Paul J. A. Borm

The potential risks of nanomaterials: a review carried out for ECETOC

During the last few years, research on toxicologically relevant properties of engineered nanoparticles has increased tremendously. A number of international research projects and additional activities are ongoing in the EU and the US, nourishing the expectation that more relevant technical and toxicological data will be published. Their widespread use allows for potential exposure to engineered nanoparticles during the whole lifecycle of a variety of products. When looking at possible exposure routes for manufactured Nanoparticles, inhalation, dermal and oral exposure are the most obvious, depending on the type of product in which Nanoparticles are used. This review shows that (1) Nanoparticles can deposit in the respiratory tract after inhalation. For a number of nanoparticles, oxidative stress-related inflammatory reactions have been observed. Tumour-related effects have only been observed in rats, and might be related to overload conditions. There are also a few reports that indicate uptake of nanoparticles in the brain via the olfactory epithelium. Nanoparticle translocation into the systemic circulation may occur after inhalation but conflicting evidence is present on the extent of translocation. These findings urge the need for additional studies to further elucidate these findings and to characterize the physiological impact. (2) There is currently little evidence from skin penetration studies that dermal applications of metal oxide nanoparticles used in sunscreens lead to systemic exposure. However, the question has been raised whether the usual testing with healthy, intact skin will be sufficient. (3) Uptake of nanoparticles in the gastrointestinal tract after oral uptake is a known phenomenon, of which use is intentionally made in the design of food and pharmacological components. Finally, this review indicates that only few specific nanoparticles have been investigated in a limited number of test systems and extrapolation of this data to other materials is not possible. Air pollution studies have generated indirect evidence for the role of combustion derived nanoparticles (CDNP) in driving adverse health effects in susceptible groups. Experimental studies with some bulk nanoparticles (carbon black, titanium dioxide, iron oxides) that have been used for decades suggest various adverse effects. However, engineered nanomaterials with new chemical and physical properties are being produced constantly and the toxicity of these is unknown. Therefore, despite the existing database on nanoparticles, no blanket statements about human toxicity can be given at this time. In addition, limited ecotoxicological data for nanomaterials precludes a systematic assessment of the impact of Nanoparticles on ecosystems.

Inhaled particles and lung cancer. Part A: Mechanisms

Both occupational and environmental exposure to particles is associated with an increased risk of lung cancer. Particles are thought to impact on genotoxicity as well as on cell proliferation via their ability to generate oxidants such as reactive oxygen species (ROS) and reactive nitrogen species (RNS). For mechanistic purposes, one should discriminate between a) the oxidant‐generating properties of particles themselves (i.e., acellular), which are mostly determined by the physicochemical characteristics of the particle surface, and b) the ability of particles to stimulate cellular oxidant generation. Cellular ROS/RNS can be generated by various mechanisms, including particle‐related mitochondrial activation or NAD(P)H‐oxidase enzymes. In addition, since particles can induce an inflammatory response, a further subdivision needs to be made between primary (i.e., particle‐driven) and secondary (i.e., inflammation‐driven) formation of oxidants. Particles may also affect genotoxicity by their ability to carry surface‐adsorbed carcinogenic components into the lung. Each of these pathways can impact on genotoxicity and proliferation, as well as on feedback mechanisms involving DNA repair or apoptosis. Although abundant evidence suggests that ROS/RNS mediate particle‐induced genotoxicity and mutagenesis, little information is available towards the subsequent steps leading to neoplastic changes. Additionally, since most of the proposed molecular mechanisms underlying particle‐related carcinogenesis have been derived from in vitro studies, there is a need for future studies that evaluate the implication of these mechanisms for in vivo lung cancer development. In this respect, transgenic and gene knockout animal models may provide a useful tool. Such studies should also include further assessment of the relative contributions of primary (inflammation‐independent) and secondary (inflammation‐driven) pathways.

Oxidative stress and calcium signaling in the adverse effects of environmental particles (PM10)

This review focuses on the potential role that oxidative stress plays in the adverse effects of PM(10). The central hypothesis is that the ability of PM(10) to cause oxidative stress underlies the association between increased exposure to PM(10) and both exacerbations of lung disease and lung cancer. Pulmonary inflammation may also underlie the cardiovascular effects seen following increased PM(10), although the mechanisms of the cardiovascular effects of PM(10) are not well understood. PM(10) is a complex mix of various particle types and several of the components of PM(10) are likely to be involved in the induction of oxidative stress. The most likely of these are transition metals, ultrafine particle surfaces, and organic compounds. In support of this hypothesis, oxidative stress arising from PM(10) has been shown to activate a number of redox-responsive signaling pathways in lung target cells. These pathways are involved in expression of genes that play a role in responses relevant to inflammation and pathological change, including MAPKs, NF-kappaB, AP-1, and histone acetylation. Oxidative stress from particles is also likely to play an important role in the carcinogenic effects associated with PM(10) and hydroxyl radicals from PM(10) cause DNA damage in vitro.

Evaluating the Toxicity of Airborne Particulate Matter and Nanoparticles by Measuring Oxidative Stress Potential—A Workshop Report and Consensus Statement

Background: There is a strong need for laboratory in vitro test systems for the toxicity of airborne particulate matter and nanoparticles. The measurement of oxidative stress potential offers a promising way forward. Objectives:Aworkshop was convened involving leading workers from the field in order to review the available test methods and to generate a Consensus Statement. Discussions: Workshop participants summarised their own research activities as well as discussion the relative merits of different test methods. Conclusions: In vitro test methods have an important role to play in the screening of toxicity in airborne particulate matter and nanoparticles. In vitro cell challenges were preferable to in vitro acellular systems but both have a potential major role to play and offer large cost advantages relative to human or animal inhalation studies and animal in vivo installation experiments. There remains a need to compare tests one with another on standardised samples and also to establish a correlation with the results of population-based epidemiology.

Multifunctional Nanocarriers for diagnostics, drug delivery and targeted treatment across blood-brain barrier: perspectives on tracking and neuroimaging

Nanotechnology has brought a variety of new possibilities into biological discovery and clinical practice. In particular, nano-scaled carriers have revolutionalized drug delivery, allowing for therapeutic agents to be selectively targeted on an organ, tissue and cell specific level, also minimizing exposure of healthy tissue to drugs. In this review we discuss and analyze three issues, which are considered to be at the core of nano-scaled drug delivery systems, namely functionalization of nanocarriers, delivery to target organs and in vivo imaging. The latest developments on highly specific conjugation strategies that are used to attach biomolecules to the surface of nanoparticles (NP) are first reviewed. Besides drug carrying capabilities, the functionalization of nanocarriers also facilitate their transport to primary target organs. We highlight the leading advantage of nanocarriers, i.e. their ability to cross the blood-brain barrier (BBB), a tightly packed layer of endothelial cells surrounding the brain that prevents high-molecular weight molecules from entering the brain. The BBB has several transport molecules such as growth factors, insulin and transferrin that can potentially increase the efficiency and kinetics of brain-targeting nanocarriers. Potential treatments for common neurological disorders, such as stroke, tumours and Alzheimers, are therefore a much sought-after application of nanomedicine. Likewise any other drug delivery system, a number of parameters need to be registered once functionalized NPs are administered, for instance their efficiency in organ-selective targeting, bioaccumulation and excretion. Finally, direct in vivo imaging of nanomaterials is an exciting recent field that can provide real-time tracking of those nanocarriers. We review a range of systems suitable for in vivo imaging and monitoring of drug delivery, with an emphasis on most recently introduced molecular imaging modalities based on optical and hybrid contrast, such as fluorescent protein tomography and multispectral optoacoustic tomography. Overall, great potential is foreseen for nanocarriers in medical diagnostics, therapeutics and molecular targeting. A proposed roadmap for ongoing and future research directions is therefore discussed in detail with emphasis on the development of novel approaches for functionalization, targeting and imaging of nano-based drug delivery systems, a cutting-edge technology poised to change the ways medicine is administered.

Inhaled particles and lung cancer, part B: Paradigms and risk assessment

Poorly soluble particles of low toxicity (PSP), such as CB, TiO2 and coal mine dust, have been demonstrated to cause lung cancer in rodents, being most pronounced in rats. Adequate epidemiologic studies do not clearly indicate increased lung cancer rates in humans exposed to such particles. This has caused controversial positions in regulatory decisions on PSP on different levels. The present review discusses the current paradigms in rodent particle carcinogenicity, i.e., (i) role of particle overload and of persistent inflammation and (ii) fibrosis as an intermediate step in particle‐induced lung cancer with regard to human risk assessment. Fibrosis, which is usually considered a precursor of lung cancer in humans, was not related to lung tumors in an animal study using 6 different particles, each at 3 dosages. Lung tumors after both inhalation and intratracheal instillation of PSP are related to particle surface dose, which forwards hazard assessment at surface‐based nonoverload concentrations and a standard setting using surface as an exposure metric. The scarce data available on humans do not support the overload concept but suggest a role for persistent lung inflammation. Differences in antioxidant protection between different rodent species correlate with susceptibility to PSP‐induced carcinogenicity and support the need for detailed studies on antioxidant response in humans. Apart from such bridging studies, further focus is also needed on surface chemistry and modifications in relation to their adverse biologic effects.

Testing Strategies to Establish the Safety of Nanomaterials: Conclusions of an ECETOC Workshop

The European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) convened a workshop in Barcelona, Spain, in November 2005 to develop testing strategies to establish the safety of nanomaterials. It brought together about 70 scientific and clinical experts from industry, academia, government agencies, research institutes, and nongovernmental organizations. The primary questions to be addressed were the following: What can we do today, and what do we need tomorrow? The three major themes of the workshop were: (1) the need for enhanced efforts in nanomaterial characterization; (2) methodologies for assessments of airborne and internal exposures to nanomaterials; and (3) evaluation of the hazard potential—primarily focusing on pulmonary or dermal routes of exposures. Some of the summary conclusions of the workshop included the following: For the development of nanoparticle characterization, the working definition of nanoparticles was defined as < 100 nm in one dimension or < 1000 nm to include aggregates and agglomerates. Moreover, it was concluded that although many physical factors can influence toxicity, including nanoparticle composition, it is dissolution, surface area and characteristics, size, size distribution, and shape that largely determine the functional, toxicological and environmental impact of nanomaterials. In addition, most of the information on potential systemic effects has thus far been derived from combustion-generated particles. With respect to the assessment of external exposures and metrics appropriate for nanoparticles, the general view of the meeting was that currently it is not possible or desirable to select one form of dose metric (i.e., mass, surface area, or particle number) as the most appropriate measure source. However, it was clear that the surface area metric was likely to be of interest and requires further development. In addition, there is a clear and immediate need to develop instruments which are smaller, more portable, and less expensive than the currently available state of the art instrumentation. With regard to a general testing approach for human health hazard evaluation of nanoparticles, a first step to determine potency may include a prioritization-related in vitro screening strategy to assess the possible reactivity, biomarkers of inflammation and cellular uptake of nanoparticles; however this process should be validated using in vivo techniques. A Tier 1 in vivo testing strategy could include a short-term inhalation or intratracheal instillation of nanoparticles as the route of exposure in the lungs of rats or mice. The endpoints that should be assessed include indices of lung inflammation, cytotoxicity, and cell proliferation, as well as histopathology of the respiratory tract and the major extrapulmonary organs. For Tier 2 in vivo testing for hazard identification, a longer term inhalation study is recommended, and this would include more substantive mechanistic endpoints such as determination of particle deposition, translocation, and disposition within the body. Additional studies could be designed with specific animal models to mimic sensitive populations. With regard to dermal exposures, currently there is little evidence that nanoparticles at a size exceeding 100 nm penetrate through the skin barrier into the living tissue (i.e., dermal compartment). The penetration of nanoparticles at a size less than 100 nm should be a topic of further investigation. Moreover, considering the impacts of dermal exposures and corresponding hazard potential of nanoparticles, it must be taken into consideration that the dermal uptake of nanoparticles will be an order of magnitude smaller than the uptake via the inhalation or oral routes of exposure. For the evaluation of the health risk of nanoparticles, it has to be determined whether they are harmful to living cells and whether, under real conditions, they penetrate through the skin barrier into the living tissue. For the evaluation of the penetration processes, in principle, three methods are available. Using the method of differential stripping, the penetration kinetics of nanoparticles in the stratum corneum and the hair follicles can be evaluated. This analysis can be carried out in vivo. Diffusion cell experiments are an efficient method for in vitro penetration studies. Also, laser scanning microscopy is well suited to test penetration kinetics, although requiring fluorescent-labeled nanoparticles. Emerging topics such as (1) environmental safety testing, (2) applications of nanoparticles for medical purposes, and (3) pathways of inhaled nanoparticles to the central nervous system were also briefly addressed during this workshop. However, it has become clear that these topics should be the subjects of separate workshops and they are not further addressed in this report.

Expert elicitation on ultrafine particles: likelihood of health effects and causal pathways

BackgroundExposure to fine ambient particulate matter (PM) has consistently been associated with increased morbidity and mortality. The relationship between exposure to ultrafine particles (UFP) and health effects is less firmly established. If UFP cause health effects independently from coarser fractions, this could affect health impact assessment of air pollution, which would possibly lead to alternative policy options to be considered to reduce the disease burden of PM. Therefore, we organized an expert elicitation workshop to assess the evidence for a causal relationship between exposure to UFP and health endpoints.MethodsAn expert elicitation on the health effects of ambient ultrafine particle exposure was carried out, focusing on: 1) the likelihood of causal relationships with key health endpoints, and 2) the likelihood of potential causal pathways for cardiac events. Based on a systematic peer-nomination procedure, fourteen European experts (epidemiologists, toxicologists and clinicians) were selected, of whom twelve attended. They were provided with a briefing book containing key literature. After a group discussion, individual expert judgments in the form of ratings of the likelihood of causal relationships and pathways were obtained using a confidence scheme adapted from the one used by the Intergovernmental Panel on Climate Change.ResultsThe likelihood of an independent causal relationship between increased short-term UFP exposure and increased all-cause mortality, hospital admissions for cardiovascular and respiratory diseases, aggravation of asthma symptoms and lung function decrements was rated medium to high by most experts. The likelihood for long-term UFP exposure to be causally related to all cause mortality, cardiovascular and respiratory morbidity and lung cancer was rated slightly lower, mostly medium. The experts rated the likelihood of each of the six identified possible causal pathways separately. Out of these six, the highest likelihood was rated for the pathway involving respiratory inflammation and subsequent thrombotic effects.ConclusionThe overall medium to high likelihood rating of causality of health effects of UFP exposure and the high likelihood rating of at least one of the proposed causal mechanisms explaining associations between UFP and cardiac events, stresses the importance of considering UFP in future health impact assessments of (transport-related) air pollution, and the need for further research on UFP exposure and health effects.

The surface area rather than the surface coating determines the acute inflammatory response after instillation of fine and ultrafine TiO2 in the rat

Alterations in the hydrophobic status of particle surfaces have been suggested to modify the toxic properties of ultrafine TiO2. We investigated the acute inflammatory responses and cell damage after intratracheal instillation of surface modified (hydrophilic and hydrophobic) fine (180 nm) and ultrafine (20-30 nm) TiO2 particles 16 h at equivalent mass (1 or 6 mg) and surface doses (100, 500, 600 and 3000 cm2) in rats. Inflammatory response and most enzyme levels were significantly related to the administered surface dose. The hydrophobic surface of the TiO2 particles, achieved by methylation, induced a lower total cell number and influx of neutrophils (PMN) compared to rats instilled with the 1 mg of the untreated, fine or ultrafine TiO2 but the outcomes were not statistically significant. No differences were observed between fine/ultrafine and hydophilic/hydrophobic TiO2 at the high dose (6 mg) or surface dose over 600 cm2. The differences in BAL cellularity at the low dose were reflected in changes in the chemokine MIP-2, but no differences were seen in levels of macrophage cytokines. Considering the large influx of PMN little cell damage was seen when studying enzyme leakage in lavage fluid, although PMNs appeared to be activated as suggested by increased myeloperoxidase (MPO) activity in the lavage fluid. We conclude that the surface area rather than the hydrophobic surface determines the acute, pulmonary inflammation induced by both fine and ultrafine TiO2.

Particle toxicology: from coal mining to nanotechnology.

Particle research has been historically closely connected to industrial activities or materials, such as coal, asbestos, man-made mineral fibers, and more recently ambient particulate matter (PM). It is the purpose of this review to combine insights and developments in particle toxicology with the historical context of exposure and organizations sponsoring such research in Europe. In supporting research on particle-induced respiratory effects and mechanisms, research programs of the European Community on Steel and Coal (ECSC) have played a tremendous role. Current particle research in Europe is dominated by PM, and funded by the World Health Organization (WHO), European Union Framework programs, and the Health Effects Institute (HEI). Differences between historical and current research in particle toxicology include the exposure concentrations, particle size, target populations, endpoints, and length of exposure. Inhaled particle effects are no longer confined to the lung, since particles are suggested to translocate to the blood while lung inflammation invokes systemic responses. Finally, the particle size and concentrations have both been reduced about 100-fold from 2-5 mg/m 3 to 20-50 mg/m 3 and from 1-2 µm to 20-100 nm (ultrafine) as domestic fuel burning has decreased and vehicle sources have increased and attention has moved from coal mining industry to general environment. There is, however, a further occupational link to nanotechnology, which continuously produces new materials in the ultrafine range. Although inhalation exposure is considered to be minimal in this technology, some particles are produced to be used for carrier purpose in medical applications. Based on our current knowledge of particle toxicology, it is highly desirable that toxicology and technology are linked in this extremely rapid developing area, to learn more about potential risks and also to develop knowledge on the role of surface and size in particle toxicity.