Lack of pulmonary fibrogenicity and carcinogenicity of titanium dioxide nanoparticles in 26-week inhalation study in rasH2 mouse model

Background With the rapid development of alternative methods based on the spirit of animal welfare, the publications of animal studies evaluating endpoints such as cancer have been extremely reduced. There have been no systemic inhalation exposure studies of titanium dioxide nanoparticles (TiO2 NPs) using CByB6F1-Tg(HRAS)2Jic (rasH2) 26-week study mice model for detecting carcinogenicity. Methods Male and female rasH2 mice were exposed to 2, 8 or 32 mg/m3 of TiO2 NPs for 6 hours/day, 5 days/week for 26 weeks using a whole-body inhalation exposure system, with reference to the Organization for Economic Co-operation and Development principles of Good Laboratory Practice. All tissues including lungs, and blood were collected and subjected to biological and histopathological analyses. Additionally, Ki67 positive index were evaluated in mice lung alveolar epithelial type 2 cell (AEC2). Results This study established a stable method for generating and exposing TiO2 NPs aerosol, and clarified the dose-response relationship by TiO2 NPs inhalation to rasH2 mice. TiO2 NPs exposure induced deposition of particles in lungs and mediastinal lymph nodes in a dose-dependent manner in each exposure group. Additionally, alveolar inflammation was only observed in 32 mg/m3 exposure group in both the sexes. Exposure to TiO2 NPs, as well as other organs, did not increase the incidence of lung tumors in any group, and pulmonary fibrosis and pre-neoplastic lesions were not observed in all groups. Finally, the cell proliferative activity of AEC2 was examined, and it was not increased by exposure to TiO2 NPs. Conclusions This is the first report showing the lack of pulmonary fibrogenicity and carcinogenicity (no evidence of carcinogenic activity) of TiO2 NPs in 26-week inhalation study in rasH2 mice exposed up to 32 mg/m3, which is considered to be a high concentration. Macrophages undergoing phagocytosis due to TiO2 NPs exposure formed inflammatory foci in the alveolar regions of exposed mice but did not develop fibrosis or hyperplasia or tumors. Moreover, the cell proliferative ability of AEC2 in lesions was not increased. In addition, no carcinogenicity was observed for any organs other than the lungs in this study. ### Competing Interest Statement The authors have declared no competing interest. * AEC2 : Alveolar epithelial type 2 cell AO : adverse outcome AOP : adverse outcome pathway σg : geometric standard deviation HE : Hematoxylin and eosin IARC : International Agency for Research on Cancer IE : initiating event KE : key event LDH : Lactate dehydrogenase LPCAT1 : Lysophosphatidylcholine acyltransferase 1 MMAD : Mass median aerodynamic diameter NBF : Neutral buffered formalin PSLT : poorly soluble, low toxicity rasH2 : Jic:CB6F1-Tg ras H2@Jcl SEM : Scanning electron microscope SUR : lesion-surrounding tissue TiO2 NPs : Titanium dioxide nanoparticles