARDskin DR assay is an in vitro test that uses human-derived cells. Just as critical, the test predicts a substance’s potential for skin sensitization and its specific potency.
Potency is key to understanding a level of exposure beneath which we can be assured that skin sensitization would not be a concern.
CDU: How does the GARDskin DR assay improve existing methods for assessing skin sensitization risk?
Isabelle Lee, PhD: The biggest advantage of this animal alternative assay is that it is quantitative rather than qualitative or semi-quantitative. A quantitative assay provides specific numerical data, allowing for precise measurement of the concentration or potency of a substance, which helps in assessing its risk more accurately.
Second, the assay uses human-derived skin cells, making it more relevant to human biology compared to animal models. This may increase the accuracy of the predictions for how a substance will behave in humans, helping to anticipate potential allergic reactions or sensitization better.
Finally, the GARDskin DR assay is relatively fast and can process many ingredients in a short timeframe. This makes it more efficient for high-throughput screening of fragrance ingredients.
CDU: What are the key takeaways from your study for the cosmetics and personal care product industry?
Isabelle Lee, PhD: The GARDskin assay is an effective tool for assessing skin sensitization potential and can be used to evaluate the quantitative potency of fragrance ingredients.
The assay is more ethical and efficient than traditional animal-based testing and validating such animal-free testing supports its acceptance by regulatory authorities.
CDU: Can you elaborate on how the GARDskin DR assay quantitatively predicts sensitization potency compared to traditional methods like local lymph node assay (LLNA)?
Isabelle Lee, PhD: The GARDskin DR assay is an in vitro (animal-free) method that quantitatively predicts skin sensitization potency through gene expression analysis. In contrast, traditional potency assessment methods like the LLNA rely on animal testing, which faces ethical and regulatory challenges due to the ban on animal testing for cosmetics in many countries.
By analyzing the level of specific gene markers associated with immune system activation (i.e., a skin sensitization response), a dose-response curve is generated, which provides a detailed, numerical prediction of the sensitization potency of an ingredient. This allows for precise comparisons of how potent a substance is in inducing sensitization at various concentrations.
The dose-response aspect allows for a clearer understanding of the strength of the reaction across a range of concentrations, making it easier to determine safe exposure thresholds. Quantitative potency in the LLNA is also determined through a dose response, but instead of analyzing gene expression, it examines the proliferation of cells in the local lymph node.
CDU: How might accuracy be improved?
Isabelle Lee, PhD: While GARDskin DR generally predicted the fragrance ingredient potency category within the correct range based on reference data, it was notable that predictions for the very weak range of sensitizers were poor.
It was challenging and beyond the scope of the study to conclusively determine if the lower predictive capacity in this specific category was due to a lower resolution of the GARDskin DR or a low resolution associated with the reference data.
While we evaluated 100 fragrance ingredients in this study, testing a larger and more diverse set of ingredients, including non-fragrance ingredients, could help elucidate the assay’s performance in the lower range and improve its accuracy.
Additionally, by including more ingredients with varying sensitization potentials, the assay can be better calibrated to account for the diversity in that fragrance and consumer product ingredient landscape.
Finally, continuous validation of the assay using real-world historical data from human cases of skin sensitization could provide additional insights and refine the assay’s predictive power. Such historical data can provide a broader understanding of how well the assay matches actual sensitization in humans.
CDU: What insights did you gain about the applicability of GARDskin DR across different chemical reactivity domains?
Isabelle Lee, PhD: The assay was shown to be effective in assessing various electrophilic chemical reactivity domains of fragrance ingredients. Electrophilic substances can react with nucleophilic sites on proteins, often a key characteristic of most allergens that trigger sensitization.
The assay measured quantitative potencies for ingredients with different types of reactivity, demonstrating its versatility in capturing a broad range of chemical types that could induce sensitization.
Whereas the GARDskin DR assay performed well with electrophilic substances, more work needs to be done to analyze non-electrophilic sensitizers and other structural chemistries not captured within the fragrance inventory.
CDU: Your study found that combining weak and very weak sensitizer categories increased accuracy significantly. How might this impact regulatory decision-making or formulation practices?
Isabelle Lee, PhD: Although the very weak sensitization range is especially important to the fragrance industry—since most sensitizers in this industry fall within this range—current cosmetic industry standards and quantitative methods like the LLNA do not differentiate between weak and very weak sensitizers. We applied a similar approach to evaluate how the assay performs in line with industry practices.
We significantly improved accuracy by reassigning very weak predictions and reference categories to the weak category. This suggests that while some reassessment may be needed, particularly for fragrance ingredients, the assay could be satisfactory when measured against current general industry and regulatory standards.
CDU: How can manufacturers integrate GARDskin DR into their existing safety and risk assessment processes?
Isabelle Lee, PhD: Manufacturers and formulators can use the GARDskin DR assay in a similar way to the traditional use of the LLNA for quantitative potency assessment.
However, it’s important to note that additional uncertainty may need to be considered when translating in vitro quantitative values into risk assessments.
CDU: What advantages does this approach offer regarding reducing animal testing and aligning with evolving regulatory frameworks?
Isabelle Lee, PhD: The assay provides a human-relevant, non-animal testing method for quantitative potency assessment, featuring a mechanistic approach, a faster testing process, and increasing alignment with international guidelines.
As regulatory bodies focus on human-relevant alternatives, GARDskin DR’s role in next-generation risk assessments will likely grow.
CDU: Do you foresee regulatory agencies adopting GARDskin DR-based data for compliance?
Isabelle Lee, PhD: It’s quite plausible that regulatory agencies could adopt GARDskin DR-based data for compliance, particularly as the method gains more validation and becomes widely recognized as a reliable animal alternative for assessing skin sensitization.
Regulatory agencies worldwide are increasingly pursuing non-animal testing methods, especially in the cosmetics and personal care industries.
The OECD (Organisation for Economic Co-operation and Development) is actively involved in validating and standardizing testing methods for skin sensitization, and it has recognized in vitro methods like the GARDskin assay, from which GARDskin DR was adopted, for their potential. OECD validation and its integration into official guidelines could drive international regulatory acceptance.
CDU: How can formulators use No Expected Sensitization Induction Level (NESIL) predictions from GARDskin DR to set safe concentration levels for fragrance ingredients?
Isabelle Lee, PhD: When necessary, safe concentration levels or maximum acceptable concentration levels are published in peer-reviewed RIFM fragrance ingredient safety assessments, which are available for free download at The Fragrance Material Safety Resource Center. This site provides open access to all of RIFM’s peer-reviewed science.
The maximum acceptable concentrations are shared with the International Fragrance Association (IFRA), and if risk management is needed, an IFRA Standard is issued. The IFRA Standards are based on RIFM’s maximum acceptable concentration levels.
In a separate collaboration, we have illustrated how the GARDskin DR NESIL can be incorporated in a next-generation risk assessment tiered approach to calculate safe concentration levels for incorporation in various consumer product types. For more information, see the study here.
CDU: What additional validation or research is needed before GARDskin DR can be widely adopted in next-generation risk assessment (NGRA)?
Isabelle Lee, PhD: The study validated a subset of substances (primarily fragrance ingredients), particularly those with well-understood electrophilic reactivity. However, for it to be more broadly applicable in NGRA, further validation is needed across a wider range of substance classes, including non-electrophilic substances that do not react in the same way as traditional sensitizers.
Additionally, more non-fragrance ingredients would need to be tested to widen the scope of applicability.
To be widely adopted, an assay must demonstrate reproducibility across different laboratories, particularly in the context of global regulatory acceptance. This means ensuring that results from other labs or different testing conditions yield consistent, reliable data.
GARDskin DR is adapted from the GARDskin assay, a qualitative assay already validated and included in the OECD guideline 442E. It remains to be determined what, if any, additional work would be required to incorporate the quantitative steps of the dose-response assay.
CDU: What are the next steps for your team to refine or advance this method?
Isabelle Lee, PhD: The next steps involve broader assay validation across different substance types, such as nucleophilic sensitizers and natural complex substances, and integrating it with other testing methods.
By addressing these areas, we aim to strengthen the GARDskin DR assay as a reliable and widely accepted tool in next-generation risk assessment for skin sensitization.
