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According to the International Organization for Standardization’s (ISO) ISO 27687:2008 Standard, a nano-object is a material having one, two, or three external dimensions in the size-range from approximately 1-100 nanometers (nm). There are three categories of nano-objects: 1) nanoplates are nano-objects with one external nanoscale dimension; 2) nanofibers are nano-objects with two external nanoscale dimensions comprising nanotubes (hollow nanofibers) and nanorods (solid nanofibers); and 3) nanoparticles with all three external dimensions in the nanoscale range. When nano-objects are incorporated into a large substrate or matrix, they are called nanomaterials.

There have been some recent developments in nanotoxicology (the study of the toxicity of nano-objects and nanomaterials).
NIOSH Current Intelligence Bulletin: Occupational Exposure to Carbon Nanotubules and Nanofibers

Following a review of available subchronic and short-term animal dose-response studies of early-stage fibrotic and inflammatory responses for carbon nanotubule exposures, NIOSH performed a risk analysis estimating the deposited or retained carbon nanotubule alveolar lung dose assuming an 8-hour time-weighted average (TWA) workshift exposure during a 40-hour work week, 50 weeks per year, for a 45 year working life. NIOSH has recommended 7 μg/m3 of elemental carbon (EC) as an 8-hour TWA respirable mass airborne concentration in a Draft for Public Comment, November 2010 (1). This recommended REL (Recommended Exposure Limit) is also set at the upper limit of quantitation (LOQ) of NIOSH Method 5040.
National Nanotechnology Initiative (NNI) 2011 Environmental, Health, and Safety Strategy

“The goals of the National Nanotechnology Initiative are fourfold: (1) to advance a world-class nanotechnology R&D program; (2) to foster the transfer of new technologies into products for commercial and public benefit; (3) to develop and sustain educational resources, a skilled workforce, and the supporting infrastructure and tools to advance nanotechnology; and (4) to support responsible development of nanotechnology.”(2)

Nanomaterials exposures may occur either unintentionally in the environment or by use of nanotechnology-enabled products. The purpose of the 2011 NNI Environmental, Health, and Safety Strategy is to ensure nanotechnology development by providing guidance to the various US Federal agencies that produce and utilize scientific information for purposes of risk assessment and risk management. The risk assessment processes consists of: 1) hazard identification; 2) exposure assessment; 3) dose-response assessment; and 4) risk characterization. The NNI risk management research framework is used to identify core research areas that provide critical scientific information on:

Nanomaterial measurement infrastructure;
Human exposure assessment;
Human health;
The environment.

Data on these areas are applied to issues such as product life-cycle, regulatory decision making, public outreach, and research planning (2).
Nanotoxicology Note from the December 2010 Medichem Newletter(3)

Dr. Katharina Klien was awarded the National Occupational Health Award at the Annual Austrian Congress in Villach, Austria for her study titled “The Genotoxic Effects of FeCoB Nanoparticles on Human Fibroblasts Assessed by Comet Assay. Dr. Klein studied the effects of iron-cobalt-boron (FeCoB) nanoparticles and two types of surface-modified FeCoB nanoparticles for their potential genotoxic effects on human fibroblasts.

All 3 types of nanoparticles had the capacity to induce genotoxicity, but they differed based on their particle surface and concentration. Uncoated and 1 type of coated FeCoB nanoparticles caused DNA damage only at the highest concentration of 10 μg/ml, while the other coated FeCoB nanoparticles caused DNA damage at both 1 μg/ml and 10 μg/ml concentrations. A dose-dependent correlation between nanoparticle concentration and genotoxicity was also observed.
Dr. Klien’s paper is to be published in German in the Austrian journal Österreichisches Forum Arbeitsmedizin.
A Virtual Special Issue on Nanotoxicology from ACSNano, Published On-Line October 26, 2010

The American Chemical Society’s on-line journal, ACSNano (www.acsnano.org), published a Virtual Special Issue on Nanotoxicology on October 26, 2010. This issue contained an editorial by Wolfgang J. Parak and a series of 12 articles on various aspects of nanotoxicology originally published between December 27, 2007 and June 18, 2010. These articles are briefly summarized below.

Xia et al (4) compared the effects of cationic nanoparticles in five different cell lines representing portal-of-entry or systemic cellular targets. 60 nm NH2-labeled polystyrene nanospheres were highly toxic in macrophages and epithelial cells, while human microvascular endothelial, hepatoma, and pheochromocytoma cells were relatively resistant to injury by nanoparticles. This study demonstrated the importance of cell-specific uptake mechanisms and pathways that can lead to either sensitivity or relative resistance to cationic nanoparticle toxicity.

Jan et al (5) noted that many nanoparticles have potential as novel drug delivery vehicles, therapeutic agents, contrast agents, and luminescent biological labels for bioimaging. In this study, they demonstrated the use of the high-content screening assay for probing the cytotoxicity of cadmium telluride quantum dots and gold nanoparticles in mouse neuroblastoma and human hepatocellular carcinoma cells. These authors found that the human hepatocellular cells represent a good model for high-content screening as they are often used as a surrogate for human hepatocytes in pharmaceutical studies. They also found that cadmium telluride quantum dots induce primarily apoptosis (programmed cell death) in a time- and dose-dependent manner in undifferentiated and differentiated neural cells.

Xia et al (6) studied three metal oxide nanoparticles currently being produced in large amounts: titanium oxide, zinc oxide, and cerium oxide. They utilized 2 cell lines. Zinc oxide nanoparticles caused cytotoxicity in both cell lines, resulting in generation of reactive oxygen species, oxidant injury, inflammation, and cell death. Cerium oxide nanoparticles were taken up into the cells, but did not cause inflammation or cytotoxicity. Titanium oxide nanoparticles were also taken up by the cells, but elicited no adverse or protective effects. This study demonstrated that metal oxide nanoparticles can cause a wide range of biological responses varying from cytotoxicity to cytoprotection.

Tarantola et al (7) noted that before in vivo utilization of nanoparticles as contrast agents or for drug delivery, in vitro cell experiments are required to determine detailed knowledge of such nanoparticles’ toxicity and biodegradation as a function of their physical and chemical properties. These authors showed that the micromotility of animal cells as monitored by electrical cell-substrate impedance analysis (ECIS) is suitable for quantifying in vitro cytotoxicity of quantum dots and gold nanorods.

AshaRani et al (8) noted that silver nanoparticles are finding increasing use in wound dressings, catheters, and a number of household products because of their antimicrobial action. They studied the toxicity of starch-coated silver nanoparticles in normal human lung fibroblast cells and human glioblastoma cells. Silver nanoparticles were found in cellular nuclei and mitochondria, implicating their direct involvement in mitochondrial toxicity to the electron transport chain and DNA damage.
Magrez et al (9) noted that nanofilaments are not just based on carbon atoms, but can be produced from many inorganic materials in the form of nanotubes or nanowires. They found that titanium oxide-based nanofilaments are cytotoxic as assayed by the MTT test in lung tumor cells and suggest that precautions should be taken during their manipulation.

Liu et al (10) studied the antibacterial activity of single-walled carbon nanotubules (SWCNTs). They found that individually-dispersed SWCNTs were more toxic to bacteria than SWCNT aggregates in Escherichia coli, Pseudomonas aeruginosa, Staphylococcus auerus, and Bacillus subtilis. They concluded that the antibacterial activity of SWCNTs can be remarkably improved by enhancing the physical puncture of bacteria.

nanoparticles developments

George et al (11) studied the use of a multiparameter cytotoxicity assay evaluating oxidative stress to compare the effects of titanium oxide, cerium oxide, and zinc oxide nanoparticles. They noted that the cytotoxicity of zinc oxide nanoparticles can be reduced by iron doping. They demonstrated that a rapid throughput, integrated biological oxidative stress pathway can be used to perform hazard ranking of metal oxide nanoparticles.

Koplosnjaj-Tabi et al (12) administered single-walled carbon nanotubulues (SWCNTs) orally to Swiss mice. At an acute dose of 1000 mg/kg body weight, no deaths or behavioral effects were noted. When administered intraperitoneally, SWCNTs coalesced and when fiber lengths exceeded 10 μm, they induced granuloma formation. Smaller aggregates did not cause granulomas, but persisted inside cells for up to 5 months. Shorter (< 300 nm) SWCNTs escaped the reticuloendothelial system and were excreted through the kidneys and in the bile.

Hutter et al (13) studied the interaction of microglia (resident brain immune cells) and neurons with gold nanoparticles of three morphologies: spheres, rods, and urchins. These authors demonstrated that the morphology and surface chemistry of gold nanoparticles strongly influence the activation status of microglia.

Zhang et al (14) studied the cellular toxicity of graphene layers and single-walled carbon nanotubes (SWCNTs) and showed that the shape of these nanomaterials is directly related to their cytotoxicity. Both graphene layers and SWCNTs induced cytotoxic effects that were concentration- and shape-dependent. Graphene layers generated reactive oxygen species resulting in oxidative stress and also apoptosis (programmed cell death).

Thubagere and Reinhard studied the impact of low chemical toxicity nanoparticles in a human intestinal membrane in vitro model. They used polystyrene nanoparticles with two different surface chemistries. These nanoparticles induced apoptosis in individual human intestinal cells, which then propagated across a monolayer through a “bystander killing effect”, mediated by generated hydrogen peroxide. These authors suggest that nonbiodegradable nanoparticles represent a potential health risk because of the detrimental impact on the intestinal membrane by destroying the barrier protection capacity over time.
The Question of Nanotoxicity in Humans

An article by Song et al (16) describing seven young hospitalized female workers who were exposed to polyacrylate nanoparticles for 5-13 months has caused some controversy since it was published in 2009. These women were admitted to hospital complaining of shortness of breath and pleural effusions. Pathological examination of their lung tissue showed nonspecific pulmonary inflammation, pulmonary fibrosis, and pleural foreign-body granulomas. On transmission electron microscopy, nanoparticles were observed in the cytoplasm and caryoplasm of pulmonary epithelial and mesothelial cells, and were also located in the pleural fluid.
These women worked from 8-12 hours per day in a poorly ventilated, small workspace, spraying large amounts of polyacrylic paste with a pressure sprayer on polystyrene boards or glass. The boards were heated to between 75 and 100 degrees C.
Whether or not the pulmonary effects seen were due to the nanoparticles can be questioned, because the total respirable dust load in the work area could have been high enough to account for the pulmonary effects. However, this article does emphasize the need for ongoing surveillance and research into the potential human health hazards of nanoparticles and nanomaterials which are becoming increasingly widely produced and used.
References

NIOSH: NIOSH Current Intelligence Bulletin: Occupational Exposure to Carbon Nanotubules and Nanofibers, November 2010 Draft.
National Institute for Occupational Safety and Health,
https://www.cdc.gov/niosh/docket/review/docket161A/pdfs/carbonNanotubeCIB/PublicReviewofDraft.pdf, accessed 04/14/2011.
NNI: National Nanotechnology Initiative 2011 Environmental, Health, and Safety Strategy, Draft for Public Comment, December 6, 2010. Subcommittee on Nanoscale Science, Engineering, and Technology, Committee on Technology, National Science and Technology Council, https://www.nano.gov, accessed 04/14/2011.
MEDICHEM: Occupational and Environmental Health in the Production and Use of Chemicals,
https://www.medichem.org, accessed 01/12/2011.
Xia T, Kovochich M, Liong M et al: Cationic polystyrene nanosphere toxicity depends on cell-specific endocytic and mitochondrial injury pathways. ACSNano 2008; 2(1):85-96.
Jan E, Byrne SJ, Cuddihy M et al: High-content screening as a universal tool for fingerprinting cytotoxicity of nanoparticles. ACSNano 2008;2(5):928-938.
Xia T, Kovochich M, Liong et al. Comparison of the Mechanism of Toxicity of zinc oxide and cerium oxide nanoparticles based on dissolution and oxidative stress properties. ACSNano 2008;2(10):2121-2134.
Tarantola M, Schneider D, Sunnick E et al: Cytotoxicity of metal and semiconductor nanoparticles indicated by cellular micromotility. ACSNano 2008;3(1):213-222.
AshaRani PV, Mun CLK, Hande MP et al: Cytotoxicity and genotoxicity of silver nanoparticles in human cells. ACSNano 2008;3(2):279-290.
Magrez A, Horváth L, Smajda R et al: Cellular toxicity of TiO2-based nanofilaments. ACSNano 2009;3(8):2274-2280.
Liu S, Wei L, Hao L et al: Sharper and faster “nano darts” kill more bacteria: A study of antibacterial activity of individually dispersed pristine single-walled carbon nanotube. ACSNano 2009;3(12):3891-3902.
George S, Pokhrel S, Xia T et al: Use of a rapid cytotoxicity screening approach to engineer a safer zinc oxide nanoparticle through iron doping. ACSNano 2010;4(1):15-29.
Kolosnjaj-Tabi J, Hartman KB, Boudjemaa S et al: In vivo behavior of large doses of ultrashort and full-length single-walled carbon nanotubes after oral and intraperitoneal administration to Swiss mice. ACSNano 2010;4(3):1481-1492.
Hutter E, Boridy S, Labrecque S et al: Microglial response to gold nanoparticles. ACSNano 2010;4(5):2595-2606.
Zhang Y, Ali SF, Dervishi E et al: Cytotoxicity effects of graphene and single-wall carbon nanotubes in neural phaechromocytoma-derived PC12 cells. ACSNano 2010;4(6):3181-3186.
Thubagere A, Reinhard BM: Nanoparticle-induced apoptosis propagates through hydrogen-peroxide mediated bystander killing: Insights from a human intestinal epithelium in vitro model. ACSNano 2010;4(7):3611-3622.
Song Y, Li X, Du X: Exposure to nanoparticles is related to pleural effusion, pulmonary fibrosis and granuloma. Eur Respir J 2009;34:559-567.

Alan H. Hall, M.D. President and Chief Medical Toxicologist
Toxicology Consulting and Medical Translating Services, Inc.
Laramie, Wyoming, USA
Clinical Assistant Professor
Colorado School of Public Health
University of Colorado-Denver
Denver, Colorado, USA

On line 05/20/2011

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