Sedimentation of Nanoparticles in in vitro Toxicity Assays

Attached extended abstract published in the conference proceedingsJRC.DG.I.5-Nanobioscience

Physicochemical characterisation of gold, silica and silver nanoparticles in water and in serum-containing cell culture media

This report presents the results from a study organised under the coordination of JRC as part of a project aiming at the adaptation of the in vitro micronucleus test (Test Guideline 487) for the assessment of manufactured NMs. The aim of the first step of the project was to evaluate the physicochemical characterisation of selected representative nanomaterials (5 nm gold, 30 nm gold, 22 nm silica, 30 nm citrate and 30 nm PVP stabilised silver nanoparticles) in pure water and in different complete culture media. The results of the study show that using a combination of different characterisation techniques is important to providing reliable information about the agglomeration behaviour of the tested nanoparticles in complete cell culture media (CCM). Most of the materials exhibited mild agglomeration in serum containing CCM. Only the PVP functionalised silver nanoparticles showed a size distribution change in all of the culture media that is so small that it could be attributed to solely protein adsorption without notable agglomeration. Silica nanoparticles were found to be the most sensitive to interaction with serum containing CCM, showing massive concentration and time dependent agglomeration strongly affected by the CCM composition. Extensive agglomeration might lead also to the accelerated sedimentation of the particles changing drastically the true, effective dose that the cells will receive under in vitro conditions1, 2. Thus, it has to be investigated in more detail and taken in account when designing in vitro experiments in the next phase of the project.JRC.F.2-Consumer Products Safet

In vitro cytotoxicity and cellular uptake evaluation of gold, silica and silver nanoparticles in five different cell lines: Caco-2, A549, CHO, V79 and TK6

The Joint Research Centre (JRC) is the science and knowledge service of the European Commission and provides scientific and technical support to a wide range of European Union policies. In particular, the use of manufactured nanomaterials (NMs) that are increasingly used in consumer and health products raised concerns regarding potential unintended risks to humans and the environment. Under the umbrella of the Organisation for Economic Co-operation and Development (OECD) Working Party on Manufactured Nanoparticles (WPMN), the JRC has contributed to the development, optimisation and harmonisation of a number of test methods incl in vitro test methods suitable for risk assessment of NMs. The present report is part of an OCED project that focuses on availability of vitro methods for the assessment of the genotoxic potential of nanomaterials. An international partnership incl. the JRC aims to adapt the in vitro micronucleus test (OECD Test Guideline 487) to specific testing requirements of nanomaterials in a stepwise approach. Within the first phase of the project, the physical-chemical properties of selected nanomaterials have been intensively characterised. This second report is now focussing on the evaluation of in vitro cytotoxicity testing and the uptake of selected representative nanomaterials. 5 nm gold, 30 nm gold, 22 nm silica, 30 nm citrate and 30 nm PVP stabilised silver nanoparticles have been analysed in 5 different cell lines.JRC.F.2 - Consumer Products Safet

Inter-laboratory comparison on the determination of the hydrophobicity index of nanomaterials through an affinity measurement

Hydrophobicity is a physico-chemical property that may influence the fate of nanomaterials in the environment and biological matrices. A method to characterise the hydrophobicity of nanomaterials was developed at the JRC and proposed as an OECD Test Guideline. In this context, the JRC led an Inter-laboratory comparison (ILC) aiming to assess the transferability of the standard operating procedure. The method is based on the measurement of the affinity of nanomaterials to engineered collectors. Nine laboratories participated to this ILC. The variability of the measurements and the reproducibility of the calculation of the Hydrophobicity Index were assessed according to the International Standard ISO 5725-2. Accordingly, with |Z-scores| < 2 for all the participants, the determination of the Hydrophobicity Index was considered satisfactory. The method was adopted by the OECD Working Party of the National Coordinators of the Test Guidelines Programme in April 2023 as Test Guideline 126.JRC.F.2 - Technologies for Healt

Interlaboratory comparison study of the Colony Forming Efficiency assay for assessing cytotoxicity of nanomaterials

Nanotechnology has gained importance in the past years as it provides opportunities for industrial growth and innovation. However, the increasing use of manufactured nanomaterials (NMs) in a number of commercial applications and consumer products raises also safety concerns and questions regarding potential unintended risks to humans and the environment. Since several years the European Commission’s Joint Research Centre (JRC) is putting effort in the development, optimisation and harmonisation of in vitro test methods suitable for screening and hazard assessment of NMs. Work is done in collaboration with international partners, in particular the Organisation for Economic Co-operation and Development (OECD). This report presents the results from an interlaboratory comparison study of the in vitro Colony Forming Efficiency (CFE) cytotoxicity assay performed in the frame of OECD's Working Party of Manufactured Nanomaterials (WPMN). Twelve laboratories from European Commission, France, Italy, Japan, Poland, Republic of Korea, South Africa and Switzerland participated in the study coordinated by JRC. The results show that the CFE assay is a suitable and robust in vitro method to assess cytotoxicity of NMs. The assay protocol is well defined and is easily and reliably transferable to other laboratories. The results obtained show good intra and interlaboratory reproducibility of the assay for both the positive control and the tested nanomaterials. In conclusion the CFE assay can be recommended as a building block of an in vitro testing battery for NMs toxicity assessment. It could be used as a first choice method to define dose-effect relationships for other in vitro assays.JRC.I.4-Nanobioscience

Interaction of magnetic nanoparticles with U87MG cells studied by synchrotron radiation X-ray fluorescence techniques

International audienceSynchrotron radiation (SR) X-ray microscopy combined with X-ray fluorescence (XRF) microspectroscopy provides unique information that have pushed the frontiers of biological research, particularly when investigating intracellular mechanisms. This work reports an SR-XRF microspectroscopy investigation on the distribution and the potential toxicity of Fe 2 O 3 and CoFe 2 O 4 nanoparticles (NPs) in U87MG glioblastoma-astrocytoma cells. The U87MG cells exposed to NPs concentrations ranging from 5 to 250 mg/ml for 24 h were analyzed in order to monitor both morphological and chemical changes. The SR-XRF maps complemented with XRM absorption and phase contrast images have revealed different intracellular distribution patterns for the two nanoparticles types allowing different mechanism of toxicity to be deduced

Agglomeration Behavior and Fate of Food-Grade Titanium Dioxide in Human Gastrointestinal Digestion and in the Lysosomal Environment

In the present study, we addressed the knowledge gaps regarding the agglomeration behavior and fate of food-grade titanium dioxide (E 171) in human gastrointestinal digestion (GID). After thorough multi-technique physicochemical characterization including TEM, single-particle ICP-MS (spICP-MS), CLS, VSSA determination and ELS, the GI fate of E 171 was studied by applying the in vitro GID approach established for the regulatory risk assessment of nanomaterials in Europe, using a standardized international protocol. GI fate was investigated in fasted conditions, relevant to E 171 use in food supplements and medicines, and in fed conditions, with both a model food and E 171-containing food samples. TiO2 constituent particles were resistant to GI dissolution, and thus, their stability in lysosomal fluid was investigated. The biopersistence of the material in lysosomal fluid highlighted its potential for bioaccumulation. For characterizing the agglomeration degree in the small intestinal phase, spICP-MS represented an ideal analytical tool to overcome the limitations of earlier studies. We demonstrated that, after simulated GID, in the small intestine, E 171 (at concentrations reflecting human exposure) is present with a dispersion degree similar to that obtained when dispersing the material in water by means of high-energy sonication (i.e., &gt;70% of particles &lt;250 nm)