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A Review of Carbon Nanotube Toxicity and Assessment of Potential Occupational and Environmental Health Risks

Posted on: Friday, 21 April 2006, 06:00 CDT

By Lam, Chiu-wing; James, John T; McCluskey, Richard; Arepalli, Sivaram; Hunter, Robert L

Nanotechnology has emerged at the forefront of science research and technology development. Carbon nanotubes (CNTs) are major building blocks of this new technology. They possess unique electrical, mechanical, and thermal properties, with potential wide applications in the electronics, computer, aerospace, and other industries. CNTs exist in two forms, single-wall (SWCNTs) and multi- wall (MWCNTs). They are manufactured predominately by electrical arc discharge, laser ablation and chemical vapor deposition processes; these processes involve thermally stripping carbon atoms off from carbon-bearing compounds. SWCNT formation requires catalytic metals. There has been a great concern that if CNTs, which are very light, enter the working environment as suspended particulate matter (PM) of respirable sizes, they could pose an occupational inhalation exposure hazard. Very recently, MWCNTs and other carbonaceous nanoparticles in fine (<2.5 m) PM aggregates have been found in combustion streams of methane, propane, and natural-gas flames of typical stoves; indoor and outdoor fine PM samples were reported to contain significant fractions of MWCNTs. Here we review several rodent studies in which test dusts were administered intratracheally or intrapharyngeally to assess the pulmonary toxicity of manufactured CNTs, and a few in vitro studies to assess biomarkers of toxicity released in CNT-treated skin cell cultures. The results of the rodent studies collectively showed that regardless of the process by which CNTs were synthesized and the types and amounts of metals they contained, CNTs were capable of producing inflammation, epithelioid granulomas (microscopic nodules), fibrosis, and biochemical/toxicological changes in the lungs. Comparative toxicity studies in which mice were given equal weights of test materials showed that SWCNTs were more toxic than quartz, which is considered a serious occupational health hazard if it is chronically inhaled; ultrafine carbon black was shown to produce minimal lung responses. The differences in opinions of the investigators about the potential hazards of exposures to CNTs are discussed here. Presented here are also the possible mechanisms of CNT pathogenesis in the lung and the impact of residual metals and other impurities on the toxicological manifestations. The toxicological hazard assessment of potential human exposures to airborne CNTs and occupational exposure limits for these novel compounds are discussed in detail. Environmental tine PM is known to form mainly from combustion of fuels, and has been reported to be a major contributor to the induction of cardiopulmonary diseases by pollutants. Given that manufactured SWCNTs and MWCNTs were found to elicit pathological changes in the lungs, and SWCNTs (administered to the lungs of mice) were further shown to produce respiratory function impairments, retard bacterial clearance after bacterial inoculation, damage the mitochondrial DNA in aorta, increase the percent of aortic plaque, and induce atherosclerotic lesions in the brachiocephalic artery of the heart, it is speculated that exposure to combustion-generated MWCNTs in fine PM may play a significant role in air pollution-related cardiopulmonary diseases. Therefore, CNTs from manufactured and combustion sources in the environment could have adverse effects on human health.

Keywords Cardiopulmonary Diseases, Fibrosis, Fullerenes, Granulomas, Intratracheal Instillation, Multi-Wall Carbon Nanotubes, Nanomaterials, Nanotechnology, Natural Gas Combustion, Particulate Matter, PM2.5, Pulmonary Toxicity, Risk Assessment, Single-Wall Carbon Nanotubes

I. INTRODUCTION

A. Nanotechnology and Carbon Nanotubes

The rapid growth in research and development involving materials of nanoscale size has propelled nanotechnology to the forefront of science and technology development. The anticipation that nanotechnology would become the strategic and dominating science and engineering field of the 21st century prompted the U.S. President and the National Science and Technology Council to promote nanotechnology and to predict that it would lead the United States to the next industrial revolution (National Science and Technology Council, 2003; White House Press secretary, 2000). Of the materials associated with the inception and progression of nanotechnology, fullerenes and carbon nanotubes (CNTs) are the two most important and noted ones. It is the potential applications of these two nanomaterials, especially CNTs, that helped trigger the rush to nanotechnology research and development.

In 1985, the discovery of C^sub 60^ molecules by Harold Kroto, James Heath, Sean O'Brien, Robert Curl, and Richard Smalley (Kroto et al., 1985) opened a whole new frontier in the chemistry of carbon. C^sub 60^ is a spherical molecule with carbon atoms arranged in a pattern like that of a geodesic dome; the molecule was given the name "buckminsterfullerene." Since the discovery of C^sub 60^, fullerenes of larger size, as well as derivatives of C^sub 60^ fullerene, have been synthesized and intensively investigated (Kroto and Walton, 1993; McKeith, 2002). These cagelike nanostructures and other related molecules have been projected to have wide industrial and medical applications. For the discovery of fullerenes, Curl, Kroto, and Smalley were awarded the Nobel Prize in Chemistry in 1996 (Kungl. Vetenskapsakademien [Royal Swedish Academy of Sciences], 1996).

B. Manufactured Carbon Nanotubes

1. Discovery of Carbon Nanotubes

In a graphite arc process that formed fullerenes from atomized carbon, Sumio Iijima in 1991 discovered multiwall CNTs (MWCNTs, Figure 1) deposited at the graphite anode (lijima, 1991, 2004). Shortly after, lijima succeeded in synthesizing single-wall CNTs (SWCNTs) (Figure 1) in the presence of metal catalyst and found that the yield of SWCNTs could be increased by increasing the amount of cobalt or other catalytic transition metals (lijima and Ichihashi, 1993). Adapting the laser ablation process that was used to make fullerenes, Richard Smalley's group at Rice University (Houston, TX) succeeded in synthesizing SWCNTs with a much higher yield and greater purity than the arc process produced (Arepalli et al., 2004a; Guo et al., 1995; Thess et al., 1996). Compared with SWCNTs, MWCNTs are more heterogeneous and difficult to characterize and study. Theoretical calculations and results of experiments on SWCNTs show that SWCNTs have highly desirable mechanical, thermal, photochemical, and electrical properties (Wikipedia, 2005).

Seeing great potential applications of the two closely related families (fullerenes and CNTs) of nanomaterials and other manufactured nanomaterials, more than a dozen government agencies under the leadership of the National Science and Technology Council (2003) jointly established the Interagency Working Group on Nanotechnology, shortly after the awarding of the Nobel Prize for fullerene research. In 2000, this federal effort was raised by President Clinton to the level of a federal initiative, which was known as the National Nanotechnology Initiative (NNI) (White House Press secretary, 2000). One of the major objectives of the NNI is "developing materials that are 10 times stronger than steel, but a fraction of the weight for making all kinds of land, sea, air and space vehicles lighter and more fuel efficient." The materials implicated in the initiative are CNTs.

FIG. 1. (A) to (D): SWCNTs; (E) to (H): MWCNTs. Scanning electron microscope (SEM) images show SWCNT (B) and MWCNT (F) aggregates; transmission electron microscope (TEM) images show raw SWCNT bundles (ropes) with metal nanoparticles (C), and individual multiwall tubes (G). High-resolution TEM images show a cross-section of a SWCNT bundle (D) consisting of >25 tubes and some amorphous carbon on the edges, and a longitudinal cross-section of a MWCNT (H) with an empty central cavity and ~20 walls on each side and some amorphous carbon. (C), (G), and (H) are courtesy of R Nikolaev of the JSC Nanomaterials Group, and (F) is courtesy of J. Rodriguez of Universitat de Barcelona, Spain.

2. Potential Wide Applications of Carbon Nanotubes

CNTs are light in weight and have the strongest tensile strength of any synthetic fiber. According to Smalley (1999), "They are expected to produce fibers 100 times stronger than steel at only 1/ 6th the weight-almost certainly the strongest fibers that will ever be made out of anything." Even composite materials containing CNTs may have incredible strength, potentially sufficient to allow the building of such things as spacecraft structures, space elevators, artificial muscles, combat jackets, and land and sea vehicles (Dresselhaus et al., 2000; Files, 2000; Wikipedia, 2005; Yowell et al., 2002).

In addition to their strength and light weight, some forms of CNTs can conduct electricity better than copper (Smalley, 1999). To harness these properties, the National Aeronautics and Space Administration (NASA) just awarded an $11-million contract to the Carbon Nanotechnology Laboratory of Rice University to produce a prototype power cable made of CNTs (Johnson Space Center, 2005). Smalley remarked that, in addition to their applications in space exploration, CNT cables will someday "rewire the world, replacing aluminum and copper in virtuall\y every application and permitting a vast increase in the capacity of the nation's electrical grid" (Boyd, 2005). Because CNTs can have conducting or semiconducting properties, depending on their nanostructures, the CNT nanofibers would have potential applications in the electronics and computer industries. These highly desirable electrical properties may allow CNT nanostructures to be formed by joining nanotubes of two different diameters end to end to form a diode, suggesting the possibility of constructing electronic computer circuits entirely out of nanotubes (Wikipedia, 2005). According to Smalley (1999), "Several decades from now we may see our current silicon-based microelectronics supplanted by a carbon-based nanoelectronics of vastly greater power and scope." Nanotubes have been shown to be superconducting at low temperature. They also have unusual thermal and optical properties. As Ajayan et al. (1999) have stated, "It is rare to come across a material that has such a range of remarkable properties."

3. Synthesis of Carbon Nanotubes and Impurities in Synthetic Products

Commercially, CNTs are produced from carbon atoms or clusters vaporized from graphite by arc discharge or by pulsed laser vaporization (Ando et al., 2004), or by a chemical-vapor deposition (CVD) process in which thermally and catalytically generated carbon atoms from hydrocarbon gaseous precursors are converted to solid structured materials (Leonhardt, 2004). Nikolaev in Smalley's laboratory developed a gas-phase catalytic growth of SWCNTs from carbon atoms generated from a continuous high-pressure stream of carbon monoxide (Nikolaev et al., 1999). This patented synthetic method is a variation of CVD and is referred to by Smalley's group as the HiPco process.

All of these synthetic processes involve thermal vaporization of carbon and metal catalysts. Carbon sources are graphite, or gaseous carbon-bearing compounds such as CO, methane, ethylene, or other hydrocarbons. The vaporized carbon atoms (or clusters) and hot metal catalysts co-condense into gas-phase molten nanoparticles where nanotubes grow (Moisala et al., 2003; Scott et al., 2001). MWCNTs can be produced without metals; however, the presence of a small amount of metal catalyst helps to align the nanotubes. An increase in the metal nanoparticles-to-carbon ratio favors the formation of SWCNTs (lijima and Ichihashi, 1993). Synthesis of CNTs is generally carried out in an inert atmosphere within a temperature range of 600 to 1200C. At the temperature of CNT synthesis, the metal(s) needs to be catalytically active and remain in the molten state, allowing dissolution of carbon atoms in the metal(s); of the metals that meet these requirements, those most commonly used are iron, nickel, cobalt, and molybdenum (Wikipedia, 2005). All of the SWCNT and MWCNT products produced by these methods contain residual metals (Arepalli et al., 2004b). Depending on the manufacturing process, an unprocessed SWCNT product may contain up to 50% of metal by weight; the metal content in a MWCNT product is much less. The metal impurities are generally undesirable; some commercial products (Figure 2) are sold in purified forms after the removal of metals (Figure 3). CNT products also contain non-NT carbon impurities, such as soot, fullerene, and graphite; the amount and type of impurities depend on the synthetic methods and manufacturers. Raw and purified SWCNT products currently sold by SES (Houston, TX) contain nanotubes at only 20-40% and ≤75%, respectively (SES Research, 1999). The HiPco process of Rice University produces CNTs with very little non-nanotube carbon (Figure 3A); a test result of a purified HiPco CNT sample (Figure 3B) showed that >99% of the carbon content was in nanotube morphology and iron accounted for 0.23% of the sample weight (Shvedova et al., 2005).

4. Physical Characteristics, Properties, and Appearance of Carbon Nanotube Products

A SWCNT is a nanosize tube or fiber formed by bonded carbon atoms arranged in a hexagonal pattern; the tube is generally capped at one end (Figure 1A). Structurally, it resembles a rolled-up single layer of graphene or graphite sheet. Both SWCNTs and C^sub 60^ fullerene have diameters of about 1 nanometer (~1/50,000 of a human hair). Fullerene has a soccer ball-like structure while a SWCNT appears like a single layer of rolled-up chicken wire. An individual SWCNT is a very thin fiber at least several micrometers long, with an aspect ratio (length/diameter) greater than 1000 (Ajayan and Ebbesen, 1997). MWCNTs have two or more concentric layers with various diameters and lengths (Figures 1G and 1H). Since the time CNTs were discovered, enormous research efforts have been devoted to making these fibrous materials longer.

SWCNTs do not naturally exist as individual tubes; van der Waals forces between the molecules cause them to aggregate into microscopic bundles or ropes, which in turn agglomerate loosely into small clumps. For example, SWCNT products made by the laser ablation process at the NASA Johnson Space Center (JSC) nanotechnology laboratory consist of aggregates of tiny ropes (Figure 1B). Each is generally formed by bundling 20 to 50 tubes (with a collective diameter of ~20 nm, Figure 1D) into a fiber several thousand nanometers long (Arepalli et al., 2004a). The ropes then aggregate (Figure 1B) into loose clumps or flakes; this unprocessed ash-like material (Figure 2A), if undisturbed, contains very little fine dust. The appearance of the bulk material depends on the processes by which it was synthesized or manufactured. Currently, SWCNTs made by the HiPco or laser process have fewest wall defects. Defects (like kinks in a straw) can diminish the effectiveness of the attraction force and subsequently reduce the formation of parallel bundles and ropes. In comparison, the arc-discharge SWCNT product (Figure 2B) made by CarboLex Inc. (Lexington, KY) and sold by Aldrich Chemical Company Inc. (Milwaukee, WI) looks like a black powder, similar to a carbon-black toner. The raw CNTs made by Rice's HiPco process are loose black clumps (Figures 2D and 2F). This unprocessed HiPco product, although it contains 30% iron by weight, is very light (Figure 2F) and the bulk density is very low (~ 1 mg/ cm^sup 3^) (Baron et al., 2003). MWCNTs also have van der Waals forces (Yu et al., 2000), but they are less effective than those of SWCNTs. Because of their weaker van der Waals forces and structural heterogeneity, MWCNTs generally exist in single tubes (like spaghetti) and form very few bundles. The tubes or bundles look like microscopic ropes (Figures 1F and 1G). However, the appearance of bulk MWCNT product (Figure 2C) is similar to that of SWCNTs (Figure 2B) made by the same company (CarboLex).

FIG. 2. (A) A sample of SWCNTs produced by the pulsed laser- vaporization process at NASA JSC Nanotube Arc Discharge Facility; this product contains very few fine particles. (B) SWCNTs produced by CarboLex's electric-arc process; the product is a fine powder. (C) MWCNTs produced by CarboLex electric arc; the product contains fine particles. (D) SWCNTs made by the Rice HiPco process; the product contains fine particles. (E) SWCNTs made by the HiPco process, purified, and produced as tiny pearl-like granules; the product contains essentially no fine particles. (F) The transfer of HiPco SWCNTs between containers shows that the product is light- weight and easily becomes airborne. (Figure F is courtesy of A. Maynard of NIOSH; Baron et al., 2003). A human hair is shown in some of the pictures for size comparison.

Because CNTs can be made by several methods and by different manufacturers who use different catalytic metals, carbon sources, and processing conditions (such as pressure and temperature), the length of individual CNTs and the purity of the raw products can vary, even from batch to batch (Cui et al., 2000). In addition to the difference in synthetic processes, differences in postmanufacture processing affect the physical characteristics of a commercial CNT product. For example, the commercial purified HiPco product of Carbon Nanotechnologies Incorporated (CNI, Houston, TX) is prepared and sold as tiny pearl-like granules, which contain few or no fine particles that would pose an inhalation risk (Figure 2E). Compression of loose clumps into a powder or preparation of granules from purified CNTs increases the compactness of the bulk products for ease of handling and shipment and would also affect the physical appearance of the CNT particles. Thus, the physical characteristics of a CNT product depend on its purity, the method by which it was synthesized, and postsynthetic processing. The difference in physical characteristics of CNTs, which depend on manufacturing processes, will affect the health risks of exposure to the product. The knowledge of these processes is important information for an industrial hygienist, toxicologist, or risk assessor who is assessing the toxicological risk of workers being exposed to a particular CNT product.

FIG. 3. TEM pictures on the top show the presence of impurities (carbon-coated metal nanoparticles, etc) in raw HiPco- and laserproduced CNT samples. The bottom pictures show the samples after purification. Notice that the purified HiPco sample contains essentially all carbon nanotubes; the purity of a final product depends on type of raw CNT product and extent of purification.

5. Potential Occupational Exposures to Manufactured Carbon Nanotubes

At present, the production volume of CNTs is still small and the products remain expensive. The price of SWCNTs was hundreds of thousands of dollars per pound in 2003, according to Richard Smalley (Bearden, 2003). Raw SWCNTs and MWCNTs are sold currently by BuckyUSA (Houston) at >$100 per gram (or >$50,000/lb) (BuckyUSA, 2005). In 2003, Baron et al. (2003) of the aerosol group of the National Institute of Occupational Safety and Health (NIOSH) visit\ed CNT synthesis laboratories at Rice University and NASA's Johnson Space Center and the CNT manufacturing facility at Carbon Nanotechnologies, Inc. (CNI, Houston), where SWCNTs are produced by the HiPco or laser processes. The laboratories were making only several grams per day at that time (Shvedova et al., 2005). These NIOSH scientists observed the recovery of CNTs from synthetic ovens and reported that handling of the collected samples was gentle, and losses of this expensive material were minimized. As expected, they found very few CNTs in the working environments of these facilities; the airborne concentrations of CNTs were less than 53 g/m^sup 3^ (Maynard et al., 2004). These facilities had implemented excellent industrial hygiene measures (Figure 4).

However, Smalley predicted that hundreds or thousands of tons of CNTs could be produced in 5 to 10 years (Ball, 2001), and "in time, millions of tonnes of nanotubes will be produced worldwide every year" (Taubes, 2002). The Department of Energy's 2010 target goal for the price of CNTs is $8/kg (or <$20/lb). The extent of industrial and commercial applications would depend on the price of CNT raw materials. If the CNT industry achieved the goal of "a couple dollars a pound" (Bearden, 2003), then millions of tons of CNTs could be produced annually. Such a large production volume would involve a very large workforce, and many workers would likely be exposed to these lightweight materials during CNT synthesis and postsynthetic material collection, handling, purification, and packaging (Figure 4). The purity of a CNT product and the manufacturing process and postsynthetic processing it undergoes determine its physical characteristics (section I.B.4). These physical characteristics will determine its ability to enter the environment. Thus, the occupational risk of exposure of a worker will depend on what product is being handled and at which phase.

FIG. 4. Photograph showing skin could be contaminated with very fine CNT dust if gloves were not worn. (Courtesy of A. Maynard of NIOSH; Baron et al., 2003).

CNTs are strong and light; these very desirable properties would make CNTs very useful in new material development and structural engineering. Progress has been made in incorporating CNTs into fabrics, plastic, rubbers, reinforced structures, composite materials, and household commodities (Laxminarayana and Jalili, 2005; Nanocyl, 2005; Smalley, 2000; Sreekumaret al, 2004; Ward et al., 2005; Wikipedia, 2005). If CNT production increases and prices drop, uses in these areas will be greatly expanded. Occupational exposures to airborne CNT dust could occur in the processes of making these products. During incorporation of CNTs into some of these types of materials, the CNT raw materials (in such forms as the clumps shown in Figure 2A and the purified granules shown in Figure 2E) will need to be pulverized and homogenized; working with pulverized CNTs, or resins or mixtures containing fine CNT particles, would pose a risk of inhalation exposure. It is important to note that some of the factories would be dustier because they would probably not have industrial hygiene standards of the same caliber as the first-class facilities that were visited by the NIOSH aerosol group, who found very little airborne CNT dust.

FIG. 5. Size distribution of particles generated from a raw HiPco carbon nanotube sample using a single-component vortex-shaker fluidized bed; data were smoothed for clarity by the original authors. (Courtesy of A. Maynard of NIOSH; Baron et al., 2003).

A study by the NIOSH aerosol group on a raw HiPco sample and a laser-synthesized CNT sample produced by Rice University revealed that gentle agitation (blowing air over the material shaken in a vortex) produced large airborne clumps (Baron et al., 2003; Maynard et al., 2004). Very few small particles were present. At high agitation levels, more airborne NT particles were generated; the particles were 10 m or less, which are mostly respirable sizes, with some being in the ultrafine range (Figure 5) (Maynard et al., 2004). Thus, if CNTs are subjected to vigorous mechanical processes (such as agitation, grinding, and pulverizing), dust of respirable sizes will be produced, and occupational inhalation exposures could occur. During manufacturing and handling, CNT particulate matter (PM) could also land on the skin of workers if it is not protected (Figure 4).

6. Environmental Health Issues of Manufactured Carbon Nanotubes

The synthesis of CNTs does not require organic solvents. It is considered a "green" industrial process. Currently, it is expected that very little of these very expensive materials will find its way to contaminate the outdoor environment as industrial waste. However, if the price drops substantially and CNTs are incorporated into industrial products and household commodities that are used world- wide, the general public could also be exposed to low levels of CNTs. In the United States we have witnessed the use of asbestos in automotive brake shoes until it was found that asbestos particles generated from abrasion contribute to environmental pollution, and the U.S. Environmental Protection Agency banned use of this carcinogenic material in automobiles (Federal Register, 1989). CNTs are light and strong, and if the price of CNTs drops to a few dollars a pound, applications of CNTs will expand to automobiles and other products that could be subject to deterioration. Wear and tear on products containing CNTs could generate fine paniculate matter that may contribute to environmental pollution. Incineration of discarded articles or wastes that contain CNTs may release some CNTs into the environment. Before the use of CNTs become more widespread, it is important that the chronic toxicity of CNT particulate matter be studied and known, and appropriate safeguards against environmental contamination be implemented.

C. Multiwall Carbon Nanotubes Generated by Fuel Combustion

As discussed in section I.B.3, CNTs could be produced from carbon atoms thermally generated from hydrocarbons (such as methane and acetylene) (Kong etal., 1998; Ren et al., 1998). The synthesis of CNTs is generally carried out with the addition of catalytic metals. In fact, it is known that the synthesis of MWCNTs can occur without metal catalysts (Cumings and Zettl, 2004; Nanotech Co. Ltd., 2003; Tomanek, 1999). MWCNTs were first reported to have been produced from carbon thermally vaporized from graphite without the presence of a metal (lijima, 1991). Che etal. (1998) observed formation of MWCNTs (diameters of about 20 nm) when ethylene/pyrene was pyrolyzed at 545C in the presence of Ni; without catalysts, MWCNT formation occurred at 900C. Many daily heat production or energy-generating activities involve burning of hydrocarbons at temperatures greater than 1000C. One would suspect such activities could produce MWCNTs.

1. Formation of Multiwall Carbon Nanotubes from Fuel-Gas Burning Activities indoors

In fact, MWCNTs and other fullerene-related multi-layer shell structures were observed in samples collected from the effluent stream of co-flowed flames of methane and air (Figures 6A and 6B) (Murr et al, 2004b). Bang et al. (2004) also found MWCNTs and carbonaceous nanoparticles in aggregates of particulate matter collected from propane or natural gas (containing 96% methane) flames generated by typical kitchen stoves; the aggregates had sizes ranging from 0.4 to 2 m, putting them in the respirable range. These aggregates were essentially pure carbon or graphene and contained several hundred to several thousand MWCNTs or other related nanocrystal forms with an average diameter of 20 nm. The individual MWCNTs ranged approximately from 3 to 30 nm in diameter (Murr et al., 2004a). These findings indicate that MWCNTs and other carbonaceous nanoparticles are produced also by water heaters, furnaces, and household appliances powered by natural gas.

2. Multiwall Carbon Nanotubes in the Outdoor Environment

MWCNTs and carbonaceous nanoparticles (Figures 6C-6F) were also observed in outdoor airborne particulate matter collected in El Paso (Texas) and Houston (Murr et al., 2004b). The aggregates were similar in structure to those collected indoors except that the outdoor particulate matter also included agglomerates with other common atmospheric mineral nanocrystals, such as silica. According to Murr et al. (2004a), about 15% of the El Paso particulate samples were carbonaceous aggregates consisting of MWCNTs and other nanoforms (shells, spheres, and other structures). Diesel-related aggregates accounted for 5% of the sample (Murr et al., 2004a). Murr's group (Bang et al., 2004) concluded that MWCNTs and carbonaceous nanoparticles are ubiquitous in the environment; they further speculated that MWCNTs are a major component of indoor and outdoor airborne particulate matter.

3. Multiwall Carbon Nanotubes Were Combustion Products of Ancient Anthropogenic or Natural Activities

MWCNTs and fullerene-like nanocrystal forms were also observed in numerous particle aggregates in a sample of ancient ice obtained from an ice core at the Greenland ice cap (Esquivel and Murr, 2004; Murr et al., 2004b). The ice sample, from a core drilled 1646 feet deep, was dated to roughly 10,000 years old (placing it in the Neolithic Stone Age). According to Murr et al. (2004b), the average diameter of the aggregates was <1 m. These findings show that MWCNTs and other carbonaceous nanoparticles were present in airborne respirable dust aggregates in the prehistoric environment.

4. Multiwall Carbon Nanotubes in Fine Particulate Matter and the Implications for Public Health

The MWCNTs generated from fuel-gas combustion and found in indoor and outdoor environments (Bang et al., 2004; Murr et al., 2004a) are components of airborne particulate aggregates less than 2.5 m (PM2.5) in size. It has been well established that the sources of PM2\.5 include fuel combustion from automobiles, power plants, wood burning, industrial processes, and diesel-powered vehicles such as buses and trucks (U.S. Environmental Protection Agency, 2003b). Combustion processes typically generate very fine particles of sizes from 0.01 to 2.5 m (Huggins et al., 2004), and combustion of fossil fuels is the greatest contributor to fine particulates in the air. The particulate matter contains elemental carbon, organic carbon, trace elements, and common ions (U.S. Environmental Protection Agency, 2003a).

Natural gas is considered an environmentally clean fuel and produces less (7 lbs/billion Btu) particulate matter than oil (84 lb) or coal (2774 lb). However, because global fuel-gas consumption is very large, the contribution of MWCNTs to air pollution is very substantial. According to the National Energy Foundation (National Energy Foundation, 2002), the United States consumed 22,096 billion Btu of natural gas in 1999; this accounted for 27% of the global consumption. The consumption of this "environmentally clean" fuel is expected to increase, and the contribution of MWCNTs to air pollution will be even greater than before.

FIG. 6. (A) A transmission electron microscope (TEM) image of a paniculate matter (PM) sample collected from a combustion stream of methane in the air. (B) High magnification of the area marked by the arrow in (A). (C) TEM image of an environmental PM sample collected from El Paso (TX). (D) High magnification of the area marked by the arrow in (C). (E) TEM image of an outdoor PM sample collected in Houston (TX). (F) High magnification of the area marked by the arrow in (E). Courtesy of L. E. Murr of University of Texas at El Paso, TX, and A. Holian of University of Montana at Missoula, with permission for reprint from Springer Publisher, Heidelberg, Germany (Murr et al., 2004a).

A study conducted by Harvard University (Boston) of mortality rates in six U.S. cities showed an association between fine paniculate air pollution and excess mortality (Dockery et al., 1993). In an American Cancer Society-sponsored epidemiological study of 1.2 million adults, Pope et al. (2002,2004) reported that fine particulate matter in ambient air is a risk factor associated with cardiopulmonary mortality and cardiovascular and pulmonary diseases. Gauderman et al. (2004) investigated the effect of air pollution on the growth of lung function of children during the period of rapid lung development that occurs between the ages of 10 and 18. They found that adverse effects on the lungs were associated with exposures to NO^sub 2^, acid vapor, fine particulate matter (PM2.5), and elemental carbon; elemental carbon was shown to have the highest correlation (p = .007). Concerns about health effects from exposure to fine particulate matter of size <2.5 m (PM2.5) prompted the U.S. Environmental Protection Agency to revise the National Ambient Air Quality Standards for particulate matter to include a standard for PM2.5 (U.S. Environmental Protection Agency, 2003b). Because MWCNTs are present in airborne particulate aggregates of respirable sizes generated from daily human activities and they are likely to be present in significant levels in our environment (Murr et al., 2004a), it is reasonable to postulate that all humans are exposed to CNTs.

It is true that much of the urban ambient PM2.5 is derived from combustion of diesel fuel and gasoline, and not from combustion of natural gas. However, if carbon atoms can be stripped off from ethylene/pyrene during pyrolysis that produced MWCNTs (Che et al., 1998), and from methane and propane during natural-gas combustion that generated MWCNTs, fullerenes, and other carbon nanoparticles (Bang et al., 2004; Murr et al., 2004a, 2004b), one could speculate that some of the carbon atoms formed in the automotive combustion chamber from gasoline (which contains mainly octane and other hydrocarbons) could form MWCNTs. One may speculate that MWCNTs are present in the soot of automotive exhaust. It is noteworthy that the fine (<2.5 m) PM samples that contained MWCNTs collected in Houston were from an area in close proximity to a road with heavy traffic (Murr et al., 2004b).

D. Comparison of Manufactured Carbon Nanotubes and Fuel Combustion-Generated Carbon Nanotubes

In production ovens, CNTs are made in large amounts, allowing van der Waals forces to be effective in driving the aggregations of nanotubes into bundles, ropes, and clumps, most of them probably larger than respirable sizes. The synthesis conditions are also optimized to produce CNTs of a high degree of uniformity and long fibers for practical applications (such as spinning into threads or ropes). However, in the environment outside the laboratory, where fuels are heterogeneous, combustion conditions are various, and the reaction environments are not confined, fuel-generated CNTs are expected to be highly irregular in size and quality (Wikipedia, 2005). These factors would reduce the effectiveness of van der Waals forces: the MWCNTs produced would be less orderly, shorter in length, fewer in numbers, and intermingled with other nanoparticles. The physical differences between MWCNTs that are manufactured and those that are combustion-generated (or found in the ambient environment) are illustrated by comparing Figure IF with Figure 6. Figure IF is an image of a laboratory sample showing manufactured MWCNTs appearing like microscopic ropes of length greater than a micrometer. Figure 6 shows environmental MWCNTs appearing like nanorods with lengths varying within several hundred nanometers, and intermingling with other nanoparticles. Murr's group showed that sizes of particulate matter containing environmental MWCNTs were in the respirable range (Murr et al., 2004a).

II. TOXICOLOGICAL STUDIES AND TOXICITY OF MANUFACTURED CARBON NANOTUBES

A. Methodology to Assess the Potential Toxicity of Carbon Nanotubes in the Lungs

When CNT synthesis moved from laboratories to factories and the products became commercial commodities, there was great concern about the toxicity of these light-weight fibrous materials and their potential effects on workers (Gorman, 2002). As pointed out earlier (section I.B.4), van der Waals forces cause SWCNTs to have a strong tendency to bundle into microscopic ropes, which may contain up to a few hundred parallel tubes (Salvetat et al., 1999). These secondary structures, in turn, form loose clumps, which can become airborne but are mostly larger than respirable size (Figure 2F). These larger particulates are not suitable for toxicology studies. To assess the potential toxicity of a dust in the lungs, particles of respirable size have to be isolated from the bulk material, or test materials have to be mechanically processed into particles in the respirable range.

Aside from direct observations of the effects of a dust on exposed human subjects, animal inhalation exposures are the most appropriate means to assess toxicity of a dust in the lung. As shown by the NIOSH aerosol scientists, it would be difficult to isolate and collect enough fine CNT particles or clumps of respirable size for an inhalation study (Baron et al., 2003). It is also difficult to generate a controlled CNT concentration in a chamber and monitor particle size and the actual exposure level. Even with more workable dusts or powders of other compounds, the technical difficulty and cost associated with inhalation toxicity experimentation have led investigators to assess the effects of these aerosols in the lungs by intratracheal instillation (ITI) (Driscoll et al., 2000; Leong et al., 1998; Sabaitis et al., 1999).

If an animal is exposed to a test dust by inhalation, histopathological changes in the upper respirable tract can be examined, in addition to assessing the effects in the pulmonary region. When dust is administered by ITI, a bolus dose of a fine dust suspension is generally instilled into the trachea of a small animal and the dust is assumed to be drawn deeper into the lung during breathing. Besides having the disadvantage that this unnatural route of administration could not be used to assess the effects of a test dust in the upper respiratory tract, ITI also produces an artificial and less even distribution of dust in the lung. Often, in an ITI study, dust aggregates in aqueous suspensions need to be dissociated or broken down to respirable sizes by ultrasonication with or without a nontoxic dispersion agent. Moreover, the instilled bolus dose may overwhelm the dust clearance mechanism, causing the results to be exaggerated. However, an ITI study, designed appropriately by including carefully chosen doses and reference compounds of known inhalation toxicities, does allow the assessment of the relative toxicity of the test material in respirable sizes (Lam et al., 2002a, 2002b). ITI is an acceptable method of screening dusts for potential pulmonary toxicity, provided that the investigators or risk assessors recognize certain limitations associated with this unnatural way of exposing animals to dust (Driscoll et al., 2000). Dusts of comparable amounts (that reached the lung parenchyma) given by inhalation or intratracheal instillation were shown to produce similar toxicity in the lungs (Henderson et al., 1995). All the animal studies conducted so far to examine CNT pulmonary toxicity have been performed by ITI or similar techniques.

B. Toxicity Studies of Carbon Nanotubes in the Lungs

The first study of S WCNT toxicity, in which lung histopathology in exposed mice was examined, was conducted to address NASA's concern that workers in occupational settings could be exposed to the airborne dust of this novel material of unknown toxicity (Lam et al., 2004). The study was also supported by the Center of Nanoscale Science and Technology of Rice University; both organizations have facilities that make SWCNTs. Parallel to the NASA study, Warheit et al. (2004) of Du \Pont Company (Wilmington, DE) conducted a toxicity study in rats of a SWCNT product made by their company. Although the two studies yielded some similar histopathological findings, the two groups reached different conclusions about the toxicological risk of exposures to SWCNTs; these have been the subject of wide debate (Shvedova et al., 2005). Shvedova et al. of NIOSH subsequently conducted a very comprehensive study in mice "to resolve this conflict" (Shvedova et al., 2005). The toxicity of MWCNTs had drawn little attention until very recently when a group led by J. Muller of Facults Universitaires Notre-Dame de la Paix in Namur, Belgium (Muller et al, 2005), published its study. All of these toxicological studies are outlined in Table 1 and are the subjects of this review.

1. Study of a Carbon Nanotube Product in Guinea Pigs by Huczko et al.

The first published report on CNT toxicity was based on a study conducted by Huczko et al. (2001) of Warsaw University on two groups of five guinea pigs. Each animal was intratracheally instilled once with 0 or 25 mg of CNT-containing soot in 0.5 ml saline (with Tween as a dispersing agent). After 4 weeks, tidal volume, breathing frequency, and pulmonary resistance were assessed. Bronchoalveolar lavage fluid (BALF) was obtained from these animals for measurement of cell differentials and total protein concentration. The investigators found no differences between groups in the measured parameters, and concluded that "the soot with a high content of CNTs does not induce any abnormalities of pulmonary function or measurable inflammation in guinea pigs treated with carbon nanotubes." The authors of this short communication, which was published in a nontoxicology journal, further concluded that "working with soot containing carbon nanotubes is unlikely to be associated with any health risk." However, examination of lung pathology, which is the most critical toxicological endpoint of any pulmonary toxicity study with dust, was not included in the study. Lung pathology was examined in the other animal studies reviewed later.

2. Study of Several Single-Wall Carbon Nanotube Products in Mice by Lam et al.

For several years, NASA has had a facility that makes SWCNTs using the laser process developed by Rice University. At the time (2000) this study was initiated, no toxicity data on this material existed and NASA was concerned about its workers being exposed to a novel material of unknown toxicity. Richard Smalley's group at Rice, also concerned about potential CNT toxicity (Gorman, 2002), joined NASA in support of our toxicity study on SWCNTs.

To assess the pulmonary toxicity of CNT particles by ITI, investigators must prepare the particles in respirable sizes. In our study, the CNTs were prepared in mouse serum by brief homogenization (shearing) and ultrasonication (Lam et al., 2004). Figure 7 shows light micrographs of CNT particles in serum suspensions and shows that the predominate size of the particles was several micrometers; smaller particles were present but could not be captured in light- microscope photographs. Particles of size about 1 m were abundant under the microscope; sizes smaller than 0.5 m could not be detected or resolved by light microscopy. Characterization of the CNT particles in serum suspensions by scanning electron microscopy (SEM) was unsuccessful because serum used as a dispersion agent for CNTs interfered with SEM detection of CNT particles. Removal of serum would change the particle dynamics and sizes in the suspension. Using a transmission electron microscope, Shvedova et al. (2005) reported that the dried images of nebulized CNT suspension droplets (mass median particle diameter of ~5 m) showed continuous mats of intertwined carbon nanoropes with varying diameters. The actual sizes of these respirable CNT particles that reached the lung remained unknown. Because we were interested only in finding out the toxicity of respirable-sized particles of SWCNTs in the lungs and not in investigating nanoparticle toxicity, no efforts were carried out to make suspensions containing CNT particles predominately in nanosizes. Moreover, CNTs are very unlikely to exist in nanosizes in the air, and the doses prepared in our animal study, by briefly (≤ l min) ultrasonicating CNTs and suspending them in mouse serum at concentrations of 0.1 mg/50 m (equivalent to 2000 mg/L) and 0.5 mg/50 l, were too concentrated for the particles to exist in nanosize (Figure 7). As is routinely done when preparing CNTs for physiochemical investigations, Smalley 's group at Rice used "aggressive sonication of purified NT samples in surfactants such as Triton X or highly polar solvents like dimethyl formamide" to make nanoparticle suspensions of 10 mg/L containing mostly individual fibers and a few small bundles (Walters et al., 2001).

TABLE 1

Pulmonary toxicity studies of CNTs in animals and characteristics of these materials

FIG. 7. Light micrographs of sonicated suspended samples of (A) raw HiPco CNTs (0.1 mg/50 l), and (B) purified HiPco CNTs (0.5 mg/ 50 l) showing the particles were predominately in respirable sizes (several micrometers or less).

Pilot Study (Lam and McCluskey, 2000 [unpublished report]) A pilot study was conducted on an early experimental SWCNT sample made at Rice University by the laser ablation process and containing about 10% nickel (by weight) and some iron (information provided by D. Colbert of Rice University). In this study, anesthetized mice (C57/BL/6J) were each intratracheally instilled with 100 l of mouse serum containing O, 0.1, or 1 mg of a CNT sample. Microscopic examination of the lungs in the lower-dose group 1 week after the treatment showed minimal tissue reactions. However, the lungs of 5 mice treated with the high CNT dose had focally severe foreign-body reactions characterized by widespread prominent foci of particle- laden macrophages and giant cells, and prominent peribronchial and perivascular lymphocytic infiltrates (Figure 8B).

This high CNT dose killed some mice immediately after the instillation; their respiratory distress apparently indicated that mechanical blockage of the airways occurred, apparently from the large volume (100 l/mouse) of a bolus instillant. Because a few animals died of suffocation in our pilot study, in our core study we reduced the volume of the dose to 50 l/mouse and modified the instillation technique (Lam et al., 2004) by adopting the intratracheal fast instillation of Sabaitis et al. (1999). We also replaced parenteral ketamine/xylazine anesthetic with isoflurane inhalation anesthetic to achieve quick anesthesia induction and reduce mucus production. No more deaths from airway blockage occurred. Because the low dose in our pilot study produced only minimal effects in the lung, we halved the high dose and used these two doses (0.1 and 0.5 mg/50 l/mouse) for our core study.

Core Study (Lam etal., 2004) Because the CNT sample used in the pilot study contained a substantial amount of nickel, and nickel is highly toxic, we could not with certainty ascribe the lung lesions to CNT itself. To more definitively characterize the intrinsic toxicity of CNTs and the influence of metals in the toxicological manifestation of the test compounds, we followed the pilot study with a core study of three CNT products made by different manufacturing processes and containing different types and/or amounts of residual metals (Lam et al., 2004). When this study was initiated, Rice had shifted from the laser process to the HiPco process for synthesizing CNTs. The study was conducted on (1) unprocessed iron-containing SWCNTs made by the new HiPco process, (2) a purified HiPco product that had been vigorously treated with concentrated acid to remove metal residues by the method published by Rinzler et al. (1998), and (3) a CarboLex SWCNT sample, made by an arc-discharge process, that contained nickel and yttrium (Table 1). Included in the core study were two standard reference materials: ultra-fine carbon black (Printex 90, a dust with relatively low toxicity in mice) and quartz (Min-U-Sil-5, a fibrogenic dust). Groups of B6C3F^sub 1^ mice (4/group for 7 days, 5/ group for 90 days) were intratracheally instilled with a test-dust suspension (0,0.1, or 0.5 mg/50 l/mouse, respectively equal to about O, 3.3, or 17 mg/kg) and were euthanized 7 or 90 days after the single treatment. The lungs were excised, fixed, and stained for histopathological study (Lam et al., 2004).

All CNT samples tested, regardless of the type and amount of metal impurities they contained, induced dose-dependent lesions characterized chiefly by interstitial granulomas (microscopic nodules) in the lungs of mice in the 7-day (Figures 8C and 8D) and 90-day groups (Figures 8E and 8F) (Lam et al., 2004). The granulomas were located beneath the bronchial epithelium and were present throughout most of the microscopic fields of lung tissue. The lungs of all the mice that received high doses of CNTs had prominent granulomas. Granulomas were less prominent but were still observed in the mice treated with the low dose of HiPco-synthesized CNTs; they were not seen in the groups treated with the low dose of graphite arcderived CNTs that contain lower content of SWCNTs. The findings were similar to those observed in the mice treated with a laser-synthesized CNT product produced in our pilot study; the pathology team of the pilot study described lung lesions as focally severe foreign-body reactions characterized by widespread prominent foci of particle-laden macrophages and giant cells (Figure 8B). The lung lesions in the 90-day high-dose groups were generally more pronounced than those in the 7-day high-dose groups (compare Figures 8C and 8D with Figures 8E and 8F). In the 90-day high-dose groups, many more lymphocytes were seen and fibrosis was more evident (Figures 8E and 8F); the lungs of some of these animals showed severe \peribronchial and interstitial inflammation, fibrosis, and necrosis that had extended into the alveolar septa.

As expected, the lungs of mice in the serum control groups were normal. The mice treated with the high dose of quartz showed mild to moderate pulmonary inflammation. This manifestation of quartz- induced toxicity was considered less severe than lesions induced by CNTs. Mice in the group treated with carbon black had black particles in alveolar regions, with minimal tissue reactions (Figure 8A).

3. Study of a Single-Wall Carbon Nanotube Product in Rats by Warheit et al.

Using a different rodent species, Warheit et al. (2004) of Du Pont Company also showed that CNTs produced granulomas in the lungs. This ITI study in Sprague-Dawley rats was conducted on a laser- synthesized SWCNT product that contained 5% nickel and 5% cobalt. The sample consisted of SWCNT ropes, each ~30 nm in diameter. According to Warheit, this CNT sample was produced by the Rice laser ablation process (Rinzler et al., 1998). In 2002, DuPont obtained this manufacturing method from Carbon Nanotechnologies, Inc., which was cofounded by Richard Smalley of Rice University (Cui et al., 2000). This same process was used to make the laser CNT sample that was provided by Rice to NASA for its pilot study (Lam and McCluskey, 2000) (unpublished report). Warheit's laboratory examined histopathology in the lungs and biomarkers of toxicity in BALF. Graphite powder (particle sizes <1 m and having the same amount of incorporated metals as CNTs) and quartz were used as negative and positive control dusts. The animals were given the test material (suspended in phosphatebuffered saline with 1 % Tween 80) at 0,1, or 5 mg/kg (0,0.25, or 1.25 mg/rat). The animals were euthanized for histopathological examination of the lungs at 1, 7, 30, or 90 days after the single treatment. Multifocal granulomas became evident 1 month after exposure (Figures 9C and 9D). The authors stated that granulomatous lesions in the lungs of the CNT-treated rats were non- dose-dependent, nonuniform, and nonprogressive. On the other hand, quartz was observed to produce cytotoxicity, inflammation, and fibrosis in a dose-dependent fashion. Graphite that contained amounts of nickel and cobalt equal to those of the CNT sample produced no signs of toxicity in the lungs.

FIG. 8. Lung tissues from mice intratracheally instilled with 0.5 mg/mouse [ 1 mg/mouse for (B)] of a test material and euthanized 7 or 90 days after the single treatment. (A) Carbon black, 7 days. Particles were scattered in alveoli, and no tissue reaction was observed. (B) Rice University laser-produced carbon nanotubes (CNTs), 7 days. Widespread prominent foci (focal granulomas) contain CNT- laden macrophages. (C) CarboLex arc-produced CNTs, 7 days. Focal granulomas were present. (D) Unprocessed HiPco CNTs, 7 days. The figure shows a well-defined granuloma with some fibrosis (thickening) of alveolar walls. (E) Unprocessed HiPco CNTs, 90 days. A picture of a large nodular lesion shows fibrosis, necrosis, and surrounding tissue, all of which represent a severe reaction. (F) Purified HiPco CNTs, 90 days. Nodular tissue shows fibrosis and necrosis; tissue in this area had very little normal parenchymal structure. Magnifications 40 to 200 (Lam et al., 2004).

The turnover of lung parenchymal cells was assessed in groups of rats euthanized at 1, 7, 30, or 90 days after the singledose dust treatment. Six hours before they were killed, the animals were injected with bromodeoxyuridine (a modified DNA base) to examine the extent of DNA synthesis in lung cells. The results showed no statistically significant change in cell-labeling indices of any CNT- treated group. Significant increases in cell proliferation indices were detected in rats treated with high doses of quartz and euthanized at 24 h and 1 month.

Study of the BALF showed that quartz at a high dose produced an increase in lactate dehydrogenase (LDH) and protein concentrations at all time points; SWCNTs induced only a transient increase in the 1 -day group. Quartz at a high dose produced an increase in LDH and protein concentrations at all time points.

The findings of no dose-dependent and no time-dependent granulomatous response, and absence of prolonged inflammation in the lungs, led Warheit et al. to conclude that the granulomatous reaction was a nonspecific response to instilled aggregates of SWCNTs and that the results "may not have physiological relevance, and may be related to the instillation of a bolus of agglomerated nanotubes" (Warheit et al., 2004).

4. Study of a Single-Wall Carbon Nanotube Product in Mice by Shvedova et al.

Both Lam et al. (2004) and Warheit et al. (2004) found that CNTs produced lung lesions, including granulomas, but the two groups reached different conclusions regarding the potential hazard of exposures to SWCNTs. To address this difference in toxicological assessments, Shvedova et al. (2005) of NIOSH carried out a pulmonary toxicity study in mice (C57CL/6). The study was conducted on a purified HiPco CNT product (>99% SWCNTs) that was exhaustively purified by the NASA JSC nanomaterials group to remove metals (final iron content 0.23% by weight) (Figure 3B). The animals were given a single treatment of CNTs, carbon black, or quartz at a dose of O, 10, 20, or 40 g/mouse (about 0, 0.5, 1, or 2 mg/kg). Aqueous suspensions of test dusts were aspirated at the pharyngeal area of mice, allowing droplets to be pulled into the lung during inspiration. The mice were then killed 1, 3, 7, 28, or 60 days after the treatment. Histopathological examination of the lungs showed an acute inflammation, early onset of formation of granulomas, and progressive fibrosis. The histopathology was characterized by SWCNT- induced granulomas, mainly associated with hypertrophied epithelial cells surrounding the dust aggregates, and diffusive interstitial fibrosis and alveolar wall thickening likely associated with dispersed SWCNTs. The total mass of granulomas in the lungs of mice in the 60-d group increased with an increase in the CNT dose (Figures 10A and B). In general, lung lesions were dose-dependent and progressive, like those reported by Lam et al. (2004). Judging by the published micrographs of lung histopathology, the granulomatous lesions in the mice treated with purified HiPco CNTs in Lam's study (Lam et al., 2004) were more prominent than those reported by Shvedova et al., who also used a purified HiPco CNT sample but with a dose range about an order of magnitude lower (Shvedova et al., 2005). Shvedova et al. further reported that there was significant damage to pulmonary cells. An increase of alveolar type II (AT-II) cells, the progenitor cells that replicate following AT-I cells' death, was detected. Pulmonary function tests showed increases in functional respiratory deficiencies with increased concentrations of CNTs, a finding consistent with fibrosis. Compared with saline-treated controls, CNT-treated mice showed slower bacterial clearance assessed 7 days after bacterial inoculation. At these test doses, quartz and carbon black did not induce granulomas or fibrosis.

To confirm Warheit's BALF findings, Shvedova et al. (2005) also examined the biomarkers of toxicity in the lavaged fluid from CNT- treated mice. The results mostly disagreed with Warheit's findings, showing increases in total protein concentration, cell counts, concentration of transforming growth factor beta (YGF-β), lactate dehydrogenase (LDH), and γ-glutamyltranspeptidase activities, and glutathione depletion; these biomarkers of inflammation, oxidative stress, or cytotoxicity in the lungs were dose-dependent. Shvedova et al. concluded that crystalline silica caused less cytotoxicity than CNTs (compared on an equal-weight basis) and recruited fewer polymorphonuclear leucocytes into the lungs. Like Lam et al. (2004), Shvedova et al. (2005) demonstrated that CNTs were intrinsically toxic and cautioned that exposure of workers to respirable SWCNT particulate matter may pose a risk of developing lung lesions.

5. Study of a Multiwall Carbon Nanotube Product in Rats by Muller et al.

MWCNTs have been shown to produce lung lesions similar to those observed in studies with SWCNTs. Muller et al. (2005) tested two forms of MWCNTs, unprocessed MWCNTs and MWCNTs that had been ground. They reported that 60 days after rats (Sprague-Dawley) were each given a single ITI dose of 0.5, 2, or 5 mg MWCNTs (sonicated and suspended in a normal saline solution containing a dispersing agent, Tween 80), their lungs showed inflammation, granulomas, and fibrosis (Figures 9E and 9F). The unground CNTs remained in the bronchial lumen and produced collagen-rich granulomas. The bronchial lumen was partly or completely blocked, likely an effect similar to that observed by Warheit et al. (2004); very few CNT particles were seen in the parenchymal (alveolar) region. The ground CNTs were "better dispersed" in the parenchyma, and in the interstitium they induced granulomas consisting of macrophages laden with particles, multinuclear giant cells, and some inflammatory cells, like those discussed earlier (Lam et al., 2004; Shvedova et al, 2005; Warheit et al, 2004) that were reported for SWCNTs. Muller et al. (2005) also showed that hydroxyproline and soluble collagen, two biomarkers of fibrosis, increased in the lung tissues in a dose-dependent fashion (Figures 10C and 10D), like that reported by Shvedova et al., 2005. BALF obtained from rats 3 days after the CNT treatment showed dosedependent increases in LDH activity, total protein concentration, and neutrophil number. Muller et al. (2005) also concluded that CNTs are potentially toxic and advocated strict industrial hygiene.

FIG. 9. (A, B) A granuloma in a mouse lung 90 days after instillation with 0.5 mg HiPco SWCNT/mouse showing CNT fibers (Lam et al., 2004). (C) Multifocal granulomas in lung tissue from a rat instilled w\ith 1 mg/kg of laser-produced SWCNTs and microscopically examined 1 month after the treatment. (D) High magnification of a granuloma in (C); the CNTs in the granuloma show fibrous structure. (E) Granulomas that appeared in lung of a rat 60 days after it was instilled with 2 mg/rat of an MWCNT suspension. (F) A granuloma at high magnification. (C) and (D) are courtesy of Warheit et al. (2004) with reprint permission from Oxford University Press (Oxford, UK); (E) and (F) are courtesy of Muller et al. (2005), with permission for reprint from Elsevier Publishing Co. (Philadelphia, PA).

FIG. 10. (A) Morphometric measurement of connective and cellular tissues of mouse lung sections 28 days after postpharyngeal aspiration of 10-40 g SWCNTs/mouse (open bar, granulomatous cellular tissue; gray bar, granulomatous connective tissue; black bar, alveolar connective tissue). Inset: morphometric changes in connective tissue of alveolar wall regions in response to SWCNT (10- 40 g/mouse) 28 and 60 days posttreatnient ([black triangle up], 28 days; [black square], 60 days); means SE (n = 6 mice/group). ^sup α^p < .05 vs. PBS-treated control mice; ^sup β^p < .05 vs. mice treated with 10 g/mouse SWCNT. (B) TEM of lung interstitium shows deposition of collagen and elastin (Shevdova et al., 2005). (C) Hydroxyproline and (D) collagen content of rat lungs 60 days after they were instilled with MWCNTs (ground or unground; 0.5, 2, or 5 mg/rat), asbestos (Asb, 2 mg/rat), or carbon black (CB, 2 mg/ rat). CNTs induced a dose-dependent increase of hydroxyproline and collagen production in the lung of treated animals (Muller et al., 2005). (A) and (B) are courtesy of A. Shvedova of NIOSH with reprint permission from the American Physiological Society (Bethesda, MD). (C) and (D) are courtesy of J. Muller, with permission for reprint from Elsevier Publishing Co. (Philadelphia, PA).

C. Effects of Single-Wall Carbon Nanotubes in the Mouse Heart

Shvedova's group at NIOSH also examined mitochondrial DNA in the aortas of animals that received CNTs by pharyngeal aspiration (Li et al., 2005); they reported dose-dependent damage to the DNA at 7, 28, and 60 days after the CNT treatment. They concluded that these oxidative changes were the result of altered expression of inflammatory genes, including MCP-1 and VCAM-1, in the heart. To further examine the effects of CNTs in the heart, 10 hypercholesterolemic (ApoE-/-) mice were each instilled pharyngeally with a total dose of 20 g SWCNTs for 8 weeks (once every other week). At the end of exposure, Li et al. (2006) observed that the percent of aortic area covered by plaque was significantly increased in the CNT-treated mice compared with the vehicle-treated controls. Morphometric analysis revealed a significant increase of atherosclerotic lesions in the brachiocephalic arteries of the CNT- treated mice. The authors concluded that the effects in the heart might be caused by cytokines released from the inflammation areas in the lungs and/or by CNTs that leave the lungs and enter the systemic circulation (Li et al., 2005, 2006).

D. Effects of Carbon Nanotubes on Skin Cells in In Vitro Culture Systems

1. Human Skin-Cell Study with a Single-Wall Carbon Nanotube Product

The effect of SWCNTs on skin cells in culture was investigated by Shvedova et al. (2003). Immortalized human epidermal keratinocytes (HEKs) were incubated (37C) with raw HiPco CNTs [containing 30% iron, and similar to the raw HiPco product used by Lam etal. (2004)] at 0.06, 0.12, or 0.24 mg/ml for 18 h; cell homogenates were then prepared and assayed. In the cells treated with SWCNTs, the authors observed formation of free-radical species, accumulation of peroxidative products, reduction of total sulfhydryls, and a decrease in content of vitamin E. The observed effects were dose dependent. Because some iron compounds are known to induce oxidation or peroxidation in cells, the authors attributed these oxidative effects to the iron in the carbon nanotubes (Shvedova et al., 2003). This conclusion seems inconsistent with the findings on MWCNTs of Monteiro-Riviere et al. (2005), discussed next. In an animal study conducted later, Shvedova et al. (2005) found iron-free CNTs were capable of producing these cytotoxic effects (section II.B.4). The role of iron in CNT-induced cytotoxicity would need further investigation.

2. Human Skin-Cell Study with a Multiwall Carbon Nanotube Product

Monteiro-Riviere et al. (2005) tested a MWCNT product on HEKs; the doses were 0.1, 0.2, and 0.4 mg/ml, and incubation time was up to 48 h. Uptake of particles by the HEKs was demonstrated by transmission electron microscopy. The cells in the 0.4-mg/ml culture were found to release the proinflammatory cytokine interleukin 8 in a time-dependent fashion. The elemental analysis of cells showed that the particles did not contain iron (iron nanofilms were used to induce MWCNT formation). Monteiro-Riviere and colleagues concluded that the effects of MWCNTs on the cells were not caused by the catalytic metal.

III. DISCUSSION

A. Highlights of the Pulmonary Toxicity Studies-Agreements and Disagreements among the Investigator Groups on Potential Health Risk of Exposure to Manufactured Carbon Nanotubes

Lam et al. (2004) conducted a study in mice given three SWCNT products made by different methods and containing different types and amounts of residual catalytic metals. The authors concluded that collectively CNTs induced dose-dependent and time-dependent interstitial inflammation, and epithelioid granulomas. They also concluded that, if CNTs reached the lungs, they could be rather toxic, even more than quartz on an equal-weight basis, and cautioned that a human exposure risk exists. Warheit et al. conducted a similar study in rats and also found that CNTs induced granulomas, but they did not observe prolonged inflammation and fibrosis. The findings that the granulomatous lesions were non-dose-dependent and nonprogressive, and that there was no prolonged inflammation, led Warheit et al. to conclude that the gr

Source: Critical Reviews in Toxicology

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