Research and development item: ②(b-2) Study on standardization of intratracheal administration testing: Comparison of single and multiple administrations

Implemented by the Japan Bioassay Research Center

Final objective: We decide the appropriate number of administration as a standard technique of intratracheal administration testing, include them in the draft of standard procedure manual developed in the course of the research and development item (2)(b-1), and release it.

Main results:
Establishing intratracheal administration testing methods for nanomaterials requires an investigation of whether the number of times of administration (single or multiple) affects the lung toxicity of the nanomaterial. Therefore, we used three types of nanomaterials with different characteristics and shapes (TiO₂, NiO, and multi-walled carbon nanotubes (MWCNTs)) to conduct single administration and multiple administrations (one-half the dose of single administration for two times administration, one-third the dose for three times administration, or one-fourth the dose for four times administration, the same total dose) and examined the effects on the lung mainly. We set the interval of multiple administrations to every other day and set the total dose to 10 mg/kg, 2 mg/kg, and 320 μg/kg for TiO₂, NiO, and MWCNTs, respectively. F344 rats (12-week-old) were kept up to three months after administration, and conducted for bronchoalveolar lavage fluid (BALF) analysis (3 days after the end of administration, 28 days after first administration, and 28 days after the end of administration). We also examined histopathologically respiratory systems at 28 days after first administration, 28 days after the end of administration, and three months after the end of administration.

All three materials, TiO₂, NiO, and MWCNTs, showed evidence of inflammation in BALF analysis. The NiO group continued to show marked inflammation up to 28 days post exposure; however, the TiO₂ and MWCNT groups showed moderate and mild inflammation, respectively. The NiO group continued to exhibit an increase in total protein, albumin, and LDH concentration in BALF up to 28 days post exposure, but the TiO₂ and MWCNT groups exhibited either the same level of increase as the vehicle control group or a slight increase. The NiO group exhibited increased lung weight 3 months after exposure, whereas the TiO₂ and MWCNT groups exhibited almost the same lung weight as that of the control group 3 months post exposure. In histopathological examination, the appearance in the alveolar space of macrophages that engulfed nanoparticles and the deposition of nanoparticles on the alveolar space and alveolar wall or in the bronchus-associated lymphoid tissue and lung-related lymph nodes were observed on 28 days and 3 months post exposure. In addition, the TiO₂ and NiO groups exhibited hyperplasia of type II pneumocytes; the NiO group also exhibited fibrosis and proteinosis in the alveolar space.

All three materials caused qualitatively similar changes in single administration and multiple administrations, although the degree of the changes differed slightly. Differences resulted from the number of the administrations were observed in BALF analysis at 3 days after the end of the administration. In TiO₂ and MWCNTs groups, the degree of responses in multiple administrations was lower than that in single administration in many items, while the degree of responses caused by NiO in multiple administrations was higher than that in single administration. Lung weight measurements and histopathological changes at 28 days and 3 months post exposure were also of approximately the same degree.

In conclusion; the degree of responses differed in the administrations depending on the material; however, all three materials were the same with respect to qualitative change of responses. We therefore understand that it is possible to obtain the effect of administration at any number of times of administration. On the basis of the aforementioned results, in consideration of the load to an animal and the additional complexity of experimental design owing to multiple administrations, single administration is sufficient for intratracheal administration screening testing. When only a suspension of low concentration can be prepared owing to limited dispersibility of the sample in the dosing vehicle because of the properties of the nanomaterials, multiple administrations may be necessary.