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, KeratinoSensTM 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 KeratinoSensTM 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 KeratinoSensTM 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.

Assessment of phototoxicity in pigmented Long-Evans rat: sparfloxacin and 8-methoxypsoralen.

The aim of the present work was to evaluate the effects of photo-activated toxicity induced after administration of two known melanin-binding phototoxic compounds, sparfloxacin (SPX) and 8-methoxypsoralen (8-MOP), followed or not by UVA/Vis exposure, in pigmented rats (Long Evans: LE) and albino rats (Sprague Dawley: SD). Groups of three rats were treated with SPX or 8-MOP by oral gavage for six consecutive days. Irradiated animals were submitted to a UVA/Vis light dose standardized to 10 J/cm2 UVA daily. Clinical signs, cutaneous reactions and body weight were monitored throughout the study period. Ear biopsy weight, lymph node weight and lymph node cell count were determined at necropsy. Ophthalmologic examinations were performed before the first treatment and on the day of sacrifice. Microscopic examinations were performed on skin biopsies and eyes. Phototoxicity was demonstrated for both SPX and 8-MOP in the pigmented and albino strains, in terms of auricular irritation, lymph node weight and proliferation index, cutaneous reactions and ocular histopathology. LE rats were less sensitive than SD rats, especially at the ocular level, supporting the notion that pigmentation may provide protection against photo-activation. The pigmented rat may be a more relevant model than the albino rat for human safety evaluation.

Allergic skin inflammation induced by chemical sensitizers is controlled by the transcription factor Nrf2.

Allergic contact dermatitis (ACD) is induced by low-molecular weight electrophilic chemicals and metal ions. Chemical contact sensitizers trigger reactive oxygen species production and provoke electrophilic stress, leading to the accumulation of the transcription factor nuclear-related factor 2 (Nrf2) in innate immune cell types. The objective of this work was to identify the role of Nrf2 in the regulation of ACD. We used the local lymph node assay (LLNA) and the mouse ear swelling test (MEST) to study the role of Nrf2 in both the sensitization and elicitation phase in nrf2 knockout (nrf2(-/-)) and wild-type (nrf2(+/+)) mice. Five chemicals were used: two compounds known to react with cysteine residues, 2,4-dinitrochlorobenzene (DNCB) and cinnamaldehyde (CinA); one sensitizer known to exhibit mixed reactivity to cysteine and lysine residues, isophorone diisocyanate; and one reacting specifically with lysine residues, trimellitic anhydride and croton oil, a well-known irritant. In the MEST assay, DNCB (1 and 2%) induced a significant increase in ear thickness in nrf2(-/-) compared with nrf2(+/+) mice, suggesting a role for Nrf2 in the control of the inflammatory process. When DNCB was used at 0.25 and 0.5% or when mice were treated with CinA, inflammation was found only in nrf2(-/-) mice. In the LLNA, all chemical sensitizers induced an increase of lymphocyte proliferation in nrf2(-/-) compared with nrf2(+/+) mice for the same chemical concentration. These results reveal an important role for Nrf2 in controlling ACD and lymphocyte proliferation in response to sensitizers.