Current guidance for the hazard assessment of nanomaterials
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Due to their small size, nanomaterials exhibit novel properties that are often vastly different from their bulk counterparts (larger sized particles with the same chemical composition). These include high tensile strength, low weight, high electrical and thermal conductivity, and unique electronic properties. This discovery has led to widespread interest in their potential commercial and industrial applications. Many applications of nanotechnology involve the use of nanomaterials including nanoparticles, nanotubes and other nano-objects and their aggregates and agglomerates. In fact, numerous products are already on the market which contain these materials such as paints, sunscreens, cosmetics, nanomedicines, self-cleaning glass, industrial lubricants, advanced tyres, semiconductors and food. This rapid proliferation has prompted concerns over the safety of engineered nanomaterials where exposure to humans and/or the environment occurs either intentionally or accidentally.
In 2004, the Royal Society and the Royal Academy of Engineering (RS/RAE) published, at the request of the UK government, a
major review of the opportunities and uncertainties of nanotechnologies. This was one of the first reports to highlight the potential risks to health and the environment that may arise from exposure to nanomaterials, especially nanoparticles, nanotubes and other nano-objects. The main conclusion of this report was that many nanosciences and nanotechnologies are unlikely to present any risks to health or the environment, but for nanoparticles and nanotubes:
- As particles get smaller, their specific surface area gets larger which is linked to increased toxicity;
- Potential to translocate (cross biological barriers) gets higher;
- Widespread usage means increased potential for exposure, people, environment;
As a result, not enough is known about exposure, toxicology and risk for adequate risk assessment; and integrated and multi-disciplinary research is needed to address these gaps.
Since then, a very large number of national and international reviews carried out by government departments, industry associations, insurance organisations and researchers (including SAFENANO) have considered nanoparticle risk issues. These reviews have provided a remarkably consistent view about the nature and the potential risks of nanoparticles, which may be summarised as follows:
- There are potential risks to human health and the environment from the manufacture and use of nanoparticles;
- There is a lack of knowledge about what these potential risks might be and how to deal with them;
- The lack of data makes it difficult for manufacturers, suppliers and users to have effective risk management processes and to comply with their regulatory duties;
- All of the stakeholders (regulators, companies) need to start to address these potential risks now.
Over the last few years there has been a significant increase in research activity in the UK and internationally, intended to fill these gaps. This activity continues to expand and is continuing to develop the evidence base around what could be considered to be the key issues that contribute to the potential for nanomaterials to demonstrate enhanced toxicity compared with their bulk counterparts. These are outlined below, followed by links to general guidance for assessing the (eco)toxicology of nanomaterials.
Plausible toxicology issues
High specific surface area
One of the main reasons nanomaterials tend to be more reactive than their corresponding bulk counterparts is that, per unit mass, they have a much higher surface area. If surface area is a driver for toxicity this clearly implies potentially increased toxic effects. Particulate surface area has been reported to play an important role in determining the biological activity of nanoparticles, with the pro-inflammatory effects of nanoparticles in the lungs demonstrated in a number of in vivo and in vitro studies to be related to their high surface area (
Stone et al., 2009).
Increased translocation potential
As a result of their small size, nanoparticles and other nano-objects can reach parts of biological systems which are not normally accessible by other larger particles. This includes the increased possibility of crossing cell boundaries, or of passing directly from the lungs into the blood stream and so on to all of the organs in the body; or even through deposition in the nose, directly to the brain. This process is known as translocation and in general nanoparticles can translocate much more easily than other larger particles. Several studies have shown that smaller particles typically have a wider tissue distribution, penetrate further within the skin and intestine, are internalised by cells to a greater extent, and have a larger toxic potency (
Stone et al., 2009). In real exposure situations, the extent to which nano-objects are agglomerated and the extent to which they de-agglomerate needs to be taken into account when considering their subsequent translocation.
Fibre analogy
Based on toxicological research on asbestos and other industrial fibres, a ‘fibre paradigm’ has been developed in which fibres that are biopersistent in the lungs and longer than 15–20 μm with a diameter less than 3 μm being considered to be hazardous to human health. Asbestos is often taken to be the benchmark fibre against which the potential toxicity of other fibres are assessed, because of the global excesses of lung diseases (namely asbestosis, lung cancer and mesothelioma) arising from asbestos fibre exposure.
A recent major review identified many similarities between high aspect ratio nanoparticles ('HARN' – which includes CNTs) and asbestos in terms of their physico-chemical properties and toxicological effects, concluded that, ‘there is sufficient evidence to suggest that HARN which have the same characteristics (diameter, length and biopersistence) as pathogenic fibres are likely to have similar pathology’ (
Tran et al., 2008). Rodent studies have demonstrated the potential for long (> 20 μm), straight MWCNTs to cause masses of immune cells termed ‘granuloma’, in the lung area associated with the peritoneal mesothelium (lining), in contrast with short tangled CNTs which did not (
Poland et al., 2008). These granulomas are known to be potential precursor to tumours such as mesothelioma). In addition, a direct comparison in vitro study has demonstrated the occurrence of frustrated phagocytosis by macrophages (an inability to clear fibres from the lungs) and pro-inflammatory cytokine expression, on exposure to long, but not short or entangled MWCNTs (
Brown et al., 2007). In this study, MWCNT reported more potent action than long fibre amosite asbestos However, further studies are required using inhalation as a route of delivery to verify these results.
Solubility
It has been shown that some types of nanoparticles become more soluble as particle size decreases. This could imply increased bioavailability for particles previously considered to be insoluble. For example bulk silver is insoluble, but nanosilver releases free silver ions in aqueous solutions. It has been hypothesised that the toxic effects of silver substances are proportional to the rate of release of free silver ions from them (
e.g. Wijnhoven et al., 2009). As such when considering the hazardous nature of a material, it is pertinent to consider both the insoluble (particle) and soluble component in the hazard assessment.
Environmental hazard
Over the past few years, research aimed at studying the ecotoxicology of nanomaterials has grown, although perhaps not to the same extent as human toxicology studies. Following a comprehensive review of the ecotoxicological literature across four groups of engineered nanomaterials (fullerenes, carbon nanotubes, metals and metal oxides),
Stone et al. (2009) observed that:
- The majority of studies have focussed on studying short-term effects while chronic effects after long-term exposures to lower concentrations have been far less studied;
- A wide range of environmentally relevant species have been used, but there is as yet no clear pattern regarding species sensitivity, suitability as test organism, or relevance of endpoints for nano-ecotoxicology;
- While some studies have carried out characterisation of the nanoparticles tested, most studies only report on chemical composition, sizes of nanoparticles (as purchased), and in some cases sizes of nanoparticles in suspension;
- The characterisation and quantification of nanoparticles in stock solutions, in media, and in biological tissues remains one of the biggest challenges in nano-ecotoxicology. This is not only needed for linking characteristics with toxicity, but also for determination of actual exposure levels and for quantification of uptake, depuration and decay of nanoparticles;
- Only for a few nanomaterials (e.g. C60 and TiO2) is data available for three trophic levels which is considered to be the base set for risk assessment (i.e., fish, crustacean, algae). However, there is a need for replication of some of the studies due to testing difficulties with regards to preparation, handling, and quantification of nanoparticle exposure;
- There is a pronounced and problematic lack of studies addressing degradability and accumulation, since quantitative knowledge of these, along with ecotoxicity, are fundamental for completing environmental hazard assessment of nanomaterials
From the literature reviewed as part of the
ENRHES project, it is evident that much more research is needed before specific properties, or combinations of properties, can be linked to the effects observed in ecotoxicity tests. For the time being only a few studies have documented links between characteristics and toxicity. As an example,
van Hoecke et al. (2008) found that when toxicity results for SiO2 nanoparticles were expressed in terms of surface area instead of mass units, the apparent differential toxicity related to nanoparticle size was eliminated.
Published Guidance
A range of guidance documents are available which provide information to support the hazard assessment of nanoparticles, including (eco)toxicity testing and sample preparation considerations. It should be acknowledged that the selection of an appropriate testing method (particularly in vivo) is a highly technical process, specific to the material type in question and dependent on the health effect or mechanism of interest. However, the following section outlines a selection of that freely available guidance which may be of some value in informing testing considerations.
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The focus of this document is on regulatory testing and how this might be adapted for nanomaterials. The document is aimed at those participating in the OECD WPMN Sponsorship Programme in the development of comprehensive and consistent dossier development plans (DDPs). However, it serves as a useful source of general guidance for the testing of manufactured nanomaterials.
The guidance manual includes appropriate protocols and methodologies for physico-chemical characterisation and the testing of toxicological and ecotoxicological endpoints, as well as outlining a number of important media considerations.
To access the document in full,
click here.
The purpose of this document is to assist sponsors as they conduct testing in support of the WPMN’s Sponsorship Programme for the Testing of Manufactured Nanomaterials, as well as other users involved in the testing of manufactured nanomaterials. The guidance includes general and common issues as well as specific considerations on sample preparation and dosimetry (measurement of dose).
The section on specific considerations is composed of four parts:
- physical chemical properties;
- ecotoxicity studies;
- degradation, transformation and accumulation; and
- health effects.
These parts may give researchers specific orientation to those issues that, at present, seem most promising for yielding meaningful and reproducible test results.
To access the document in full,
follow this link.
NanoImpactNet is a multidisciplinary European network on the health and environmental impact of nanomaterials to facilitate collaboration between research projects, communicate results to stakeholders and their needs back to researchers and help implement the EU's Action Plan for Nanotechnology. In addition to networking activity, the project’s aims also include the development of best practice to ensure that studies are comparable in terms of basic parameters such as particle type, cell types, dispersion and characterisation protocols and appropriate testing methodologies. NanoImpactNet currently provides an online-space for members to share research protocols with other members with the view that laboratories can easily compare their methods and subsequently develop common protocols and strategies for testing of nanomaterials. Once the protocols are tested by other members and further amended, the aim is to develop a set of “methods recommended by NanoImpactNet” for publication and provision to international bodies (e.g. ISO, OECD, etc.).
This activity is on-going at present and no protocols have yet been published as “methods recommended by NanoImpactNet”. Draft protocols currently available cover nanoparticle preparation, characterisation, ecotoxicology and toxicity testing, and usage. Registration to access the protocols is free through the
NanoImpactNet website.
The NanoCare project (running from 2006 - 2009) aimed to broaden knowledge about synthetic nanomaterials with regard to the potential impacts of nanomaterials on human health. As part of this project, several (non-validated) standard operating procedures (SOPs) were developed to support toxicology testing.