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Nanotoxicology is the study of the toxicity of nanomaterials. Because of quantum size effects and large surface area to volume ratio, nanomaterials have unique properties compared with their larger counterparts.
Nanotoxicology is a branch of bionanoscience which deals with the study and application of toxicity of nanomaterials. Nanomaterials, even when made of inert elements like gold, become highly active at nanometer dimensions. Nanotoxicological studies are intended to determine whether and to what extent these properties may pose a threat to the environment and to human beings. For instance, Diesel nanoparticles have been found to damage the cardiovascular system in a mouse model.
- 1 Human health and safety
- 2 Toxicology of nanoparticles
- 2.1 Background
- 2.2 Reactive oxygen species
- 2.3 Biodistribution
- 2.4 Nanotoxicity studies
- 2.5 Immunogenicity of nanoparticles
- 2.6 Complications with nanotoxicity studies
- 3 See also
- 4 References
- 5 Further reading
- 6 External links
Human health and safety
Calls for tighter regulation of nanotechnology have arisen alongside a growing debate related to the human health and safety risks associated with nanotechnology.  The Royal Society identifies the potential for nanoparticles to penetrate the skin, and recommends that the use of nanoparticles in cosmetics be conditional upon a favorable assessment by the relevant European Commission safety advisory committee. Andrew Maynard also reports that ‘certain nanoparticles may move easily into sensitive lung tissues after inhalation, and cause damage that can lead to chronic breathing problems’. 
Carbon nanotubes – characterized by their microscopic size and incredible tensile strength – are frequently likened to asbestos, due to their needle-like fiber shape. In a recent study that introduced carbon nanotubes into the abdominal cavity of mice, results demonstrated that long thin carbon nanotubes showed the same effects as long thin asbestos fibers, raising concerns that exposure to carbon nanotubes may lead to pleural abnormalities such as mesothelioma (cancer of the lining of the lungs caused by exposure to asbestos). Given these risks, effective and rigorous regulation has been called for to determine if, and under what circumstances, carbon nanotubes are manufactured, as well as ensuring their safe handling and disposal.
The Woodrow Wilson Centre’s Project on Emerging Technologies conclude that there is insufficient funding for human health and safety research, and as a result there is currently limited understanding of the human health and safety risks associated with nanotechnology. While the US National Nanotechnology Initiative reports that around four percent (about $40 million) is dedicated to risk related research and development, the Woodrow Wilson Centre estimate that only around $11 million is actually directed towards risk related research. They argued in 2007 that it would be necessary to increase funding to a minimum of $50 million in the following two years so as to fill the gaps in knowledge in these areas.
The potential for workplace exposure was highlighted by the 2004 Royal Society report which recommended a review of existing regulations to assess and control workplace exposure to nanoparticles and nanotubes. The report expressed particular concern for the inhalation of large quantities of nanoparticles by workers involved in the manufacturing process.
Stakeholders concerned by the lack of a regulatory framework to assess and control risks associated with the release of nanoparticles and nanotubes have drawn parallels with bovine spongiform encephalopathy (‘mad cow’s disease'), thalidomide, genetically modified food, nuclear energy, reproductive technologies, biotechnology, and asbestosis. In light of such concerns, the Canadian based ETC Group have called for a moratorium on nano-related research until comprehensive regulatory frameworks are developed that will ensure workplace safety.
In October 2008, the Department of Toxic Substances Control (DTSC), within the California Environmental Protection Agency, announced its intent to request information regarding analytical test methods, fate and transport in the environment, and other relevant information from manufacturers of carbon nanotubes. The term "manufacturers” includes persons and businesses that produce nanotubes in California, or import carbon nanotubes into California for sale. This information request is meant to identify information gaps and to develop further knowledge about the health and safety of carbon nanotubes.
DTSC exercises its authority under California Health and Safety Code, Chapter 699, sections 57018-57020. These sections were added as a result of the adoption of Assembly Bill AB 289 (2006). They are intended to make information on the fate and transport, detection and analysis, and other information on chemicals more available. The law places the responsibility to provide this information to the Department on those who manufacture or import the chemicals. On January 22, 2009, a formal information request letter was sent to manufacturers who produce or import carbon nanotubes in California, or who may export carbon nanotubes into the State. This letter constitutes the first formal implementation of the authorities placed into statute by AB 289 (2006) and is directed to manufacturers of carbon nanotubes, both industry and academia within the State, and to manufacturers outside California who export carbon nanotubes to California. This request for information must be met by the manufacturers within one year.
On January 22, 2010, California manufacturers and importers of carbon nanotubes were required to submit their responses. On January 25, 2010, DTSC posted the responses received to date along with a list of companies that had failed to respond to the information request. On February 16, 2010, DTSC issued a follow-up letter to the companies that failed to submit a response.
DTSC is indicating interest in expanding the Specific Chemical Information Call-in to members of the brominated flame retardants, members of the methyl siloxanes, and other nanometals and nanometal oxides such as vanadium oxide, aluminum oxide, silicon dioxide, titanium dioxide, zinc oxide, cerium oxide, nano platinum, nano silver, and nano zerovalent iron. DTSC is also planning to include quantum dots, ocean plastics, and nanoclay into the list of chemicals of interest.
Toxicology of nanoparticles
Nanotoxicology is a sub-specialty of particle toxicology. It addresses the toxicology of nanoparticles (particles <100 nm diameter) which appear to have toxicity effects that are unusual and not seen with larger particles. Nanoparticles can be divided into combustion-derived nanoparticles (like diesel soot), manufactured nanoparticles like carbon nanotubes and naturally occurring nanoparticles from volcanic eruptions, atmospheric chemistry etc. Typical nanoparticles that have been studied are titanium dioxide, alumina, zinc oxide, carbon black, and carbon nanotubes, and "nano-C60". Nanoparticles have much larger surface area to unit mass ratios which in some cases may lead to greater pro-inflammatory effects (in, for example, lung tissue). In addition, some nanoparticles seem to be able to translocate from their site of deposition to distant sites such as the blood and the brain. This has resulted in a sea-change in how particle toxicology is viewed- instead of being confined to the lungs, nanoparticle toxicologists study the brain, blood, liver, skin and gut. Nanotoxicology has revolutionised particle toxicology and rejuvenated it.
Reactive oxygen species
For some types of particles, the smaller they are, the greater their surface area to volume ratio and the higher their chemical reactivity and biological activity. The greater chemical reactivity of nanomaterials can result in increased production of reactive oxygen species (ROS), including free radicals. ROS production has been found in a diverse range of nanomaterials including carbon fullerenes, carbon nanotubes and nanoparticle metal oxides. ROS and free radical production is one of the primary mechanisms of nanoparticle toxicity; it may result in oxidative stress, inflammation, and consequent damage to proteins, membranes and DNA.
The extremely small size of nanomaterials also means that they much more readily gain entry into the human body than larger sized particles. How these nanoparticles behave inside the body is still a major question that needs to be resolved. The behavior of nanoparticles is a function of their size, shape and surface reactivity with the surrounding tissue. In principle, a large number of particles could overload the body's phagocytes, cells that ingest and destroy foreign matter, thereby triggering stress reactions that lead to inflammation and weaken the body’s defense against other pathogens. In addition to questions about what happens if non-degradable or slowly degradable nanoparticles accumulate in bodily organs, another concern is their potential interaction or interference with biological processes inside the body. Because of their large surface area, nanoparticles will, on exposure to tissue and fluids, immediately adsorb onto their surface some of the macromolecules they encounter. This may, for instance, affect the regulatory mechanisms of enzymes and other proteins.
Nanomaterials are able to cross biological membranes and access cells, tissues and organs that larger-sized particles normally cannot. Nanomaterials can gain access to the blood stream via inhalation or ingestion. At least some nanomaterials can penetrate the skin; even larger microparticles may penetrate skin when it is flexed. Broken skin is an ineffective particle barrier, suggesting that acne, eczema, shaving wounds or severe sunburn may accelerate skin uptake of nanomaterials. Then, once in the blood stream, nanomaterials can be transported around the body and be taken up by organs and tissues, including the brain, heart, liver, kidneys, spleen, bone marrow and nervous system. Nanomaterials have proved toxic to human tissue and cell cultures, resulting in increased oxidative stress, inflammatory cytokine production and cell death. Unlike larger particles, nanomaterials may be taken up by cell mitochondria and the cell nucleus. Studies demonstrate the potential for nanomaterials to cause DNA mutation and induce major structural damage to mitochondria, even resulting in cell death.
Since there is no authority to regulate nanotech-based products, there are many products that could possibly be dangerous to humans. Scientific research has indicated the potential for some nanomaterials to be toxic to humans or the environment. In March 2004 tests conducted by environmental toxicologist Eva Oberdörster, Ph.D. working with Southern Methodist University in Texas, found extensive brain damage to fish exposed to fullerenes for a period of just 48 hours at a relatively moderate dose of 0.5 parts per million (commensurate with levels of other kinds of pollution found in bays). The fish also exhibited changed gene markers in their livers, indicating their entire physiology was affected. In a concurrent test, the fullerenes killed water fleas, an important link in the marine food chain. The extremely small size of fabricated nanomaterials also means that they are much more readily taken up by living tissue than presently known toxins. Nanoparticles can be inhaled, swallowed, absorbed through skin and deliberately or accidentally injected during medical procedures. They might be accidentally or inadvertently released from materials implanted into living tissue.
Researcher Shosaku Kashiwada of the National Institute for Environmental Studies in Tsukuba, Japan, in a more recent study, intended to further investigate the effects of nanoparticles on soft-bodied organisms. His study allowed him to explore the distribution of water-suspended fluorescent nanoparticles throughout the eggs and adult bodies of a species of fish, known as the see-through medaka (Oryzias latipes). See-through medaka were used because of their small size, wide temperature and salinity tolerances, and short generation time. Moreover, small fish like the see-through medaka have been popular test subjects for human diseases and organogenesis for other reasons as well, including their transparent embryos, rapid embryo development, and the functional equivalence of their organs and tissue material to that of mammals. Because the see-through medaka have transparent bodies, analyzing the deposition of fluorescent nanoparticles throughout the body is quite simple. For his study, Dr. Kashiwada evaluated four aspects of nanoparticle accumulation. These included the overall accumulation and the size-dependent accumulation of nanoparticles by medaka eggs, the effects of salinity on the aggregation of nanoparticles in solution and on their accumulation by medaka eggs, and the distribution of nanoparticles in the blood and organs of adult medaka. It was also noted that nanoparticles were in fact taken up into the bloodstream and deposited throughout the body. In the medaka eggs, there was a high accumulation of nanoparticles in the yolk; most often bioavailibility was dependent on specific sizes of the particles. Adult samples of medaka had accumulated nanoparticles in the gills, intestine, brain, testis, liver, and bloodstream. One major result from this study was the fact that salinity may have a large influence on the bioavailibility and toxicity of nanoparticles to penetrate membranes and eventually kill the specimen.
As the use of nanomaterials increases worldwide, concerns for worker and user safety are mounting. To address such concerns, the Swedish Karolinska Institute conducted a study in which various nanoparticles were introduced to human lung epithelial cells. The results, released in 2008, showed that iron oxide nanoparticles caused little DNA damage and were non-toxic. Zinc oxide nanoparticles were slightly worse. Titanium dioxide caused only DNA damage. Carbon nanotubes caused DNA damage at low levels. Copper oxide[disambiguation needed] was found to be the worst offender, and was the only nanomaterial identified by the researchers as a clear health risk. The latest toxicology studies on mice involving exposure to carbon nanotubes (CNT) showed a limited pulmonary inflammatory potential of MWCNT at levels corresponding to the average inhalable elemental carbon concentrations observed in U.S.-based CNT facilities. The study estimated that considerable years of exposure are necessary for significant pathology to occur. 
No fullerene toxicity reported
Nanoparticles can also be made of C60, as is the case with almost any room temperature solid, and several groups have done this and studied toxicity of such particles. The results in the work of Oberdörster at Southern Methodist University, published in "Environmental Health Perspectives" in July 2004, in which questions were raised of potential cytotoxicity, has now been shown by several sources to be likely caused by the tetrahydrofuran used in preparing the 30 nm–100 nm particles of C60 used in the research. Isakovic, et al., 2006, who review this phenomenon, gives results showing that removal of THF from the C60 particles resulted in a loss of toxicity. Sayes, et al., 2007, also show that particles prepared as in Oberdorster caused no detectable inflammatory response when instilled intratracheally in rats after observation for 3 months, suggesting that even the particles prepared by Oberdorster do not exhibit markers of toxicity in mammalian models. This work used as a benchmark quartz particles, which did give an inflammatory response.
A comprehensive and recent review of work on fullerene toxicity is available in "Toxicity Studies of Fullerenes and Derivatives," a chapter from the book "Bio-applications of Nanoparticles". In this work, the authors review the work on fullerene toxicity beginning in the early 1990s to present, and conclude that the evidence gathered since the discovery of fullerenes overwhelmingly points to C60 being non-toxic. As is the case for toxicity profile with any chemical modification of a structural moiety, the authors suggest that individual molecules be assessed individually.
Immunogenicity of nanoparticles
Very little attention has been directed towards the potential immunogenicity of nanostructures. Nanostructures can activate the immune system, inducing inflammation, immune responses, allergy, or even affect to the immune cells in a deleterious or beneficial way (immunosuppression in autoimmune diseases, improving immune responses in vaccines). More studies are needed in order to know the potential deleterious or beneficial effects of nanostructures in the immune system. In comparison to conventional pharmaceutical agents, nanostructures have very large sizes, and immune cells, especially phagocytic cells, recognize and try to destroy them.
Complications with nanotoxicity studies
Size is therefore a key factor in determining the potential toxicity of a particle. However it is not the only important factor. Other properties of nanomaterials that influence toxicity include: chemical composition, shape, surface structure, surface charge, aggregation and solubility, and the presence or absence of functional groups of other chemicals. The large number of variables influencing toxicity means that it is difficult to generalise about health risks associated with exposure to nanomaterials – each new nanomaterial must be assessed individually and all material properties must be taken into account.
In addition, standarization of toxicology tests between laboratories are needed. Díaz, B. et al. from the University of Vigo (Spain) has shown (Small, 2008) that many different cell lines should be studied in order to know if a nanostructure induces toxicity, and human cells can internalize aggregated nanoparticles. Moreover, it is important to take into account that many nanostructures aggregate in biological fluids, but groups manufacturing nanostructures do not care much about this matter. Many efforts of interdisciplinary groups are strongly needed in order to progress in this field.
Effect of aggregation or agglomeration of nanoparticles
Many nanoparticles agglomerate or aggregate when they are placed in environmental or biological fluids. The terms agglomeration and aggregation have distinct definitions according to the standards organizations ISO and ASTM, where agglomeration signifies more loosely bound particles and aggregation signifies very tightly bound or fused particles (typically occurring during synthesis or drying). Nanoparticles frequently agglomerate due to the high ionic strength of environmental and biological fluids, which shields the repulsion due to charges on the nanoparticles. Unfortunately, agglomeration has frequently been ignored in nanotoxicity studies, even though agglomeration would be expected to affect nanotoxicity since it changes the size, surface area, and sedimentation properties of the nanoparticles. In addition, many nanoparticles will agglomerate to some extent in the environment or in the body before they reach their target, so it is desirable to study how toxicity is affected by agglomeration.
A method was published that can be used to produce different mean sizes of stable agglomerates of several metal, metal oxide, and polymer nanoparticles in cell culture media for cell toxicity studies. Different mean sizes of agglomerates are produced by allowing the nanoparticles to agglomerate to a particular size in cell culture media without protein, and then adding protein to coat the agglomerates and "freeze" them at that size. By waiting different amounts of time before adding protein, different mean sizes of agglomerates of a single type of nanoparticle can be produced in an otherwise identical solution, allowing one to study how agglomerate size affects toxicity. In addition, it was found that vortexing while adding a high concentration of nanoparticles to the cell culture media produces much less agglomerated nanoparticles than if the dispersed solution is only mixed after adding the nanoparticles.
With comparison to more conventional toxicology studies, the nanotoxicology field is however suffering from a lack of easy characterisation of the potential contaminants, the "nano" scale being a scale difficult to comprehend. The biological systems are themselves still not completely known at this scale. Ultimate Atomic visualisation methods such as Electron microscopy (SEM and TEM) and Atomic force Microscopy (AFM) analysis allow visualisation of the nano world. Further nanotoxicology studies will require precise characterisation of the specificities of a given nano-element : size, chemical composition, detailed shape, level of aggregation, combination with other vectors, etc. Above all, these properties would have to be determined not only on the nanocomponent before its introduction in the living environment but also in the (mostly acqueous) biological environment.
It is not easy to determine to what extent a given nanoparticle has a dramatic effect when compared to comparable nanoparticles already present in our environment either through natural/biological origin or through human activity.
AEM - Analytical Electron Microscopy was used over 40 years ago to investigate amphibole asbestos bodies in Lake Superior from the Reserve Mining operations. This could non-destructively characterise sub micron particles. Today AEM can fully characterise to atom dimensions. Perhaps AEM should be employed to attempt to achieve those goals discussed above?
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