Nanomaterials (NMs) display many unique and useful physico-chemical properties. However, reliable approaches are needed for risk assessment of NMs. The present study was performed in the FP7-MARINA project, with the objective to identify and evaluate in vitro test methods for toxicity assessment in order to facilitate the development of an intelligent testing strategy (ITS). Six representative oxide NMs provided by the EC-JRC Nanomaterials Repository were tested in nine laboratories. The in vitro toxicity of NMs was evaluated in 12 cellular models representing 6 different target organs/systems (immune system, respiratory system, gastrointestinal system, reproductive organs, kidney and embryonic tissues). The toxicity assessment was conducted using 10 different assays for cytotoxicity, embryotoxicity, epithelial integrity, cytokine secretion and oxidative stress. Thorough physico-chemical characterization was performed for all tested NMs. Commercially relevant NMs with different physico-chemical properties were selected: two TiO₂ NMs with different surface chemistry – hydrophilic (NM-103) and hydrophobic (NM-104), two forms of ZnO – uncoated (NM-110) and coated with triethoxycapryl silane (NM-111) and two SiO₂ NMs produced by two different manufacturing techniques – precipitated (NM-200) and pyrogenic (NM-203). Cell specific toxicity effects of all NMs were observed; macrophages were the most sensitive cell type after short-term exposures (24-72h) (ZnO>SiO₂>TiO₂). Longer term exposure (7 to 21 days) significantly affected the cell barrier integrity in the presence of ZnO, but not TiO₂ and SiO₂, while the embryonic stem cell test (EST) classified the TiO₂ NMs as potentially ‘weak-embryotoxic’ and ZnO and SiO₂ NMs as ‘non-embryotoxic’. A hazard ranking could be established for the representative NMs tested (ZnO NM-110 > ZnO NM-111 > SiO₂ NM-203 > SiO₂ NM-200 > TiO₂ NM-104 > TiO₂ NM-103). This ranking was different in the case of embryonic tissues, for which TiO₂ displayed higher toxicity compared with ZnO and SiO₂. Importantly, the in vitro methodology applied could identify cell- and NM-specific responses, with a low variability observed between different test assays. Overall, this testing approach, based on a battery of cellular systems and test assays, complemented by an exhaustive physico-chemical characterization of NMs, could be deployed for the development of an ITS suitable for risk assessment of NMs. This study also provides a rich source of data for modeling of NM effects.

Current European (EU) policies, such as the Green Deal, envisage safe and sustainable by design (SSbD) practices for the management of chemicals, which cogently entail nanomaterials (NMs) and advanced materials (AdMa). These practices, applied at the earliest stages of innovation and throughout the life-cycle of chemicals, materials and products, could prevent and/or minimise their environmental, health and safety (EHS) and sustainability impacts. This requires a shift from the established risk control paradigms towards prevention-based approaches at the design stage that accelerate the development of safer and more sustainable chemicals, materials, products and processes, while promoting a transition towards a circular economy and a more sustainable future. The EU commission has funded several Horizon 2020 projects applying the concepts of SSbD to nanotechnologies, biotechnologies and advanced materials. This article is inspired from the answers and opinions shared during a stakeholders meeting arranged throughout the workshop entitled ‘Safe and Sustainable by Design Paradigms applied to NMs and AdMa’, held in Venice, Italy, in September 2022. The goal of the workshop was to identify differences and overlaps between the SSbD approaches and to provide common messages on the progress towards the implementation of concrete SSbD concepts, and to reveal challenges faced in their realistic and straightforward execution. In this article, we provide insights into the intersecting industrial domains, the technical and organisational challenges to the practical implementation of the SSbD, and future financial directions in supporting and maintaining the digital products currently under development within the H2020 projects, in order to ultimately enable their uptake by industry and regulators.