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PARTICLE_RISK was one of the first multidisciplinary projects funded by the European Commission's Framework Programme that was responsible for evaluating the implications of nanomaterial (NM) exposure on human health. This project was the basis for this review which identifies the challenges that exist within the assessment of NM risk. We have retrospectively reflected on the findings of completed nanotoxicology studies to consider what progress and advances have been made within the risk assessment of NMs, as well as discussing the direction that nanotoxicology research is taking and identifying the limitations and failings of existing research. We have reflected on what commonly encountered challenges exist and explored how these issues may be resolved. In particular, the following is discussed (i) NM selection (ii) NM physico-chemical characterisation; (iii) NM dispersion; (iv) selection of relevant doses and concentrations; (v) identification of relevant models, target sites and endpoints; (vi) development of alternatives to animal testing; and (vii) NM risk assessment. These knowledge gaps are relatively well recognised by the scientific community and recommendations as to how they may be overcome in the future are provided. It is hoped that this will help develop better defined hypothesis driven research in the future that will enable comprehensive risk assessments to be conducted for NMs. Importantly, the nanotoxicology community has responded and adapted to advances in knowledge over recent years to improve the approaches used to assess NM hazard, exposure and risk. It is vital to learn from existing information provided by ongoing or completed studies to avoid unnecessary duplication of effort, and to offer guidance on aspects of the experimental design that should be carefully considered prior to the start of a new study.

One of the first lines of defence to inhaled toxins is the barrier formed by the tracheobronchial epithelium, making this the ideal region for studying the toxicity of inhaled substances. This study utilises a highly differentiated, three-dimensional, in vitro model of human upper respiratory tract epithelium (EpiAirway-100) to measure the acute toxicological responses to well-characterised tobacco smoke components. To determine the suitability of this model for screening inhaled toxicants, the EpiAirway tissue model (ETM) was treated apically with tobacco smoke components (nicotine, formaldehyde, cadmium, urethane) which are known to induce a variety of toxic effects (e.g. cytotoxic, thrombogenic, carcinogenic). A range of concentrations were used to model different mechanisms and severity of toxicity which were then compared to known in vivo responses. Similar trends in stress response occurred, with distinct alterations to the tissue in response to all four toxins. At high concentrations, cell viability decreased and tight junctions were degraded, but at sub-toxic concentrations epithelial resistance (indicating tissue integrity) increased 20–60% from control. This peak in resistance coincided with an increase in secreted protein levels, elevated cytokine release and goblet cell hyperplasia and hypertrophy. In conclusion, acute exposure to tobacco smoke components induces measurable toxic responses within human respiratory epithelium. Sub-toxic concentrations appear to illicit a protective response by increasing mucus secretion and mediating immune responses via cytokine release. These responses are comparable to human in vivo responses, indicating potential for the ETM as a tool for screening the toxicity of inhaled compounds.

Engineered nanomaterials (NMs) offer great technological advantages but their risks to human health are still not fully understood. An increasing body of evidence suggests that some NMs are capable of distributing from the site of exposure to a number of secondary organs. The research into the toxicity posed by the NMs in these secondary organs is expanding due to the realisation that some materials may reach and accumulate in these target sites. The translocation to secondary organs includes, but is not limited to, the hepatic, central nervous, cardiovascular and renal systems. Current data indicates that pulmonary exposure is associated with low (inhalation route-0.00001-1% of total applied dose-24 h) translocation of virtually insoluble NMs such as iridium, carbon black, gold and polystyrene, while slightly higher translocation has been observed for NMs with either slow (e.g., silver, cerium dioxide and quantum dots) or fast (e.g., zinc oxide) solubility. The translocation of NMs following intratracheal, intranasal and pharyngeal aspiration is higher (up to 10% of administered dose), however the relevance of these routes for risk assessment is questionable. Uptake of the materials from the gastrointestinal tract seems to follow the same pattern as inhalation translocation, whereas the dermal uptake of NMs is generally reported to be low. The toxicological effects in secondary organs include oxidative stress, inflammation, cytotoxicity and dysfunction of cellular and physiological processes. For toxicological and risk evaluation, further information on the toxicokinetics and persistence of NMs is crucial. The overall aim of this review is to outline the data currently available in the literature on the biokinetics, accumulation, toxicity and eventual fate of NMs in order to assess the potential risks posed by NMs to secondary organs.

BACKGROUND:To assess the risk of all nanomaterials (NMs) on a case-by-case basis is challenging in terms of financial, ethical and time resources. Instead a more intelligent approach to knowledge gain and risk assessment is required.METHODS:A framework of future research priorities was developed from the accorded opinion of experts covering all major stake holder groups (government, industry, academia, funders and NGOs). It recognises and stresses the major topics of physicochemical characterisation, exposure identification, hazard identification and modelling approaches as key components of the current and future risk assessment of NMs.RESULTS:The framework for future research has been developed from the opinions of over 80 stakeholders, that describes the research priorities for effective development of an intelligent testing strategy (ITS) to allow risk evaluation of NMs. In this context, an ITS is a process that allows the risks of NMs to be assessed accurately, effectively and efficiently, thereby reducing the need to test NMs on a case-by-case basis.For each of the major topics of physicochemical characterisation, exposure identification, hazard identification and modelling, key-priority research areas are described via a series of stepping stones, or hexagon diagrams structured into a time perspective. Importantly, this framework is flexible, allowing individual stakeholders to identify where their own activities and expertise are positioned within the prioritisation pathway and furthermore to identify how they can effectively contribute and structure their work accordingly. In other words, the prioritisation hexagon diagrams provide a tool that individual stakeholders can adapt to meet their own particular needs and to deliver an ITS for NMs risk assessment. Such an approach would, over time, reduce the need for testing by increasing the reliability and sophistication of in silico approaches.The manuscript includes an appraisal of how this framework relates to the current risk assessment approaches and how future risk assessment could adapt to accommodate these new approaches. A full report is available in electronic format (pdf) at http://www.nano.hw.ac.uk/research-projects/itsnano.html.CONCLUSION:ITS-NANO has delivered a detailed, stakeholder driven and flexible research prioritisation (or strategy) tool, which identifies specific research needs, suggests connections between areas, and frames this in a time-perspective.

Engineered nanoparticles are increasingly used in medical applications and day-to-day consumer products, leading to concerns about the potential environmental and human health impacts. Silver nanoparticles are particularly prevalent because of their use as anti-bacterial agents in many commonly available products. Nanoparticles (NPs) are believed to accumulate, often preferentially, in the liver. This study therefore investigates the effect of a silver NP (20 nm) on the liver, and in particular, the role of Kupffer cells (KCs; resident liver macrophages) in the overall inflammatory response in the organ. Cytokine expression in the normal liver was measured in terms of IL2, IL4, TNF-α, IFN-γ and IL10 released from the organ with significant up-regulation of TNF-α and IL10 being observed. For livers in which the KC population was specifically targeted and destroyed this cytokine increase was significantly decreased in comparison to the normal tissue. IL10 was secreted at approximately three times the concentration of TNF-α in all the test cases. The high levels of IL10 released from the normal tissue in comparison to the KC depleted livers suggest that the cytokine may help to protect against a pro-inflammatory response to these Ag NPs. This may indicate a potentially important role for KCs in the anti-inflammatory response and suggests that tolerance to the Ag NPs is favoured over a fully activated immune response. In addition, albumin production was measured as an indicator of hepatic function. It was noted that the liver function was unaffected by the Ag NPs.

This study was undertaken primarily to identify genetic markers of oedema and inflammation. Mild pulmonary injury was induced following the instillation of the oedema-producing agent, bleomycin (0.5 units). Oedema was then confirmed by conventional toxicology (lavage protein levels, free cell counts and lung/body weight ratios) and histology 3 days post-bleomycin instillation. The expression profile of 1176 mRNA species was determined for bleomycin-exposed lung (Clontech Atlas macroarray, n=9). To obtain pertinent results from these data, it was necessary to develop a simple, effective method for bioinformatic analysis of altered gene expression. Data were log10 transformed followed by global normalisation. Differential gene expression was accepted if: (a) genes were statistically significant (P < or = 0.05) from a two-tailed t test; (b) genes were consistently outside a two standard deviation (SD) range from control levels. A combination of these techniques identified 31 mRNA transcripts (approximately 3%) which were significantly altered in bleomycin treated tissue. Of these genes, 26 were down-regulated whilst only five were up-regulated. Two distinct clusters were identified, with 17 genes classified as encoding hormone receptors, and nine as encoding ion channels. Both these clusters were consistently down-regulated. The magnitude of the changes in gene expression were quantified and confirmed by Q-PCR (n = 6), validating the macroarray data and the bioinformatic analysis employed. In conclusion, this study has developed a suitable macroarray analysis procedure and provides the basis for a better understanding of the gene expression changes occurring during the early phase of drug-induced pulmonary oedema. This work has been presented orally, in part at the British Association for Lung Research Summer Meeting, University of Brighton, 3-5 September, 2003 and in full at the British Toxicology Society Annual Congress, Heriot Watt University, Edinburgh, 21-24 April 2004.