Assessment of the toxicity and carcinogenicity of double-walled carbon nanotubes in the rat lung after intratracheal instillation: a two-year study

Our plain-English summary of Assessment of the toxicity and carcinogenicity of double-walled carbon nanotubes in the rat lung after intratracheal instillation: a two-year study, by D.M. Saleh, S. Luo, O.H.M. Ahmed, et al (Particle and Fibre Toxicology, 2022).

The study

• Authors: D.M. Saleh, S. Luo, O.H.M. Ahmed, D.B. Alexander, W.T. Alexander, S. Gunasekaran, A.M. El-Gazzar, M. Abdelgied, T. Numano, H. Takase, M. Ohnishi, S. Tomono, R.H.A.E. Hady, K. Fukamachi, J. Kanno, A. Hirose, J. Xu, S. Suzuki, A. Naiki-Ito, S. Takahashi, H. Tsuda
• Journal: Particle and Fibre Toxicology
• Published: 2022;19(1):30
• Cited by: 28 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: Considering the expanding industrial applications of carbon nanotubes (CNTs), safety assessment of these materials is far less than needed. Very few long-term in vivo studies have been carried out. This is the first 2-year in vivo study to assess the effects of double walled carbon nanotubes (DWCNTs) in the lung and pleura of rats after pulmonary exposure.

Methods: Rats were divided into six groups: untreated, Vehicle, 3 DWCNT groups (0.12 mg/rat, 0.25 mg/rat and 0.5 mg/rat), and MWCNT-7 (0.5 mg/rat). The test materials were administrated by intratracheal-intrapulmonary spraying (TIPS) every other day for 15 days. Rats were observed without further treatment until sacrifice.

Results: DWCNT were biopersistent in the rat lung and induced marked pulmonary inflammation with a significant increase in macrophage count and levels of the chemotactic cytokines CCL2 and CCL3. In addition, the 0.5 mg DWCNT treated rats had significantly higher pulmonary collagen deposition compared to the vehicle controls. The development of carcinomas in the lungs of rats treated with 0.5 mg DWCNT (4/24) was not quite statistically higher (p = 0.0502) than the vehicle control group (0/25), however, the overall incidence of lung tumor development, bronchiolo-alveolar adenoma and bronchiolo-alveolar carcinoma combined, in the lungs of rats treated with 0.5 mg DWCNT (7/24) was statistically higher (p < 0.05) than the vehicle control group (1/25). Notably, two of the rats treated with DWCNT, one in the 0.25 mg group and one in the 0.5 mg group, developed pleural mesotheliomas. However, both of these lesions developed in the visceral pleura, and unlike the rats administered MWCNT-7, rats administered DWCNT did not have elevated levels of HMGB1 in their pleural lavage fluids. This indicates that the mechanism by which the mesotheliomas that developed in the DWCNT treated rats is not relevant to humans.

Conclusions: Our results demonstrate that the DWCNT fibers we tested are biopersistent in the rat lung and induce chronic inflammation. Rats treated with 0.5 mg DWCNT developed pleural fibrosis and lung tumors. These findings demonstrate that the possibility that at least some types of DWCNTs are fibrogenic and tumorigenic cannot be ignored.

© 2022. The Author(s).

In plain English

This was a two-year animal experiment in Japan testing double-walled carbon nanotubes (DWCNT), a manufactured carbon fiber, not asbestos. 180 male F344 rats were divided into groups: untreated, vehicle-only, DWCNT at total doses of 0.125, 0.25 or 0.5 mg per rat, and a comparison carbon nanotube, MWCNT-7, at 0.5 mg. The material was sprayed into the lungs eight times over 15 days. DWCNT stayed in the lungs, and inflammation markers were significantly raised in all treated groups. In the 0.5 mg DWCNT group, lung collagen deposition (fibrosis) was significantly increased. Combined benign and malignant lung tumors occurred in 7 of 24 of these rats versus 1 of 25 vehicle rats. Lung carcinomas alone (4 of 24 versus 0 of 25) fell just short of statistical significance (p = 0.0502). Two DWCNT-treated rats developed pleural mesothelioma on the visceral pleura, which the authors judged unlikely to be relevant to humans. In the MWCNT-7 group, 16 of 25 rats developed malignant pleural mesothelioma. The authors concluded that the possibility that DWCNTs may be toxic to humans cannot be ignored. They also stated that their study lacks unambiguous evidence that DWCNT is carcinogenic in rats.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Two-year animal experiment (rats; test materials instilled into the lungs by intratracheal intrapulmonary spraying, TIPS)
• Who was studied: 180 male F344 rats, 11 weeks old at the start, divided into six groups (untreated, vehicle, three DWCNT doses, MWCNT-7), at Nagoya City University, Japan; interim sacrifice at week 52, final sacrifice at week 104 (week 91 for the MWCNT-7 group)
• Exposure: Double-walled carbon nanotubes (DWCNT, brand Tocana, Toray Industries; thin and tangled, mean diameter 14.32 nm) at total doses of 0.125, 0.25 or 0.5 mg per rat, and MWCNT-7 (multi-walled carbon nanotube, thicker and straight) at 0.5 mg per rat as a reference material; one dose every other day over 15 days (8 doses). No asbestos was tested.
• Main results:
  • Total lung tumors (bronchiolo-alveolar adenoma plus carcinoma) in the 0.5 mg DWCNT group: 7/24, significantly higher (p < 0.05) than the vehicle group, 1/25
  • Lung carcinomas in the 0.5 mg DWCNT group: 4/24 versus 0/25 in vehicle controls, not quite statistically significant (p = 0.0502)
  • One rat in the 0.25 mg DWCNT group and one in the 0.50 mg DWCNT group developed pleural mesothelioma, both in the visceral pleura; the authors considered these unlikely to be relevant to human mesothelioma
  • Malignant pleural mesothelioma in the MWCNT-7 group: 16/25, significantly higher (p < 0.001) than the vehicle group (0/25)
  • HMGB1 (a mesothelioma marker) in pleural lavage fluid at final sacrifice was not elevated in DWCNT-treated rats but was significantly elevated (p < 0.001) in MWCNT-7 treated rats
  • Pulmonary collagen deposition (fibrosis) was significantly higher than in vehicle controls in the 0.5 mg DWCNT and MWCNT-7 groups
  • Subpleural collagen deposition was not significantly increased in the 0.5 mg DWCNT group compared with controls
  • Macrophage counts were significantly increased in all treated groups compared with vehicle, at 52 weeks and at final sacrifice; CCL2 and CCL3 levels were also significantly higher in all treated groups
  • DWCNT, like MWCNT-7, remained in the rat lung (biopersistent) through the final sacrifice
  • Two rats (one in the 0.125 mg and one in the 0.50 mg DWCNT group) developed malignant peritoneal mesothelioma; the authors considered these not treatment related because aged male F344 rats are prone to spontaneous peritoneal mesothelioma
  • No significant differences in average survival time between the untreated, vehicle and DWCNT groups
  • No DNA adducts specific to any treated group were found at 52 weeks
• The authors' conclusion: The authors concluded that DWCNT at 0.5 mg/rat caused pulmonary fibrosis and lung tumors, most likely because the fibers formed agglomerates that persisted in the lung. They concluded that DWCNT did not cause toxic or carcinogenic effects in the pleural cavity. In their view, the possibility that DWCNTs may be toxic to humans cannot be ignored.
• Limitations the authors state:
  • The study does not give unambiguous evidence that the tested DWCNT is carcinogenic in rats; a future study with more animals in the DWCNT groups could be done
  • Persistence of DWCNT in the rat lung could be viewed as particle overload, a mechanism generally specific to rats
  • A second test animal species, such as mice, is needed
  • Instillation can be used for hazard identification but not for risk characterization; inhalation studies would be needed

Topics

The National Library of Medicine indexes this study under subjects our site covers: Lung cancer, Mesothelioma.

Medical terms in this study, explained in our Lexicon: Pleura, Inflammation, Macrophage, Cytokine, Collagen, Deposition, Carcinoma, Pleural Mesothelioma.

Related studies

• Comparative carcinogenicity study of a thick, straight-type and a thin, tangled-type multi-walled carbon nanotube administered by intra-tracheal instillation in the rat — D.M. Saleh, W.T. Alexander, et al. (2020)
• Comparative Carcinogenicity of Double-Walled Carbon Nanotubes of Different Lengths Administered by Intratracheal Installation into Rat Lungs — O.H.M. Ahmed, D.M. Saleh, et al. (2025)
• Two-year intermittent exposure of a multiwalled carbon nanotube by intratracheal instillation induces lung tumors and pleural mesotheliomas in F344 rats — M. Hojo, A. Maeno, et al. (2022)
• MWCNT-7 administered to the lung by intratracheal instillation induces development of pleural mesothelioma in F344 rats — T. Numano, H. Higuchi, et al. (2019)
• A single intratracheal instillation of single-walled carbon nanotubes induced early lung fibrosis and subchronic tissue damage in mice — E.J. Park, J. Roh, et al. (2011)
• Dose-Response Relationships for Lung Tumor and Pleural Mesothelioma Induction by Repetitive Intratracheal Instillation of a Multiwalled Carbon Nanotube, MWCNT-7, in Rats: Comparison with Inhalation Carcinogenicity Based on Lung Burden — A. Maeno, M. Hojo, et al. (2026)

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