Knowledge, Information, and Data Readiness Levels (KaRLs) for Risk Assessment, Communication, and Governance of Nano-, New, and Other Advanced Materials.

The obvious benefits derived from the increasing use of engineered nano-, new, and advanced materials and associated products have to be weighed out by a governance process against their possible risks. Differences in risk perception (beliefs about potential harm) among stakeholders, in particular nonscientists, and low transparency of the underlying decision processes can lead to a lack of support and acceptance of nano-, new, and other advanced material enabled products. To integrate scientific outcomes with stakeholders needs, this work develops a new approach comprising a nine-level, stepwise categorization and guidance system entitled "Knowledge, Information, and Data Readiness Levels" (KaRLs), analogous to the NASA Technology Readiness Levels. The KaRL system assesses the type, extent, and usability of the available data, information, and knowledge and integrates the participation of relevant and interested stakeholders in a cocreation/codesign process to improve current risk assessment, communication, and governance. The novelty of the new system is to communicate and share all available and relevant elements on material related risks in a user/stakeholder-friendly, transparent, flexible, and holistic way and so stimulate reflection, awareness, communication, and a deeper understanding that ultimately enables the discursive process that is needed for the sustainable risk governance of new materials.

Risk Governance of Emerging Technologies Demonstrated in Terms of its Applicability to Nanomaterials.

Nanotechnologies have reached maturity and market penetration that require nano-specific changes in legislation and harmonization among legislation domains, such as the amendments to REACH for nanomaterials (NMs) which came into force in 2020. Thus, an assessment of the components and regulatory boundaries of NMs risk governance is timely, alongside related methods and tools, as part of the global efforts to optimise nanosafety and integrate it into product design processes, via Safe(r)-by-Design (SbD) concepts. This paper provides an overview of the state-of-the-art regarding risk governance of NMs and lays out the theoretical basis for the development and implementation of an effective, trustworthy and transparent risk governance framework for NMs. The proposed framework enables continuous integration of the evolving state of the science, leverages best practice from contiguous disciplines and facilitates responsive re-thinking of nanosafety governance to meet future needs. To achieve and operationalise such framework, a science-based Risk Governance Council (RGC) for NMs is being developed. The framework will provide a toolkit for independent NMs' risk governance and integrates needs and views of stakeholders. An extension of this framework to relevant advanced materials and emerging technologies is also envisaged, in view of future foundations of risk research in Europe and globally.

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.

Transient DNA damage following exposure to gold nanoparticles.

Due to their interesting physicochemical properties, gold nanoparticles (Au-NPs) are the focus of increasing attention in the field of biomedicine and are under consideration for use in drug delivery and bioimaging, or as radiosensitizers and nano-based vaccines. Thorough evaluation of the genotoxic potential of Au-NPs is required, since damage to the genome can remain undetected in standard hazard assessments. Available genotoxicity data is either limited or contradictory. Here, we examined the influence of three surface modified 3-4 nm Au-NPs on human A549 cells, according to the reactive oxygen species (ROS) paradigm. After 24 h of Au-NP treatment, nanoparticles were taken up by cells as agglomerates; however, no influence on cell viability or inflammation was detected. No increase in ROS production was observed by H2-DCF assay; however, intracellular glutathione levels reduced over time, indicating oxidative stress. All three types of Au-NPs induced DNA damage, as detected by alkaline comet assay. The strongest genotoxic effect was observed for positively charged Au-NP I. Further analysis of Au-NP I by neutral comet assay, fluorimetric detection of alkaline DNA unwinding assay, and γH2AX staining, revealed that the induced DNA lesions were predominantly alkali-labile sites. As highly controlled repair mechanisms have evolved to remove a wide range of DNA lesions with great efficiency, it is important to focus on both acute cyto- and genotoxicity, alongside post-treatment effects and DNA repair. We demonstrate that Au-NP-induced DNA damage is largely repaired over time, indicating that the observed damage is of transient nature.

The DaNa2.0 Knowledge Base Nanomaterials-An Important Measure Accompanying Nanomaterials Development.

Nanotechnology is closely related to the tailored manufacturing of nanomaterials for a huge variety of applications. However, such applications with newly developed materials are also a reason for concern. The DaNa2.0 project provides information and support for these issues on the web in condensed and easy-to-understand wording. Thus, a key challenge in the field of advanced materials safety research is access to correct and reliable studies and validated results. For nanomaterials, there is currently a continuously increasing amount of publications on toxicological issues, but criteria to evaluate the quality of these studies are necessary to use them e.g., for regulatory purposes. DaNa2.0 discusses scientific results regarding 26 nanomaterials based on actual literature that has been selected after careful evaluation following a literature criteria checklist. This checklist is publicly available, along with a selection of standardized operating protocols (SOPs) established by different projects. The spectrum of information is rounded off by further articles concerning basics or crosscutting topics in nanosafety research. This article is intended to give an overview on DaNa2.0 activities to support reliable toxicity testing and science communication alike.

Removal of Cells from Body Fluids by Magnetic Separation in Batch and Continuous Mode: Influence of Bead Size, Concentration, and Contact Time.

The magnetic separation of pathogenic compounds from body fluids is an appealing therapeutic concept. Recently, removal of a diverse array of pathogens has been demonstrated using extracorporeal dialysis-type devices. The contact time between the fluid and the magnetic beads in such devices is limited to a few minutes. This poses challenges, particularly if large compounds such as bacteria or cells need to be removed. Here, we report on the feasibility to remove cells from body fluids in a continuous dialysis type of setting. We assessed tumor cell removal efficiencies from physiological fluids with or without white blood cells using a range of different magnetic bead sizes (50-4000 nm), concentrations, and contact times. We show that tumor cells can be quantitatively removed from body fluids within acceptable times (1-2 min) and bead concentrations (0.2 mg per mL). We further present a mathematical model to describe the minimal bead number concentration needed to remove a certain number of cells, in the presence of competing nonspecific uptake. The present study paves the way for investigational studies to assess the therapeutic potential of cell removal by magnetic blood purification in a dialysis-like setting.

Uptake of label-free graphene oxide by Caco-2 cells is dependent on the cell differentiation status.

BACKGROUND: Understanding the interaction of graphene-related materials (GRM) with human cells is a key to the assessment of their potential risks for human health. There is a knowledge gap regarding the potential uptake of GRM by human intestinal cells after unintended ingestion. Therefore the aim of our study was to investigate the interaction of label-free graphene oxide (GO) with the intestinal cell line Caco-2 in vitro and to shed light on the influence of the cell phenotype given by the differentiation status on cellular uptake behaviour. RESULTS: Internalisation of two label-free GOs with different lateral size and thickness by undifferentiated and differentiated Caco-2 cells was analysed by scanning electron microscopy and transmission electron microscopy. Semi-quantification of cells associated with GRM was performed by flow cytometry. Undifferentiated Caco-2 cells showed significant amounts of cell-associated GRM, whereas differentiated Caco-2 cells exhibited low adhesion of GO sheets. Transmission electron microscopy analysis revealed internalisation of both applied GO (small and large) by undifferentiated Caco-2 cells. Even large GO sheets with lateral dimensions up to 10 µm, were found internalised by undifferentiated cells, presumably by macropinocytosis. In contrast, no GO uptake could be found for differentiated Caco-2 cells exhibiting an enterocyte-like morphology with apical brush border. CONCLUSIONS: Our results show that the internalisation of GO is highly dependent on the cell differentiation status of human intestinal cells. During differentiation Caco-2 cells undergo intense phenotypic changes which lead to a dramatic decrease in GRM internalisation. The results support the hypothesis that the cell surface topography of differentiated Caco-2 cells given by the brush border leads to low adhesion of GO sheets and sterical hindrance for material uptake. In addition, the mechanical properties of GRM, especially flexibility of the sheets, seem to be an important factor for internalisation of large GO sheets by epithelial cells. Our results highlight the importance of the choice of the in vitro model to enable better in vitro-in vivo translation.

Transcriptomic Analysis of Intestinal Tissues from Two 90-Day Feeding Studies in Rats Using Genetically Modified MON810 Maize Varieties.

Background: Global as well as specific expression profiles of selected rat tissues were characterized to assess the safety of genetically modified (GM) maize MON810 containing the insecticidal protein Cry1Ab. Gene expression was evaluated by use of Next Generation Sequencing (NGS) as well as RT-qPCR within rat intestinal tissues based on mandatory 90-day rodent feeding studies. In parallel to two 90-day feeding studies, the transcriptional response of rat tissues was assessed as another endpoint to enhance the mechanistic interpretation of GM feeding studies and/or to facilitate the generation of a targeted hypothesis. Rats received diets containing 33% GM maize (MON810) or near-isogenic control maize. As a site of massive exposure to ingested feed the transcriptomic response of ileal and colonic tissue was profiled via RT-qPCR arrays targeting apoptosis, DNA-damage/repair, unfolded protein response (UPR). For global RNA profiling of rat ileal tissue, we applied NGS. Results: No biological response to the GM-diet was observed in male and in female rat tissues. Transcriptome wide analysis of gene expression by RNA-seq confirmed these findings. Nevertheless, gene ontology (GO) analysis clearly associated a set of distinctly regulated transcripts with circadian rhythms. We confirmed differential expression of circadian clock genes using RT-qPCR and immunoassays for selected factors, thereby indicating physiological effects caused by the time point of sampling. Conclusion: Prediction of potential unintended effects of GM-food/feed by transcriptome based profiling of intestinal tissue presents a novel approach to complement classical toxicological testing procedures. Including the detection of alterations in signaling pathways in toxicity testing procedures may enhance the confidence in outcomes of toxicological trials. In this study, no significant GM-related changes in intestinal expression profiles were found in rats fed GM-maize MON810. Relevant alterations of selected cellular pathways (apoptosis, DNA damage and repair, UPR) pointing toward intestinal toxicity of the diets were not observed. Transcriptomic profiles did not reveal perturbations of pathways associated with toxicity, underlining the study results revealed by classical OECD endpoints.

One-year oral toxicity study on a genetically modified maize MON810 variety in Wistar Han RCC rats (EU 7th Framework Programme project GRACE).

The GRACE (GMO Risk Assessment and Communication of Evidence; www.grace-fp7.eu ) project was funded by the European Commission within the 7th Framework Programme. A key objective of GRACE was to conduct 90-day animal feeding trials, animal studies with an extended time frame as well as analytical, in vitro and in silico studies on genetically modified (GM) maize in order to comparatively evaluate their use in GM plant risk assessment. In the present study, the results of a 1-year feeding trial with a GM maize MON810 variety, its near-isogenic non-GM comparator and an additional conventional maize variety are presented. The feeding trials were performed by taking into account the guidance for such studies published by the EFSA Scientific Committee in 2011 and the OECD Test Guideline 452. The results obtained show that the MON810 maize at a level of up to 33 % in the diet did not induce adverse effects in male and female Wistar Han RCC rats after a chronic exposure.

Caveolin-1 and CDC42 mediated endocytosis of silica-coated iron oxide nanoparticles in HeLa cells.

Nanomedicine is a rapidly growing field in nanotechnology, which has great potential in the development of new therapies for numerous diseases. For example iron oxide nanoparticles are in clinical use already in the thermotherapy of brain cancer. Although it has been shown, that tumor cells take up these particles in vitro, little is known about the internalization routes. Understanding of the underlying uptake mechanisms would be very useful for faster and precise development of nanoparticles for clinical applications. This study aims at the identification of key proteins, which are crucial for the active uptake of iron oxide nanoparticles by HeLa cells (human cervical cancer) as a model cell line. Cells were transfected with specific siRNAs against Caveolin-1, Dynamin 2, Flotillin-1, Clathrin, PIP5Kα and CDC42. Knockdown of Caveolin-1 reduces endocytosis of superparamagnetic iron oxide nanoparticles (SPIONs) and silica-coated iron oxide nanoparticles (SCIONs) between 23 and 41%, depending on the surface characteristics of the nanoparticles and the experimental design. Knockdown of CDC42 showed a 46% decrease of the internalization of PEGylated SPIONs within 24 h incubation time. Knockdown of Dynamin 2, Flotillin-1, Clathrin and PIP5Kα caused no or only minor effects. Hence endocytosis in HeLa cells of iron oxide nanoparticles, used in this study, is mainly mediated by Caveolin-1 and CDC42. It is shown here for the first time, which proteins of the endocytotic pathway mediate the endocytosis of silica-coated iron oxide nanoparticles in HeLa cells in vitro. In future studies more experiments should be carried out with different cell lines and other well-defined nanoparticle species to elucidate possible general principles.