The overall goal of this project is to identify correlations between particle component/properties (metrics) with adverse outcome pathways that are associated with the induction of acute and chronic health effects within the European population. This will be delivered through the development of a new method for studying in vitro cytotoxicity based on the use of ‘tailored’ synthetic ambient aerosols combined with high-resolution optical imaging and state-of-the-art cell analysis methods. The specific objectives of the project are: To develop a stable and reproducible laboratory-based source of well-controlled and chemically defined, synthetic reference aerosol mixtures that mimic real ambient aerosols at high concentrations (at around the limit values of the EU Air Quality Directive and up to a few mg/m3). The aerosol properties should be tunable and the source should be coupled to an oxidation flow reactor (OFR) to mimic atmospheric photochemical ‘ageing’. To improve traceability for the physical and chemical characterisation of the synthetic aerosols using EU reference methods (target uncertainty in mass concentration 15 %, number concentration <15 %, analysis of major chemical components <15 %). Moreover, to quantify uncertainties for emerging techniques, such as Aerosol Mass Spectrometry (AMS) for chemical analysis and Brunauer–Emmett–Teller (BET) for surface area analysis and develop new approaches to ensure reproducibility and quality assurance. To apply novel methods for cell exposure at the air-liquid interface (ALI) in order to mimic and quantify the effects of the in vivo aerosol inhalation routes. To study phenotypic effects using lung organoids. To compare these novel methods with the conventional cell-exposure techniques relying on submerged cell systems, where aerosol particles are collected in water with high-volume samplers. To assess how the composition of the collected aerosols and their ageing impacts on their acellular and cellular oxidative characteristics, both in simple chemical models simulating human respiratory tract lining fluids (in health and disease) and in representative cell lines maintained under near physiologic conditions. To evaluate adverse outcome pathways using proteomics and transcriptomics (high throughput sequencing), to examine known causal pathways, such as pro-/anti-inflammatory responses, cytotoxicity and genotoxicity, as well as novel ‘component-specific’ pathways. The project will work toward improved, validated protocols for harmonising/standardising cell analysis studies, as well as on the integration of multi-omic approaches for statistical analysis of complex data sets on a European level./li> To push the frontiers of optical imaging and biological image analysis to quantify the effects of particle uptake on single cells and cell populations by using various types of optical microscopy including confocal, structured illumination, light sheet and fluorescence lifetime imaging. To facilitate the take up of the technology and measurement infrastructure developed in the project by the measurement supply chain (accredited laboratories, instrumentation manufacturers), standards developing organisations (CEN, ISO) and end users (e.g. hospitals and health centres).