Exposure and Hazard
8.3 Potential Hazard
8.3.1 Distribution: Target Organs
The diffusion rate of particulate materials through the GI mucus is dependent on a number of factors, predominantly size, charge30and surface coating.31The translocation from the GI tract has been found to be greater for ENPs than the larger particles.32,33ENPs in the smaller nanometer range have been found to cross the mucus layer faster than the larger ones.34Again, the translocation of smaller ENPs has been shown to be comparatively greater than the ENPs in the larger nanometer range.35 Within the GI tract, the rate uptake of ENPs has been reported to be between 2 to 200 times greater in the Peyer’s patches compared with the enterocytes.33This is despite the fact that Peyer’s patches represent only around 1% of the total intestinal surface area.
Following oral administration, the translocation and distribution of certain ENPs to different organs and tissues has been reported. Oral administration of colloidal gold ENPs (58, 28, 10 and 4 nm) to mice resulted in an increasing distribution of smaller ENPs to different organs.35 Jani et al.36 administered 12.5 mg kg 1of titanium dioxide (TiO2, rutile, 500 nm), in daily oral gavage for
10 days to female Sprague Dawley rats, and assessed their translocation to target organs and tissues such as Peyer’s patches, small intestine, colon, mesentery network and nodes, peritoneal tissue, liver, spleen, heart and kidney. The authors have demonstrated that TiO2particles were translocated to sys-
temic organs, such as the liver and spleen. The ENPs were also detected in the lungs and peritoneal tissues, but not in the heart and kidney. Similarly, accu- mulation of silver (60 nm) was observed in the stomach, followed by kidney and liver, lungs, testes, brain and blood.37The highest amount of intraperitoneally administered 13 nm colloidal gold beads was observed in liver and spleen.35
Fullerene (C60) appears to pass through the placental barrier as shown after
intraperitoneal administration of C60 fullerenes, solubilised with polyvinyl 127 Engineered Nanoparticles and Food: An assessment of Exposure and Hazard
pyrrolidone (50 mg kg 1; day 18 of gestation). The C60fullerenes were shown to
be distributed throughout the embryo, and the yolk sac was impaired 18 hours after injection.38 Another study, however, provides contradictory evidence. Gold ENPs, injected intravenously (2 and 40 nm) or intraperitoneally (40 nm), do not seem to penetrate the placental barrier.39
The conventional form of silica (SiO2) is a permitted food additive (E551),
which is used as an anti-caking and anti-foaming agent, and for clearing beers and wines. It is also used in coatings in food packaging. In an in vitro study on human epithelial cell cultures using fluorescence-labelled silica (SiO2) nano-
particles, Chen and Mikecz40 have shown that particles smaller than 70 nm could enter cell nuclei. The study also found protein accumulation in the nuclei and indication for impairment of DNA replication and transcription. It is, however, not known whether the intake of nano-silica through the GI route can lead to similar harmful effects in vivo.
8.3.2
Toxicity
Understanding the potential harmful and/or toxic effects of ENPs is complex. This is because the ability of ENPs to penetrate cellular barriers adds a new dimension to particulate toxicology. Thus ENPs can potentially reach new targets in the body, which are normally protected against the entry of larger particulates.
Current methods of toxicity testing rely on a range of in vitro and in vivo methods that have been developed for assessing conventional chemicals and consumer products. Whilst comparisons can be drawn from these testing regimes, many ENPs have distinctly different physicochemical properties, behaviour and interactions, which make measuring their potential toxic effects more problematic. As a consequence, it is difficult, if not impossible, to predict the effects and impacts of ENPs by extrapolating the existing knowledge on risks for larger sized particles having the same chemical composition. A major stumbling block in this regard is the lack of ability to establish the physico- chemical nature and the dose of a nanomaterial to which the cells are actually exposed. One of the fundamental principles of pharmacology is that chemical activity (response) is proportional to the concentration of the affecter substance at the site of action. Unlike conventional soluble chemicals, ENPs are rarely in a homogeneous form and can aggregate or diffuse according to differences in their density, size and surface chemistry, all of which can change over time during an exposure. Consequently, there is a need to develop robust dose metrics that give a better understanding of how ENPs and different biological parameters affect both in vitro and in vivo cellular dose and corresponding toxicity profiles.
Many studies have been published which demonstrate the potential cyto- toxicity of ENPs using in vitro cell models. Despite these reports, there are still no standardised or validated methods established for nanotoxicity testing. This has led to the publication of conflicting and confusing data and is hindering the
development of ENP risk assessment strategies. Measuring the in vitro dose of ENPs both at the cell surface and within cells presents analytical challenges that are hard to address with a single technology platform. Nanoscale imaging methods can measure ENP localisation and agglomeration state with varying levels of resolution, but lack the ability to quantify cell dose at the macro scale. Analytical mass spectrometry methods such as ICP-MS, LC-MS and stable isotope tracing have greater sensitivity and can quantify dose but lack infor- mation on ENP characteristics. A combinatorial approach would precipitate a more complete understanding of the factors, which influence nanoparticle dosimetry and allow for improved in vitro dose–response assessments.
Of the most commonly used measures of ENP cytotoxicity developed for chemical and pharmaceutical testing, only one method, the neutral red uptake (NRU) assay, is actually validated for testing chemicals under the REACH directive. Most of these ‘classic’ cytotoxicity and stress response assays rely on colorimetric or fluorescent outputs, which may be impacted by ENPs through absorbance of assay reagent, scattering light or quenching of the fluorescent signals. The suitability of such assays needs to be evaluated and standardised methods and protocols developed. This may, however, be further complicated by the properties of the culture system. A few studies have attempted to measure the impact of these changes. Despite such shortcomings, the in vitro assay systems still represent the best methods for high throughput analysis of ENP cytotoxicity, and an understanding the limitations will allow development of better methods. This has been recognised in a number of international reports by the UK’s Nanotechnologies Research Coordination Group, NRCG, and recently at a European Union workshop on Research Projects on the Safety of Nanomaterials: Reviewing the Knowledge Gaps (2008), which recognised the specific knowledge gaps in the field of nanotoxicology and called for urgent action to be taken to develop validated test methods for nanoparticle toxicity testing; rapid, user friendly, in vitro toxicity screens, and guidelines for toxicity testing.
Whilst there is a growing literature on the inhalation toxicity of ENPs, only a limited number of studies have so far been published on the toxicity of ENPs ingested through the oral route. In an oral toxicity study, Zhang et al.41 fed selenite or nano elemental selenium to healthy mice at 2, 4 and 6 mg kg 1bw for 12 days and found that, over a short-term, a high dose of selenite caused more pronounced oxidative stress, greater liver injury and prominent retardation of growth as compared to nano-selenium. Wang et al.42 administered an equal mass dose of 5 g kg 1bw of nano- and micro-sized zinc to groups of healthy mice. From the results of their experiments, the authors concluded that severe renal damage could occur in the nano-zinc treated mice, although no significant change of blood biochemical levels occurred. Nano-zinc powder could also cause severe anaemia. Wang et al.43 evaluated the toxicity of ultrafine TiO2
(155 nm) and nano-TiO2 (25 and 80 nm) using a single dose of 5 g kg 1 bw
delivered by gavage. The authors demonstrated that TiO2 particles induced
significant lesions of liver and kidneys in female mice. TiO2 particles were
mainly retained in liver, kidneys, spleen and lung. The hepatic injury and renal 129 Engineered Nanoparticles and Food: An assessment of Exposure and Hazard
lesions were observed in the histopathological examination. Wang et al.44 evaluated the toxicity of 20 and 120 nm ZnO particles, which indicates that 20 and 120 nm ZnO, similar to the normal ZnO compound, are relatively non- toxic for mice according to GHS classification criteria. Combined with the results of zinc accumulation, pathological examination and the biological indicators assays, the target organs for 20 and 120 nm ZnO acute oral administration were demonstrated as liver, heart, spleen, pancreas and bone. The biochemical and pathological investigation showed that the toxic effects between the 20 and 120 nm ZnO particles were little different. For example, the blood viscosity could be induced by low and median dose of 20 nm ZnO, but only at a high dose of the ultrafine ZnO after oral administration. The oedema and degeneration of hepatocytes, and inflammation of pancreas could be observed in most of the 20 nm ZnO-treated mice. The 120 nm ZnO-treated mice were found having a dose–effect dependent pathological damage in gastric, liver, heart and spleen, although the 20 nm ZnO-treated mice showed less liver, spleen and pancreas damage with the increase of treated dose. Therefore, more attention needs to be paid to the potential toxicity induced by low oral doses of small-sized ZnO (e.g. 20 nm).
Silver nanoparticles (60 nm) were administered orally to groups of male and female Sprague-Dawley rats repeatedly for 28 days following the OECD test guideline 407 according to three different dosing regimens: low dose (30 mg kg 1), middle dose (300 mg kg 1) and high dose (1000 mg kg 1).37This resulted in some significant dose-dependent changes in the alkaline phosphatase and cholesterol values in either the male or female rats indicating that exposure to over more than 300 mg of silver nanoparticles may result in slight liver damage.
8.4
Discussion
In this chapter we have presented an overview of the state-of-the-art issues in regard to nanomaterials and food. Since there are currently a very few estab- lished methods for detection and characterisation of ENPs in the food matrix, there is little available information on translocation from the GI tract to other organs. The available information is also very sparse in relation to dose– response data following the oral exposure. The current knowledge gaps will need studies on the following lines.
1. Analytical tools for characterisation of ENP in bulk materials and food matrices. There is a need to select appropriate reference ENPs in con- sideration of their likely use in food, and determine physicochemical properties, interactions, behaviour and fate in food and in the GI tract. A number of analytical methods are being developed in different research projects but they will need validating and adapting for detecting ENPs in complex food matrices as part of the routine safety testing procedures. 2. Dosimetry studies, with radio-labelled ENPs are needed. These studies
will contribute relevant data regarding the translocation of ENPs,
following single or repeated oral administration, in different target organs such as liver and kidneys.
3. Dose–response studies for a range of realistic doses reflecting consumer exposure delivered by single or repeated oral administration to healthy animals to demonstrate the health effects on different target organs (e.g. lung, brain, liver, heart/circulation, etc.) and the mechanisms of action. In view of the current uncertainties over the potential toxicity hazard and exposure of ENP, it is important that these studies are carried out over prolonged exposures, and are followed by histopathological investiga- tions on multiple organs. Other important endpoints in respect to risk assessment of ENPs include mutagenicity, carcinogenicity and repro- ductive toxicity. In vitro tests can provide a preliminary indication of the inflammatory and mutagenicity potential of the tested ENP, and if necessary, these can be followed by in vivo studies.
In this chapter we have also given a short overall description of the issues regarding the use of ENPs in food and food additives, and the available tox- icological studies in orally exposed animals. A conceptual categorisation of different ENPs has also been presented (Figure 8.1), based on the factors that are likely to contribute to the overall risk potential of different ENPs. However, there may be other yet-unknown factors that might influence the properties and effects of ENPs. The data generated to date are sparse, and indicate major data gaps for a rigorous risk assessment to be made. The ongoing and future studies, on the lines suggested above, are likely to generate relevant data that will enable adequate risk assessment of the oral exposure to nanoparticles through the consumption of food and drinks.
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