Hepato(Geno)toxicity assessment of nanoparticles in a HepG2 liver spheroid model

(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), 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 3.8 µg/cm 2) than in 3D cultures (EC > 30 µg/cm 2), and ZnO-NPs induced cytotoxicity to a similar extent in both models (EC 10.1-16.2 µg/cm 2). Ag- and ZnO-NPs showed a concentration-dependent genotoxic effect, but the effect was not statistically significant. TiO-NPs showed no toxicity (EC > 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

Versailles project on advanced materials and standards (VAMAS) interlaboratory study on measuring the number concentration of colloidal gold nanoparticles

We describe the outcome of a large international interlaboratory study of the measurement of particle number concentration of colloidal nanoparticles, project 10 of the technical working area 34, "Nanoparticle Populations" of the Versailles Project on Advanced Materials and Standards (VAMAS). A total of 50 laboratories delivered results for the number concentration of 30 nm gold colloidal nanoparticles measured using particle tracking analysis (PTA), single particle inductively coupled plasma mass spectrometry (spICP-MS), ultraviolet-visible (UV-Vis) light spectroscopy, centrifugal liquid sedimentation (CLS) and small angle X-ray scattering (SAXS). The study provides quantitative data to evaluate the repeatability of these methods and their reproducibility in the measurement of number concentration of model nanoparticle systems following a common measurement protocol. We find that the population-averaging methods of SAXS, CLS and UV-Vis have high measurement repeatability and reproducibility, with between-labs variability of 2.6%, 11% and 1.4% respectively. However, results may be significantly biased for reasons including inaccurate material properties whose values are used to compute the number concentration. Particle-counting method results are less reproducibile than population-averaging methods, with measured between-labs variability of 68% and 46% for PTA and spICP-MS respectively. This study provides the stakeholder community with important comparative data to underpin measurement reproducibility and method validation for number concentration of nanoparticles

Versailles project on advanced materials and standards (VAMAS) interlaboratory study on measuring the number concentration of colloidal gold nanoparticles

We describe the outcome of a large international interlaboratory study of the measurement of particle number concentration of colloidal nanoparticles, project 10 of the technical working area 34, "Nanoparticle Populations" of the Versailles Project on Advanced Materials and Standards (VAMAS). A total of 50 laboratories delivered results for the number concentration of 30 nm gold colloidal nanoparticles measured using particle tracking analysis (PTA), single particle inductively coupled plasma mass spectrometry (spICP-MS), ultraviolet-visible (UV-Vis) light spectroscopy, centrifugal liquid sedimentation (CLS) and small angle X-ray scattering (SAXS). The study provides quantitative data to evaluate the repeatability of these methods and their reproducibility in the measurement of number concentration of model nanoparticle systems following a common measurement protocol. We find that the population-averaging methods of SAXS, CLS and UV-Vis have high measurement repeatability and reproducibility, with between-labs variability of 2.6%, 11% and 1.4% respectively. However, results may be significantly biased for reasons including inaccurate material properties whose values are used to compute the number concentration. Particle-counting method results are less reproducibile than population-averaging methods, with measured between-labs variability of 68% and 46% for PTA and spICP-MS respectively. This study provides the stakeholder community with important comparative data to underpin measurement reproducibility and method validation for number concentration of nanoparticles

Versailles project on advanced materials and standards (VAMAS) interlaboratory study on measuring the number concentration of colloidal gold nanoparticles

We describe the outcome of a large international interlaboratory study of the measurement of particle number concentration of colloidal nanoparticles, project 10 of the technical working area 34, "Nanoparticle Populations" of the Versailles Project on Advanced Materials and Standards (VAMAS). A total of 50 laboratories delivered results for the number concentration of 30 nm gold colloidal nanoparticles measured using particle tracking analysis (PTA), single particle inductively coupled plasma mass spectrometry (spICP-MS), ultraviolet-visible (UV-Vis) light spectroscopy, centrifugal liquid sedimentation (CLS) and small angle X-ray scattering (SAXS). The study provides quantitative data to evaluate the repeatability of these methods and their reproducibility in the measurement of number concentration of model nanoparticle systems following a common measurement protocol. We find that the population-averaging methods of SAXS, CLS and UV-Vis have high measurement repeatability and reproducibility, with between-labs variability of 2.6%, 11% and 1.4% respectively. However, results may be significantly biased for reasons including inaccurate material properties whose values are used to compute the number concentration. Particle-counting method results are less reproducibile than population-averaging methods, with measured between-labs variability of 68% and 46% for PTA and spICP-MS respectively. This study provides the stakeholder community with important comparative data to underpin measurement reproducibility and method validation for number concentration of nanoparticles

Different Sensitivity of Advanced Bronchial and Alveolar Mono- and Coculture Models for Hazard Assessment of Nanomaterials

<p>For the next-generation risk assessment (NGRA) of chemicals and nanomaterials, new approach methodologies (NAMs) are needed for hazard assessment in compliance with the 3R's to reduce, replace and refine animal experiments. This study aimed to establish and characterize an advanced respiratory model consisting of human epithelial bronchial BEAS-2B cells cultivated at the air–liquid interface (ALI), both as monocultures and in cocultures with human endothelial EA.hy926 cells. The performance of the bronchial models was compared to a commonly used alveolar model consisting of A549 in monoculture and in coculture with EA.hy926 cells. The cells were exposed at the ALI to nanosilver (NM-300K) in the VITROCELL® Cloud. After 24 h, cellular viability (alamarBlue assay), inflammatory response (enzyme-linked immunosorbent assay), DNA damage (enzyme-modified comet assay), and chromosomal damage (cytokinesis-block micronucleus assay) were measured. Cytotoxicity and genotoxicity induced by NM-300K were dependent on both the cell types and model, where BEAS-2B in monocultures had the highest sensitivity in terms of cell viability and DNA strand breaks. This study indicates that the four ALI lung models have different sensitivities to NM-300K exposure and brings important knowledge for the further development of advanced 3D respiratory in vitro models for the most reliable human hazard assessment based on NAMs.</p&gt

Different Sensitivity of Advanced Bronchial and Alveolar Mono- and Coculture Models for Hazard Assessment of Nanomaterials

For the next-generation risk assessment (NGRA) of chemicals and nanomaterials, new approach methodologies (NAMs) are needed for hazard assessment in compliance with the 3R’s to reduce, replace and refine animal experiments. This study aimed to establish and characterize an advanced respiratory model consisting of human epithelial bronchial BEAS-2B cells cultivated at the air–liquid interface (ALI), both as monocultures and in cocultures with human endothelial EA.hy926 cells. The performance of the bronchial models was compared to a commonly used alveolar model consisting of A549 in monoculture and in coculture with EA.hy926 cells. The cells were exposed at the ALI to nanosilver (NM-300K) in the VITROCELL® Cloud. After 24 h, cellular viability (alamarBlue assay), inflammatory response (enzyme-linked immunosorbent assay), DNA damage (enzyme-modified comet assay), and chromosomal damage (cytokinesis-block micronucleus assay) were measured. Cytotoxicity and genotoxicity induced by NM-300K were dependent on both the cell types and model, where BEAS-2B in monocultures had the highest sensitivity in terms of cell viability and DNA strand breaks. This study indicates that the four ALI lung models have different sensitivities to NM-300K exposure and brings important knowledge for the further development of advanced 3D respiratory in vitro models for the most reliable human hazard assessment based on NAMs

Different Sensitivity of Advanced Bronchial and Alveolar Mono- and Coculture Models for Hazard Assessment of Nanomaterials

For the next-generation risk assessment (NGRA) of chemicals and nanomaterials, new approach methodologies (NAMs) are needed for hazard assessment in compliance with the 3R’s to reduce, replace and refine animal experiments. This study aimed to establish and characterize an advanced respiratory model consisting of human epithelial bronchial BEAS-2B cells cultivated at the air–liquid interface (ALI), both as monocultures and in cocultures with human endothelial EA.hy926 cells. The performance of the bronchial models was compared to a commonly used alveolar model consisting of A549 in monoculture and in coculture with EA.hy926 cells. The cells were exposed at the ALI to nanosilver (NM-300K) in the VITROCELL® Cloud. After 24 h, cellular viability (alamarBlue assay), inflammatory response (enzyme-linked immunosorbent assay), DNA damage (enzyme-modified comet assay), and chromosomal damage (cytokinesis-block micronucleus assay) were measured. Cytotoxicity and genotoxicity induced by NM-300K were dependent on both the cell types and model, where BEAS-2B in monocultures had the highest sensitivity in terms of cell viability and DNA strand breaks. This study indicates that the four ALI lung models have different sensitivities to NM-300K exposure and brings important knowledge for the further development of advanced 3D respiratory in vitro models for the most reliable human hazard assessment based on NAMs

The colony forming efficiency assay for toxicity testing of nanomaterials—Modifications for higher-throughput

To cope with the high number of nanomaterials manufactured, it is essential to develop high-throughput methods for in vitro toxicity screening. At the same time, the issue with interference of the nanomaterial (NM) with the read-out or the reagent of the assay needs to be addressed to avoid biased results. Thus, validated label-free methods are urgently needed for hazard identification of NMs to avoid unintended adverse effects on human health. The colony forming efficiency (CFE) assay is a label- and interference-free method for quantification of cytotoxicity by cell survival and colony forming efficiency by CFE formation. The CFE has shown to be compatible with toxicity testing of NMs. Here we present an optimized protocol for a higher-throughput set up

The colony forming efficiency assay for toxicity testing of nanomaterials – Modifications for higher-throughput

To cope with the high number of nanomaterials manufactured, it is essential to develop high-throughput methods for in vitro toxicity screening. At the same time, the issue with interference of the nanomaterial (NM) with the read-out or the reagent of the assay needs to be addressed to avoid biased results. Thus, validated label-free methods are urgently needed for hazard identification of NMs to avoid unintended adverse effects on human health. The colony forming efficiency (CFE) assay is a label- and interference-free method for quantification of cytotoxicity by cell survival and colony forming efficiency by CFE formation. The CFE has shown to be compatible with toxicity testing of NMs. Here we present an optimized protocol for a higher-throughput set up

The colony forming efficiency assay for toxicity testing of nanomaterials—Modifications for higher-throughput

To cope with the high number of nanomaterials manufactured, it is essential to develop high-throughput methods for in vitro toxicity screening. At the same time, the issue with interference of the nanomaterial (NM) with the read-out or the reagent of the assay needs to be addressed to avoid biased results. Thus, validated label-free methods are urgently needed for hazard identification of NMs to avoid unintended adverse effects on human health. The colony forming efficiency (CFE) assay is a label- and interference-free method for quantification of cytotoxicity by cell survival and colony forming efficiency by CFE formation. The CFE has shown to be compatible with toxicity testing of NMs. Here we present an optimized protocol for a higher-throughput set up