Third Virtual Summer School 3Rs for ONE Science: Alternative methods: From complexity to predictivity.

710 a multiple time exposure. This is the first test method submitted for regulatory acceptance to OECD for discriminating on its own the three UN GHS categories; it was adopted as OECD TG 492B in June 2022. The potential to directly identify eye irritants is important for the cosmetic industry as well as for the pharmaceutical, chemical, and pesticide sectors and will improve and speed up their safety assessment. Arno C. Gutleb, Environmental Research and Innovation (ERIN) Department, Luxembourg Institute of Science and Technology, presented “Necessary or unnecessary complexity of in vitro lung models?” The classical approach for cell culture is that in vitro models are based on single cell types cultured submerged in medium. In recent years, complex models using multiple cell types cultured at the air-liquid interphase have added a new dimension of complexity to in vitro models. Such complex models need careful characterization of the properties of the cell types, especially when culture conditions change such as in co-culture. The similarities and discrepancies among 3D-in vitro models, human tissue and animal models used in the past need to be understood. Overall, models should be as simple as possible and as complex as necessary to mimic physiological responses. Hassan Rashidi, NIHR Great Ormond Street Hospital Biomedical Research Centre, UCL Great Ormond Street Institute of Child Health, University College London, presented a lecture entitled “Human pluripotent stem cell-derived hepatocyte-like cells as a tool to predict drug-induced liver toxicity”. The use of primary human hepatocytes (PHHs) is considered the gold standard in vitro model for drug toxicity testing to predict drug-induced liver toxicity (DILI) (Gomez-Lechon et al., 2014). However, their scarcity and transient ex vivo phenotype limit their use (Lauschke et al., 2016). A variety of alternative 2-dimensional (2D) models have been developed, but none has been able to predict DILI more accurately. Recent advances in the generation of 3D liver organoids and the development of microfluidic platforms, including liver-on-chip and human-on-chip, have opened new avenues to develop more sophisticated in vitro platforms to predict DILI more accurately. Recently developed tools were discussed. Giulia Ranaldi, Food and Nutrition Research Centre, Council for Agricultural Research and Economics, CREA-AN, Rome gave a presentation entitled “Application of Confocal Laser ScanThe international Third Virtual Summer School1 was focused on the application and predictivity of complex methods and approaches in different disciplines of science. The event, held on June 8-9, 2022 and chaired by Francesca Caloni, Università degli Studi di Milano, was attended by young researchers from all over the world. Francesca Caloni, opened the summer school, emphasizing that new approach methodologies (NAMs) are useful predictive and investigative tools for a multifaceted science with a global perspective. Helena Kandarova, CEM & FChFT Bratislava, gave a lecture entitled “Everything you ever wanted to know about successful validation of alternative methods but were afraid to ask – The secrets revealed”. The goal of any validation trial is to show that a method or model is relevant, reproducible, and predictive, ultimately leading to its regulatory acceptance. In the field of NAMs, it also has the ethical aspect of replacing the use of experimental animals. The presentation summarized critical aspects of the validation process and illustrated some practical examples of prospective and retrospective validation trials. Tips and tricks for designing a successful validation study were provided. Laura Ceriotti, in collaboration with Marisa Meloni, VitroScreen, Milan, Italy, gave a lecture entitled “Eye hazard classification: the road to discriminate between UN GHS category 1 and 2”. According to the Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS), test chemicals can be classified as inducing serious eye damage (UN GHS Category 1), inducing eye irritation (UN GHS Category 2), and not classified for eye irritation or serious eye damage (UN GHS No Category). Considerable progress has been made towards replacement of the in vivo Draize eye test, and several non-animal test methods have been validated. Due to the complexity of the eye responses towards chemical exposure, it is generally accepted that no single in vitro test method is sufficient, but a combination of several alternative test methods within integrated approaches to testing and assessment (IATA) may be able to fully replace the Draize eye test. Up to now, such methods or testing strategies allow to directly identify Cat. 1 and/or No Category, while Cat. 2 is indirectly identified through a testing strategy. Recently the SkinEthicTM HCE test method was developed for the evaluation of liquids and solids using a time-to-toxicity approach based on Meeting Report