Absence of phototoxicity/photoirritation potential of bergamottin determined In Vitro using OECD TG 432.

The phototoxic potential of a number of furocoumarins is well established. On the other hand, studies have shown that bergamottin, a furocoumarin containing a bulky, hydrophobic side chain, has significantly less or is even absent of phototoxicity potential. The OECD Test Guideline 432 3T3/Neutral Red Uptake (NRU) in vitro phototoxicity test has shown to be a highly predictive test for identifying compounds that exhibit no phototoxicological potential. In this study using OECD 432, the established phototoxic furocoumarin 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP) and psoralen were phototoxic, whereas bergamottin showed no phototoxic potential. When compared to 5-MOP, 8-MOP and psoralen, bergamottin was clearly negative at molar-adjusted concentrations that were more than 9 times higher than those that produced phototoxicity in 8-MOP; nearly 16 times than those for psoralen and more than 36 times higher than those for 5-MOP. These data using in vitro 3T3 NRU Phototoxicity Test (OECD 432) are supportive of earlier studies showing bergamottin does not exhibit phototoxicological properties. The detection and quantification of bergamottin should therefore not contribute to the potential marker furocoumarins for risk management interventions intended to reduce the phototoxicity of natural furocoumarin containing preparations.

Non-animal methods to predict skin sensitization (II): an assessment of defined approaches *.

Skin sensitization is a toxicity endpoint of widespread concern, for which the mechanistic understanding and concurrent necessity for non-animal testing approaches have evolved to a critical juncture, with many available options for predicting sensitization without using animals. Cosmetics Europe and the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods collaborated to analyze the performance of multiple non-animal data integration approaches for the skin sensitization safety assessment of cosmetics ingredients. The Cosmetics Europe Skin Tolerance Task Force (STTF) collected and generated data on 128 substances in multiple in vitro and in chemico skin sensitization assays selected based on a systematic assessment by the STTF. These assays, together with certain in silico predictions, are key components of various non-animal testing strategies that have been submitted to the Organization for Economic Cooperation and Development as case studies for skin sensitization. Curated murine local lymph node assay (LLNA) and human skin sensitization data were used to evaluate the performance of six defined approaches, comprising eight non-animal testing strategies, for both hazard and potency characterization. Defined approaches examined included consensus methods, artificial neural networks, support vector machine models, Bayesian networks, and decision trees, most of which were reproduced using open source software tools. Multiple non-animal testing strategies incorporating in vitro, in chemico, and in silico inputs demonstrated equivalent or superior performance to the LLNA when compared to both animal and human data for skin sensitization.

Non-animal methods to predict skin sensitization (I): the Cosmetics Europe database.

Cosmetics Europe, the European Trade Association for the cosmetics and personal care industry, is conducting a multi-phase program to develop regulatory accepted, animal-free testing strategies enabling the cosmetics industry to conduct safety assessments. Based on a systematic evaluation of test methods for skin sensitization, five non-animal test methods (DPRA (Direct Peptide Reactivity Assay), KeratinoSensTM, h-CLAT (human cell line activation test), U-SENSTM, SENS-IS) were selected for inclusion in a comprehensive database of 128 substances. Existing data were compiled and completed with newly generated data, the latter amounting to one-third of all data. The database was complemented with human and local lymph node assay (LLNA) reference data, physicochemical properties and use categories, and thoroughly curated. Focused on the availability of human data, the substance selection resulted nevertheless resulted in a high diversity of chemistries in terms of physico-chemical property ranges and use categories. Predictivities of skin sensitization potential and potency, where applicable, were calculated for the LLNA as compared to human data and for the individual test methods compared to both human and LLNA reference data. In addition, various aspects of applicability of the test methods were analyzed. Due to its high level of curation, comprehensiveness, and completeness, we propose our database as a point of reference for the evaluation and development of testing strategies, as done for example in the associated work of Kleinstreuer et al. We encourage the community to use it to meet the challenge of conducting skin sensitization safety assessment without generating new animal data.

State-of-the-art and new options to assess T cell activation by skin sensitizers: Cosmetics Europe Workshop.

Significant progress has been made in the development and validation of non-animal test methods for skin sensitization assessment. At present, three of the four key events of the Adverse Outcome Pathway (AOP) are assessable by OECD-accepted in vitro methods. The fourth key event describes the immunological response in the draining lymph node where activated dendritic cells present major histocompatibility complex-bound chemically modified peptides to naive T cells, thereby priming the proliferation of antigen-specific T cells. Despite substantial efforts, modelling and assessing this adaptive immune response to sensitizers with in vitro T cell assays still represents a challenge. The Cosmetics Europe Skin Tolerance Task Force organized a workshop, bringing together academic researchers, method developers, industry representatives and regulatory stakeholders to review the scientific status of T cell-based assays, foster a mutual scientific understanding and conceive new options to assess T cell activation. Participants agreed that current T cell assays have come a long way in predicting immunogenicity, but that further investment and collaboration is required to simplify assays, optimize their sensitivity, better define human donor-to-donor variability and evaluate their value to predict sensitizer potency. Furthermore, the potential role of T cell assays in AOP-based testing strategies and subsequent safety assessment concepts for cosmetic ingredients was discussed. It was agreed that it is currently difficult to anticipate uses of T cell assay data for safety assessment and concluded that experience from case studies on real-life risk assessment scenarios is needed to further consider the usefulness of assessing the fourth AOP key event.

Cosmetics Europe compilation of historical serious eye damage/eye irritation in vivo data analysed by drivers of classification to support the selection of chemicals for development and evaluation of alternative methods/strategies: the Draize eye test Reference Database (DRD).

A thorough understanding of which of the effects assessed in the in vivo Draize eye test are responsible for driving UN GHS/EU CLP classification is critical for an adequate selection of chemicals to be used in the development and/or evaluation of alternative methods/strategies and for properly assessing their predictive capacity and limitations. For this reason, Cosmetics Europe has compiled a database of Draize data (Draize eye test Reference Database, DRD) from external lists that were created to support past validation activities. This database contains 681 independent in vivo studies on 634 individual chemicals representing a wide range of chemical classes. A description of all the ocular effects observed in vivo, i.e. degree of severity and persistence of corneal opacity (CO), iritis, and/or conjunctiva effects, was added for each individual study in the database, and the studies were categorised according to their UN GHS/EU CLP classification and the main effect driving the classification. An evaluation of the various in vivo drivers of classification compiled in the database was performed to establish which of these are most important from a regulatory point of view. These analyses established that the most important drivers for Cat 1 Classification are (1) CO mean ≥ 3 (days 1-3) (severity) and (2) CO persistence on day 21 in the absence of severity, and those for Cat 2 classification are (3) CO mean ≥ 1 and (4) conjunctival redness mean ≥ 2. Moreover, it is shown that all classifiable effects (including persistence and CO = 4) should be present in ≥60 % of the animals to drive a classification. As a consequence, our analyses suggest the need for a critical revision of the UN GHS/EU CLP decision criteria for the Cat 1 classification of chemicals. Finally, a number of key criteria are identified that should be taken into consideration when selecting reference chemicals for the development, evaluation and/or validation of alternative methods and/or strategies for serious eye damage/eye irritation testing. Most important, the DRD is an invaluable tool for any future activity involving the selection of reference chemicals.