Research and development item: ②(a) Comparative study of inhalation toxicity testing and intratracheal administration testing

Implemented by the University of Occupational and Environmental Health

Final objective: To obtain basic hazard information through comparison of response between inhalation toxicity and intratracheal administration tests, we compile and release a technical instruction manual detailing points of data interpretation using intratracheal administration test.

Main results:
For comparison of inhalation toxicity testing and intratracheal administration test, we used nanoparticles with high and low lung toxicities to conduct inhalation toxicity and intratracheal administration tests. We studied the reactivity of lung inflammation as an endpoint in both tests. We used NiO and TiO₂ as nanoparticles that exhibit high and low lung toxicities among nanoparticles, respectively. In inhalation toxicity test, we performed inhalation exposure for four weeks, with a maximum concentration of approximately 2 mg/m³; analyzed the cells in bronchoalveolar lavage fluid (BALF); measured the concentration of cytokine; and performed a pathological study at 3 days, 1 month, and 3 months after exposure. In intratracheal administration testing, we injected 0.2 and 1 mg of nanoparticles as low and high doses, respectively, into the trachea of rats (distilled water was injected into negative controls); and evaluated the same endpoint as that used in inhalation toxicity test at 3 days, 1 week, 1 month, 3, and 6 months later.

In inhalation toxicity test, results for NiO nanoparticles showed infiltration of neutrophils and increase in cytokine-induced neutrophil chemoattractant (CINC)-1 and CINC-2, chemokine of neutrophils, and heme oxygenase (HO)-1, a marker of oxidative stress. In contrast, the results for TiO₂ nanoparticles did not show lung inflammation or an increase in chemokine, such as increased levels of (CINC)-1, CINC-2, and HO-1. In intratracheal administration test, results for NiO nanoparticles showed infiltration of neutrophils and a continuous increase in (CINC)-1, CINC-2, and HO-1 concentrations, whereas results for TiO₂ nanoparticles showed no increase in these endpoints or only a transient increase in the acute phase. In summary, in inhalation toxicity test, particles with high lung toxicity induced inflammation, whereas those with low lung toxicity did not; in intratracheal administration test on rats, particles with high lung toxicity induced continuous lung inflammation, whereas those of low lung toxicity induced only a transient lung inflammation.

Hence, inhalation toxicity testing and intratracheal administration testing showed similar results with respect to ranking particles of different lung toxicity, although the degree of inflammation varied. The results suggested that intratracheal administration test is a useful screening for hazard assessment of manufactured nanomaterials (Table ②(a)-1).

To evaluate the chronic effect of TiO₂ nanoparticles, we administered TiO₂ nanoparticles into the trachea of rats and observed the pathology in the lung for a maximum observation period of two years, which generally is the lifespan of a rat. The TiO₂ nanoparticles used in this testing were P90, and their crystal structures were rutile and anatase. We administered 0.2 and 1 mg of nanoparticles as low and high doses, respectively, intratracheally to rats and injected distilled water into the trachea of negative controls. We analyzed data at 3 days, one week, 1 month, 3, 6, 12, and 24 months after intratracheal administration. As the endpoint, from 3 days to 6 months after administration (the acute phase), an endpoint centered on inflammation was used, and from 12 to 24 months (the chronic phase), the degree of fibrosis and the incidence rate of tumor were used. In the acute phase, the 1-mg administration group exhibited a temporary increase in the total cell count and neutrophil count in BALF and macrophage count 3 days after intratracheal administration; however, these counts decreased over time to the same level as those of the negative control group. The 0.2-mg administration group showed no difference compared with the negative control group. The cytokine concentration in BALF also showed a similar tendency. In histopathological features, an increase in transient infiltration of macrophage was observed from 3 days after administration to one week after administration, but it decreased with time to the level of the negative control group. In the chronic phase, fibrosis and tumors were not observed throughout the observation period. In intratracheal administration test of TiO₂ nanoparticles in rats, transient inflammation and an associated change in cytokine were observed, but continuous inflammation, fibrosis, and tumor formation were not observed. These results suggested that the TiO₂ nanoparticles used in this test had low effect on the lung (Table ②(a)-2).

To study the biological effects of CeO₂ nanoparticles, we performed inhalation toxicity and intratracheal administration tests using lung inflammation as an endpoint. In inhalation toxicity test, we performed inhalation exposure to F344 rats at a low exposure concentration of 2.1 mg/m³ and a high concentration of 10.2 mg/m³ for four weeks (6 h per day, five days per week). The primary particle size was approximately 8 nm. We analyzed the cells in BALF at 3 days, 1 month, and 3 months after exposure. In intratracheal administration testing, we used the same CeO₂ suspension as that in inhalation toxicity testing to conduct intratracheal administration to rats with doses of 0.2 and 1 mg/rat. We analyzed the cells in BALF similar to inhalation toxicity test at 3 days, one week, 1 month, 3 months, and 6 months after administration. In inhalation toxicity test, CeO₂ at both high and low concentrations induced an increase in neutrophil count in BALF. In intratracheal administration test, a continuous increase in neutrophil count was observed (Table ②(a)-3).

In addition, we are conducting inhalation toxicity and intratracheal administration tests of new manufactured nanomaterials. To obtain basic hazard information, we will study similarities and differences between the results of inhalation toxicity and intratracheal administration tests to compile points on data interpretation using intratracheal administration test.

Table②(a)-1 Summary of results of intratracheal administration testing and inhalation toxicity testing of NiO and TiO₂ nanoparticles

Table②(a)-2 Intratracheal administration testing of TiO₂ nanoparticles (P90)

Table②(a)-3 Summary of results of intratracheal administration testing and inhalation toxicity testing of CeO₂ nanoparticles