Information on particle deposition, retention and clearance are important for the evaluation of the risk of inhaled nanomaterials to human health. Recent revised OECD inhalation toxicity test guidelines require to evaluate the lung burden of nanomaterials after rodent subacute and subchronic inhalation exposure (OECD 412, OECD 413). These revised test guidelines require additional post-exposure observation (PEO) periods that include lung burden measurements that can inform on lung clearance behavior and translocation. The latter being particularly relevant when the testing chemical is a solid poorly soluble nanomaterial. Therefore, in the spirit of 3 R’s, we investigated whether measurement of retained lung burden of inhaled nanoparticles (NPs) in individual lung lobes is sufficient to determine retained lung burden in the total lung. If it is possible to use only one lobe, it will reduce animal use and maximize the number of endpoints evaluated. To achieve these goals, rats were exposed nose-only for 1 or 5 days (6 h/day) to an aerosol of 20 nm well-dispersed silver nanoparticles (AgNPs), which is the desired particle diameter resulting in maximum deposition in the pulmonary region when inhaled as singlets. After exposure, the five lung lobes were separated and silver concentration was measured using inductively coupled plasma-mass spectrophotometer (ICP-MS). The results showed that the retention of deposited silver nanoparticle in the different lung lobes did not show any statistically significant difference among lung lobes in terms of silver mass per gram lung lobe. This novel finding of evenness of retention/deposition of inhaled 20 nm NPs in rats for all five lobes in terms of mass per unit tissue weight contrasts with earlier studies reporting greater apical lobe deposition of inhaled micro-particles in rodents. The difference is most likely due to preferred and efficient deposition of inhaled NPs by diffusion vs. additional deposition by sedimentation and impaction for micron-sized particles. AgNPs following acute inhalation by rats are evenly retained in each lung lobe in terms of mass per unit lung tissue weight. Accordingly, we suggest sampling any of the rat lung lobes for lung burden analysis can be used to determine deposited or retained total lung burden after short-term inhalation of NPs and using the other lobes for collecting and analyzing bronchoalveolar lavage fluid (BALF) and for histopathological analysis. Therefore, by combining lung burden measurement, histopathological tissue preparation, and BALF assay in the same rat will reduce the number of animals used and maximize the number of endpoints measured.

The inhalation toxicity of carbon nanofibers (CNFs) is not clearly known due to relatively few related studies reported. An acute inhalation study and short-term inhalation study (5 days) were therefore conducted using Sprague-Dawley rats. In the acute inhalation study, the rats were grouped and exposed to a fresh air control or to low (0.238 ± 0.197), moderate (1.935 ± 0.159), or high (24.696 ± 6.336 mg/m³) CNF concentrations for 6 h and thereafter sacrificed at 14 days. For the short-term inhalation study, the rats were grouped and exposed to a fresh air control or low (0.593 ± 0.019), moderate (2.487 ± 0.213), or high (10.345 ± 0.541 mg/m³) CNF concentrations for 6 h/day for 5 days and sacrificed at 1, 3, and 21 days post-exposure. No mortality was observed in the acute inhalation study. Thus, the CNF LC₅₀ was higher than 25 mg/m³. No significant body or organ weight changes were noted during the 5 days short-term inhalation study or during the post-exposure period. No significant effects of toxicological importance were observed in the hematological, blood biochemical, and coagulation tests. In addition, the bronchoalveolar lavage (BAL) fluid cell differential counts and BAL inflammatory markers showed no CNF-exposure-relevant changes. The histopathological examination also found no CNF-exposure-relevant histopathological lesions. Thus, neither acute nor 5 days inhalation exposure to CNFs induced any noticeable toxicological responses.