Nanomaterials

9. Where does risk assessment for nanomaterials now stand?

While risk assessment methodologies for the evaluation of potential risks of substances and conventional materials to man and the environment are widely used and are generally applicable to nanomaterials, specific aspects related to nanomaterials still require further development. This will remain so until there is sufficient scientific information available to characterise the harmful effects of nanomaterials on humans and the environment. The methodology for both exposure estimations and hazard identification needs to be further developed, validated and standardised. The highest risk, and thus concern, is considered to be associated with the presence or occurrence of free (non bound) insoluble nanoparticles either in a liquid dispersion or airborne dusts.

Characterization of manufactured nanomaterials

For the characterisation of manufactured nanomaterials, several issues are important. A consensus is now emerging about what properties need to be determined for risk assessment purposes. In biological test systems nanomaterials may change their properties. In particular, they may partly dissolve or agglomerate/aggregate so that the particle size distribution changes. For (partially) soluble nanomaterials the toxicity may be governed at least in part by the soluble species/fraction released from the nanomaterial. For low solubility or a slow release, the particulate nature of the substance may be relevant with regard to potential tissue distribution and local release of toxic species which should then be considered in the risk assessment of such nanomaterials. When using nanomaterials, an extensive characterisation is necessary, including the nanomaterial as produced and the nanomaterials as used in test systems and the nanomaterial as present in final products. The characterisation ‘as manufactured’ provides information for the material safety data sheet (MSDS) of the product itself. The characterisation ‘as used’ in biological systems is needed as properties of nanomaterials may considerably change, notably the size distribution due to agglomeration/aggregation of the particles. An issue of specific importance are the properties of the nanomaterial as it is actually used in products and to which consumers may be exposed. For the risk assessment the latter characterization is of highest relevance.

Legally, in the EU, nanomaterials are covered by the definition of substance within the REACH regulation (Regulation (EC) No. 1907/2006) (European Commission 2006). However, the definition of nanoscale is still under debate. Various organisations have proposed definitions of nanoscale using an upper limit of about 100nm. It should be noted that most currently proposed definitions use the size of the primary particle/structure as a starting point. However, when a nanomaterial is in particulate form, the particles may be present either as single particles or as agglomerates or aggregates. Depending on the nanomaterial the majority of the particles may be agglomerates or aggregates. This may lead to the misinterpretation that agglomerates or aggregates of nanoparticles that have external dimensions well beyond 100nm are not considered nanomaterials. Yet they retain specific physicochemical properties which are characteristic of nanomaterials, most likely due to their large specific surface area (SSA). Therefore, when describing a nanomaterial it is important to describe not only the mean particle size but also the size of the primary particles. In addition, information on the presence of agglomerates or aggregates should be presented. Besides size, the specific surface area as determined by the BET method is a good metric to describe particulates as it is independent of the primary versus the agglomerated state. Hence, it should be considered to complement the current definition based on physical size by adding a limit of the specific surface area. Solid spheres of 100 nm with unit density have a specific surface area of 60 m²/g.

There is currently a need for reference nanomaterials. Some are available but they are spherical model materials which are certified primarily for size and are used mainly to calibrate instruments which measure particle size. The absence of well-defined parameters to measure and of standardised test protocols is identified as a major obstacle for reference material production. It should be noted that for use in biological systems certain compounds need to be added which may have an effect on nanomaterial composition and properties resulting in changes in (toxic) behaviour.

Human exposure

One of the main limitations in the risk assessment of nanomaterials is the general lack of high quality exposure and dosimetry data both for humans and the environment. One of the issues is the difficulty to determine the presence on nanomaterials and properly measure them. In contrast to the situation for other exposure routes, for air-borne nanomaterials, analytical instruments are generally available to determine exposure (size distribution of mass and number). This is particularly true in the context of test atmospheres. However, differentiation between background and incidental exposure is generally not possible in real life situations as the methods employed mainly measure the presence of (ultrafine) particles and do not discriminate between the different types of particles that may be present. To date most information on particle measurements comes from airborne measurements at the workplace. No quantitative or qualitative measurements of manufactured nanomaterials in ambient air outside of workplaces have been identified. Even when outside measurements are available these may be confounded, an example being carbon nanotubes that also may originate from general combustion processes and thus can be found in ambient locations. This illustrates the difficulty of identifying exposure levels of airborne manufactured nanomaterials. There is a need to establish reliable and standardised measurement techniques, to develop measurement strategies, and to implement screening/monitoring of nanoscale particles in sensitive work areas. Challenges are currently especially seen in the detection and assessment of manufactured nanoparticles in the environment. This is even more urgent for exposure of humans and ecosystems via natural water, sediment and soil.

Exposure estimates for consumers from food and consumer products remains difficult. Information on the presence of manufactured nanomaterials solely relies on information (claims) provided by manufacturers. In addition, exposure estimation is also hampered by lack of information on product use and use of multiple products containing manufactured nanomaterials. In a similar fashion to air measurements, determination of manufactured nanomaterials in consumer products suffers from the difficulty of discrimination between background and intentionally added manufactured nanomaterials. Coordinated efforts and research strategies for a comprehensive exposure assessment of manufactured nanomaterials still have to be defined. The main issues may be summarised as problems in replicating actual exposure conditions in laboratory tests and the lack of general availability of robust and specific measurement methods. Exposure assessment needs to consider each stage in the life-cycle.

Human hazard

When nanomaterials come into contact with a biological fluid they may become coated with proteins and other biomolecules. The coating may then influence the outcome of the biological response to the nanoparticle. Coating proteins have been most widely studied in mammalian systems. The significance of nanomaterial coating for nanomaterial safety and nanomaterial risk assessment is clear, as it implies that detailed characterisation of the nanoparticles in the relevant biological environment is necessary. The coating of nanoparticles may be used for therapeutic purposes to prolong circulation time (by PEGylation) or to target specific locations (e.g. apolipoprotein E for brain, immunoglobulins for tumors).

As the protein coating may affect the nanomaterial behaviour including its biological effect, it may be anticipated that nanomaterials may have an effect on protein behaviour. Some nanoparticles have been found to have the potential to promote and to retard protein assembly into amyloid fibrils in vitro. These experiments were performed using an incubation of various nanoparticles with purified β2-microglobulin or β- amyloid protein. Whether the observed nucleation process also occurs in an in vivo situation or in more complex biological fluids where competitive binding may take place remains to be determined.

Existing data show that nanoparticles can enter the circulation from the respiratory tract or the gastro-intestinal tract but typically in minimal amounts (less than 1% percentage of the dose as expressed in mass units). However, although minimal in percentage this may result in a systemic availability of a considerable number of nanoparticles. Nanoparticle migration is likely to depend on the physico-chemical properties of the nanoparticles such as size and on the physiological state of the organs of entry. When the nanoparticles reach the blood circulation, the liver and the spleen are the two major organs for distribution. Circulation time increases drastically when the nanoparticles are hydrophilic and their surface is positively charged. A coating like polyethylene glycol (PEGylation) also increases the residence time in the circulation. For certain nanoparticles all organs may be at some risk. For all organs investigated so far, either the chemical component of the nanoparticles or the nanoparticles themselves could be detected, as demonstrated for the brain and the testes. In the case of distribution to the foetus in utero, contradicting results were observed. The knowledge on toxicokinetics has been increased showing that, for a given substance, the smaller nanoparticles do have a much wider organ distribution than the larger nanoparticles.

There are indications that after deposition at the olfactory mucosa of the nose nanoparticles may translocate into the brain. This may offer a potential route of entry for medicinal products into the brain. Because of the potential of nanoparticles to cause protein fibrillation in vitro, this observation may raise some concern in view of the amyloid diseases of the brain. This is certainly an area for which additional research is urgently needed.

Based on the observations on the effects of particulate matter present in air pollution some concern exists on the possible effect of manufactured nanoparticles on the cardiovascular system. However, this has not been clearly demonstrated to be the case for manufactured nanoparticles so far. Overall the information on the possible hazard of nanoparticles for cardiovascular effects is rather limited and needs expansion.

When carbon nanotubes have physico-chemical and biopersistence characteristics similar to those of hazardous asbestos fibres, it was demonstrated that they can induce similar inflammatory reactions. The main characteristics for this to occur are a long thin fibrous form (length >20 μm), rigidity, and no degradability (biopersistence). Whether inhalation exposure to such carbon nanotubes would pose a risk to humans is unknown. Thus, manufacturers of nanotubes (possibly of any chemical composition) should be aware that certain characteristics (e.g. length, rigidity, biopersistence) may pose a risk and the possibility for chronic inflammation and mesothelioma induction and consequently should be considered in the safety evaluation.

The genotoxic effects of conventional particles are driven by two mechanisms: direct and indirect (mediated by inflammatory processes). Nanoparticles may act via either of these pathways since they can cause inflammation and can also enter cells and cause oxidative stress. There is some evidence that the small size may allow nanoparticles to penetrate into sub-cellular compartments like the mitochondria and nucleus . The presence of nanomaterials in mitochondria and the nucleus opens the possibility for oxidative stress mediated genotoxicity, and/or direct interaction with DNA. For some manufactured nanomaterials genotoxic activity has been reported, mainly associated to reactive oxygen species (ROS) generation, while for others contradicting results were obtained. Besides oxidative stress, additional mechanisms of genotoxicity which may be specific for nanomaterials also need to be considered, such as possible mechanical interferences during cell division, and other sources of genotoxic effects (i.e. metal release by nanomaterials).

The main issues for human hazard identification may be summarised as a need to ensure that each test system is appropriate for nanomaterials and to ensure that endpoints of potential particular concern (e.g. cardio-vascular effects) are properly addressed.

Environmental exposure

The increasing production, use and disposal of nanomaterials will lead to an increase in environmental exposure. Similar to human health risks, fate and behaviour of the manufactured nanomaterials in the environment itself is crucial for the potential ecotoxic effects in various environmental species. Estimation of relevant exposure concentrations is seriously hampered by lack of the two most important pieces of information/knowledge. Firstly, there is no quantitative knowledge on the rates of release of nanomaterials to the environment. Secondly, there is hardly any knowledge on the concentrations of nanomaterials in the ambient environment. There is also no theory that can be used to estimate such concentrations from release rates. The main problem is that the well-established knowledge of distribution and fate of chemical substances, as it is applied in the current EU guidelines for environmental risk assessment of conventional chemicals, cannot be used for nanomaterials without modification. Most certainly, the Kow is of limited use as a predictor of the extent to which nanomaterials adhere to solid surfaces.

A hypothesis to describe fate and distribution of nanomaterials is slowly being developed, mostly from classical knowledge of colloid science. It is recognised that the main factors that influence the colloidal behaviour of nanoparticles (aggregation/agglomeration, sedimentation) are, besides the physical and chemical properties of the nanomaterial, the properties of the receiving environment: pH, ionic strength, prensence of natural organic matter. Depending of the nanomaterial characteristics either an increased sedimentation or improved dispersion of nanomaterials in water may occur. Exposure estimates are hampered by difficulties in distinguishing manufactured nanomaterials from background levels of naturally occurring nanomaterials. For the environmental risk assessment the estimation of water concentrations is essential. The assessment of exposure concentrations of dispersed nanomaterials requires detailed insight into the processes that act on the particles in the environment. However, currently available knowledge of these processes is insufficient to allow quantitative predictions of the environmental fate of nanomaterials.

The solubility of the nanomaterials is an important property that needs to be addressed. Knowledge of the extent to which nanomaterials dissolve and the rate at which this takes place is essential in two respects: (i) it is a direct control of the concentrations of nanomaterials in the environment and of the time that the nanomaterials reside in the environment and in organisms, and (ii) it determines the concentrations of dissolved species that originate from the nanomaterials. Knowledge of the extent to which nanomaterials dissolve in water, and of the rate at which this occurs, is essential to predicting the environmental fate and the effects of nanomaterials. It is doubtful whether currently available standard methods for measuring the (rate of) dissolution can adequately deliver this knowledge.

Unlike in the assessment of exposure concentrations of conventional (dissolved) chemical substances, the octanol-water partition coefficient Kow is likely to have a limited role in predicting water-solids partitioning. An alternative theory to predict the exposure levels of nanomaterials in water is yet to be developed. Based on well-established knowledge of colloid science, it is expected that pH, ionic strength and presence of natural organic matter in the water compartment (freshwater versus marine environments) are important factors influencing the residual levels of nanomaterials in suspension. Depending on these factors and the chemistry of the manufactured nanomaterial increased aggregation and thus sedimentation or in contrast enhanced dispersion may occur .

For some nanomaterials (i.e. quantum dots) the transfer across environmental species was demonstrated indicating a potential for bioaccumulation in the species at the end of that part of the food chain. The main issues may be summarised as the development of suitable methods to assess the distribution of nanomaterials in the environment and the lack of portable monitoring equipment to measure levels of nanomaterials in different environmental media.

In addition, for many manufactured nanomaterials the methods currently used (carbon dioxide production, integration into biomass) for determining biological degradation will not be applicable.

Environmental hazard

Ecotoxic effects on environmental species have been demonstrated; aquatic species have been most studied. One of the major problems in ecotoxicological fate and effects testing is the absence of consistent and broadly-applicable information on how nanomaterials are to be suspended in various exposure media used in testing. Exposure media, mixing of materials with the media and consideration of realistic exposures, need a particular focus of attention. In this context, the characterisation of the nanomaterials in the eco(toxico)logical studies is important. Mixing of nanomaterials with sediments/soils, as well as characterisation over time, are areas which are still at a very early stage of development. In addition, there is the problem of the presence of background levels of nanomaterials and how to distinguish them from the nanomaterials being tested.

The common endpoints used in ecotoxicology such as mortality, growth, feeding, and reproduction can also be used for the evaluation of ecotoxicity by nanomaterials. In addition, some biomarkers similar to those used in the assessment of mammalian toxicity, such as oxidative stress, genetic damage and gene expression, may provide some insight in toxic mechanisms of nanomaterials.

The main issues for environmental hazard assessment may be summarised as the need for validation of laboratory test systems for characterising the effects of nanomaterials and the need for studies of the impacts of specific nanomaterials on ecosystems.

Risk assessment

Health and environmental hazards have been demonstrated for a variety of manufactured nanomaterials. The identified hazards indicate potential toxic effects of nanomaterials for man and the environment. However, it should be noted that not all nanomaterials induce toxic effects. Some manufactured nanomaterials have already been in use for a long time (e.g. carbon black, TiO2) showing low toxicity. Therefore, the hypothesis that smaller means more reactive, and thus more toxic, cannot be substantiated by the published data. In this respect nanomaterials are similar to normal chemicals/substances in that some may be toxic and some may not. As there is not yet a generally applicable paradigm for nanomaterial hazard identification, a case-by-case approach for the risk assessment of nanomaterials is still recommended.