BackgroundWith the increasing use of nanomaterials, the need for methods and assays to examine their immunosafety is becoming urgent, in particular for nanomaterials that are deliberately administered to human subjects (as in the case of nanomedicines). To obtain reliable results, standardised in vitro immunotoxicological tests should be used to determine the effects of engineered nanoparticles on human immune responses. However, before assays can be standardised, it is important that suitable methods are established and validated.ResultsIn a collaborative work between European laboratories, existing immunological and toxicological in vitro assays were tested and compared for their suitability to test effects of nanoparticles on immune responses. The prototypical nanoparticles used were metal (oxide) particles, either custom-generated by wet synthesis or commercially available as powders. Several problems and challenges were encountered during assay validation, ranging from particle agglomeration in biological media and optical interference with assay systems, to chemical immunotoxicity of solvents and contamination with endotoxin.ConclusionThe problems that were encountered in the immunological assay systems used in this study, such as chemical or endotoxin contamination and optical interference caused by the dense material, significantly affected the data obtained. These problems have to be solved to enable the development of reliable assays for the assessment of nano-immunosafety.

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BackgroundThe increasing production of nanoplastics and the fragmentation of microplastics into smaller particles suggest a plausible yet unclear hazard in the natural environment, such as soil. We investigated the short-term effects (28 days) of polystyrene nanoparticles (PS-NPs) on the activity and biomass of soil microbiota, and the functional diversity of soil enzymes at environmental relevant low levels in an incubation experiment.ResultsOur results showed a significant decrease in microbial biomass in treatments of 100 and 1000 ng PS-NP g−1 DM throughout the incubation period. Dehydrogenase activity and activities of enzymes involved in N-(leucine-aminopeptidase), P-(alkaline-phosphatase), and C-(β-glucosidase and cellobiohydrolase) cycles in the soil were significantly reduced at day 28 suggesting a broad and detrimental impact of PS-NPs on soil microbiota and enzymes. Leucine-aminopeptidase and alkaline-phosphatase activities tended to decrease consistently, while β-glucosidase and cellobiohydrolase activities increased at high concentrations (e.g., PS-NP-1000) in the beginning of the incubation period, e.g., at day 1. On the other hand, basal respiration and metabolic quotient increased with increasing PS-NP application rate throughout the incubation period possibly due to increased cell death that caused substrate-induced respiration (cryptic growth).ConclusionsWe herewith demonstrated for the first time the potential antimicrobial activity of PS-NPs in soil, and this may serve as an important resource in environmental risk assessment of PS-NPs in the soil environment.Electronic supplementary materialThe online version of this article (10.1186/s12302-018-0140-6) contains supplementary material, which is available to authorized users.

1) In compliance with the 3Rs policy to reduce, refine and replace animal experiments, the development of advanced in vitro models is needed for nanotoxicity assessment. Cells cultivated in 3D resemble organ structures better than 2D cultures. This study aims to compare cytotoxic and genotoxic responses induced by titanium dioxide (TiO 2 ), silver (Ag) and zinc oxide (ZnO) nanoparticles (NPs) in 2D monolayer and 3D spheroid cultures of HepG2 human liver cells. (2) NPs were characterized by electron microscopy, dynamic light scattering, laser Doppler anemometry, UV-vis spectroscopy and mass spectrometry. Cytotoxicity was investigated by the alamarBlue assay and confocal microscopy in HepG2 monolayer and spheroid cultures after 24 h of NP exposure. DNA damage (strand breaks and oxidized base lesions) was measured by the comet assay. (3) Ag-NPs were aggregated at 24 h, and a substantial part of the ZnO-NPs was dissolved in culture medium. Ag-NPs induced stronger cytotoxicity in 2D cultures (EC 50 3.8 µg/cm 2 ) than in 3D cultures (EC 50 > 30 µg/cm 2 ), and ZnO-NPs induced cytotoxicity to a similar extent in both models (EC 50 10.1-16.2 µg/cm 2 ). Agand ZnO-NPs showed a concentration-dependent genotoxic effect, but the effect was not statistically significant. TiO 2 -NPs showed no toxicity (EC 50 > 75 µg/cm 2 ). (4) This study shows that the HepG2 spheroid model is a promising advanced in vitro model for toxicity assessment of NPs.Nanomaterials 2020, 10, 545 2 of 21 and silver (Ag) is used as a disinfection agent in medical equipment and consumer products, on account of its antimicrobial activity [5]. Thus, humans are likely to be exposed to NPs, either intentionally or accidentally, during production and usage [6]. Transport of NPs across biological barriers has been observed by elemental analysis in both rodents and humans [7][8][9][10][11]. As an example, gold NPs have been reported to reach the systemic circulation in humans, after inhalation, and translocate to other organs [8,9].Several in vivo studies show that NPs accumulate in the liver, which is an important target organ for NPs and other xenobiotics due to its metabolic activity [12][13][14][15][16][17][18]. Induction of hepatotoxicity is one of the most common reasons for a medicine to be rejected or removed from the market [19,20]. Therefore, there is a need for sensitive hepatotoxicity screening methods for drug development and hazard assessment of chemicals or new materials, such as NPs. When considering the 3Rs-replacement, reduction and refinement-to minimize the use of animal experiments, hepatotoxicity should be assessed by reliable in vitro models. A great advantage of in vitro hepatocellular models for studying hepatotoxicity is the possibility of using human cells, either as primary cells or cell lines. The use of human hepatocyte cell lines, such as HepG2, C3A, Huh7 and HepaRG, has many advantages compared to primary cells. They are relatively easy to culture and have an unlimited life span, a relatively stable phenotype, high availability and ...

Changes in the genetic material can lead to serious human health defects, as mutations in somatic cells may cause cancer and can contribute to other chronic diseases. Genotoxic events can appear at both the DNA, chromosomal or (during mitosis) whole genome level. The study of mechanisms leading to genotoxicity is crucially important, as well as the detection of potentially genotoxic compounds. We consider the current state of the art and describe here the main endpoints applied in standard human in vitro models as well as new advanced 3D models that are closer to the in vivo situation. We performed a literature review of in vitro studies published from 2000–2020 (August) dedicated to the genotoxicity of nanomaterials (NMs) in new models. Methods suitable for detection of genotoxicity of NMs will be presented with a focus on advances in miniaturization, organ-on-a-chip and high throughput methods.