The DaNa projects: public communication of (nano)material safety data-from conspiracy theories to study quality.

In this perspective, the authors give their view on the developments and experiences on communicating on (nano)materials safety. We would like to share our experiences with the scientific community in order to make them useful for future communication activities. We present the long-term work of the science communication projects DaNa, DaNa2.0 and DaNa4.0, running from 2009 to 2023. Starting in the early 2000s with the beginnings of nanotechnology research, communication on the safety of nanomaterials with the public was still very new and faced the projects with many challenges. Today, science communication is indispensable for the dissemination of scientific findings and a fact-based approach like the DaNa "Knowledge Base Materials" creates a trustworthy dialogue with the public. This long-term project series has made a significant contribution to communication on the safety of nanomaterials, perhaps even the largest among publicly funded project series worldwide.

A weight of evidence review of the genotoxicity of titanium dioxide (TiO2).

Titanium dioxide is a ubiquitous white material found in a diverse range of products from foods to sunscreens, as a pigment and thickener, amongst other uses. Titanium dioxide has been considered no longer safe for use in foods (nano and microparticles of E171) by the European Food Safety Authority (EFSA) due to concerns over genotoxicity. There are however, conflicting opinions regarding the safety of Titanium dioxide. In an attempt to clarify the situation, a comprehensive weight of evidence (WoE) assessment of the genotoxicity of titanium dioxide based on the available data was performed. A total of 192 datasets for endpoints and test systems considered the most relevant for identifying mutagenic and carcinogenic potential were reviewed and discussed for both reliability and relevance (by weight of evidence) and in the context of whether the physico-chemical properties of the particles had been characterised. The view of an independent panel of experts was that, of the 192 datasets identified, only 34 met the reliability and quality criteria for being most relevant in the evaluation of genotoxicity. Of these, 10 were positive (i.e. reported evidence that titanium dioxide was genotoxic), all of which were from studies of DNA strand breakage (comet assay) or chromosome damage (micronucleus or chromosome aberration assays). All the positive findings were associated with high cytotoxicity, oxidative stress, inflammation, apoptosis, necrosis, or combinations of these. Considering that DNA and chromosome breakage can be secondary to physiological stress, it is highly likely that the observed genotoxic effects of titanium dioxide, including those with nanoparticles, are secondary to physiological stress. Consistent with this finding, there were no positive results from the in vitro and in vivo gene mutation studies evaluated, although it should be noted that to definitively conclude a lack of mutagenicity, more robust in vitro and in vivo gene mutation studies would be useful. Existing evidence does not therefore support a direct DNA damaging mechanism for titanium dioxide (nano and other forms).

A Systematic Review on the Hazard Assessment of Amorphous Silica Based on the Literature From 2013 to 2018.

Background: Nanomaterials are suspected of causing health problems, as published studies on nanotoxicology indicate. On the other hand, some of these materials, such as nanostructured pyrogenic and precipitated synthetic amorphous silica (SAS) and silica gel, have been used for decades without safety concerns in industrial, commercial, and consumer applications. However, in addition to many in vivo and in vitro studies that have failed to demonstrate the intrinsic toxicity of SAS, articles periodically emerge, in which biological effects of concern have been described. Even though most of these studies do not meet high-quality standards and do not always use equivalent test materials or standardized test systems, the results often trigger substance re-evaluation. To put the results into perspective, an extensive literature study was carried out and an example of amorphous silica will be used to try to unravel the reliability from the unreliable results. Methods: A systematic search of studies on nanotoxicological effects has been performed covering the years 2013 to 2018. The identified studies have been evaluated for their quality regarding material and method details, and the data have been curated and put into a data collection. This review deals only with investigations on amorphous silica. Results: Of 18,162 publications 1,217 have been selected with direct reference to experiments with synthetically produced amorphous silica materials. The assessment of these studies based on defined criteria leads to a further reduction to 316 studies, which have been included in this systematic review. Screening for quality with well-defined quantitative criteria following the GUIDE nano concept reveals only 27.3% has acceptable quality. Overall, the in vitro and in vivo data showed low or no toxicity of amorphous silica. The data shown do not support the hypothesis of dependency of biological effects on the primary particle size of the tested materials. Conclusion: This review demonstrates the relatively low quality of most studies published on nanotoxicological issues in the case of amorphous silica. Moreover, mechanistic studies are often passed off or considered toxicological studies. In general, standardized methods or the Organization for Economic Cooperation and Development (OECD) guidelines are rarely used for toxicological experiments. As a result, the significance of the published data is usually weak and must be reevaluated carefully before using them for regulatory purposes.

Collection of Controlled Nanosafety Data-The CoCoN-Database, a Tool to Assess Nanomaterial Hazard.

Hazard assessment is the first step in nanomaterial risk assessment. The overall number of studies on the biological effects of nanomaterials or innovative materials is steadily increasing and is above 40,000. Several databases have been established to make the amount of data manageable, but these are often highly specialized or can be used only by experts. This paper describes a new database which uses an already existing data collection of about 35,000 publications. The collection from the first phase between the years 2000 and 2013 contains about 11,000 articles and this number has been reduced by specific selection criteria. The resulting publications have been evaluated for their quality regarding the toxicological content and the experimental data have been extracted. In addition to material properties, the most important value to be extracted is the no-observed-adverse-effect-level (NOAEL) for in vivo and the no-observed-effect-concentration (NOEC) for in vitro studies. The correlation of the NOAEL/NOEC values with the nanomaterial properties and the investigated endpoints has been tested in projects such as the OECD-AOP project, where the available data for inflammatory responses have been analysed. In addition, special attention was paid to titanium dioxide particles and this example is used to show with searches for in vitro and in vivo experiments on possible lung toxicity what a typical result of a database query can look like. In this review, an emerging database is described that contains valuable information for nanomaterial hazard estimation and should aid in the progress of nanosafety research.

Non-Animal Strategies for Toxicity Assessment of Nanoscale Materials: Role of Adverse Outcome Pathways in the Selection of Endpoints.

Faster, cheaper, sensitive, and mechanisms-based animal alternatives are needed to address the safety assessment needs of the growing number of nanomaterials (NM) and their sophisticated property variants. Specifically, strategies that help identify and prioritize alternative schemes involving individual test models, toxicity endpoints, and assays for the assessment of adverse outcomes, as well as strategies that enable validation and refinement of these schemes for the regulatory acceptance are needed. In this review, two strategies 1) the current nanotoxicology literature review and 2) the adverse outcome pathways (AOPs) framework, a systematic process that allows the assembly of available mechanistic information concerning a toxicological response in a simple modular format, are presented. The review highlights 1) the most frequently assessed and reported ad hoc in vivo and in vitro toxicity measurements in the literature, 2) various AOPs of relevance to inhalation toxicity of NM that are presently under development, and 3) their applicability in identifying key events of toxicity for targeted in vitro assay development. Finally, using an existing AOP for lung fibrosis, the specific combinations of cell types, exposure and test systems, and assays that are experimentally supported and thus, can be used for assessing NM-induced lung fibrosis, are proposed.

A methodology for developing key events to advance nanomaterial-relevant adverse outcome pathways to inform risk assessment.

Significant advances have been made in the development of Adverse Outcome Pathways (AOPs) over the last decade, mainly focused on the toxicity mechanisms of chemicals. These AOPs, although relevant to manufactured nanomaterials (MNs), do not currently capture the reported roles of size-associated properties of MNs on toxicity. Moreover, some AOs of relevance to airborne exposures to MNs such as lung inflammation and fibrosis shown in animal studies may not be targeted in routine regulatory decision making. The primary objective of the present study was to establish an approach to advance the development of AOPs of relevance to MNs using existing, publicly available, nanotoxicology literature. A systematic methodology was created for curating, organizing and applying the available literature for identifying key events (KEs). Using a case study approach, the study applied the available literature to build the biological plausibility for 'tissue injury', a KE of regulatory relevance to MNs. The results of the analysis reveal the various endpoints, assays and specific biological markers used for assessing and reporting tissue injury. The study elaborates on the limitations and opportunities of the current nanotoxicology literature and provides recommendations for the future reporting of nanotoxicology results that will expedite not only the development of AOPs for MNs but also aid in application of existing data for decision making.

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.

The uncertainty with nanosafety: Validity and reliability of published data.

Use and production of chemicals and new materials are always reasons for concern especially with regard to human health and the environmental impacts. Over the past few decades occupational safety is a greater focus for toxicologists and of national and international registration programs for new products. Thus, the careful investigation of the biological effects of new chemicals and materials is critical. However, the hype around "The Nanotechnology" has boosted a competition for public funds and thereby the number of publications on this "nanotoxicology" topic has exploded. For more than two decades the public discussion around the special effects of nanomaterials or nanoparticles is ongoing without a final conclusion regarding an existing issue of a "nano-specific effect". Facing the situation of a dramatic increase in the number of publications (>4400 PubMed references in 2017 alone!); the quality of the findings appears to be questionable, particularly with regard to the implementation of risk assessment for nanomaterials. Most of the published nanotoxicology studies are associated with fundamental deficiencies in the experimental design of these investigations, including 1) a lack of rigorous and adequate physicochemical characterization of the test materials; 2) the absence of adequate particle controls; and 3) the implementation of high dose experiments, designed to produce toxicological effects - which are publishable (and sensational). As a consequence, the "toxicology" results have limited utility, and therefore must be critically (re)evaluated. This service is provided by the internet knowledge base DaNa (www.nanoobjects.info). On this website a criteria catalogue for the re-evaluation of scientific publications has been published and if these criteria are utilized > 60 70% of reported study findings are not acceptable and cannot be taken into consideration for risk assessment criteria.

Environmental Impacts of Engineered Nanomaterials-Imbalances in the Safety Assessment of Selected Nanomaterials.

For this Editorial, we, the guest editors, performed a brief review with the aim of setting the framework for the Special issue on the "Environmental Impacts of Nanomaterials" [...].

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.

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.

From in vivo to in vitro: The medical device testing paradigm shift.

Amid growing efforts to advance the replacement, reduction, and refinement of the use of animals in research, there is a growing recognition that in vitro testing of medical devices can be more effective, both in terms of cost and time, and also more reliable than in vivo testing. Although the technological landscape has evolved rapidly in support of these concepts, regulatory acceptance of alternative testing methods has not kept pace. Despite the acceptance by regulators of some in vitro tests (cytotoxicity, gene toxicity, and some hemocompatibility assays), many toxicity tests still rely on animals (irritation, sensitization, acute toxicity, reproductive/developmental toxicity), even where other industrial sectors have already abandoned them. Bringing about change will require a paradigm shift in current approaches to testing - and a concerted effort to generate better data on risks to human health from exposure to leachable chemicals from medical devices, and to boost confidence in the use of alternative methods to test devices. To help advance these ideas, stir debate about best practices, and coalesce around a roadmap forward, the JHU-Center for Alternatives to Animal Testing (CAAT) hosted a symposium believed to be the first gathering dedicated to the topic of in vitro testing of medical devices. Industry representatives, academics, and regulators in attendance presented evidence to support the unique strengths and challenges associated with the approaches currently in use as well as new methods under development, and drew next steps to push the field forward from their presentations and discussion.

Green Toxicology: a strategy for sustainable chemical and material development.

Green Toxicology refers to the application of predictive toxicology in the sustainable development and production of new less harmful materials and chemicals, subsequently reducing waste and exposure. Built upon the foundation of "Green Chemistry" and "Green Engineering", "Green Toxicology" aims to shape future manufacturing processes and safe synthesis of chemicals in terms of environmental and human health impacts. Being an integral part of Green Chemistry, the principles of Green Toxicology amplify the role of health-related aspects for the benefit of consumers and the environment, in addition to being economical for manufacturing companies. Due to the costly development and preparation of new materials and chemicals for market entry, it is no longer practical to ignore the safety and environmental status of new products during product development stages. However, this is only possible if toxicologists and chemists work together early on in the development of materials and chemicals to utilize safe design strategies and innovative in vitro and in silico tools. This paper discusses some of the most relevant aspects, advances and limitations of the emergence of Green Toxicology from the perspective of different industry and research groups. The integration of new testing methods and strategies in product development, testing and regulation stages are presented with examples of the application of in silico, omics and in vitro methods. Other tools for Green Toxicology, including the reduction of animal testing, alternative test methods, and read-across approaches are also discussed.

Diverse Applications of Nanomedicine.

The design and use of materials in the nanoscale size range for addressing medical and health-related issues continues to receive increasing interest. Research in nanomedicine spans a multitude of areas, including drug delivery, vaccine development, antibacterial, diagnosis and imaging tools, wearable devices, implants, high-throughput screening platforms, etc. using biological, nonbiological, biomimetic, or hybrid materials. Many of these developments are starting to be translated into viable clinical products. Here, we provide an overview of recent developments in nanomedicine and highlight the current challenges and upcoming opportunities for the field and translation to the clinic.

Toward achieving harmonization in a nano-cytotoxicity assay measurement through an interlaboratory comparison study.

Development of reliable cell-based nanotoxicology assays is important for evaluation of potentially hazardous engineered nanomaterials. Challenges to producing a reliable assay protocol include working with nanoparticle dispersions and living cell lines, and the potential for nano-related interference effects. Here we demonstrate the use of a 96-well plate design with several measurement controls and an interlaboratory comparison study involving five laboratories to characterize the robustness of a nanocytotoxicity MTS cell viability assay based on the A549 cell line. The consensus EC50 values were 22.1 mg/L (95% confidence intervals 16.9 mg/L to 27.2 mg/L) and 52.6 mg/L (44.1 mg/L to 62.6 mg/L) for positively charged polystyrene nanoparticles for the serum-free and serum conditions, respectively, and 49.7 µmol/L (47.5 µmol/L to 51.5 µmol/L) and 77.0 µmol/L (54.3 µmol/L to 99.4 µmol/L) for positive chemical control cadmium sulfate for the serum-free and serum conditions, respectively. Results from the measurement controls can be used to evaluate the sources of variability and their relative magnitudes within and between laboratories. This information revealed steps of the protocol that may need to be modified to improve the overall robustness and precision. The results suggest that protocol details such as cell line ID, media exchange, cell handling, and nanoparticle dispersion are critical to ensure protocol robustness and comparability of nanocytotoxicity assay results. The combination of system control measurements and interlaboratory comparison data yielded insights that would not have been available by either approach by itself.

Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology.

Models of the outer epithelia of the human body - namely the skin, the intestine and the lung - have found valid applications in both research and industrial settings as attractive alternatives to animal testing. A variety of approaches to model these barriers are currently employed in such fields, ranging from the utilization of ex vivo tissue to reconstructed in vitro models, and further to chip-based technologies, synthetic membrane systems and, of increasing current interest, in silico modeling approaches. An international group of experts in the field of epithelial barriers was convened from academia, industry and regulatory bodies to present both the current state of the art of non-animal models of the skin, intestinal and pulmonary barriers in their various fields of application, and to discuss research-based, industry-driven and regulatory-relevant future directions for both the development of new models and the refinement of existing test methods. Issues of model relevance and preference, validation and standardization, acceptance, and the need for simplicity versus complexity were focal themes of the discussions. The outcomes of workshop presentations and discussions, in relation to both current status and future directions in the utilization and development of epithelial barrier models, are presented by the attending experts in the current report.

Bidirectional Transfer Study of Polystyrene Nanoparticles across the Placental Barrier in an ex Vivo Human Placental Perfusion Model.

BACKGROUND: Nanoparticle exposure in utero might not be a major concern yet, but it could become more important with the increasing application of nanomaterials in consumer and medical products. Several epidemiologic and in vitro studies have shown that nanoparticles can have potential toxic effects. However, nanoparticles also offer the opportunity to develop new therapeutic strategies to treat specifically either the pregnant mother or the fetus. Previous studies mainly addressed whether nanoparticles are able to cross the placental barrier. However, the transport mechanisms underlying nanoparticle translocation across the placenta are still unknown. OBJECTIVES: In this study we examined which transport mechanisms underlie the placental transfer of nanoparticles. METHODS: We used the ex vivo human placental perfusion model to analyze the bidirectional transfer of plain and carboxylate modified polystyrene particles in a size range between 50 and 300 nm. RESULTS: We observed that the transport of polystyrene particles in the fetal to maternal direction was significantly higher than for the maternal to fetal direction. Regardless of their ability to cross the placental barrier and the direction of perfusion, all polystyrene particles accumulated in the syncytiotrophoblast of the placental tissue. CONCLUSIONS: Our results indicate that the syncytiotrophoblast is the key player in regulating nanoparticle transport across the human placenta. The main mechanism underlying this translocation is not based on passive diffusion, but is likely to involve an active, energy-dependent transport pathway. These findings will be important for reproductive toxicology as well as for pharmaceutical engineering of new drug carriers.