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The risk assessment of ingested nanomaterials (NMs) is an important issue. Here we present nine integrated approaches to testing and assessment (IATAs) to group ingested NMs following predefined hypotheses. The IATAs are structured as decision trees and tiered testing strategies for each decision node to support a grouping decision. Implications (e.g., regulatory or precautionary) per group are indicated. IATAs integrate information on durability and biopersistence (dissolution kinetics) to specific hazard endpoints, e.g., inflammation and genotoxicity, which are possibly indicative of toxicity. Based on IATAs, groups of similar nanoforms (NFs) of a NM can be formed, such as very slow dissolving, highly biopersistent and systemically toxic NFs. Reference NMs (ZnO, SiO 2 and TiO 2 ) along with related NFs are applied as case studies to testing the oral IATAs. Results based on the Tier 1 level suggest a hierarchy of biodurability and biopersistence of TiO 2 > SiO 2 > ZnO, and are confirmed by in vivo data (Tier 3 level). Interestingly, our analysis suggests that TiO 2 and SiO 2 NFs are able to induce both local and systemic toxicity along with microbiota dysbiosis and can be grouped according to the tested fate and hazard descriptors. This supports that the decision nodes of the oral IATAs are suitable for classification and assessment of the toxicity of NFs.

Grouping approaches of nanomaterials have the potential to facilitate high throughput and cost effective nanomaterial screening. However, an effective grouping of nanomaterials hinges on the application of suitable physicochemical descriptors to identify similarities. To address the problem, we developed an integrated testing approach coupling acellular and cellular phases, to study the full life cycle of ingested silver nanoparticles (NPs) and silver salts in the oro-gastrointestinal (OGI) tract including their impact on cellular uptake and integrity. This approach enables the derivation of exposure-dependent physical descriptors (EDPDs) upon biotransformation of undigested nanoparticles, digested nanoparticles and digested silver salts. These descriptors are identified in: size, crystallinity, chemistry of the core material, dissolution, high and low molecular weight Ag-biomolecule soluble complexes, and are compared in terms of similarities in a grouping hypothesis. Experimental results indicate that digested silver nanoparticles are neither similar to pristine nanoparticles nor completely similar to digested silver salts, due to the presence of different chemical nanoforms (silver and silver chloride nanocrystals), which were characterized in terms of their interactions with the digestive matrices. Interestingly, the cellular responses observed in the cellular phase of the integrated assay (uptake and inflammation) are also similar for the digested samples, clearly indicating a possible role of the soluble fraction of silver complexes. This study highlights the importance of quantifying exposure-related physical descriptors to advance grouping of NPs based on structural similarities.

In this contribution, we show the suitability of a 3D airway model, when coupled with a nebulizer system, for simulating workplace exposure to nanoparticles. As a proof of concept, workplace exposure to silica nanoparticles was experimentally measured in an occupational facility where nanoparticles are produced weekly, and compared with the official limit value for bulk silica materials. These values of potential exposure were simulated in a 3D airway model by nebulizing low doses (from 0.90 to 55 µg/cm 2 ) of silica nanoparticles over a prolonged period (12 weeks of repeated exposure, 5 days per week). Overall, the results suggest the efficiency of the defense mechanisms of the respiratory system and the clearance of the breathed silica nanoparticles by the mucociliary apparatus in accordance with the recent in vivo data. This in vitro platform shows that the doses tested may correlate with the occupational exposure limit values. Such relationship could provide regulatory-oriented data useful for risk classification of nanomaterials.

Multi-walled carbon nanotubes (MWCNT) have many outstanding physical and chemical properties that make them useful in many applications in nanotechnology. However, these properties are reported to be potentially harmful for the human body. The effects of low and realistic doses of three well-characterized preparations of MWCNT, obtained from the Joint Research Centre (JRC) (NM-400, NM-401, and NM-402), were assessed in two murine macrophage lines, Raw264.7, of peritoneal origin, and MH-S, derived from alveolar macrophages. Macrophage viability, evaluated with two distinct methods, was significantly lowered by NM-401 (needle-like, average length 4 μm, diameter 67 nm) with IC 50 values of 10 μg/cm 2 , whereas NM-400 and NM-402 (tangled, average lengths 846-1372 nm, diameter 11 nm) had much smaller effects. In contrast, at 10 μg/cm 2 , NM-400 and NM-402 induced the M1 marker Nos2 and, consistently, a sizable accumulation of nitrites in the medium, whereas NM-401 had no significant effect. None of the MWCNT preparations induced the M2 marker Arg1 . Phagocytic activity, assessed in Raw264.7 macrophages, was significantly reduced in cells exposed to NM-401, but not to NM-400 or NM-402. When tested on Calu-3 bronchial epithelial cell monolayers, the three MWCNT preparations did not affect cell viability, but decreased the trans-epithelial electrical resistance at the maximal dose tested (80 μg/cm 2 ), with the most evident effect detected for NM-401, even at 10 μg/cm 2 . In conclusion, among the possible structural determinants of the toxic effects exerted by MWCNT towards macrophages and airway epithelial cells, shape and length appear the most relevant at low, realistic doses., Competing Interests: The authors declare no conflict of interest.