Nanomaterial Characterization in Complex Media-Guidance and Application.

A broad range of inorganic nanoparticles (NPs) and their dissolved ions possess a possible toxicological risk for human health and the environment. Reliable and robust measurements of dissolution effects may be influenced by the sample matrix, which challenges the analytical method of choice. In this study, CuO NPs were investigated in several dissolution experiments. Two analytical techniques (dynamic light scattering (DLS) and inductively-coupled plasma mass spectrometry (ICP-MS)) were used to characterize NPs (size distribution curves) time-dependently in different complex matrices (e.g., artificial lung lining fluids and cell culture media). The advantages and challenges of each analytical approach are evaluated and discussed. Additionally, a direct-injection single particle (DI sp)ICP-MS technique for assessing the size distribution curve of the dissolved particles was developed and evaluated. The DI technique provides a sensitive response even at low concentrations without any dilution of the complex sample matrix. These experiments were further enhanced with an automated data evaluation procedure to objectively distinguish between ionic and NP events. With this approach, a fast and reproducible determination of inorganic NPs and ionic backgrounds can be achieved. This study can serve as guidance when choosing the optimal analytical method for NP characterization and for the determination of the origin of an adverse effect in NP toxicity.

Correction: Peters et al. Benchmarking the ACEnano Toolbox for Characterisation of Nanoparticle Size and Concentration by Interlaboratory Comparison. Molecules 2021, 26, 5315.

Due to miscommunication a number of potential co-authors were not listed in the original publication [...].

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.

Automation and Standardization-A Coupled Approach towards Reproducible Sample Preparation Protocols for Nanomaterial Analysis.

Whereas the characterization of nanomaterials using different analytical techniques is often highly automated and standardized, the sample preparation that precedes it causes a bottleneck in nanomaterial analysis as it is performed manually. Usually, this pretreatment depends on the skills and experience of the analysts. Furthermore, adequate reporting of the sample preparation is often missing. In this overview, some solutions for techniques widely used in nano-analytics to overcome this problem are discussed. Two examples of sample preparation optimization by automation are presented, which demonstrate that this approach is leading to increased analytical confidence. Our first example is motivated by the need to exclude human bias and focuses on the development of automation in sample introduction. To this end, a robotic system has been developed, which can prepare stable and homogeneous nanomaterial suspensions amenable to a variety of well-established analytical methods, such as dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), field-flow fractionation (FFF) or single-particle inductively coupled mass spectrometry (sp-ICP-MS). Our second example addresses biological samples, such as cells exposed to nanomaterials, which are still challenging for reliable analysis. An air-liquid interface has been developed for the exposure of biological samples to nanomaterial-containing aerosols. The system exposes transmission electron microscopy (TEM) grids under reproducible conditions, whilst also allowing characterization of aerosol composition with mass spectrometry. Such an approach enables correlative measurements combining biological with physicochemical analysis. These case studies demonstrate that standardization and automation of sample preparation setups, combined with appropriate measurement processes and data reduction are crucial steps towards more reliable and reproducible data.

ICP-MS-based Approach to Determine Nanoparticle Recovery After Hollow Fiber Flow Field Flow Fractionation.

Compared to the classical chemicals, nanoparticles (NPs) exhibit unique properties, which lead to challenges in sample preparation and analysis. Fractionation techniques and, in particular, hollow fiber flow field flow fractionation (HF5) have recently become popular in the characterization and quantification of nanomaterials, because of their fine fractionation capability in the nanoscale-range. When dealing with NPs, a great drawback during fractionation is the loss of particles in the fractionation devices, tubing and connectors. There is a need for studies to systematically explore and assess the quality of the fractionation process. A combination of two complementary mass-based setups was used to determine particle loss in HF5. Inductively coupled plasma mass spectrometry (ICP-MS) enabled the estimation of recovery rates for NPs after HF5 separation. Reciprocally, laser ablation ICP-MS (LA-ICP-MS) permitted the evaluation of particles retained on the hollow fiber. 15 nm Au-NPs in different concentrations were evaluated in this study and showed a recovery level for Au-NPs of 50 - 65% based on the applied concentrations after a complete HF5 separation run. Detection of sample deposition on the hollow fiber by LA-ICP-MS indicated a sample loss of about 8%. These findings are important for experiments relying on fractionation of low concentrated nanoparticulate samples.

UV-Vis Spectroscopic Characterization of Nanomaterials in Aqueous Media.

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 NM-bearing products. There are several methods available to monitor and achieve reliable measurement of nanoscale-related properties, one example of which is Ultraviolet-Visible 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.

Benchmarking the ACEnano Toolbox for Characterisation of Nanoparticle Size and Concentration by Interlaboratory Comparisons.

ACEnano is an EU-funded project which aims at developing, optimising and validating methods for the detection and characterisation of nanomaterials (NMs) in increasingly complex matrices to improve confidence in the results and support their use in regulation. Within this project, several interlaboratory comparisons (ILCs) for the determination of particle size and concentration have been organised to benchmark existing analytical methods. In this paper the results of a number of these ILCs for the characterisation of NMs are presented and discussed. The results of the analyses of pristine well-defined particles such as 60 nm Au NMs in a simple aqueous suspension showed that laboratories are well capable of determining the sizes of these particles. The analysis of particles in complex matrices or formulations such as consumer products resulted in larger variations in particle sizes within technologies and clear differences in capability between techniques. Sunscreen lotion sample analysis by laboratories using spICP-MS and TEM/SEM identified and confirmed the TiO2 particles as being nanoscale and compliant with the EU definition of an NM for regulatory purposes. In a toothpaste sample orthogonal results by PTA, spICP-MS and TEM/SEM agreed and stated the TiO2 particles as not fitting the EU definition of an NM. In general, from the results of these ILCs we conclude that laboratories are well capable of determining particle sizes of NM, even in fairly complex formulations.

Use of Cause-and-Effect Analysis to Optimize the Reliability of In Vitro Inhalation Toxicity Measurements Using an Air-Liquid Interface.

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.

Parametric Optimization of an Air-Liquid Interface System for Flow-Through Inhalation Exposure to Nanoparticles: Assessing Dosimetry and Intracellular Uptake of CeO2 Nanoparticles.

Air-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&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.

Versatile Dual-Inlet Sample Introduction System for Multi-Mode Single Particle Inductively Coupled Plasma Mass Spectrometry Analysis and Validation.

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.

Preparation of Nanoparticles for ToF-SIMS and XPS Analysis.

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.

Metabolomics profiling to investigate nanomaterial toxicity in vitro and in vivo.

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.

Tackling Complex Analytical Tasks: An ISO/TS-Based Validation Approach for Hydrodynamic Chromatography Single Particle Inductively Coupled Plasma Mass Spectrometry.

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.

Organ burden of inhaled nanoceria in a 2-year low-dose exposure study: dump or depot?

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 Vitamin A and D Exposure of Cells Affects the Intracellular Uptake of Aluminum Nanomaterials and its Agglomeration Behavior: A Chemo-Analytic Investigation.

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'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.

Aluminum and aluminum oxide nanomaterials uptake after oral exposure - a comparative study.

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.

Improved validation for single particle ICP-MS analysis using a pneumatic nebulizer / microdroplet generator sample introduction system for multi-mode nanoparticle determination.

This study reports on the development of a single-particle (sp) inductively coupled plasma mass spectrometry (ICP-MS) technique suitable for the multi-mode determination of nanoparticle (NP) metal mass fraction and number concentration. The described technique, which is based on a dual inlet system consisting of a pneumatic nebulizer (PN) and a microdroplet generator (MDG), allows for the sequential introduction of ionic metal calibrant solutions and nanoparticle suspensions via all combinations of the two inlets; thus allowing for a combination of three independent modes of analysis. A novel interface, assembled using standard analytical components (a demountable quartz ICP-MS torch, flexible non-conducting silicon tubing and various connectors), was used to interface the dual inlet system to an ICP-MS. The interface provided improved functionality, compared to a previous design. It is now possible to conveniently exchange and introduce standard solutions and samples via all inlet combinations, analyze them, and also wash the sample inlet systems while the whole setup is still connected to an operating ICP-MS. This setup provided seamless and robust operation in a total of three analysis modes, i.e. three ways to independently determine the metal mass fraction and NP number concentration. All three analyses modes could be carried out within a single analytical run lasting approximately 20 min. The unique feature of the described approach is that each analysis mode is based on a different calibration principle, thus constituting an independent way to determine metal mass fractions and nanoparticle number concentrations. Conducting the three independent state-of-the-art analysis, within a single analytical run, improves substantially the validation capabilities of sp-ICP-MS for NP analysis. To assess the technique's analytical performance, Au, Ag and CeO2 nanoparticles were analyzed. The determined average diameters for Au (56.7 ± 1.5 nm), Ag (72.8 ± 3.4 nm) and CeO2 (69.0 ± 6.4 nm) NPs were in close agreement for all three modes of analysis, as well as with the values provided by suppliers' for Au and Ag NPs (56.0 ± 0.5 for Au, 74.6 ± 3.8 nm for Ag). However, the determined average value for CeO2 was much higher than the expected 28.4 ± 10.4 nm, possibly due to NP agglomeration and the inability to detect NPs existing within the lower size range. The determined NP number concentrations, using analysis modes -I and -II, gave recoveries between 91 and 100% for the Au and Ag NP number concentrations. Whereas analysis mode -III showed a recovery of 70-88% for the same materials. Because of the polydispersity, the small size and polyhedral shape of the CeO2 NPs it was not possible to make NP number concentration comparisons for this material.

ToF-SIMS 3D imaging unveils important insights on the cellular microenvironment during biomineralization of gold nanostructures.

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.

Aggregated aluminium exposure: risk assessment for the general population.

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.