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 ...