ToxSci Advance Access originally published online on September 25, 2007
Toxicological Sciences 2008 101(1):181-182; doi:10.1093/toxsci/kfm250
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To the Editor
* Lovelace Respiratory Research Institute, Albuquerque, New Mexico 87108
University of New Mexico, College of Pharmacy, Albuquerque, New Mexico 87131
Received September 13, 2007; accepted September 17, 2007
We appreciate the comments brought by Lison and Muller, as they not only reaffirm many of our major conclusions and caveats of the manuscript but they have given us an opportunity to clarify an important point regarding the nature of the carbon nanotubes (CNT) used. We found seven major points to the Letter to the Editor, and we have addressed each of them below. Recapitulations of the claims from Muller are shown in italics.
- (1) CNT used were nanofibers, not multiwalled carbon nanotubes (MWCNT). We concede that the magnified image shown in the manuscript (and Letter to the Editor) is not a MWCNT, but a nanofiber. We regret using this image alone for that magnification, as after this point was brought up we looked closer at our transmission electron microscopy (TEM) analysis, the claims of the manufacturer regarding the nature of the MWCNT, and in addition conducted additional characterization of the MWCNT used for these studies. First, a look at the lesser magnification images in the manuscript reveals the hollow structured concentric MWCNT that are similar in nature to the image reported by Muller et al. (2005)
. An additional TEM scan of the CNT used in our studies is provided below. As shown, the CNT used in Mitchell et al. (2007) actually contain a mixture of structures that include some graphitic nanofibers and MWCNT that contain the hollow concentric structure. A qualitative assessment suggests that the hollow concentric MWCNT are the majority, but it is indeed a mixture.
In addition to this investigation, we looked back at the manufacturer characterization and specifications of the material that were purchased for these studies. The manufacturer, which is among the largest producers of MWCNT in the world, reaffirms their definition of these MWCNT by their additional characterization. Last, it is important to note that although some of the nanofibers used for these studies may not meet the definition of MWCNT defined by Muller (and others), the precise classification of MWCNT is blurred in the literature. Indeed, because carbon nanofibers are synthesized by similar processes to MWCNT they are in many cases called (perhaps incorrectly) MWCNT. We thank Lison and Muller for the opportunity to better clarify the nature of the MWCNT used for these studies, and agree that this illustrates the importance of characterization for all toxicology studies, including nanotoxicology.
- (2) Dose used by Mitchell is at the low range of the doses used by Muller, and dose should be confirmed by metal analysis. We concur that the calculated dose given the mice is substantially less then the doses used in the previous studies, and that the lower dose may explain (at least in part) the absence of pulmonary injury. We note that the concentration used is at the OSHA nuisance dust standard, and is significantly higher than airborne concentrations that have been reported in simulated release of CNT (Maynard et al., 2004). We were hesitant even to go to the high concentrations we did, for fear of artifacts associated with overdosing and lack of relevance to human exposure.
We also note that the measurement of CNT content by measurement of metal catalyst has been warned against by Lam et al. (2006) and recent observations of metals leaching from tubes in biological fluid (Hurt, 2007) limit the utility of this assay for quantitative determination of tubes.
- (3) The surface area of the MWCNT used (100 m2/g) was 3x lower than used by Muller (300 m2/g). This is certainly true, and may in part explain the difference in response between the two studies.
- (4) The MWCNT were evaluated in different species, and the mice used by Mitchell may be less sensitive to particle effects than rats used by Muller. Rats are more sensitive to particle induced effects than mice, as was shown initially with studies comparing their relative response to diesel exhaust (Mauderly, 1994
). Indeed, years of study showed particle pathogenesis in high-dose rat studies could not be repeated in several other species. It is not clear which is the best model for humans, but the particle response in rats does not agree with most other species.
- (5) Observation period differed between studies, and this may account for differences. This reaffirms our caveat, but we also mention that in some of the single walled carbon nanotubes studies (summarized in Lam, 2006
) significant pathology was observed at early time points. Longer-term studies, especially by inhalation, are certainly warranted.
- (6) Biopersistence may be low by the apparent low amount of material in lung images. We note that clearance of these materials is unknown, and certainly would be related to the toxicity. We also note that the image of lower amounts of material in the lung may be a little misleading, as the material delivered by an aerosol is distributed throughout a much larger surface area then when administered by instillation. The bolus delivery not only makes the material easier to see, it may also impact the ability to clear the CNT.
- (7) Although the results of intratracheally administered CNT may prove to be of little relevance to health risk, the issue is not resolved on the basis of existing data. We concur with the statement, and admit that although the absence of pulmonary injury by inhalation reported for MWCNT complements some of our previous studies showing pulmonary injury by instillation of diesel soot versus limited or no injury by inhalation, more work is necessary to place the results of all studies conducted to date in context.
- (4) The MWCNT were evaluated in different species, and the mice used by Mitchell may be less sensitive to particle effects than rats used by Muller. Rats are more sensitive to particle induced effects than mice, as was shown initially with studies comparing their relative response to diesel exhaust (Mauderly, 1994
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REFERENCES
Hurt R. Presentation at the Interagency Workshop on Environmental Implications of Nanotechnology. (2007).
Lam CW, James JT, McCluskey R, Arepalli S, Hunter RL. A review of carbon nanotube toxicity and assessment of potential occupational and environmental health risks. Crit. Rev. Toxicol. (2006) 36:189–217.[CrossRef][Web of Science][Medline]
Mauderly JL. Toxicological and epidemiological evidence for health risks from inhaled engine emissions. Environ. Health Perspect. (1994) 102(Suppl. 4):165–171.
Maynard AD, Baron PA, Foley M, Shvedova AA, Kisin ER, Castranova V. Exposure to carbon nanotube material: Aerosol release during the handling of unrefined single-walled carbon nanotube material. J. Toxicol. Environ. Health A (2004) 67:87–107.[Web of Science][Medline]
Mitchell LM, Gao J, Vander Wal R, Gigliotti A, Burchiel SW, McDonald JD. Pulmonary and systemic immune response to inhaled multiwalled carbon nanotubes. Toxicol. Sci. (2007) 100:203–214.
Muller J, Huaux F, Moreau N, Misson P, Heilier JF, Delos M, Arras M, Fonseca A, Nagy JB, Lison D. Respiratory toxicity of multi-wall carbon nanotubes. Toxicol. Appl. Pharmacol. (2005) 207:221–231.[Web of Science][Medline]
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