A central challenge for the safe design of nanomaterials (NMs) is the inherent variability of NM properties, both as produced and as they interact with and evolve in, their surroundings. This has led to uncertainty in the literature regarding whether the biological and toxicological effects reported for NMs are related to specific NM properties themselves, or rather to the presence of impurities or physical effects such as agglomeration of particles. Thus, there is a strong need for systematic evaluation of the synthesis and processing parameters that lead to potential variability of different NM batches and the reproducible production of commonly utilized NMs. The work described here represents over three years of effort across 14 European laboratories to assess the reproducibility of nanoparticle properties produced by the same and modified synthesis routes for four of the OECD priority NMs (silica dioxide, zinc oxide, cerium dioxide and titanium dioxide) as well as amine-modified polystyrene NMs, which are frequently employed as positive controls for nanotoxicity studies. For 46 different batches of the selected NMs, all physicochemical descriptors as prioritized by the OECD have been fully characterized. The study represents the most complete assessment of NMs batch-to-batch variability performed to date and provides numerous important insights into the potential sources of variability of NMs and how these might be reduced.

Ship engine emissions are important with regard to lung and cardiovascular diseases especially in coastal regions worldwide. Known cellular responses to combustion particles include oxidative stress and inflammatory signalling. To provide a molecular link between the chemical and physical characteristics of ship emission particles and the cellular responses they elicit and to identify potentially harmful fractions in shipping emission aerosols. Through an air-liquid interface exposure system, we exposed human lung cells under realistic in vitro conditions to exhaust fumes from a ship engine running on either common heavy fuel oil (HFO) or cleaner-burning diesel fuel (DF). Advanced chemical analyses of the exhaust aerosols were combined with transcriptional, proteomic and metabolomic profiling including isotope labelling methods to characterise the lung cell responses. The HFO emissions contained high concentrations of toxic compounds such as metals and polycyclic aromatic hydrocarbon, and were higher in particle mass. These compounds were lower in DF emissions, which in turn had higher concentrations of elemental carbon (“soot”). Common cellular reactions included cellular stress responses and endocytosis. Reactions to HFO emissions were dominated by oxidative stress and inflammatory responses, whereas DF emissions induced generally a broader biological response than HFO emissions and affected essential cellular pathways such as energy metabolism, protein synthesis, and chromatin modification. Despite a lower content of known toxic compounds, combustion particles from the clean shipping fuel DF influenced several essential pathways of lung cell metabolism more strongly than particles from the unrefined fuel HFO. This might be attributable to a higher soot content in DF. Thus the role of diesel soot, which is a known carcinogen in acute air pollution-induced health effects should be further investigated. For the use of HFO and DF we recommend a reduction of carbonaceous soot in the ship emissions by implementation of filtration devices.

Reliable and predictive in vitro assays for hazard assessments of manufactured nanomaterials (MNMs) are still limited. Specifically, exposure systems which more realistically recapitulate the physiological conditions in the lung are needed to predict pulmonary toxicity. To this end, air-liquid interface (ALI) systems have been developed in recent years which might be better suited than conventional submerged exposure assays. However, there is still a need for rigorous side-by-side comparisons of the results obtained with the two different exposure methods considering numerous parameters, such as different MNMs, cell culture models and read outs. In this study, human A549 lung epithelial cells and differentiated THP-1 macrophages were exposed under submerged conditions to two abundant types of MNMs i.e., ceria and titania nanoparticles (NPs). Membrane integrity, metabolic activity as well as pro-inflammatory responses were recorded. For comparison, A549 monocultures were also exposed at the ALI to the same MNMs. In the case of titania NPs, genotoxicity was also investigated. In general, cells were more sensitive at the ALI compared to under classical submerged conditions. Whereas ceria NPs triggered only moderate effects, titania NPs clearly initiated cytotoxicity, pro-inflammatory gene expression and genotoxicity. Interestingly, low doses of NPs deposited at the ALI were sufficient to drive adverse outcomes, as also documented in rodent experiments. Therefore, further development of ALI systems seems promising to refine, reduce or even replace acute pulmonary toxicity studies in animals.