Comparative Carcinogenicity of Double-Walled Carbon Nanotubes of Different Lengths Administered by Intratracheal Installation into Rat Lungs

Our plain-English summary of Comparative Carcinogenicity of Double-Walled Carbon Nanotubes of Different Lengths Administered by Intratracheal Installation into Rat Lungs, by O.H.M. Ahmed, D.M. Saleh, W.T. Alexander, et al (Nanomaterials, 2025).

The study

• Authors: O.H.M. Ahmed, D.M. Saleh, W.T. Alexander, H. Takase, Y. Taquahashi, M. Hojo, A. Maeno, K. Fukamachi, M. Gi, A. Hirose, S. Tsuruoka, S. Takahashi, H. Tsuda, A. Naiki-Ito
• Journal: Nanomaterials
• Published: 2025;15(18)
• Cited by: 3 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

We previously carried out an in vivo 2-year study to assess the potential toxicity/carcinogenicity of double-walled carbon nanotubes (DWCNTs) in a rat lung. We found that administration of DWCNTs by intratracheal-intrapulmonary spraying (TIPS) at a dose of 0.5 mg/rat induced the development of lung tumors in 7 of 24 treated rats while 1 of 21 untreated rats and 1 of 25 vehicle treated rats developed lung tumors. In the current study, we administered DWCNTs of different lengths, 1.5 µm, 7 µm, and 15 µm, to rats by TIPS to investigate the possible effect of the length of this thin, flexible CNT on toxicity/carcinogenicity in rat lungs. Rats were administered DWCNTs with lengths of 1.5 µm (D1.5), 7 µm (D7), and 15 µm (D15) by TIPS once every other day over the course of two weeks for a total of eight administrations. The total dose administered was approximately 22 × 1012 fibers per rat, corresponding to 0.0504 mg for D1.5, 0.232 mg for D7, and 0.504 mg for D15. Another group of rats was administered 0.5 mg MWCNT-7, a known carcinogen. Animals were killed at weeks 6 and 104 (4 and 102 weeks after the final TIPS administration). The mean survival time of the rats in the untreated, vehicle, D1.5, D7, and D15 groups was 99 to 104 weeks. One rat in the D1.5 group and one rat in the D15 group died before week 75. The remaining rats in the untreated, vehicle, D1.5, D7, and D15 groups were included in the final assessment of lung toxicity/carcinogenicity. In contrast, 11 rats in the MWCNT-7 group died before week 75 due to the development of malignant mesothelioma. Due to the much shorter survival time of the rats treated with MWCNT-7, accurate assessment of lung proliferative lesions in this group was not possible. At week 6, an increase in alveolar macrophages and granulation tissue foci in the alveoli was observed in all DWCNT administered groups. The alveolar epithelial cell PCNA index was also significantly increased in the D7 and D15 groups. Increases in alveolar macrophages, granulation tissue foci, and the alveolar epithelial cell PCNA index were observed in all DWCNT-treated groups at the final sacrifice. The incidences of lung tumors were 0/13, 0/12, 4/12, 3/8, and 2/10 in the untreated, vehicle, D1.5, D7, and D15 groups, respectively. In agreement with our previous study, the DWCNTs tested in the present study were carcinogenic in the rat lung. In addition, we present evidence that DWCNT fiber length may possibly have an effect on DWCNT-induced carcinogenicity in rat lungs.

In plain English

Male F344 rats had double-walled carbon nanotubes (DWCNTs) 1.5, 7 or 15 µm long sprayed into their lungs eight times over two weeks. With matched fiber numbers, doses were about 0.050, 0.232 and 0.504 mg per rat. Controls were untreated or got carrier liquid; one group got 0.5 mg of another nanotube, MWCNT-7. Five rats per group were examined at week 6, the rest up to 104 weeks; MWCNT-7 rats died (11 of 15 from mesothelioma) or were killed by week 75, so their lung lesions could not be accurately assessed. Lung carcinomas occurred in 0 of 13 untreated, 0 of 12 vehicle, 4 of 12 (1.5 µm), 3 of 8 (7 µm) and 2 of 10 (15 µm) rats. Only the 1.5 and 7 µm increases were significant versus vehicle; DWCNT groups did not differ significantly. Among DWCNT and control rats, preneoplastic hyperplasia occurred only at 7 and 15 µm (5 rats each). The authors concluded DWCNTs were mildly carcinogenic in rat lungs and fiber length may possibly matter. Their major stated limitation was few 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: Animal experiment: long-term (up to 104 weeks) lung toxicity and carcinogenicity study in rats, with interim sacrifice at week 6
• Who was studied: Male F344 rats from Charles River Laboratories Japan, housed at Nagoya City University. They were split into 6 groups of 16 to 20: untreated 18, vehicle 18, DWCNT 1.5 µm 18, DWCNT 7 µm 16, DWCNT 15 µm 16, MWCNT-7 20. One vehicle rat and 3 D7 rats died at weeks 29 to 30 from a drinking-water system failure and were removed. Five rats per group were killed at week 6. Lung tumor assessment covered rats living longer than 75 weeks: 13, 12, 12, 8 and 10 rats in the untreated, vehicle, D1.5, D7 and D15 groups. In the MWCNT-7 group, 11 of 15 rats died before week 75 and the remaining 4 were sacrificed at week 75.
• Exposure: Double-walled carbon nanotubes 1.5, 7 and 15 µm long (Neura Inc.) and the multi-walled carbon nanotube MWCNT-7 (Mitsui Chemicals). Doses were given by intratracheal–intrapulmonary spraying (TIPS) every other day for 2 weeks (8 doses). Total doses per rat were 50.4 µg (2.13 × 10^13 fibers) of D1.5, 232.3 µg (2.18 × 10^13 fibers) of D7, 504.1 µg (2.21 × 10^13 fibers) of D15, and 504.0 µg of MWCNT-7.
• Main results:
  • Bronchioloalveolar carcinoma was found in 4 of 12 rats in the D1.5 group (p less than 0.05 vs vehicle), 3 of 8 in the D7 group (p less than 0.05), and 2 of 10 in the D15 group (not marked significant). It was found in 0 of 13 untreated and 0 of 12 vehicle rats. No adenomas were found in any group.
  • Tumor development did not differ significantly between the three DWCNT length groups.
  • Bronchioloalveolar hyperplasia was found in 5 rats in the D7 group and 5 rats in the D15 group (each p less than 0.01 vs vehicle). None was found in the untreated, vehicle or D1.5 groups.
  • The authors counted proliferative lung lesions (carcinoma plus hyperplasia) as 4 of 12 rats (D1.5), 8 of 8 (D7) and 7 of 10 (D15). They interpreted this as potential tumor development increasing with fiber length.
  • In the MWCNT-7 group, 11 of 15 rats developed pleural mesothelioma and died before week 75. One of the remaining 4 rats, killed at week 75, also had pleural mesothelioma (12 of 15 in Table 3, p less than 0.001 vs vehicle).
  • The 4 MWCNT-7 rats examined for lung lesions were reported separately, not as an accurate assessment. One had bronchioloalveolar hyperplasia and two had bronchioloalveolar carcinoma.
  • One rat in the D15 group developed a malignant mesothelioma on the visceral pleura. The authors stated this single case is not evidence that D15 fibers induce pleural mesothelioma by a mechanism relevant to humans.
  • At week 6, lung granulomas were significantly increased in all DWCNT groups and in the MWCNT-7 group. Among the DWCNT groups, granuloma numbers increased with fiber length.
  • At week 6, the alveolar epithelial cell PCNA count (a marker of cell proliferation) was significantly increased in the 7 µm and 15 µm DWCNT groups and in the MWCNT-7 group.
  • Retention of DWCNT fibers in the lung increased with fiber length: 37% (1.5 µm), 68% (7 µm) and 100% (15 µm) at week 104 compared with week 6. MWCNT-7 retention was 80% at week 75.
• The authors' conclusion: The authors concluded that the DWCNTs were mildly carcinogenic in the rat lung, consistent with their earlier study. They also concluded that fiber length, and the size and form of fiber agglomerates, may possibly influence carcinogenic potential.
• Limitations the authors state:
  • The number of rats given each DWCNT was small, because the precise-length DWCNTs were made specifically for the study and available only in very limited quantities.
  • Only a single dose level of each DWCNT was tested.
  • Because most MWCNT-7 rats died early, lung proliferative lesions in that group could not be accurately assessed.
  • The D15 rat with visceral mesothelioma was found dead and could not be tested for HMGB1 in the pleural lavage.

Topics

Medical terms in this study, explained in our Lexicon: Mesothelioma, Alveolar Macrophage, Alveoli, Carcinogenic, Fiber Length.

Related studies

• Assessment of the toxicity and carcinogenicity of double-walled carbon nanotubes in the rat lung after intratracheal instillation: a two-year study — D.M. Saleh, S. Luo, et al. (2022)
• 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 Gene Expression Analysis of Malignant Mesothelioma and Lung Adenocarcinomas Induced by Multi-Walled Carbon Nanotube-7 and Double-Walled Carbon Nanotubes in Rats: Distinct Molecular Signatures and Canonical Pathways — M. Gi, S. Suzuki, et al. (2025)
• 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)
• Multi-walled carbon nanotubes translocate into the pleural cavity and induce visceral mesothelial proliferation in rats — J. Xu, M. Futakuchi, et al. (2012)
• Multiwalled carbon nanotubes intratracheally instilled into the rat lung induce development of pleural malignant mesothelioma and lung tumors — M. Suzui, M. Futakuchi, et al. (2016)

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