ToxSci Advance Access originally published online on September 26, 2009
Toxicological Sciences 2009 112(2):273-275; doi:10.1093/toxsci/kfp237
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Long-term Inhalation Toxicity Studies with Multiwalled Carbon Nanotubes: Closing the Gaps or Initiating the Debate?
DuPont Haskell Global Centers for Health and Environmental Sciences, Newark, Delaware 19714
1 To whom correspondence should be addressed. Fax: (302) 366-5211. E-mail: david.warheit{at}gmail.com.
Received September 18, 2009; accepted September 21, 2009
Carbon nanotubes (CNTs) are allotropes of carbon with a graphene cylindrical nanostructure and have novel physical properties that make them potentially useful for multiple commercial applications. In many ways, CNTs have recently become the material science "posterchild" for the emerging field of nanotechnology—based upon their seemingly unlimited potential applications, including in industrial and biomedical products. The CNT physical characteristics that are most frequently cited include extraordinary strength, unique electrical properties, efficient conductors of heat, and compatibility with biological systems—owing to their ease of functionality. It is important to note, however, that the future successful development of products with CNTs may be undermined by serious concerns regarding their potential toxicity. In this regard, the results of numerous pulmonary toxicity studies in animals or cell culture systems have indicated that exposures to single-walled carbon nanotubes (SWCNTs, single cylinder) (Lam et al., 2004
; Shvedova et al., 2008; Warheit et al., 2004) or multiwalled carbon nanotubes (MWCNTs, multiple cylinders) (Muller et al., 2005) have produced significant lung injury and often at relatively low doses or concentrations. Virtually all the studies, however, have utilized nonphysiological routes of pulmonary exposures (intratracheal instillation or nasopharyngeal aspiration) or have implemented inhalation studies of short-term duration (Shvedova et al., 2008). Thus, the longer term, 90-day inhalation study reported by Ma-Hock et al. (2009) highlighted in this volume of Toxicological Sciences has special significance.
The Ma-Hock paper represents the first long-term inhalation study in rodents with CNTs, either of the single-walled (SWCNT) or multiwalled (MWCNT) variety. In this study, Wistar rats were exposed to aerosols of MWCNT for 13 weeks at exposure concentrations of 0, 0.1, 0.5, or 2.5 mg/m3. The physicochemical characteristics of the MWCNT test samples were reported to be the following: (1) MWCNT composition = 90% carbon–10% metal oxide catalyst; (2) BET surface area = 250–300 m2/g; and (3) MWCNT dimensions—length range = 0.1–10.0 µm and diameter range = 5–15 nm.
Following 13 weeks of aerosol exposures, the authors reported no (extrapulmonary) systemic organ toxicity, based upon histopathological evaluation criteria. This may be a particularly important finding as it relates to any potential effects of MWCNT particulates that may have transmigrated from alveolar regions to systemic vasculature following deposition in the distal lungs. Indeed, the absence of any pathological responses in major organs such as the liver, kidney, or heart following 90 days of exposure is noteworthy. However, as might be expected, significant impacts in the respiratory tract were evident and included hyperplastic responses in the nasal cavity and upper airways (larynx and trachea) concomitant with increased lung weights, multifocal granulomatous inflammation, histiocytic and neutrophilic inflammation, lipoproteinosis in alveoli, and lung-associated lymph nodes. It is interesting to note that the investigators did not report pulmonary fibrotic effects in exposed rats.
At the lowest exposure level, namely 0.1 mg/m3, there was minimal granulomatous-type inflammation in the lungs and lung-associated lymph nodes. As a consequence, a no-observed-effect concentration could not be established. These findings confirm the potency of MWCNT as a pneumotoxicant and provide the hazard criteria basis for establishing risk assessment determinations. Moreover, the study results, by a "physiological" route of exposure (i.e., inhalation), confirm in principle the findings of adverse pulmonary effects reported in earlier studies employing nonphysiological routes or short-term inhalation exposures.
While the inhalation study by Ma-Hock et al. has set an important precedent for gauging the pulmonary effects of CNTs, future studies should be expanded to address additional relevant safety issues that were not fully investigated in this study. Some of these topics include the following:
- Sustainability or reversibility of measured effects
- Physicochemical characteristics
- Potential cardiovascular effects
- Potential pleural effects
Numerous reports have emerged over the past few years which have raised concerns regarding the potential health risks associated with CNT inhalation exposures. Donaldson et al. (2006) have noted in particular that CNTs share three properties that traditionally have been associated with the pathogenicity of inhaled particles and fibers. These include the presumption that (1) nanoparticles are more hazardous than fine sized particles of similar composition, (2) that the fiber-like shapes of CNT suggest potential similarities to asbestos fibers, and (3) the potential biopersistence of CNTs in the lung following deposition could be problematic. Thus, it has been postulated that repeated exposures to either SWCNT and/or MWCNT could be consistent with the "3 D’s" (i.e., dose, dimension, and durability) fiber paradigm of dose, dimension, and durability, which are known to promote mechanisms of lung disease. Perhaps, a fourth health-related concern relates to the emerging opinion that inhaled air pollutants, in the form of ultrafine particulate matter (PM), could produce significant adverse impacts on the heart and cardiovascular system and have been postulated to correlate with increased mortality during high pollution days (Dockery et al., 1993; Pope et al., 1995; Seaton et al., 2009).
SUSTAINABILITY OR REVERSIBILITY OF MEASURED EFFECTS
The study reported by Ma-Hock et al. did not contain a postexposure recovery period in the experimental design. In future studies, it will be very important to determine whether the pulmonary effects observed at the three exposure concentrations are sustainable in the absence of continuing exposures. This is an important parameter to assess and would serve to ascertain whether the CNT-related measured effects are persistent and progressive as evidenced in quartz and asbestos-exposed workers; or whether the effects are transient or reversible, as has been described with low-toxicity particulates. Accordingly, it is recommended that a 90-day exposure study contain a 1- or 3-month postexposure recovery period.
PHYSICOCHEMICAL CHARACTERISTICS
It is widely known that physical factors such as length dimension and durability play important roles in the development of fiber-related pathological effects. In addition, transition metals, such as bioavailable iron and nickel are known to mediate toxic effects to lung cells. The synthesis of either SWCNT or MWCNTs generally requires the contribution of metal catalysts, and these transition metals may trigger metal-mediated toxicity, particularly if the metal is bioavailable (Liu et al., 2008). The Nanocyl MWCNT test material in the Ma-Hock study contains 10% metal oxides, including traces of iron and cobalt. Finally, the large surface area (250–300 mg/m2) of CNT may also play a significant role in the development of pulmonary effects. Surface area dose metrics have been implicated as important factors in facilitating inflammatory effects when comparing ultrafine (nano) versus fine particle types. Therefore, it will be interesting to assess the results of longer term inhalation studies with CNTs of different compositions, dimensions, and surface characteristics to determine whether some forms of CNT are less reactive in the lungs when compared to others.
POTENTIAL CARDIOVASCULAR EFFECTS
Air pollution epidemiological studies have provided evidence that inhaled PM is associated with the development of heart disease. Two distinct mechanisms of cardiovascular toxicity have been postulated: (1) oxidative stress, emanating in part, from pulmonary inflammation (direct or indirect effects) leading to atherothrombosis; and (2) translocation of inhaled particles to the vasculature and direct effects on endothelial cells (Seaton et al., 2009). As a consequence, for future CNT inhalation studies, a focus on potential cardiovascular effects should be a major component of the experimental design regimen. In the Ma-Hock study, no pathological effects were observed in the heart muscle of exposed rats. However, more extensive investigations could include cell proliferation studies on cardiac tissue, a more comprehensive evaluation of coagulation factors in the hematology and clinical chemistry panel of parameters, along with conventional histopathological analyses.
POTENTIAL PLEURAL EFFECTS
As discussed above, CNTs have physical characteristics which are consistent with other fibrous particulates. Donaldson et al. (2006) have postulated that exposures to CNTs could produce toxicological properties similar to asbestos fibers. Experimental results from their laboratory have indicated that intraperitoneal (ip) injection exposures in mice to "long" but not "short" MWCNT produced inflammatory and granulomatous effects in the abdominal cavity similar to amosite asbestos fibers (Poland et al., 2008). Ip injection studies in rodents have been employed as screening assays for potential mesotheliogenic activity in humans. Thusfar, exposures to only a few fiber types are known to produce mesotheliomas in humans. These include amphibole asbestos fiber types and erionite fibers. Exposures to other biopersistent fibrous particulates such as refractory ceramic fibers produce pleural plaques but have not been associated with the development of mesotheliomas in humans. Accordingly, it is conceivable that a more rigorous surveillance of potential pleural activity could be augmented in future subchronic inhalation studies with CNTs in rodents by inclusion of cell proliferation evaluations of subpleural and mesothelial regions (Warheit et al., 1996), as complements to standard histopathological evaluations.
In summary, the Ma-Hock study represents an excellent starting barometer for studying the toxicological effects of aerosolized MWCNT. The 90-day study carried out under Organization for Economic Co-operation and Development guidelines was well conducted and reaffirmed the lung hazard potency of CNT exposures. The results generated from this study, concomitant with relevant exposure data, form the basis for conducting reliable risk assessment determinations. It is also important to note that 13-week exposures did not produce any significant systemic effects beyond the respiratory tract. Thus, this study has set a useful precedent for evaluating CNTs. Yet, there are several additional questions that should be addressed in the future testing of CNTs. These include the sustainability of the measured responses in the respiratory tract to delineate between quartz-type (i.e., sustainable and progressive) effects versus transient effects; the role of physicochemical characteristics including composition, size distributions, surface area determinations, and transition metals; and a more intensively focused investigation into the potential cardiovascular and pleural effects. Integration of these additional parameters/evaluations should provide more comprehensive information on hazard effects of inhaled CNT types and may serve to better delineate the more potent forms of CNTs from the more benign particle types.
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