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In vitro inhalation toxicology methods are increasingly being used for research and regulatory purposes. Although the opportunity for increased human relevance of in vitro inhalation methods compared to in vivo tests has been established and discussed, how to systematically account for variability and maximize the reliability of these in vitro methods, especially for assays that use cells cultured at an air-liquid interface (ALI), has received less attention. One tool that has been used to evaluate the robustness of in vitro test methods is cause-and-effect (C&E) analysis, a conceptual approach to analyze key sources of potential variability in a test method. These sources of variability can then be evaluated using robustness testing and potentially incorporated into in-process control measurements in the assay protocol. There are many differences among in vitro inhalation test methods including the use of different types of biological test systems, exposure platforms/conditions, substances tested, and end points, which represent a major challenge for use in regulatory testing. In this manuscript, we describe how C&E analysis can be applied using a modular approach based on the idea that shared components of different test methods (e.g., the same exposure system is used) have similar sources of variability even though other components may differ. C&E analyses of different in vitro inhalation methods revealed a common set of recommended exposure systems and biological in-process control measurements. The approach described here, when applied in conjunction with Good Laboratory Practices (GLP) criteria, should help improve the inter- and intralaboratory agreement of in vitro inhalation test results, leading to increased confidence in these methods for regulatory and research purposes.

Nanomaterials (NMs) can be produced in plenty of variants posing several challenges for NM hazard and risk assessment. Metabolomic profiling of NM-treated cells and tissues allows for insights into underlying Mode-of-Action (MoA) and offers several advantages in this context. It supports the description of Adverse Outcome Pathways (AOPs) and, therefore, tailored AOP-based hazard testing strategies. Moreover, it bears great potential for biomarker discovery supporting toxicity prediction. Here, we applied metabolomics profiling to cells treated with four well-selected SiO2 variants, differing in structure, size and surface charge. TiO2 NM-105 served as a benchmark. Responses were studied in vitro in rat lung epithelial cells (RLE-6TN) and alveolar macrophages (NR8383) and compared to in vivo responses in rat lung tissues obtained from in vivo instillation and short-term inhalation studies (STIS). Time- and concentration-dependent changes were observed in both in vitro models but with cell-type specific responses. Overall, the levels of lipids and biogenic amines (BAs) tended to increase in epithelial cells but decreased in macrophages. Many identified metabolites like Met-SO, hydroxy-Pro and spermidine were related to oxidative stress, indicating that oxidative stress contributes to the MoA for the selected NMs. Several biomarker candidates such as Asp, Asn, Ser, Pro, spermidine, putrescine and LysoPCaC16:1 were identified in vitro and verified in vivo. In this study, we successfully applied a metabolomics workflow for in vitro and in vivo samples, which proved to be well suited to identify potential biomarkers, to gain insights into NM structure–activity relationship and into the underlying MoA.

No detailed information on in vivo biokinetics of CeO2 nanoparticles (NPs) following chronic low-dose inhalation is available. The CeO2 burden for lung, lung-associated lymph nodes, and major non-pulmonary organs, blood, and feces, was determined in a chronic whole-body inhalation study in female Wistar rats undertaken according to OECD TG453 (6 h per day for 5 days per week for a 104 weeks with the following concentrations: 0, 0.1, 0.3, 1.0, and 3.0 mg/m3, animals were sacrificed after 3, 12, 24 months). Different spectroscopy methods (ICP-MS, ion-beam-microscopy) were used for the quantification of organ burden and for visualization of NP distribution patterns in tissues. After 24 months of exposure, the highest CeO2 lung burden (4.41 mg per lung) was associated with the highest aerosol concentration and was proportionally lower for the other groups in a dose-dependent manner. Imaging techniques confirmed the presence of CeO2 agglomerates of different size categories within lung tissue with a non-homogenous distribution. For the highest exposure group, after 24 months in total 1.2% of the dose retained in the lung was found in the organs and tissues analyzed in this study, excluding lymph nodes and skeleton. The CeO2 burden per tissue decreased from lungs > lymph nodes > hard bone > liver > bone marrow. For two dosage groups, the liver organ burden showed a low accumulation rate. Here, the liver can be regarded as depot, whereas kidneys, the skeleton, and bone marrow seem to be dumps due to steadily increasing NP burden over time.

The advancement of analytical methods for nanoparticle measurements is critical both for the growing industrial exploitation of engineered nanoparticles and for developing robust strategies to understand and control the concentration of nanomaterials in humans and the environment. For high value nanoparticles, the measurement of nanoparticle number concentration in a liquid directly impacts the ability to assess the scale and reproducibility of the production process, it allows optimisation of efficiency and supports regulatory compliance. This measurement is also useful to monitor and control the intentional or accidental release of engineered nanoparticles into the environment at the production plant, as well as by end-users., peer-reviewed

The physicochemical characterization of nanomaterials (NMs) is often an analytical challenge, due to their small size (at least one dimension in the nanoscale, i.e. 1–100 nm), dynamic nature, and diverse properties. At the same time, reliable and repeatable characterization is paramount to ensure safety and quality in the manufacturing of NMbearing products. There are several methods available to monitor and achieve reliable measurement of nanoscale-related properties, one example of which is UltravioletVisible Spectroscopy (UV-Vis). This is a well-established, simple, and inexpensive technique that provides non-invasive and fast real-time screening evaluation of NM size, concentration, and aggregation state. Such features make UV-Vis an ideal methodology to assess the proficiency testing schemes (PTS) of a validated standard operating procedure (SOP) intended to evaluate the performance and reproducibility of a characterization method. In this paper, the PTS of six partner laboratories from the H2020 project ACEnano were assessed through an interlaboratory comparison (ILC). Standard gold (Au) colloid suspensions of different sizes (ranging 5–100 nm) were characterized by UV-Vis at the different institutions to develop an implementable and robust protocol for NM size characterization., peer-reviewed

The biomolecular imaging of cell-nanoparticle (NP) interactions using time-of-flight secondary ion mass spectrometry (ToF-SIMS) represents an evolving tool in nanotoxicology. In this study we present the three dimensional (3D) distribution of nanomaterials within biomolecular agglomerates using ToF-SIMS imaging. This novel approach was used to model the resistance of human alveolar A549 cells against gold (Au) ion toxicity through intra- and extracellular biomineralization. At low Au concentrations (≤1 mM HAuCl4) 3D-ToF-SIMS imaging reveals a homogenous intracellular distribution of Au-NPs in combination with polydisperse spherical NPs biomineralized in different layers on the cell surface. However, at higher precursor concentrations (≥2 mM) supplemented with biogenic spherical NPs as seeds, cells start to biosynthesize partially embedded long aspect ratio fiber-like Au nanostructures. Most interestingly, A549 cells seem to be able to sense the enhanced Au concentration. They change the chemical composition of the extracellular NP agglomerates from threonine-O-3-phosphate aureate to an arginine-Au(I)-imine. Furthermore they adopt the extracellular mineralization process from spheres to irregular structures to nanoribbons in a dose-dependent manner with increasing Au concentrations. The results achieved regarding size, shape and chemical specificity were cross checked by SEM-EDX and single particle (sp-)ICP-MS. Our findings demonstrate the potential of ToF-SIMS 3D imaging to better understand cell-NP interactions and their impact in nanotoxicology.

Air&ndash, liquid interface (ALI) systems have been widely used in recent years to investigate the inhalation toxicity of many gaseous compounds, chemicals, and nanomaterials and represent an emerging and promising in vitro method to supplement in vivo studies. ALI exposure reflects the physiological conditions of the deep lung more closely to subacute in vivo inhalation scenarios compared to submerged exposure. The comparability of the toxicological results obtained from in vivo and in vitro inhalation data is still challenging. The robustness of ALI exposure scenarios is not yet well understood, but critical for the potential standardization of these methods. We report a cause-and-effect (C&amp, E) analysis of a flow through ALI exposure system. The influence of five different instrumental and physiological parameters affecting cell viability and exposure parameters of a human lung cell line in vitro (exposure duration, relative humidity, temperature, CO2 concentration and flow rate) was investigated. After exposing lung epithelia cells to a CeO2 nanoparticle (NP) aerosol, intracellular CeO2 concentrations reached values similar to those found in a recent subacute rat inhalation study in vivo. This is the first study showing that the NP concentration reached in vitro using a flow through ALI system were the same as those in an in vivo study.

Metal-containing nanoparticles (NP) can be characterized with inductively coupled plasma mass spectrometers (ICP-MS) in terms of their size and number concentration by using the single-particle mode of the instrument (spICP-MS). The accuracy of measurement depends on the setup, operational conditions of the instrument and specific parameters that are set by the user. The transport efficiency of the ICP-MS is crucial for the quantification of the NP and usually requires a reference material with homogenous size distribution and a known particle number concentration. Currently, NP reference materials are available for only a few metals and in limited sizes. If particles are characterized without a reference standard, the results of both size and particle number may be biased. Therefore, a dual-inlet setup for characterizing nanoparticles with spICP-MS was developed to overcome this problem. This setup is based on a conventional introduction system consisting of a pneumatic nebulizer (PN) for nanoparticle solutions and a microdroplet generator (µDG) for ionic calibration solutions. A new and flexible interface was developed to facilitate the coupling of µDG, PN and the ICP-MS system. The interface consists of available laboratory components and allows for the calibration, nanoparticle (NP) characterization and cleaning of the arrangement, while the ICP-MS instrument is still running. Three independent analysis modes are available for determining particle size and number concentration. Each mode is based on a different calibration principle. While mode I (counting) and mode III (µDG) are known from the literature, mode II (sensitivity), is used to determine the transport efficiency by inorganic ionic standard solutions only. It is independent of NP reference materials. The µDG based inlet system described here guarantees superior analyte sensitivities and, therefore, lower detection limits (LOD). The size dependent LODs achieved are less than 15 nm for all NP (Au, Ag, CeO2) investigated.

Nano-carrier systems such as liposomes have promising biomedical applications. Nevertheless, characterization of these complex samples is a challenging analytical task. In this study a coupled hydrodynamic chromatography-single particle-inductively coupled plasma mass spectrometry (HDC-spICP-MS) approach was validated based on the technical specification (TS) 19590:2017 of the international organization for standardization (ISO). The TS has been adapted to the hyphenated setup. The quality criteria (QC), e.g., linearity of the calibration, transport efficiency, were investigated. Furthermore, a cross calibration of the particle size was performed with values from dynamic light scattering (DLS) and transmission electron microscopy (TEM). Due to an additional Y-piece, an online-calibration routine was implemented. This approach allows the calibration of the ICP-MS during the dead time of the chromatography run, to reduce the required time and enhance the robustness of the results. The optimized method was tested with different gold nanoparticle (Au-NP) mixtures to investigate the characterization properties of HDC separations for samples with increasing complexity. Additionally, the technique was successfully applied to simultaneously determine both the hydrodynamic radius and the Au-NP content in liposomes. With the established hyphenated setup, it was possible to distinguish between different subpopulations with various NP loads and different hydrodynamic diameters inside the liposome carriers.

International audience; Aluminum (Al) is one of the most common elements in the earth crust and increasingly used in food, consumer products and packaging. Its hazard potential for humans is still not completely understood. Besides the metallic form, Al also exists as mineral, including the insoluble oxide, and in soluble ionic forms. Representatives of these three species, namely a metallic and an oxidic species of Al-containing nanoparticles and soluble aluminum chloride, were applied to human intestinal cell lines as models for the intestinal barrier. We characterized physicochemical particle parameters, protein corona composition, ion release and cellular uptake. Different in vitro assays were performed to determine potential effects and molecular modes of action related to the individual chemical species. For a deeper insight into signaling processes, microarray transcriptome analyses followed by bioinformatic data analysis were employed. The particulate Al species showed different solubility in biological media. Metallic Al nanoparticles released more ions than AlO nanoparticles, while AlCl showed a mixture of dissolved and agglomerated particulate entities in biological media. The protein corona composition differed between both nanoparticle species. Cellular uptake, investigated in transwell experiments, occurred predominantly in particulate form, whereas ionic Al was not taken up by intestinal cell lines. Transcellular transport was not observed. None of the Al species showed cytotoxic effects up to 200 µg Al/mL. The transcriptome analysis indicated mainly effects on oxidative stress pathways, xenobiotic metabolism and metal homeostasis. We have shown for the first time that intestinal cellular uptake of Al occurs preferably in the particle form, while toxicological effects appear to be ion-related.

NanoImpact : an international journal of nanosafety research 6, 69-80 (2017). doi:10.1016/j.impact.2017.03.003, Published by Elsevier, Amsterdam

Aluminum (Al) is extensively used for the production of different consumer products, agents, as well as pharmaceuticals. Studies that demonstrate neurotoxicity and a possible link to Alzheimer&rsquo, s disease trigger concern about potential health risks due to high Al intake. Al in cosmetic products raises the question whether a possible interaction between Al and retinol (vitamin A) and cholecalciferol (vitamin D3) metabolism might exist. Understanding the uptake mechanisms of ionic or elemental Al and Al nanomaterials (Al NMs) in combination with bioactive substances are important for the assessment of possible health risk associated. Therefore, we studied the uptake and distribution of Al oxide (Al2O3) and metallic Al0 NMs in the human keratinocyte cell line HaCaT. Possible alterations of the metabolic pattern upon application of the two Al species together with vitamin A or D3 were investigated. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging and inductively coupled plasma mass spectrometry (ICP-MS) were applied to quantify the cellular uptake of Al NMs.

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Nanoparticles have gained increasing attention in recent years due to their potential and application in different fields including medicine, cosmetics, chemistry, and their potential to enable advanced materials. To effectively understand and regulate the physico-chemical properties and potential adverse effects of nanoparticles, validated measurement procedures for the various properties of nanoparticles need to be developed. While procedures for measuring nanoparticle size and size distribution are already established, standardized methods for analysis of their surface chemistry are not yet in place, although the influence of the surface chemistry on nanoparticle properties is undisputed. In particular, storage and preparation of nanoparticles for surface analysis strongly influences the analytical results from various methods, and in order to obtain consistent results, sample preparation must be both optimized and standardized. In this contribution, we present, in detail, some standard procedures for preparing nanoparticles for surface analytics. In principle, nanoparticles can be deposited on a suitable substrate from suspension or as a powder. Silicon (Si) wafers are commonly used as substrate, however, their cleaning is critical to the process. For sample preparation from suspension, we will discuss drop-casting and spin-coating, where not only the cleanliness of the substrate and purity of the suspension but also its concentration play important roles for the success of the preparation methodology. For nanoparticles with sensitive ligand shells or coatings, deposition as powders is more suitable, although this method requires particular care in fixing the sample.

The knowledge about a potential in vivo uptake and subsequent toxicological effects of aluminum (Al), especially in the nanoparticulate form, is still limited. This paper focuses on a three day oral gavage study with three different Al species in Sprague Dawley rats. The Al amount was investigated in major organs in order to determine the oral bioavailability and distribution. Al-containing nanoparticles (NMs composed of Al0 and aluminum oxide (Al2O3)) were administered at three different concentrations and soluble aluminum chloride (AlCl3·6H2O) was used as a reference control at one concentration. A microwave assisted acid digestion approach followed by inductively coupled plasma mass spectrometry (ICP-MS) analysis was developed to analyse the Al burden of individual organs. Special attention was paid on how the sample matrix affected the calibration procedure. After 3 days exposure, AlCl3·6H2O treated animals showed high Al levels in liver and intestine, while upon treatment with Al0 NMs significant amounts of Al were detected only in the latter. In contrast, following Al2O3 NMs treatment, Al was detected in all investigated organs with particular high concentrations in the spleen. A rapid absorption and systemic distribution of all three Al forms tested were found after 3-day oral exposure. The identified differences between Al0 and Al2O3 NMs point out that both, particle shape and surface composition could be key factors for Al biodistribution and accumulation.

Aluminium is one of the most abundant elements in earth’s crust and its manifold uses result in an exposure of the population from many sources. Developmental toxicity, effects on the urinary tract and neurotoxicity are known effects of aluminium and its compounds. Here, we assessed the health risks resulting from total consumer exposure towards aluminium and various aluminium compounds, including contributions from foodstuffs, food additives, food contact materials (FCM), and cosmetic products. For the estimation of aluminium contents in foodstuff, data from the German “Pilot-Total-Diet-Study” were used, which was conducted as part of the European TDS-Exposure project. These were combined with consumption data from the German National Consumption Survey II to yield aluminium exposure via food for adults. It was found that the average weekly aluminium exposure resulting from food intake amounts to approx. 50% of the tolerable weekly intake (TWI) of 1 mg/kg body weight (bw)/week, derived by the European Food Safety Authority (EFSA). For children, data from the French “Infant Total Diet Study” and the “Second French Total Diet Study” were used to estimate aluminium exposure via food. As a result, the TWI can be exhausted or slightly exceeded—particularly for infants who are not exclusively breastfed and young children relying on specially adapted diets (e.g. soy-based, lactose free, hypoallergenic). When taking into account the overall aluminium exposure from foods, cosmetic products (cosmetics), pharmaceuticals and FCM from uncoated aluminium, a significant exceedance of the EFSA-derived TWI and even the PTWI of 2 mg/kg bw/week, derived by the Joint FAO/WHO Expert Committee on Food Additives, may occur. Specifically, high exposure levels were found for adolescents aged 11–14 years. Although exposure data were collected with special regard to the German population, it is also representative for European and comparable to international consumers. From a toxicological point of view, regular exceedance of the lifetime tolerable aluminium intake (TWI/PTWI) is undesirable, since this results in an increased risk for health impairments. Consequently, recommendations on how to reduce overall aluminium exposure are given.

International audience; The application of appropriate analytical techniques is essential for nanomaterial (NM) characterization. In this study, we compared different analytical techniques for NM analysis. Regarding possible adverse health effects, ionic and particulate NM effects have to be taken into account. As NMs behave quite differently in physiological media, special attention was paid to techniques which are able to determine the biosolubility and complexation behavior of NMs. Representative NMs of similar size were selected: aluminum (Al 0) and aluminum oxide (Al 2 O 3), to compare the behavior of metal and metal oxides. In addition, titanium dioxide (TiO 2) was investigated. Characterization techniques such as dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) were evaluated with respect to their suitability for fast characterization of nanoparticle dispersions regarding a particle's hydrodynamic diameter and size distribution. By application of inductively coupled plasma mass spectrometry in the single particle mode (SP-ICP-MS), individual nanoparticles were quantified and characterized regarding their size. SP-ICP-MS measurements were correlated with the information gained using other characterization techniques, i.e. transmission electron microscopy (TEM) and small angle X-ray scattering (SAXS). The particle surface as an important descriptor of NMs was analyzed by X-ray diffraction (XRD). NM impurities and their co-localization with biomolecules were determined by ion beam microscopy (IBM) and confocal Raman microscopy (CRM). We conclude advantages and disadvantages of the different techniques applied and suggest options for their complementation. Thus, this paper may serve as a practical guide to particle characterization techniques.

Environmental science / Nano 5(1), 48-63 (2018). doi:10.1039/C7EN00594F, Published by Cambridge

International audience; Development and market introduction of new nanomaterials trigger the need for an adequate risk assessment of such products alongside suitable risk communication measures. Current application of classical and new nanomaterials is analyzed in context of regulatory requirements and standardization for chemicals, food and consumer products. The challenges of nanomaterial characterization as the main bottleneck of risk assessment and regulation are presented. In some areas, e.g., quantification of nanomaterials within complex matrices, the establishment and adaptation of analytical techniques such as laser ablation inductively coupled plasma mass spectrometry and others are potentially suited to meet the requirements. As an example, we here provide an approach for the reliable characterization of human exposure to nanomaterials resulting from food packaging. Furthermore, results of nanomaterial toxicity and ecotoxicity testing are discussed, with concluding key criteria such as solubility and fiber rigidity as important parameters to be considered in material development and regulation. Although an analysis of the public opinion has revealed a distinguished rating depending on the particular field of application, a rather positive perception of nanotechnology could be ascertained for the German public in general. An improvement of material characterization in both toxicological testing as well as end-product control was concluded as being the main obstacle to ensure not only safe use of materials, but also wide acceptance of this and any novel technology in the general public

International audience; Useful properties render titanium dioxide nanomaterials (NMs) to be one of the most commonly used NMs worldwide. TiO2 powder is used as food additives (E171), which may contain up to 36% nanoparticles. Consequently, humans could be exposed to comparatively high amounts of NMs that may induce adverse effects of chronic exposure conditions. Visualization and quantification of cellular NM uptake as well as their interactions with biomolecules within cells are key issues regarding risk assessment. Advanced quantitative imaging tools for NM detection within biological environments are therefore required. A combination of the label-free spatially resolved dosimetric tools, microresolved particle induced X-ray emission and Rutherford backscattering, together with high resolution imaging techniques, such as time-of-flight secondary ion mass spectrometry and transmission electron microscopy, are applied to visualize the cellular translocation pattern of TiO2 NMs and to quantify the NM-load, cellular major, and trace elements in differentiated Caco-2 cells as a function of their surface properties at the single cell level. Internalized NMs are not only able to impair the cellular homeostasis by themselves, but also to induce an intracellular redistribution of metabolically relevant elements such as phosphorus, sulfur, iron, and copper.

Aluminum has gathered toxicological attention based on relevant human exposure and its suspected hazardous potential. Nanoparticles from food supplements or food contact materials may reach the human gastrointestinal tract. Here, we monitored the physicochemical fate of aluminum-containing nanoparticles and aluminum ions when passaging an in vitro model of the human gastrointestinal tract. Small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), ion beam microscopy (IBM), secondary ion beam mass spectrometry (TOF-SIMS), and inductively coupled plasma mass spectrometry (ICP-MS) in the single-particle mode were employed to characterize two aluminum-containing nanomaterials with different particle core materials (Al0, γAl2O3) and soluble AlCl3. Particle size and shape remained unchanged in saliva, whereas strong agglomeration of both aluminum nanoparticle species was observed at low pH in gastric fluid together with an increased ion release. The levels of free aluminum ions decreased in i...

International audience; Nanosized titanium dioxide (TiO2) is one of the most interesting and valuable nanomaterials for the construction industry but also in health care applications, food, and consumer goods, e.g., cosmetics. Therefore, the properties associated with this material are described in detail. Despite its widespread use, the analytical determination and characterization of nanosized metal oxides is not as straightforward as the comparatively easy-to-detect metallic nanoparticles (e.g., silver or gold). This study presents the method development and the results of the determination of tissue titanium (Ti) levels after treatment of rats with the nanosized TiO2. Total Ti levels were chosen to evaluate the presence and distribution of TiO2 nanoparticles. A procedure consisting of incubation with a mixture of nitric acid (HNO3) and hydrofluoric acid (HF), and heating was developed to digest tissues and TiO2 nanomaterials in order to determine the total Ti content by inductively coupled plasma mass spectrometry (ICPMS). For the inter-laboratory comparison, altogether four laboratories analyzed the same samples upon digestion using the available ICPMS equipment. A major premise for any toxicokinetic study is the possibility to detect the chemical under investigation in biological samples (tissues). So, the study has to be performed with a dose high enough to allow for subsequent tissue level measurement of the chemical under investigation. On the other hand, dose of the chemical applied should not induce over toxicity in the animal as this may affect its absorption, distribution, metabolism, and excretion. To determine a non-toxic TiO2 dosage, an acute toxicity study in rats was performed, and the organs obtained were evaluated for the presence of Ti by ICPMS. Despite the differences in methodology and independent of the sample preparation and the ICPMS equipment used, the results obtained for samples with Ti concentrations >4 µg Ti/g tissue agreed well.

Silver nanoparticles (SNP) are the subject of worldwide commercialization because of their antimicrobial effects. Yet only little data on their mode of action exist. Further, only few techniques allow for visualization and quantification of unlabeled nanoparticles inside cells. To study SNP of different sizes and coatings within human macrophages, we introduce a novel laser postionization secondary neutral mass spectrometry (Laser-SNMS) approach and prove this method superior to the widely applied confocal Raman and transmission electron microscopy. With time-of-flight secondary ion mass spectrometry (TOF-SIMS) we further demonstrate characteristic fingerprints in the lipid pattern of the cellular membrane indicative of oxidative stress and membrane fluidity changes. Increases of protein carbonyl and heme oxygenase-1 levels in treated cells confirm the presence of oxidative stress biochemically. Intriguingly, affected phagocytosis reveals as highly sensitive end point of SNP-mediated adversity In macrophages. The cellular responses monitored are. hierarchically linked, but follow individual kinetics and are partially reversible.

Silver nanoparticles (SNP) are among the most commercialized nanoparticles worldwide. They can be found in many diverse products, mostly because of their antibacterial properties. Despite its widespread use only little data on possible adverse health effects exist. It is difficult to compare biological data from different studies due to the great variety in sizes, coatings or shapes of the particles. Here, we applied a novel synthesis approach to obtain SNP, which are covalently stabilized by a small peptide. This enables a tight control of both size and shape. We applied these SNP in two different sizes of 20 or 40 nm (Ag20Pep and Ag40Pep) and analyzed responses of THP-1-derived human macrophages. Similar gold nanoparticles with the same coating (Au20Pep) were used for comparison and found to be non-toxic. We assessed the cytotoxicity of particles and confirmed their cellular uptake via transmission electron microscopy and confocal Raman microscopy. Importantly a majority of the SNP could be detected as individual particles spread throughout the cells. Furthermore we studied several types of oxidative stress related responses such as induction of heme oxygenase I or formation of protein carbonyls. In summary, our data demonstrate that even low doses of SNP exerted adverse effects in human macrophages.

In light of the broad spectrum of products containing nanosilver, the harmfulness of nanosilver to human health and the environment was intensively discussed at a conference held in February 2012 at the BfR. The conference agenda covered the aspects of analytics of nanosilver materials, human exposure and toxicology as well as effects on microorganisms and the environment. The discussion recovered major gaps related to commonly agreed guidelines for sample preparation and central analytical techniques. In particular, the characterization of the nanoparticles in complex matrices was regarded as a challenge which might become a pitfall for further innovation and application. Historical and anecdotal records of colloidal silver have been sometimes taken as empirical proof for the general low toxicity of nanosilver. Yet as reported herein, a growing number of animal studies following modern performance standards of toxicity testing have been carried out recently revealing well-characterized adverse effects on different routes of exposure in addition to argyria. Furthermore, recent approaches in exposure assessment were reported. However, consumer exposure scenarios are only starting to be developed and reliable exposure data are still rare. It was further widely agreed on the workshop that the use of silver may lead to the selection of silver resistant bacteria. With respect to its environmental behavior, it was suggested that nanosilver released to wastewater may have negligible ecotoxicological effects. Finally, the presentations and discussion on risk assessment and regulation of nanosilver applications gave insights into different approaches of risk assessment of nanomaterials to be performed under the various regulatory frameworks.

In Europe, the data requirements for the hazard and exposure characterisation of chemicals are defined according to the REACH regulation and its guidance on information requirements and chemical safety assessment (Regulation (EC) No 1907/2006 of the European Parliament and of the Council of 18 December 2006 concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), and its guidance documents; available at: http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2006:396:0001:0849:EN:PDF ; and at: http://guidance.echa.europa.eu/docs/guidance_document/information_requirements_en.htm ). This is the basis for any related risk assessment. The standard reference for the testing of cosmetic ingredients is the SCCP's 'Notes of Guidance for the Testing of Cosmetic Ingredients and their Safety Evaluation' (The SCCP's Notes of Guidance for the testing of cosmetic ingredients and their safety evaluation (2006); available at: http://ec.europa.eu/health/ph_risk/committees/04_sccp/docs/sccp_o_03j.pdf ), which refers to the OECD guidelines for the testing of chemicals (The OECD Guidelines for the Testing of Chemicals as a collection of the most relevant internationally agreed testing methods used by government, industry and independent laboratories to assess the safety of chemical products; available at: http://www.oecd.org/topic/0,2686,en_2649_34377_1_1_1_1_37407,00.html ). According to the cosmetics directive [76/768/EEC], compounds that are classified as mutagenic, carcinogenic or toxic to reproduction are banned for the use in cosmetic products. Since December 2010, the respective labelling is based on the rules of regulation (EC) No. 1272/2008 (Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006, Official Journal L 353, 31/12/2008, pages 1-1355; available at: http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2008:353:0001:1355:en:PDF ) on classification, labelling and packaging of substances and mixtures (CLP). There is no further impact from the CLP regulation on cosmetic products, because regulation (EC) No. 1223/2009 on cosmetic products defines its own labelling rules (Regulation (EC) No 1223/2009 of the European Parliament and of the Council of 30 November 2009 on cosmetic products; available at: http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2009:342:0059:0209:en:PDF ). Special notification procedures are mandatory for preservatives, colourants and UV-filters where a safety approval from the European 'Scientific Committee on Consumer Safety' (SCCS) is needed prior to marketing. The risk assessment of nanomaterials in consumer products still poses a significant challenge as highlighted by the example of UV-filters in sunscreens since nanomaterials cannot be classified as a homogenous group of chemicals but still need to be addressed in risk characterisation on a case by case basis.

Background Safety assessment of nanoparticles (NPs) requires techniques that are suitable to quantify tissue and cellular uptake of NPs. The most commonly applied techniques for this purpose are based on inductively coupled plasma mass spectrometry (ICP-MS). Here we apply and compare three different ICP-MS methods to investigate the cellular uptake of TiO2 (diameter 7 or 20 nm, respectively) and Ag (diameter 50 or 75 nm, respectively) NPs into differentiated mouse neuroblastoma cells (Neuro-2a cells). Cells were incubated with different amounts of the NPs. Thereafter they were either directly analyzed by laser ablation ICP-MS (LA-ICP-MS) or were lysed and lysates were analyzed by ICP-MS and by single particle ICP-MS (SP-ICP-MS). Results All techniques confirmed that smaller particles were taken up to a higher extent when values were converted in an NP number-based dose metric. In contrast to ICP-MS and LA-ICP-MS, this measure is already directly provided through SP-ICP-MS. Analysis of NP size distribution in cell lysates by SP-ICP-MS indicates the formation of NP agglomerates inside cells. LA-ICP-MS imaging shows that some of the 75 nm Ag NPs seemed to be adsorbed onto the cell membranes and were not penetrating into the cells, while most of the 50 nm Ag NPs were internalized. LA-ICP-MS confirms high cell-to-cell variability for NP uptake. Conclusions Based on our data we propose to combine different ICP-MS techniques in order to reliably determine the average NP mass and number concentrations, NP sizes and size distribution patterns as well as cell-to-cell variations in NP uptake and intracellular localization. Electronic supplementary material The online version of this article (doi:10.1186/s12951-016-0203-z) contains supplementary material, which is available to authorized users.