We compared long-term pulmonary toxicities after a single intratracheal instillation of two types of dispersed single-wall carbon nanotubes (SWCNTs), namely, those with relatively long or short linear shapes with average lengths of 8.6 and 0.55 mm, respectively. Both types of SWCNTs were instilled intratracheally in male F344 rats at 0.2 or 1.0 mg/kg (long SWCNTs) or 1.0 mg/kg (short SWCNTs). Pulmonary responses were characterized at 26, 52 and 104 weeks after a single instillation. Inflammatory changes, test substance deposition, test substance engulfment by macrophages, and alveolar wall fibrosis were observed in the lungs of almost all test rats at 52 and 104 weeks after short nanotube instillation. The incidences of these changes were much lower in the long nanotube-treated groups. In almost all rats of the long nanotube-treated groups, fibrosis and epithelium loss in the terminal bronchiole with test substance deposition were observed. These bronchiolar changes were not observed after administering short nanotubes. Both bronchiolo-alveolar adenoma and carcinoma were found in the negative-control group, the high-dose long-nanotube group, and the short-nanotube group at 104 weeks post-instillation, although the incidences were not statistically different. The genotoxicity of the SWCNTs was also evaluated by performing in vivo comet assays with lung cells obtained 26 weeks post-instillation. No significant changes in the percent tail deoxyribonucleic acid were found in any group. These findings suggested that most long SWCNTs were deposited at the terminal bronchioles and that a considerable amount of short SWCNTs reached the alveolus, resulting in chronic inflammatory responses, but no genotoxicity in the lungs. ARTICLE HISTORY

-We aimed to evaluate the effects of the length of single-walled carbon nanotubes (SWCNTs) on pulmonary toxicity in rats. Each rat received a single intratracheal instillation of short (S-) (average length of 0.40 μm) or long (L-) (average length of 2.77 μm) SWCNTs at a dose of 1 mg/kg and was observed for the next 6 months. Neither S-nor L-SWCNTs affected clinical signs, body weight, or autopsy findings. An increase in lung weight was observed after instillation of S-or L-SWCNTs; however, lung weights were slightly higher in the rats that were administered the S-SWCNTs. Distinct differences in bronchoalveolar lavage fluid (BALF) composition were observed between the S-and L-SWCNTtreated rats as early as 7 days after the intratracheal instillations of the SWCNTs. The S-SWCNTs caused persistent lung injury and inflammation during the 6-month observational period. However, the L-SWCNTs induced minimal lung injury and inflammation. Although the S-and L-SWCNTs changed BALF parameters and histopathological features of the lung, the magnitudes of the changes observed after the S-SWCNT treatment were greater than the respective changes observed after the L-SWCNT treatment. These findings indicate that the severity of the pulmonary toxicity caused after intratracheal instillation of SWCNT depends on the length of the SWCNTs. It appears that shorter SWCNTs induce greater pulmonary toxicity than longer SWCNTs do.

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-Usefulness of bronchoalveolar lavage fluid (BALF) and pleural cavity lavage fluid (PLF) as an experimental material was evaluated for the assessment of pulmonary toxicity of chemicals in rats. From the viewpoint of safety, isoflurane can be used for euthanasia/anesthesia because there was no difference in biological properties of BALF between diethyl ether and isoflurane. Here, we also recognized phosphate buffered saline (PBS) and distilled water equally as a solvent/vehicle for negative control. PLF is also provided as a useful target material as well as BALF for assessing chemical lung toxicity. To evaluate the method, we used zinc chloride as a model chemical and obtained the expected and satisfied results. We may conclude that the intratracheal treatment and combination usage of BALF and PLF as a target material is a good method for assessment of chemical pulmonary (lung and plural cavity) toxicity in rats.