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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.

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.

Three-dimensional (3D) printing as a type of additive manufacturing shows continuing increase in application and consumer popularity. The fused filament fabrication (FFF) is an inexpensive method used most frequently by consumers. Studies with 3D printers have shown that during the printing process particulate and volatile substances are released. Handheld 3D printing pens also use the FFF method but the consumer's proximity to the 3D pens gives reason to higher exposure compared to a 3D printer. At the same time, 3D printing pens are often marketed for children who could be more sensitive to the printing emission. The aim of this study was to implement a low cost method to analyze the emissions of 3D printing pens. Polylactide (PLA) and acrylonitrile butadiene styrene (ABS) filaments of different colors were tested. In addition, filaments containing metal and carbon nanotubes (CNTs) were analyzed. An 18.5 L chamber and sampling close to the emission source was used to characterize emissions and concentrations near the breathing zone of the user. Particle emissions and particle size distributions were measured and the potential release of metal particles and CNTs investigated. Particle number concentrations were found in a range of 105 - 106 particles/cm3, which is comparable to previous reports from 3D printers. Transmission electron microscopy (TEM) analysis showed nanoparticles of the different thermoplastic materials as well as of metal particles and CNTs. High contents of metal were observed by inductively coupled plasma mass spectrometry (ICP-MS). These results call for a cautious use of 3D pens due to potential risk to the consumers.

During the last few decades, nanotechnology has evolved into a success story, apparent from a steadily increasing number of scientific publications as well as a large number of applications based on engineered nanomaterials (ENMs). Its widespread uses suggest a high relevance for consumers, workers and the environment, hence justifying intensive investigations into ENM-related adverse effects as a prerequisite for nano-specific regulations. In particular, the inhalation of airborne ENMs, being assumed to represent the most hazardous type of human exposure to these kinds of particles, needs to be scrutinized. Due to an increased awareness of possible health effects, which have already been seen in the case of ultrafine particles (UFPs), research and regulatory measures have set in to identify and address toxic implications following their almost ubiquitous occurrence. Although ENM properties differ from those of the respective bulk materials, the available assessment protocols are often designed for the latter. Despite the large benefit ensuing from the application of nanotechnology, many issues related to ENM behavior and adverse effects are not fully understood or should be examined anew. The traditional hypothesis that ENMs exhibit different or additional hazards due to their “nano” size has been challenged in recent years and ENM categorization according to their properties and toxicity mechanisms has been proposed instead. This review summarizes the toxicological effects of inhaled ENMs identified to date, elucidating the modes of action which provoke different mechanisms in the respiratory tract and their resulting effects. By linking particular mechanisms and adverse effects to ENM properties, grouping of ENMs based on toxicity-related properties is supposed to facilitate toxicological risk assessment. As intensive studies are still required to identify these “ENM classes”, the need for alternatives to animal studies is evident and advances in cell-based test systems for pulmonary research are presented here. We hope to encourage the ongoing discussion about ENM risks and to advocate the further development and practice of suitable testing and grouping methods.

In a proof of concept study, metal-coded affinity tags based on click chemistry (MeCAT-Click) were used to analyze the proteome of Escherichia coli (E. coli) in response to heat stress. This allows high labeling efficiency, high detection sensitivity, and multiplex capabilities, which are pivotal for its application to protein quantification. Two approaches are presented for relative quantification of differentially lanthanide-labeled proteins. The first approach uses isotope-labeling, where ESI–MS was utilized to quantify the differentially labeled proteins from different states of E. coli. With this approach, 14 proteins were found with changed abundance, among them five proteins upregulated. In the second approach, differentially labeled samples were separated by two dimensional gel electrophoresis (2-DE) and scanned by laser ablation inductively coupled plasma mass spectrometry (LA–ICP–MS). Comparison of the signal intensities of the different lanthanides was used to quantify different sample states. Based on this information, ESI–MS was used to identify the proteins with different abundance. The sensitivity of LA–ICP–MS allowed us to find one upregulated protein that was nearly invisible by silver staining (“Probable replication endonuclease from retron EC67”). The advantage of this approach is to locate low abundant proteins with differential expression using LA–ICP–MS, which may be overlooked otherwise. Biological significance This paper demonstrates the successful application of a novel metal labeling strategy to quantify the proteins from complex biological samples. In comparison with former metal labeling strategies, it reduces the steric hindrance and improves the labeling efficiency during the labeling process, which ensure its successful application. This methodology is compatible with both molecular and elemental mass spectrometry. ESI–MS/MS in combination with software-based search allows the identification and relative quantification of labeled proteins. In addition, LA–ICP–MS helps to locate the labeled proteins in 2-DE gels with superior detection capability, thus, target proteins with low abundance can be precisely followed. Its excellent sensitivity allows one to track the proteins of interest that are barely visible by silver staining.

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.