Skin sensitizing effects of sulfur mustard and other alkylating agents in accordance to OECD guidelines.
Vesicants cause a multitude of cutaneous reactions like erythema, blisters and ulcerations. After exposure to sulfur mustard (SM) and related compounds, patients present dermal symptoms typically known for chemicals categorized as skin sensitizer (e.g. hypersensitivity and flare-up phenomena). However, although some case reports led to the assumption that SM and other alkylating compounds represent sensitizers, a comprehensive investigation of SM-triggered immunological responses has not been conducted so far. Based on a well-structured system of in chemico and in vitro test methods, the Organization for Economic Co-operation and Development (OECD) established procedures to categorize agents on their skin sensitizing abilities. In this study, the skin sensitizing potential of SM and three related alkylating agents (AAs) was assessed following the OECD test guidelines. Besides SM, investigated AAs were chlorambucil (CHL), nitrogen mustard (HN3) and 2-chloroethyl ethyl sulfide (CEES). The methods are described in detail in the EURL ECVAM DataBase service on ALternative Methods to animal experimentation (DB-ALM). In accordance to OECD recommendations, skin sensitization is a pathophysiological process starting with a molecular initiating step and ending with the in vivo outcome of an allergic contact dermatitis. This concept is called adverse outcome pathway (AOP). An AOP links an adverse outcome to various key events which can be assayed by established in chemico and in vitro test methods. Positive outcome in two out of three key events indicates that the chemical can be categorized as a skin sensitizer. In this study, key event 1 "haptenation" (covalent modification of epidermal proteins), key event 2 "activation of epidermal keratinocytes" and key event 3 "activation of dendritic cells" were investigated. Covalent modification of epidermal proteins measured by using the DPRA-assay provided distinct positive results for all tested substances. Same outcome was seen in the KeratinoSens assay, investigating the activation of epidermal keratinocytes. The h-CLAT assay performed to determine the activation of dendritic cells provided positive results for SM and CEES but not for CHL and HN3. Altogether, following OECD requirements, our results suggest the classification of all investigated substances as skin sensitizers. Finally, a tentative AOP for SM-induced skin sensitization is suggested.
Steinritz D
，Lang S
，Popp T
，Siegert M
，Rothmiller S
，Kranawetvogl A
，Schmidt A
，John H
，Gudermann T
，Thiermann H
，Kehe K
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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.
Bergal M
，Puginier M
，Gerbeix C
，Groux H
，Roso A
，Cottrez F
，Milius A
... -
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Application of Defined Approaches for Skin Sensitization to Agrochemical Products.
Skin sensitization testing is a regulatory requirement for safety evaluations of pesticides in multiple countries. Globally harmonized test guidelines that include in chemico and in vitro methods reduce animal use, but no single assay is recommended as a complete replacement for animal tests. Defined approaches (DAs) that integrate data from multiple non-animal methods are accepted; however, the methods that comprise them have been evaluated using monoconstituent substances rather than mixtures or formulations. To address this data gap, we tested 27 agrochemical formulations in the direct peptide reactivity assay (DPRA), the KeratinoSens™ assay, and the human cell line activation test (h-CLAT). These data were used as inputs to evaluate three DAs for hazard classification of skin sensitization potential and two DAs for potency categorization. When compared to historical animal results, balanced accuracy for the DAs for predicting in vivo skin sensitization hazard (i.e., sensitizer vs. nonsensitizer) ranged from 56 to 78%. The best performing DA was the "2 out of 3 (2o3)" DA, in which the hazard classification was based on two concordant results from the DPRA, KeratinoSens, or h-CLAT. The KE 3/1 sequential testing strategy (STS), which uses h-CLAT and DPRA results, and the integrated testing strategy (ITSv2), which uses h-CLAT, DPRA, and an in silico hazard prediction from OECD QSAR Toolbox, had balanced accuracies of 56-57% for hazard classification. Of the individual test methods, KeratinoSens had the best performance for predicting in vivo hazard outcomes. Its balanced accuracy of 81% was similar to that of the 2o3 DA (78%). For predicting potency categories defined by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS), the correct classification rate of the STS was 52% and that of the ITSv2 was 43%. These results demonstrate that non-animal test methods have utility for evaluating the skin sensitization potential of agrochemical formulations as compared to animal reference data. While additional data generation is needed, testing strategies such as DAs anchored to human biology and mechanistic information provide a promising approach for agrochemical formulation testing.
Strickland J
，Truax J
，Corvaro M
，Settivari R
，Henriquez J
，McFadden J
，Gulledge T
，Johnson V
，Gehen S
，Germolec D
，Allen DG
，Kleinstreuer N
... -
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ResearchGate
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