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The immense diversity and constant development of nanomaterials (NMs) increase the need for facilitated risk assessment, which requires knowledge of the modes of action (MoAs) of NMs. This necessitates a comprehensive data basis, which can be obtained using omics. Furthermore, the establishment of suitable in vitro test systems is essential to follow the 3R concept and to cope with the high number of NMs.In the present study, we aimed to compare NM effects in vitro and in vivo using a multi-omics approach. We applied an integrated data analysis strategy based on proteomics and metabolomics to four silica NMs and one titanium dioxide-based NM. For the in vitro investigations, rat alveolar epithelial cells (RLE-6TN) and rat alveolar macrophages (NR8383) were treated with different doses of NMs, and the results were compared with the effects on rat lungs after short-term inhalations and instillations.Since reactive oxygen species (ROS) production has been described as a critical biological effect of NMs, we focused on different levels of oxidative stress. Thus, we found opposite changes in proteins and metabolites related to the production of reduced glutathione in alveolar epithelial cells and alveolar macrophages, demonstrating that the MoAs of NMs depend on the model system used. Interestingly, in vivo, pathways related to inflammation were more affected than oxidative stress responses. Hence, the assignment of the observed effects to levels of oxidative stress was also different in vitro and in vivo.However, the overall classification of “active” and “passive” NMs was consistent in vitro and in vivo, suggesting that both cell lines tested are suitable for the assessment of NM toxicity. In summary, the results presented here highlight the need to carefully review model systems to decipher the extent to which they can replace in vivo assays.

In respect to the high number of released nanomaterials and their highly variable properties, novel grouping approaches are required based on the effects of nanomaterials. Proper grouping calls for a combination of an experimental setup with a higher number of structurally similar nanomaterials and for employing integrated omics approaches to identify the mode of action. Here, we analyzed the effects of seven well-characterized NMs comprising different chemical compositions, sizes and chemical surface modifications on the rat alveolar macrophage cell line NR8383. The NMs were investigated at three doses ranging from 2.5 to 10 µg/cm2 after 24 h incubation using an integrated multi-omics approach involving untargeted proteomics, targeted metabolomics, and src homology 2 (SH2) profiling. By using Weighted Gene Correlation Network Analysis (WGCNA) for the integrative data, we identified correlations of molecular pathways with physico-chemical properties and toxicological endpoints. The three investigated SiO2 variants induced strong alterations in all three omics approaches and were, therefore, be classified as “active.” Two organic phthalocyanines showed minor responses and Mn2O3 induced a different molecular response pattern than the other NMs. WGCNA revealed that agglomerate size and surface area as well as LDH release are among the most important parameters correlating with nanotoxicology. Moreover, we identified key drivers that can serve as representative biomarker candidates, supporting the value of multi-omics approaches to establish integrated approaches to testing and assessment (IATAs).

The project nanoGRAVUR (BMBF, 2015–2018) developed a framework for grouping of nanomaterials. Different groups may result for each of the three distinct perspectives of occupational, consumer and environmental safety. The properties, methods and descriptors are harmonised between the three perspectives and are based on: Tier 1 intrinsic physico-chemical properties (what they are) or GHS classification of the non-nano-form (human tox, ecotox, physical hazards); Tier 2 extrinsic physico-chemical properties, release from nano-enabled products, in vitro assays with cells (where they go; what they do); Tier 3 case-specific tests, potentially in vivo studies to substantiate the similarity within groups or application-specific exposure testing. Amongst all properties, dissolution and transformation are least modulated by different nanoforms within one substance, whereas dustiness, dispersion stability, abiotic and especially in vitro surface reactivity vary more often between different nanoforms. The methods developed or selected by nanoGRAVUR fill several gaps highlighted in the ProSafe reviews, and are useful to implement (i) the concept of nanoforms of the European Chemicals Agency (ECHA) and (ii) the concept of discrete forms of the United States Environmental Protection Agency (EPA). One cannot assess the significance of a dissimilarity, if the dynamic range of that property is unknown. Benchmark materials span dynamic ranges that enable us to establish bands, often with order-of-magnitude ranges. In 34 case studies we observed high biological similarity within each substance when we compared different (nano)forms of SiO2, BaSO4, kaolin, CeO2, ZnO, organic pigments, especially when we compared forms that are all untreated on the surface. In contrast, different Fe2O3 or TiO2 (nano)forms differ more significantly. The same nanoforms were also integrated in nano-enabled products (NEPs) for automotive coatings, clinker-reduced cements, cosmetic sunscreen, and lightweight

© 2018 The Royal Society of Chemistry. The likelihood of exposure to antimicrobial silver nanoparticles continues to grow with their increasing ubiquity in various medical and consumer products. While translocation of silver nanoparticles to major organs has been examined, the in situ location and concentration in the organs is not well characterised. Here we have used laser ablation-inductively coupled plasma-mass spectrometry to quantitatively image serial sections to construct a three-dimensional representation of the distribution of silver in rat spleen following respiratory tract exposure via intratracheal instillation of silver nanoparticles. Silver was distributed predominantly in the white pulp of the spleen at concentrations greater than 300 ng g-1. Imaging tissue sections via laser-ablation-inductively coupled plasma-mass spectrometry is an excellent tool for the visualisation and quantification of metals attributed to nanoparticles in organs allowing investigation of silver nanoparticle exposure in vivo.

Lung lining fluid is the first biological barrier nanoparticles (NPs) encounter during inhalation. As previous inhalation studies revealed considerable differences between surface functionalized NPs with respect to deposition and toxicity, our aim was to investigate the influence of lipid and/or protein binding on these processes. Thus, we analyzed a set of surface functionalized NPs including different SiO2 and ZrO2 in pure phospholipids, CuroSurfTM and purified native porcine pulmonary surfactant (nS). Lipid binding was surprisingly low for pure phospholipids and only few NPs attracted a minimal lipid corona. Additional presence of hydrophobic surfactant protein (SP) B in CuroSurfTM promoted lipid binding to NPs functionalized with Amino or PEG residues. The presence of the hydrophilic SP A in nS facilitated lipid binding to all NPs. In line with this the degree of lipid and protein affinities for different surface functionalized SiO2 NPs in nS followed the same order (SiO2 Phosphate ∼ unmodified SiO2 < SiO2 PEG < SiO2 Amino NPs). Agglomeration and biomolecule interaction of NPs in nS was mainly influenced by surface charge and hydrophobicity. Toxicological differences as observed in short-term inhalation studies (STIS) were mainly influenced by the core composition and/or surface reactivity of NPs. However, agglomeration in lipid media and lipid/protein affinity appeared to play a modulatory role on short-term inhalation toxicity. For instance, lipophilic NPs like ZrO2, which are interacting with nS to a higher extent, exhibited a far higher lung burden than their hydrophilic counterparts, which deserves further attention to predict or model effects of respirable NPs.

Presents the introductory welcome message from the conference proceedings. May include the conference officers' congratulations to all involved with the conference event and publication of the proceedings record., QC 20221004