Steffen Foss
Hansen
*a,
Kåre Nolde
Nielsen
b,
Nina
Knudsen
c,
Khara D.
Grieger
d and
Anders
Baun
ae
aDepartment of Environmental Engineering, Technical University of Denmark, DTU Building 113, Kgs. Lyngby, DK-2800. E-mail: sfh@env.dtu.dk; Tel: +45 45251593
bGenØk, Centre for Biosafety, Breivika, 9294 Tromsø, Norway. E-mail: kare.nolde.nielsen@genok.no; Tel: +47 77645478
cLEO Pharma A/S, Industriparken 55, 2730 Ballerup, Denmark. E-mail: nina.knudsen@leo-pharma.com; Tel: +45 7226 2680
dRTI International 3040 E. Cornwallis Rd. Research Triangle Park, North Carolina 27709, USA. E-mail: kgrieger@rti.org; Tel: +1 919 541-7243
eDepartment of Environmental Engineering, Technical University of Denmark, DTU Building 113, Kgs. Lyngby, DK-2800. E-mail: anb@env.dtu.dk; Tel: +45 45251567
First published on 23rd November 2012
In 2001 the European Environment Agency (EEA) published a report that analyzed 14 cases of technological developments that later on turned out to have negative side-effects and they identified 12 “late lessons” for current and future policy-makers to bear in mind when initiating new technological endeavors. This paper explores how the first lesson – “Acknowledge and respond to ignorance, uncertainty and risk in technology appraisal” could be applied to screen nanomaterials. In cases of ignorance, uncertainty and risk, the EEA recommends paying particular attention to important warning signs such as novelty, persistency, whether materials are readily dispersed in the environment, and whether they bioaccumulate or lead to potentially irreversible action. Through an analysis of these criteria using five well-known nanomaterials (titanium dioxide, carbon nanotubes, liposomes, poly(lactic-co-glycolic acid) and nanoscale zero-valent iron), it was found that only nanoTiO2 fulfils all the five criteria. Depending on the length of the nanotubes, carbon nanotubes fulfil 3 or 4 criteria whereas liposomes, poly(lactic-co-glycolic acid), nanoscale zero-valent iron fulfil only one criteria. Finally, we discuss how these warning signs can be used by different stakeholders such as nanomaterial researchers and developers, companies and regulators to design benign nanomaterials, communicate what is known about nano-risks and decide on whether to implement precautionary regulatory measures.
Environmental impactThere is an urgent need for a set of simple and robust warning signs for evaluating the risks of nanomaterials without complete information to inform risk governance efforts. In 2001 the European Environment Agency (EEA) published a report, Late Lessons from Early Warnings: The Precautionary Principle 1896–2000, which explored 14 case studies, all of which demonstrated how not heeding early warnings had led to a failure to protect human health and the environment (EEA 2001). The EEA recommended paying particular attention to important warning signs such as novelty, persistency, whether the material is readily dispersed, whether it bioaccumulates and whether its use leads to potentially irreversible action. In this article we explore how the EEA warnings signs may be used to screen nanomaterials for human health and environmental risks and we use liposomes, poly(lactic-co-glycolic acid) (PLGA), nanoscale zero-valent iron (nZVI), titanium dioxide (TiO2), and carbon nanotubes (CNT) as illustrative examples. Based on our analysis, we conclude that the five warning signs can easily be used to screen nanomaterials for harmful properties and we furthermore show that these warning signs can be used by different stakeholders to design benign nanomaterials, communicate what is known about risks related to nanomaterials and decide on whether to implement precautionary regulatory measures. |
In 2001 the European Environment Agency (EEA) published a report, Late Lessons from Early Warnings: The Precautionary Principle 1896–2000, which explored 14 case studies, all of which demonstrated how not heeding early warnings had led to a failure to protect human health and the environment.2 Covering topics as diverse as asbestos, chlorofluorocarbons, non-ionizing radiation and ‘mad cow disease’, the EEA report examined the delay between the emergence of scientific evidence of harm and the action being taken to reduce risks in each case. To avoid repeating past mistakes as new technologies are developed, the expert group identified 12 “late lessons”. This paper explores how the first lesson – “Acknowledge and respond to ignorance, uncertainty and risk in technology appraisal” could be applied in the emerging technological field of nanotechnology. In cases of ignorance, the EEA recommends being proactive, alert and humble about the state of the scientific evidence indicating harm as well as paying particular attention to important warning signs such as novelty, persistency, whether the material is readily dispersed, whether it bioaccumulates and whether its use leads to potentially irreversible action.
The potential risks of especially nanomaterials are as diverse as the field of nanotechnology, and it has yet to be determined as to which inherent properties of nanomaterials determine their hazard potential and fate and behavior in the environment. Current risk assessment procedures for chemicals require substantial information about physico-chemical properties that we know are not applicable or meaningful for nanomaterials. Furthermore, the generation of hazard and exposure information based on guidelines and standard for chemicals has repetitively been identified as being cumbersome and difficult.4–7
There is an urgent need for a set of simple and robust warning signs for evaluating the risks of nanomaterials without complete information to inform risk governance efforts including, among other things, efforts to assess hazards, exposure and design regulations and monitoring and prioritization of research efforts.
In this article we explore how the EEA warnings signs may be used to screen nanomaterials for human health and environmental risks and we use liposomes, poly(lactic-co-glycolic acid) (PLGA), nanoscale zero-valent iron (nZVI), titanium dioxide (TiO2), and carbon nanotubes (CNT) as illustrative examples. These nanomaterials were chosen in order to capture the diversity of nanomaterials and challenge the applicability of the five warning signs suggested by the EEA. For each warning sign we will first discuss how it can be understood and operationalized as limited guidance is provided in this regard in the original publication by the EEA and the process by which one can determine whether a given nanomaterial fulfils the warning sign or not. We then describe how the warning sign applies to one illustrative nanomaterial in depth and to the four other nanomaterials in less detail. Finally, we will discuss how these warning signs can be used by different stakeholders such as researchers, developers, companies and regulators to design benign nanomaterials, communicate what is known about nanorisks and decide on whether and how to implement precautionary regulatory measures.
In the case of halocarbons and PCBs and methyl tert-butyl ether, “novelty” lies in the fact that huge amounts of these chemicals were suddenly used for new applications (spray, refrigerants, PVC plastics, paints, adhesives, lubricants, anti-knocking agent, etc.) and subsequently released into the atmosphere and the environment.2
The EEA does not define the term “novelty”; however this is of key importance in order to determine whether an application of nanotechnology or a given nanomaterial is indeed novel. Without specifying how the term “novelty” should be defined, many definitions of nanotechnology require either “novel applications” and/or that nanomaterials exhibit “novel” properties compared to bulk materials.8–10 No single exhaustive taxonomy exists for novel materials and as noted by UK Royal Commission on Environmental Pollution4 it is unlikely that one is possible or even necessarily desirable. Nevertheless, the UK Royal Commission on Environmental Pollution4 distinguished between four types of novel materials:
1. new materials hitherto unused or rarely used on an industrial scale;
2. new forms of existing materials with characteristics that differ significantly from familiar or naturally occurring forms e.g. silver and gold;
3. new applications for existing materials or existing technological products formulated in a new way e.g. cerium oxide used as a fuel additive;
4. new pathways and destinations for familiar materials that may enter the environment in forms different from their manufacture and envisaged use.4
In the following we intend to categorize five exemplary materials according to these four overall criteria of novelty.
Liposomes can be used to change the physicochemical properties of active pharmaceutical drugs, either by encapsulation of a hydrophilic drug into the aqueous core or by incorporation of a lipophilic drug into the lipid bilayer. Encapsulation or incorporation protects the drug from degradation and can increase the half-life of the drug in the organism.13 Additional liposomes can control drug delivery to a specific area of the body, and in this way increase the therapeutic index of the drug.13 A lot of focus has also been in the development of vaccines using liposomes, since liposomes can be used to induce adjuvant effects.14 Epaxal, a vaccine for hepatitis A, was the first commercially available liposomal drug product to be approved in the 1990's. Today several drug loaded nano-sized liposomes for intravenous administration are on the market, e.g. AmBisome for treatment of fungal infection, where amphotericin B is formulated in 100 nm conventional liposomes. Another available product is Doxil for treatment of cancer, where doxorubicin citrate is formulated in 100 nm liposomes modified with polymer coating. The liposomes are associated with the cells by endocytosis. During endocytosis the liposomes enter the endosomes, where the liposomes are cleaved enzymatically, the phospholipids are hydrolysed and the drug is released into the cytoplasm of the cell.15 Another widespread application of nano-sized liposomes is in topically applied creams and ointments, where liposomes are utilized to deliver active ingredients into the skin, and increase hydration of the skin. Liposomes have been explored for delivery of calcipotriol into barrier impaired skin for treatment of skin diseases.16 Although, it has been reported that several cosmetic creams and gels containing liposomes are commercially available, it is unclear how widespread use of liposomes actually is, as claims regarding the presence of intact liposomes in the final product are difficult to validate.17
A good example of a “new form of existing materials with characteristics that differ significantly from familiar or naturally occurring forms” (RCEP category 2) is the use of nanoscale zero-valent iron (nZVI) for in situ remediation, which has gained increased attention in recent years as a promising environmentally beneficial nanotechnology. For nZVI the nanoscale size provides significantly increased surface area, leading to higher reactivity per mass unit compared to larger iron particles. This has been found to subsequently enhance contaminant degradation reactions. Furthermore, the nano-sized particles can be prepared as suspensions to be pumped directly into contamination plumes in groundwater aquifers allowing for in situ remediation without the often very costly excavations needed for traditional remediation with zero-valent iron. Today nZVI has been applied for in situ soil and groundwater remediation at contaminated sites in order to degrade a range of contaminants (e.g. PCBs, chlorinated organic solvents, organochlorine pesticides).21 Scientists and engineers are increasingly interested in developing various coatings of nZVI in order to better control its reactivity and migration to the environment as discussed further in subsequent sections.
TiO2 nanoparticles are a good example of a nanomaterial that would fall into the third RCEP category of novel categories, as the use of TiO2 is nothing new whereas new applications are enabled by formulating TiO2 in the nanoscale. TiO2 nanomaterials have the ability to scatter electro-magnetic radiation, which can be manipulated with by changing size, surface treatment and lattice doping. A wide range of applications already exists for TiO2 nanoparticles e.g. nanoscale TiO2 is widely used in sunscreens and cosmetics due to the UV-absorption of the material. It is also used in paints and for water treatment as a photo-catalyst producing reactive oxygen species that degrades organic contaminants. Other uses include ointments, toothpaste, catalysts, semi-conductors, etc.22,23
Finally, biodegradable polymeric particles such as poly(lactide-co-glycolides) (PLGA) could be categorized as “(4) new pathways and destinations for familiar materials that may enter the environment in forms different from their manufacture and envisaged use”4 since polymer particles can be engineered into the nanoscale to protect them from premature degradation in vivo, have a specific drug-release rate, or target specific tissues or cells (e.g. specific immunological cell types).24–27 PLGA has been used in clinical applications at least since 1975,28 the use of the material in itself is not particularly novel. However, a major potential advantage of using PLGA nanoparticles (as compared to microparticles) in drug delivery is that their small size enables them to transgress biological membranes (e.g. epithelial lining and cell membranes), allowing for (passive or active) distribution to desired tissues and cell types.25 Recent immunological research indicates that nanoparticles, depending on their size, may target different immunologically significant cell types that are regarded as important in the design of improved vaccines (i.e. dendritic cells),29 and that PLGA nanoparticles – but not microparticles – allow for the targeting of such cell types.30
However, since most nanomaterials are inorganic, degradation based criteria for persistency is in most cases not relevant. On the one hand inorganic nanomaterials can be claimed to be persistent per se since the elements cannot be degraded. In this way all inorganic nanomaterials would be classified as persistent; however attention should be given to the fact that the definition of persistency also involves “chemical transformations”. A number of inorganic nanomaterials may be reactive and be transformed to other materials or other forms of the same element depending on the redox conditions and ionic strength of the environmental media into which they are released or where they end up (e.g. uncoated nZVI will be transformed to iron-oxides under aerobic conditions, nano-silver may dissociate to silver-ions in freshwater). These “new” forms may or may not be nanoscale, due to processes like dissolution, oxidation/reduction, agglomeration and aggregation. Thus, only for non-reactive inorganic nanomaterials should applying the term “persistent” be considered whereas reactive inorganic nanomaterials that undergo transformation in the environment or in the human body should not be called persistent. For trace organic contaminants the term “pseudo-persistency” has been introduced to describe the occurrence of ready biodegradable compounds that, because of a continuous (low) discharge, remain in the environment.33 It may be tempting to describe “pseudo-persistency” as an early warning sign for a number of nanomaterials, since these are expected to be emitted continuously in low concentrations as a result of leaching from consumer products. This term is, however, not suited for this purpose in our view as “pseudo-persistency” depends more on the exposure scenario than on the inherent properties of the material. For nanomaterials the exposure scenarios are at present very uncertain and there is a risk of generating too many false positives if “pseudo-persistency” were to be introduced as a generally applicable early warning sign for nanomaterials.34
Little research seems to have been conducted to establish degradation rates for PLGA in the environment under realistic environmental conditions, including at temperatures below 5 °C. Dunne et al.44 reported no polymer mass loss, but a loss in shape or coalescence of PLGA micro-/nanoparticles was observed after 175 days at 5 °C indicating that PLGA may degrade much less rapidly in low temperature aquatic environments (e.g. as compared to in mammalian bodies or at equivalent temperatures). While the hydrolysis of PLGA may be rather slow in cold water, it nevertheless seems likely that PLGA nanoparticles in an aquatic environment should not be termed as persistent.
Liposomes and PLGA represent examples of nanotechnology designs that are not persistent and for which increased stability (or persistency) is what developers are trying to achieve in order to increase longer circulation time in the body and/or shelf life of e.g. a pharmaceutical or a cosmetic product. Liposomes mainly consist of biodegradable phospholipids, but different components might be added to adjust the properties of the bilayer such as biodegradable polymer, ethanol, surfactants or cholesterol. One way to increase the stability of liposomes is to coat them with hydrophilic lipopolymers, and long chains of polyethylene glycol (PEG) are commonly used to increase in vivo and in vitro stability.14,48In vitro PEG chains introduce a sterical barrier between adjacent liposomal lipid bilayers which prevent fusion and aggregation upon storage.49In vivo PEG chains reduce the interaction with plasma proteins in the blood, thereby increasing the circulation of liposomes in the blood stream after intravenous (i.v.) administration.50,51
CNTs are often said to be among the least biodegradable man-made materials known.4 Only one paper refers to the possible degradation of SWCNTs via enzymatic catalysis.52 After incubation of SWCNTs with a natural horseradish peroxidase (HRP) and low concentrations of H2O2 (40 μM) at 4 °C over 12 weeks under static conditions Allen et al.52 found indications of degradation of the nanotube structure. These results lead to the suggestion that plant peroxidases may have a role in CNT degradation along with material type and physico-chemical conditions. CNTs can be coated and functionalized and although the CNTs might not be degradable, the coating and functionalization might be so, which again may alter the degradability behavior significantly.6 In a study on Daphnia magna Roberts et al.53 showed that water-soluble, lysophosphatidylcholine-coated SWCNTs underwent biological modification upon ingestion as the Daphnia stripped off the lysophosphatidylcholine from the particle surface thereby decreasing the solubility of the SWCNTs. In the presence of Daphnia, initial concentrations of 2.5–20 mg L−1 were decreased by around 50% whereas SWCNTs remained in solution without the D. magna. This finding may be of importance for the evaluation of the fate of modified CNTs after release into the environment.6
Whether a given nanomaterial is readily dispersed depends on a number of factors such as distribution in the air, water, and soil including issues of water soluble versus insoluble materials. Soluble materials generally move through aqueous environments, whereas insoluble particles have different transport mechanisms and may be more easily retained in soil or sediments. In this regard it is important to notice that one of the greatest opportunities of nanotechnology is that it enables engineering of increased solubility through manipulation of surface chemistry and surface charge of nanomaterials.54 Under laboratory conditions it has been found that hydrophobic nanoparticles such as C60, CNTs, pure nanometals and nanometal oxides aggregate rapidly in water55,56 as a result of electric double layer compression.57 This indicates that these will precipitate out of the air or water column and end up in the soil or the sediment and hence they would not be readily dispersed. Knowledge to date indicates that in many cases, rather than remaining intact, nanomaterials will tend to aggregate, agglomerate or become associated with other dissolved, colloidal or particulate matter present in the environment.58
Due to these limited migration distances, nZVI has been intentionally engineered to have different applied coatings which would enable increased migration distances in order to have better contact with the environmental contaminants. From the perspective of successfully remediating contaminated soil and groundwater, it is advantageous to ensure better contact between the nZVI nanoparticles and the contaminants in order to allow for sufficient degradation processes to occur. In some cases, where coated or ‘stabilized’ nZVI has been used, it has been estimated that nZVI may migrate up to tens or hundreds of meters in certain environments (i.e. consolidated sandy aquifers),59 and possibly remain mobile up to 8 months under some hydrogeological conditions.47 It should be stated, however, that these studies have been conducted under laboratory settings as opposed to field-scale conditions, which have natural heterogeneities, which would also likely affect migration and dispersivity in the environment.21
In addition to the use of engineered coatings, natural coatings has to be considered as well.63 However there are no studies, which have investigated this for nZVI thus far.
The factors affecting the stability of MWCNTs in natural waters are similar to those identified for C60 (ref. 64 and 65) and in particular, NOM (e.g. humic acids) in water can stabilize MWCNT dispersions for over 1 month, but the NOM adsorption efficiency is dependent on the aromatic hydrocarbon content.66 Finally, absorption of other materials present within water bodies (such as bacteria) has also been shown to affect solubility of CNTs. For instance Roberts et al.53 have found that daphnids decreased the solubility of water-soluble, lysophosphatidylcholine-coated SWCNT upon ingestion by stripping off the lysophosphatidylcholine from the particle surface. With regard to soil transport, Lecoanet and Wiesner67 have observed that SWCNTs display fairly high mobility compared to other nanomaterials such as functionalized C60 and nanometals as they migrated about 10 m in unfractured sand aquifers. Jaisi et al.68 found carboxyl-modified SWCNT deposition to be correlated with ionic strength or the addition of calcium ions when studying SWCNT transport in quartz.
Only one study has so far reported quantitative information on transport of nanomaterials in the environment, tracing leached synthetic TiO2 nanoparticles from paint on house facades into receiving water bodies.69 Kaegi et al.69 compared the leaching of TiO2 nanoparticles from a newly painted model facade and a real facade. Leaching from a newly painted model facade resulted in a titanium concentration of 600 μg L−1 with 90% of the nanoparticles with a size range between 20 and 300 nm. In contrast, the real facade exposed for two years to weather resulted in a leaching of 10 μg L−1 for titanium, while the urban run-off contained 8 μg L−1. The size of particles in the real facade was around 20–300 nm for 90% of the particles, while in urban run-off 50% of particles are <300 nm. As with CNTs, absorption of other materials present within water bodies (such as bacteria) has also been shown to affect transport of metal oxide nanoparticles.70 Clearance of metal oxide nanoparticle within aquatic systems is also significantly altered by surface charge and addition of dispersion stabilizing surfactant.70 TiO2 transport was investigated by Lecoanet and Wieser67 who found that transport is a function of Darcy velocity, with 55% and 77% of the particles being transported through the porous media under low and high flow velocities, respectively.
Although liposomes might affect the dispersibility of a given drug, the likelihood of being readily dispersed is low as the liposomes can degrade before they are excreted from or washed off the human body or a short while after they enter the environment. Little is known about dispersibility of liposomes in cosmetics, but it is not likely that the liposomes enter the environment as intact nanosized vesicles as surfactants during wash will disturb the hydrophobic interactions.
Little is known about the environmental fate and behavior of PLGA nanoparticles. These issues become particularly pertinent for potential applications of PLGA nanoparticles in agriculture71,72 or aquaculture73 for which large-scale usage would be conceivable. The dispersibility of PLGA nanoparticles depends on issues like their tendency to aggregate in aquatic environments,74 issues of which little or no information is currently available.73
The bioconcentration factor for benthic and terrestrial invertebrates is expressed as biota-to-soil/sediment accumulation factor (BSAF). As a general rule BSAF values of 0.5 and higher are interpreted as an indication of high bioaccumulation. Lower BSAF values should however not be used to conclude that a substance has a low bioaccumulation potential as a low uptake from sediment or soil does not imply a low aquatic BCF value.32
Whether or not a given nanomaterial will bioaccumulate will depend on properties such as environmental distribution (e.g. solubility in lipid or water) and degradability. For instance, carbon nanotubes are known to be non-biodegradable, insoluble in water and lipophilic (i.e. preference for entering fatty cell membranes) which indicates that carbon nanotubes are likely to bioaccumulate.4
Petersen et al. (2008) observed the biota-sediment accumulation factors (BSAFs) for worms (Lumbriculus variegatus) to be almost an order of magnitude lower for SWCNTs and MWCNTs compared to pyrene, which is known to be accumulative.77 No indications of any systematic differences were observed between the SWCNTs and MWCNTs. When decreasing the organic carbon content by a factor of 8 a decrease in the BSAF value from 0.51 ± 0.09 to 0.035 ± 0.015 was observed after 14 days of exposure. Petersen et al.78 observed that bioaccumulation factors (BAF) of the SWCNTs (99 ± 1% C) and MWCNTs (91.1 ± 0.2% C) by Eisenia foetida were almost 2 orders of magnitude lower than those for pyrene, but a soil dependent difference was observed between SWCNTs and MWCNTs. Bio-uptake of MWCNTs after 14 days was larger than that for SWCNTs in the Chelsea soil and in versa in Ypsilanti soil.
Even though the EEA in their publication “Late Lessons from Early: The Precautionary Principle 1896–2000” mainly focused on bioaccumulation in the environment, accumulation in the human body is relevant as well for a wide range of biomedical applications of nanoparticles and materials including liposomes. In this relation, accumulation is perceived as a positive. One major advantage of nanosized liposomes is their ability to encapsulate the active drug or incorporate the drug in the lipid bilayer to change its physicochemical properties.12
The challenge in this regard is to get drugs to accumulate at the injured site.79Via i.v. injection of drug-loaded nanosized liposomes a fast clearance by macrophages in obtained whereby drug accumulation is enabled in the liver and spleen.11 A decreased liposomal uptake by macrophages is promoted through stealth liposomes stabilized by polymer coating on the surface. A protective layer is formed over the liposomal surface, which increases circulation time in the bloodstream.51 Upon tumour growth or inflammation the venous become more permeate, and consequently nano-sized liposomes accumulate at injured sites. Targeting of liposomes to a site can be more specific by conjugation of ligands for specific receptors on the surface of liposomes. This increased delivery of drug into cells expressing certain receptors on the surface, and especially, cells important for cancer progression are extensively studied for a specific delivery using liposomes.80 Endocytosis is the main mechanism by which the liposomes enter into the endosomes of the cell. In the endosomes the liposomes are enzymatically cleaved, and the phospholipids are hydrolysed.
Since PLGA nanoparticles and liposomes degrade rather rapidly in mammals, it seems unlikely that uptake rates of PLGA and liposomes from the environment can exceed their rates of degradation.81 In low temperature environments, degradation rates in poikilothermic animals (e.g. fish) may in addition to the physical hydrolyzation process depend significantly on the immunological properties of the organism. While PLGA nanoparticles might degrade at a lower rate in low temperature environments, it appears unlikely that concentrations of bioavailable PLGA particles can build up to an extent to which bioaccumulation in poikilothermic organisms becomes available. However, this suggestion needs to be experimentally tested under different relevant environmental conditions.
Lam et al.86 were the first to report on the pulmonary toxicity of carbon nanotubes after having tested SWCNTs of different purities, i.e. raw and purified nanotubes. Lam et al.86 found that all the tested nanotubes induced dose-dependent granulomas and interstitial inflammation in mice. The results by Lam were supported by observations made by Warheit et al.87 who also observed pulmonary granulomas in rats after exposure to SWCNT soot (5% Ni and Co).
Although a granuloma could be everything from harmless nevus to malignant tumors, their mere presence is a matter of concern, as inflammation can be seen as a forerunner to lung fibrosis. The fact that purified nanotubes produced prominent granulomas in the study by Lam et al.86 indicated that nanotubes were the cause for the observed lesions and not the residue metals often present in batches of non-purified carbon nanotubes. This finding has since been confirmed by others.88,89
In a direct comparison between long and short MWCNTs, Muller et al.89 compared 0.7 μm ground and 5.9 μm unground MWCNTs with chrysotile asbestos and ultrafine carbon black. Muller et al.89 found that ground MWCNTs produced a similar inflammatory and fibrogenic response as chrysotile asbestos in rats by equal mass dose and a greater response than ultrafine carbon black.89
The finding that length of CNTs affect the biological activity has since then been supported by findings by Takagi et al.90 and Muller et al.91 Takagi et al.90 found that long >5 μm MWCNT) caused fibrotic peritoneal adhesions and peritoneal tumors after intraperitoneal injection whereas Poland et al.84 observed granulomatous lesions similar to those observed in crocidolite asbestos-treated animals. In contrast, Poland et al.84 and Muller et al.91 observed only acute inflammation when animals were exposed to short <1 μm MWCNTs by intraperitoneal injection.92 Finally, mesothelial tumors have also been reported in a susceptible strain of mice after intraperitoneal injection of 10–20 μm MWCNTs90 but not by short <1 μm MWCNTs.91,92
Most studies have used intratracheal or intraperitoneal administration. However, questions have been raised about the biological relevance of intratracheal and intraperitoneal instillation as it bypasses upper respiratory tract defenses and does not deposit particles evenly in the lung similar to what would be observed after inhalation.93 During the last couple of years, a number of nose-only inhalation studies have been published that support previous findings such as Ellinger-Ziegelbauer and Pauluhn,94 Ma-Hock et al.88 and Pauluhn.95 For instance, in a 90-day nose-only inhalation toxicity study in MWCNTs, Ma-Hock et al.88 observed an increased lung weight, pronounced multifocal granulomatous inflammation, diffuse histiocytic and neutrophilic inflammation and intra-alveolar lipoproteinosis in lung and lung-associated lymph nodes at 0.5 and 2.5 mg m3. The incidence and severity of granulomatous inflammation of the lung and the lung draining lymph nodes were concentration-dependent, which has previously been demonstrated for intratracheally instilled SWCNTs86 and MWCNTs.89 Exposure via inhalation also revealed inflammation in the nasal cavity, larynx, and trachea, where the particles deposited during the inhalation exposure as well as alveolar lipoproteinosis, which was not observed while using intratracheal or intraperitoneal administration.88
In regard to carcinogenicity, epidemiological evidence remains inconclusive, although one study by Boffetta et al.99have observed an elevation in lung cancer mortality among male TiO2 workers in the latter study when compared to the general population. However, there was no indication of an exposure–response relationship in that study. Heinrich et al.100 described a statistically significant increase observed in lung cancer in rats following chronic inhalation after repeated exposure. Based on this study the National Institute for Occupational Safety and Health (NIOSH) in the United States has determined that exposure to ultrafine TiO2 should be considered a potentially occupational carcinogen. NIOSH furthermore “concluded that TiO2 is not a direct-acting carcinogen, but acts through a secondary genotoxicity mechanism that is not specific to TiO2 but primarily related to particle size and surface area” and surface area was found to be the critical metric for occupational inhalation exposure to TiO2. Besides respiratory tract toxicity and carcinogenicity, a number of studies have connected nanoTiO2 to cardiovascular toxicity101 and neurotoxicity.102,103 Finally, 35 nm titanium dioxide nanoparticles have recently been found to cause smaller uteri and smaller fetuses after being injected intravenously into pregnant mice. Titanium dioxide nanoparticles were found in the placenta, fetal liver and fetal brain.104
The in situ use of nZVI for environmental remediation involves the direct injection of nZVI into the environment, and therefore definite environmental exposures occur through its intended use. The question of whether the environment around injection sites – and possibly other areas if nZVI migrates in the environment – may be returned to previous states before introduction of nZVI is not yet known. On one hand, if nZVI may be degraded or transformed through abiotic or biotic mechanisms including biological degradation then it is plausible that the introduction of nZVI into the environment may be essentially ‘reversed’ and the original state(s) of the environment may be returned. On the other hand, if degradation of nZVI is not possible or if the introduction of nZVI results in other unforeseen consequences leading to irreversible changes to the ecosystem, then irreversible consequences may follow nZVI introduction. Furthermore, it is plausible that these irreversible changes may be more problematic if nZVI was found to migrate widely in the environment. In addition to natural or biological degradation mechanisms, there may in theory be technical mechanisms to remove nZVI from the environment, although this possibility is not yet feasible.
Since liposomes can be made from naturally occurring substances it is often said that they are nontoxic, biodegradable and non-immunogenic.17 However a number of side effects have been observed upon i.v. injection of liposomes in both humans and pigs. In several cases i.v. injection of liposomes loaded with doxorubicin cause an immediate hypersensitivity reaction in the patients.105 Temporary symptoms include signs of cardiopulmonary distress (dyspnea, tachypnea, hypertension/hypotension); chest and back pain are noted to be rather common among sensitive individuals.106,107 Although an element of concern, these side effects are not classified as potentially irreversible harmful to humans and environment in general as many of the effects seem to be less severe compared to death, cancer, reproductive toxicity, etc. in addition to being temporary and limited to human patients undergoing medical treatment i.v. under doctor's advice.
As in the case of liposomes, PLGA has not been associated with adverse effects such carcinogenicity, reproductive toxicity, etc., and PLGA has been approved by the US Food and Drug Administration for drug delivery and other clinical applications.25 A number of cytotoxicity studies have been performed on PLGA and have observed no significant adverse effects on several non-carcinogen cell lines.108,109 In one of the few in vivo studies on PLGA, Semete et al.109 did detect translocation of rhodamine- and coumarin-labelled 200 and 350 nm PLGA particles in the brain, heart, kidney, liver, lungs and spleen of Balb/C mice 7 days after oral administration of concentrations between 1 and 100 mg mL−1. However no histopathological or tissue damage was observed even at the highest dose.109
Nanomaterial | EEA warning signs | ||||
---|---|---|---|---|---|
Novelty | Persistency | Readily dispersed in the environment | Bioaccumulation | Potentially irreversible action | |
a “Yes” for short CNTs, “no” for longer CNTs based on Poland et al. (2008). b “Yes” if the enhanced reactivity due to larger surface area is considered to be a “nano-related property”; “no” if compared to reaction processes for conventional ZVI particles. c “Yes” if the study by Dunne et al. (2000) carried out at non-standardized conditions (5 °C) is used; “no” if the Dunne et al. (2000) is rejected. | |||||
CNTs | Yes | Yes | No | Yes | Yes/noa |
TiO2 | Yes | Yes | Yes | Yes | Yes |
nZVI | Yes/nob | No | No | Unknown | Unknown |
Liposomes | Yes | No | No | No | No |
PLGA | Yes | Yes/noc | Unknown | No | No |
In the case of CNTs, it was evaluated that they can be categorized as novel, persistent and bioaccumulative. No CNTs seem to be readily dispersible in the environment at least in their non-functionalized form. There are indications in the literature that only short and entangled CNTs fulfil the warning signs of use leading to potentially irreversible action, whereas long CNTs might not110. This is indicated in Table 1 by using “yes/no” to indicate either/or. This apparently inconclusive outcome of the evaluation illustrates the important fact that individual kinds of nanomaterials within the same overall material type may have very different hazard profiles. Thus different outcomes can be anticipated when applying the five EEA warning sign indicators to the same nanomaterial. Keeping this in mind, the five warning signs can be used to do a comparative analysis between different types of the same nanomaterial (short CNTs versus long entangled CNTs) but also between various types of nanomaterials (e.g. CNTs, TiO2).
TiO2 was found to fulfil all five of the warning signs. The novelty of TiO2 is primarily related to the ability of nanoTiO2 to scatter electro-magnetic radiation, which can be manipulated by changing the size, surface treatment and lattice doping.22,23 TiO2 was also found to be persistent per se since it is an inorganic nanomaterial that will not be chemically or biologically transformed under normal environmental conditions or in organisms (including mammals). TiO2 nanoparticles will most likely agglomerate in the aquatic and terrestrial environment; however since part of the size distribution may still be <100 nm and agglomeration is a reversible process, the evaluation as “persistent” is retained. It was also found that TiO2 is “readily dispersible in the environment” as nanoforms of TiO2 are used in a range of products that are dispersive in nature (e.g., sunscreens, cosmetics, paints) and will lead to direct environmental exposure. However, it has been shown that TiO2 and other metal oxide nanoparticles are absorbed by e.g. bacteria present within water bodies, but it is currently not clear whether that increases or decreases the dispersibility of TiO2 over time and how it is affected by the surface charge and stabilizing surfactant of TiO2.70 Finally, it has been found that TiO2 nanoparticles can be transported through porous media.67
Significant bioaccumulation of TiO2 has been observed in carp and in regard to potentially irreversible harm, the nanoforms of TiO2 have consistently been observed to be more toxic than micro-TiO2 in regard to respiratory tract toxicity.6,111 Although epidemiological evidence on lung carcinogenicity remains inconclusive,99,111 the nanoforms of TiO2 have been associated with cardiovascular toxicity, neurotoxicity as well as reproductive toxicity.101,102,104
In contrast to CNT and TiO2, nZVI fulfilled only one of the five warning signs and even for this one (novelty) it may be questioned whether nano-sized iron particles are in fact novel since the nZVI particles are not reported to gain specific properties due to the nanoscale (besides the ease of injecting them in contamination plumes compared to larger particles). The transformation of nZVI to first larger particles through agglomeration and afterwards oxidation to iron particles is the reason why nZVI in this study are categorized as “not persistent”. It is at present unclear whether nZVI are bioaccumulative and there is no evidence that the use of nZVI could lead to potentially irreversible action. Even in the case that it would later turn out that it does, this would be local as there seems to be no evidence that nZVI are readily dispersive in the environment, at least when evaluating the uncoated nanoparticles.
Being novel was the only warning sign being raised in regard to liposomes and PLGA and considering that indications are that both of these nanomaterials are not persistent and degrade when used it follows that they are not bioaccumulative, are readily dispersive or might lead to potentially irreversible action. For PLGA there is one study44 that indicates that PLGA is persistent according to the ECHA's persistency criteria. However, the study was carried out at 5 °C, which is not in compliance with international guidelines and a slower degradation at lower temperatures is to be expected for PLGA as well as other substances.
Except for the final EEA warning sign on leading to potentially irreversible action, it is important to realize that a material or process is not inherently dangerous just because it triggers one specific warning sign e.g. novelty or persistency.
In the initial R & D process, nanomaterial researchers and developers can use the warning signs to screen their materials for adverse effects and use this to develop nanomaterials that are “safe by design” from the outset whereas companies that develop and produce nanomaterials can use the warning signs to communicate what they know about the nanospecific risks of the material. Independent parties and regulators can use the warning signs to validate claims about one or more nanomaterials being benign to humans and the environment even in the case of a lack of knowledge. Finally, regulators can use the warning signs to evaluate and prioritize research efforts as well as to trigger default regulatory actions such as bans, moratoriums, etc., depending on how many and which of the warning signs are fulfilled.
NM | Novelty | Persistency | Readily dispersed | Bioaccumulation | Potentially irreversible action |
---|---|---|---|---|---|
a Scatter electro-magnetic radiation. b Inorganic. c Disperse use, transported through porous media. d Significant bioaccumulation has been observed in carp, enhances the bioaccumulation of Cd and As. e Respiratory tract toxicant inconclusive evidence on lung carcinogenicity remains inconclusive, associated with cardiovascular toxicity, neurotoxicity and as well as reproductive toxicity. | |||||
TiO2 | Yesa | Yesb | Yesc | Yesd | Yese |
For liposomes in a medical product, the text box would look as follows (see Table 3).
NM | Novelty | Persistency | Readily dispersed | Bioaccumulation | Potentially irreversible action |
---|---|---|---|---|---|
a ∼100 nm liposomes more easily penetrate biological barriers and the cell membrane. b Not dispersed. Consist of biodegradable phospholipids and degrade in the human body. c Degrade before they are excreted. d Not bioaccumulative. In the endosomes of the cell, the liposomes are enzymatically cleaved, and the phospholipids are hydrolysed. e No potentially irreversible effect observed. Acute adverse allergic-type reactions have been reported to occur as well, within 5–10 min of liposome infusion. Reproducible physiological signs of shock and significant pulmonary hypertension have been observed in pigs after intravenous administration. | |||||
Liposomes | Yesa | Nob | Unknownc | Nod | Noe |
Res. prior. | NM | Novelty | Persistency | Readily dispersed | Bioaccumulation | Potentially irreversible action |
---|---|---|---|---|---|---|
1. | nZVI | Yes | Yes | No | Unknown | No |
2. | Liposomes | X | No | Unknown | No | No |
3. | PLGA | X | No | Unknown | No | No |
4. | CNTs | Yes | Yes | No | Yes | Yes/no |
5. | TiO2 | Yes | Yes | Yes | Yes | Yes |
It might be worth noting that some combinations of warning signs are more worrisome than others e.g. persistent and potentially irreversible action might be more worrisome that other combinations of two.
This application of the five warnings signs to guide research prioritize is similar to the approach suggested by Tervonen et al.113 using MultiCriteria Decision Analysis. Tervonen et al.113 set forward a number of criteria, both in terms of nanoparticle properties as well bioavailability, bioaccumulation and toxic potential. Quantitative criteria were either measured or based on expert judgments whereas qualitative criteria were established in terms of ordinal classes: 1 was the most favorable (least risk) value class, while 5 the least favorable (highest risk). Weight bounds were then assigned to the criteria by the authors e.g. toxic potential 0.3–0.5, bioavailability and bioaccumulation potentials 0.02–0.08 and the rest of the criteria were assigned weight bounds of 0.05–0.15. A cutting level within the range of 0.65–0.85 was then used to define the minimum sum of weights for the criteria that must be in concordance with the outranking relation to hold.
In regard to the application of the five EEA warning signs, we do not suggest attempting to assign a numeric score on qualitative criteria such as novelty, potentially irreversible harm, etc. In this case, we find that assigning a value of e.g. “2” to a given nanomaterials in regard to e.g. novelty lowers the overall level of transparency as there must be a large level of expert subjectivity when assigning quantitative scores between 1 and 5 to properties that are qualitative in nature.
NM | Novelty | Persistency | Readily dispersed | Bioaccumulation | Potentially irreversible action | Default regulatory action |
---|---|---|---|---|---|---|
TiO2 | Yes | Yes | Yes | Yes | Yes | Ban |
CNTs | Yes | Yes | No | Yes | Yes | Moratorium |
nZVI | Yes | Yes | No | Unknown | No | Controlled uses only |
Liposomes | Yes | No | Unknown | No | No | Invest in risk research |
PLGA | Yes | No | Unknown | No | No | Invest in risk research |
There is currently a substantial risk of “paralysis by analysis” in the field of risk assessment and management and regulation of nanomaterials115 and calls for more research are often made. It could seem somewhat too simplistic to base complex regulatory decision on such simple decision-making rules and principles that again would trigger the default implementation of precautionary measures. However, having such default decision-making rules and principles has a number of advantages. First of all, it would make it clear as to which criteria and principles are those on which the decisions to implement precautionary measures are based and it would also initiate discussions in cases where regulators decided to deviate from the default decision making processes and they would have to explain why they decided not to implement precautionary measures, something regulators hardly ever have to do currently. It would furthermore be possible to overcome the limitations of a case-by-case risk assessment of nanomaterials and expose any attempt by various stakeholders to delay the implementation of risk reducing measures.
This journal is © The Royal Society of Chemistry 2013 |