In vitro testing strategy for assessing the skin sensitizing potential of "difficult to test" cosmetic ingredients

Before placing a new cosmetic ingredient on the market, manufacturers must establish its safety profile, in particular assessing the skin sensitization potential, which is a mandatory requirement for topical applications. Since the ban on animal testing in Europe, and its extension to many parts of the world, a battery of in vitro tests covering the key steps of the Adverse Outcome Pathway (AOP) for skin sensitization is recommended. To date, three in vitro methods are validated in the OECD guidelines (442C, 442D, 442E), and many others are under validation by OECD (2019) and ECVAM. However, there is still no official strategy. Some industrial manufacturers have proposed in vitro strategies with good predictivity, but their studies were mainly based on the testing of simple and "easy to test" substances. This work therefore focused on "difficult to test" ingredients with particular physicochemical properties (i.e. poorly water-soluble components) or with particular intrinsic properties placing them outside the applicability domains of most in vitro models (irritants or cytotoxic like surfactants, complex substances). Furthermore a particular focus was made on weak to moderate sensitizers. The objective was to develop a robust, quick and straightforward testing strategy enabling the evaluation of the skin sensitization potential of "difficult to test" ingredients. In this context, four in vitro test models were used: three validated methods and the Sens-Is® assay, currently in the work plan of the OECD, chosen for its ability to overcome solubility issues and to discriminate irritants from sensitizers. 25 ingredients with particular physicochemical properties were evaluated, chosen among positive or negative sensitizers according to in vivo data (M&K and/or LLNA). Such ingredients, including cleansers, solubilizers, emulsifiers, emollients, active ingredients, preservatives, and antioxidants are indeed essential constituents of cosmetic and dermopharmaceutical formulations. The results analysis on each in vitro test demonstrated that the DPRA model was the less predictive on the chosen ingredients, resulting especially in many false negative responses compared to animal studies, or being unsuited to the mode of action of the selected ingredients. On the contrary, the Sens-Is® assay revealed a real capability to discriminate sensitizers from non-sensitizers. The two other models, KeratinoSens^TM and h-CLAT, showed a lower ability to classify the materials correctly than in previously published studies, linked to the particular physicochemical and intrinsic properties of the chosen ingredients and the applicability domains of these in vitro tests. The KeratinoSens^TM model tended to overestimate the sensitization potential of the tested ingredients, whereas the h-CLAT model tended to underestimate the sensitizers. Based on these results a new sequential testing strategy was set up combining 1 to 3 models to cover the main key events of the skin sensitization AOP. Sens-Is® model, assessing the first two AOP Key Events with consideration of the ingredient dermal penetration, is chosen as a starting point. The approach is completed, depending on the first response, by the h-CLAT model, assessing Key Event 3, and then potentially KeratinoSens^TM assessing Key Event 2, but with a more direct application mode. This new testing strategy increases the accuracy to 88% on the selected ingredients and minimizes the risk of a false negative conclusion, which is crucial from the perspective of the ingredients' users and cosmetic consumers.