Potential Artifacts and Control Experiments in Toxicity Tests of Nanoplastic and Microplastic Particles.

To fully understand the potential ecological and human health risks from nanoplastics and microplastics (NMPs) in the environment, it is critical to make accurate measurements. Similar to past research on the toxicology of engineered nanomaterials, a broad range of measurement artifacts and biases are possible when testing their potential toxicity. For example, antimicrobials and surfactants may be present in commercially available NMP dispersions, and these compounds may account for toxicity observed instead of being caused by exposure to the NMP particles. Therefore, control measurements are needed to assess potential artifacts, and revisions to the protocol may be needed to eliminate or reduce the artifacts. In this paper, we comprehensively review and suggest a next generation of control experiments to identify measurement artifacts and biases that can occur while performing NMP toxicity experiments. This review covers the broad range of potential NMP toxicological experiments, such as in vitro studies with a single cell type or complex 3-D tissue constructs, in vivo mammalian studies, and ecotoxicity experiments testing pelagic, sediment, and soil organisms. Incorporation of these control experiments can reduce the likelihood of false positive and false negative results and more accurately elucidate the potential ecological and human health risks of NMPs.

Cause-and-Effect Analysis as a Tool To Improve the Reproducibility of Nanobioassays: Four Case Studies.

One of the challenges in using in vitro data to understand the potential risks of engineered nanomaterials (ENMs) is that results often differ or are even contradictory among studies. While it is recognized that numerous factors can influence results produced by nanobioassays, there has not yet been a consistently used conceptual framework to identify key sources of variability in these assays. In this paper, we use cause-and-effect analysis to systematically describe sources of variability in four key in vitro nanobioassays: the 2',7'-dichlorofluorescein assay, an enzyme-linked immunosorbent assay for measuring interleukin-8, a flow cytometry assay (Annexin V/propidium iodide), and the Comet assay. These assays measure end points that can occur in cells impacted by ENMs through oxidative stress, a principle mechanism for ENM toxicity. The results from this analysis identify control measurements to test for potential artifacts or biases that could occur during conduct of these assays with ENMs. Cause-and-effect analysis also reveals additional measurements that could be performed either in preliminary experiments or each time the assay is run to increase confidence in the assay results and their reproducibility within and among laboratories. The approach applied here with these four assays can be used to support the development of a broad range of nanobioassays.

Bridging communities in the field of nanomedicine.

An early dialogue between nanomedicine developers and regulatory authorities are of utmost importance to anticipate quality and safety requirements for these innovative health products. In order to stimulate interactions between the various communities involved in a translation of nanomedicines to clinical applications, the European Commission's Joint Research Centre hosted a workshop titled "Bridging communities in the field of Nanomedicine" in Ispra/Italy on the 27th -28th September 2017. Experts from regulatory bodies, research institutions and industry came together to discuss the next generation of nanomedicines and their needs to obtain regulatory approval. The workshop participants came up with recommendations highlighting methodological gaps that should be addressed in ongoing projects addressing the regulatory science of nanomedicines. In addition, individual opinions of experts relevant to progress of the regulatory science in the field of nanomedicine were summarised in the format of a survey.

Cytotoxic effects of nanosilver are highly dependent on the chloride concentration and the presence of organic compounds in the cell culture media.

BACKGROUND: Nanosilver shows great promise for use in industrial, consumer or medical products because of its antimicrobial properties. However, the underlying mechanisms of the effects of silver nanoparticles on human cells are still controversial. Therefore, in the present study the influence of the chloride concentration and different serum content of culture media on the cytotoxic effects of nanosilver was systematically evaluated. RESULTS: Our results show that nanosilver toxicity was strongly affected by the composition of the culture media. The chloride concentration, as well as the carbon content affected the silver agglomeration and the complex formation. But also the dissolution of nanosilver and the availability of free silver ions (Ag+) were severely affected by the compositions of the culture media. Cells, only exposed to silver particles in suspension and dissolved silver complexes, did not show any effects under all conditions. Nanosilver agglomerates and silver complexes were not very soluble. Thus, cells growing on the bottom of the culture dishes were exposed to sedimented nanosilver agglomerates and precipitated silver complexes. Locally, the concentration of silver on the cell surface was very high, much higher compared the silver concentration in the bulk solution. The cytotoxic effects of nanosilver are therefore a combination of precipitated silver complexes and organic silver compounds rather than free silver ions. CONCLUSIONS: Silver coatings are used in health care products due to their bacteriostatic or antibacterial properties. The assessment of the toxicity of a certain compound is mostly done using in vitro assays. Therefore, cytotoxicity studies of nanosilver using human cell cultures have to be undertaken under well controlled and understood cultivations conditions in order to improve the compatibility of different studies. Especially when eukaryotic versus prokaryotic systems are compared for the evaluation of the use of nanosilver as antibacterial coatings for implants in order to prevent bacterial colonization.

Comparability of in vitro tests for bioactive nanoparticles: a common assay to detect reactive oxygen species as an example.

The release of reactive oxygen species (ROS) during the electron transport of mitochondrial aerobic respiration is the major source of ROS. However, contact between cells and nanoparticles (NPs) can also induce release of ROS, leading to an imbalance towards the pro-oxidative state. At low levels of ROS production, cells initiate a protective response to guarantee their survival, but an excess of ROS can damage cellular compounds such as membranes and various organelles, or directly cause genotoxicity. Thus an elevated level of ROS is an important indicator of cellular stress and an accurate recording of this parameter would be very informative. ROS can be measured by various assays, but all known assays measuring and quantifying ROS possess certain weaknesses. The problems and challenges of quantitatively detecting ROS in vitro using the 2',7'-dichlorodihydrofluorescein (DCF) assay is discussed as an example. In addition, we debate the difficulties in finding a suitable and stable chemical reaction control for the DCF assay (or other ROS-detecting assays). As a conclusion, we believe that using 3-morpholinosydnonimine hydrochloride (Sin-1) as a ROS inducer in the DCF assay is feasible only qualitatively. However, a quantitative measurement of the absolute amount of ROS produced and a quantitative comparison between experiments is (at the moment) impossible.

Future perspectives for advancing regulatory science of nanotechnology-enabled health products.

The identification of regulatory challenges for nanotechnology-enabled health products, followed by discussions with the involved stakeholders, is the first step towards a strategic planning of how such challenges can be successfully addressed in the future. In order to better understand whether the identified regulatory needs are sector-specific for health products or might also hinder the progress in other domains, the REFINE consortium reached out to communities representing other sectors that also exploit the potential of nanotechnology, i.e. industrial chemicals, food and cosmetics. Through a series of trans-sectorial workshops, REFINE partners identified common as well as sector-specific challenges and discussed possible ways forward. Potential solutions lie in a more strengthen collaboration between regulatory and research communities resulting in a targeted production and exploitation of academic data for the regulatory decision-making. Furthermore, a coordinated use of knowledge sharing platforms and databases, trans-sectorial standardisation activities and harmonisation of regulatory activities between geographical regions are possible ways forward, in line with the upcoming European political initiatives such as the Chemical Strategy for Sustainability (CSS). Finally, we also discuss the perspectives for further development and sustainability of methods and tools developed in the REFINE project.

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.

Characteristics to consider when selecting a positive control material for an in vitro assay

The use of in vitro assays to inform decision-making requires robust and reproducible results across studies, laboratories, and time. Experiments using positive control materials are an integral component of an assay procedure to demonstrate the extent to which the measurement system is performing as expected. This paper reviews ten characteristics that should be considered when selecting a positive control material for an in vitro assay: 1) the biological mechanism of action, 2) ease of preparation, 3) chemical purity, 4) verifiable physical properties, 5) stability, 6) ability to generate responses spanning the dynamic range of the assay, 7) technical or biological interference, 8) commercial availability, 9) user toxicity, and 10) disposability. Examples and a case study of the monocyte activation test are provided to demonstrate the application of these characteristics for identification and selection of potential positive control materials. Because specific positive control materials are often written into testing standards for in vitro assays, selection of the positive control material based on these characteristics can aid in ensuring the long-term relevance and usability of these standards.

Emerging Standards and Analytical Science for Nanoenabled Medical Products.

Development and application of nanotechnology-enabled medical products, including drugs, devices, and in vitro diagnostics, are rapidly expanding in the global marketplace. In this review, the focus is on providing the reader with an introduction to the landscape of commercially available nanotechnology-enabled medical products as well as an overview of the international documentary standards and reference materials that support and facilitate efficient regulatory evaluation and reliable manufacturing of this diverse group of medical products. We describe the materials, test methods, and standards development needs for emerging medical products. Scientific and measurement challenges involved in the development and application of innovative nanoenabled medical products motivate discussion throughout this review.

A novel approach to increase robustness, precision and high-throughput capacity of single cell gel electrophoresis.

The routine use of single cell gel electrophoresis assay in medical diagnostics and biomonitoring is prevented by its high variability. Several factors have been identified and can be grouped into four main categories: 1) the biological sample, 2) the assay protocol, 3) the physical parameters during electrophoresis and 4) the analysis. Even though the scientific knowledge on assay variability is available, not much has been done so far to tackle the issues from the technological side. Therefore, this study addresses the question in how far the precise and accurate control over the physical parameters of electrophoresis is able to reduce variability of single cell gel electrophoresis assay results. All four above mentioned categories make up the overall assay variability. To resolve the contribution from a single category, the remaining three have to be kept as constant as possible. To achieve this we generated a set of x-ray treated control cells, worked according to a well-defined standard operating procedure and one single operator performed the analysis. Thereby variability resulting from the electrophoresis tank could be elucidated. We compared assay performance in two such tank systems: a newly developed electrophoresis tank that accurately controls voltage, temperature during the electrophoretic run and the homogeneity of the electric field, and a widely used commercially available standard platform tank. In summary, our results demonstrate that, irrespective of the cellular sample and its intrinsic biological variability, accurate control over physical parameters considerably increases repeatability, reproducibility and precision of single cell gel electrophoresis.

Interference of engineered nanomaterials in flow cytometry: A case study.

Nanotechnology is regarded as the enabling technology of the 21st century. However, only a relatively small number of nano-enabled medical and healthcare products finally made their way to the market. There are several reasons why such innovative approaches fail in translation, with one key factor being the uncertainty surrounding their safety assessment. Although well described, interference reactions of engineered nanomaterials (ENM) with classical cytotoxicity assays remain a major source of uncertainty. Flow cytometry is a powerful, widely used, in vitro technique. Its readout is based on the detection of refracted laser light and fluorescence signals. It is therefore susceptible to ENM interference. Here we investigated possible interferences of ENM in the Annexin V/propidium iodide (PI) assay, which quantifies apoptotic and necrotic cell populations by flow cytometry. Two case studies were conducted using either silica or gold nanoparticles differing in size, specific surface area and surface chemistry. Both ENM types were found to cause distinct interference reactions at realistic concentrations. Silica particles induced false-positive signals; however only in the absence of a protein corona and in conjunction with a particular fluorophore combination (FITC/PI). In contrast, gold particles led to complex quenching effects which were only marginally influenced by the presence of proteins and occurred for both fluorophore combinations analyzed. We present a versatile spike-in approach which is applicable to all ENM and cell types. It further allows for the identification of a broad range of different interference phenomena, thereby increasing the reliability and quality of flow cytometry and ENM hazard assessment.

Human health risk of ingested nanoparticles that are added as multifunctional agents to paints: an in vitro study.

Microorganisms growing on painted surfaces are not only an aesthetic problem, but also actively contribute to the weathering and deterioration of materials. A widely used strategy to combat microbial colonization is the addition of biocides to the paint. However, ecotoxic, non-degradable biocides with a broad protection range are now prohibited in Europe, so the paint industry is considering engineered nanoparticles (ENPs) as an alternative biocide. There is concern that ENPs in paint might be released in run-off water and subsequently consumed by animals and/or humans, potentially coming into contact with cells of the gastrointestinal tract and affecting the immune system. Therefore, in the present study we evaluated the cytotoxic effects of three ENPs (nanosilver, nanotitanium dioxide and nanosilicon dioxide) that have a realistic potential for use in paints in the near future. When exposed to nanotitanium dioxide and nanosilicon dioxide in concentrations up to 243 µg/mL for 48 h, neither the gastrointestinal cells (CaCo-2) nor immune system cells (Jurkat) were significantly affected. However, when exposed to nanosilver, several cell parameters were affected, but far less than by silver ions used as a control. No differences in cytotoxicity were observed when cells were exposed to ENP-containing paint particles, compared with the same paint particles without ENPs. Paint particles containing ENPs did not affect cell morphology, the release of reactive oxygen species or cytokines, cell activity or cell death in a different manner to the same paint particles without ENPs. The results suggest that paints doped with ENPs do not pose an additional acute health hazard for humans.

Interlaboratory comparison of size measurements on nanoparticles using nanoparticle tracking analysis (NTA).

One of the key challenges in the field of nanoparticle (NP) analysis is in producing reliable and reproducible characterisation data for nanomaterials. This study looks at the reproducibility using a relatively new, but rapidly adopted, technique, Nanoparticle Tracking Analysis (NTA) on a range of particle sizes and materials in several different media. It describes the protocol development and presents both the data and analysis of results obtained from 12 laboratories, mostly based in Europe, who are primarily QualityNano members. QualityNano is an EU FP7 funded Research Infrastructure that integrates 28 European analytical and experimental facilities in nanotechnology, medicine and natural sciences with the goal of developing and implementing best practice and quality in all aspects of nanosafety assessment. This study looks at both the development of the protocol and how this leads to highly reproducible results amongst participants. In this study, the parameter being measured is the modal particle size.

Nanomaterial cell interactions: are current in vitro tests reliable?

New properties of engineered nanomaterials raise great expectations for industrial, scientific as well as medical applications. At the same time concerns among consumers regarding the safety aspects of this new technology emerge. Furthermore, among the multitude of published studies, a considerable number do not reveal reliable data. Thus, standardized, validated, reliable, robust, reproducible and intelligent testing strategies are urgently needed that address nanomaterial toxicity. This article discusses the reliability of currently used in vitro toxicity assays. It covers major problems, pitfalls and challenges of assay performance and validation. We recommend a series of different controls to improve the experimental quality and, thus, also the reliability and reproducibility of current in vitro systems. These recommendations consequently applied in the future will increase the safe and sustainable use of nanotechnology.