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.

中文翻译：

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吸入银纳米粒子在大鼠肺部沉积/保留的叶叶均匀性：一种减少动物使用同时最大化终点的方法

有关颗粒沉积、保留和清除的信息对于评估吸入纳米材料对人类健康的风险非常重要。最近修订的 OECD 吸入毒性测试指南要求评估啮齿动物亚急性和亚慢性吸入暴露后纳米材料的肺部负担（OECD 412、OECD 413）。这些修订后的测试指南要求额外的暴露后观察（PEO）期，其中包括肺负荷测量，可以了解肺清除行为和易位。当测试化学品是固体难溶性纳米材料时，后者尤其相关。因此，本着 3R 的精神，我们研究了测量单个肺叶中吸入纳米粒子 (NP) 的保留肺负荷是否足以确定整个肺中的保留肺负荷。如果可以仅使用一个叶，则将减少动物的使用并最大限度地增加评估的终点数量。为了实现这些目标，大鼠仅通过鼻子接触 20 nm 分散良好的银纳米粒子 (AgNP) 气溶胶 1 或 5 天（6 小时/天），这是导致肺部最大沉积的所需粒径。当作为单线吸入时的区域。暴露后，分离五个肺叶并使用电感耦合等离子体质谱仪（ICP-MS）测量银浓度。结果表明，沉积的银纳米颗粒在不同肺叶中的保留量在每克肺叶的银质量方面没有显示出任何肺叶之间的统计学显着差异。这一新发现表明，大鼠吸入的 20 nm 纳米粒子在所有五个叶中的保留/沉积均匀性（以每单位组织重量的质量计）与早期研究报告的啮齿类动物中吸入微粒的顶叶沉积量更大形成鲜明对比。这种差异很可能是由于通过扩散对吸入的纳米颗粒进行优先且有效的沉积，而不是通过沉降和撞击微米级颗粒进行额外沉积。大鼠急性吸入后，AgNPs 以每单位肺组织重量的质量均匀地保留在每个肺叶中。因此，我们建议对任何大鼠肺叶进行采样进行肺负荷分析，可用于确定短期吸入 NP 后沉积或保留的总肺负荷，并使用其他肺叶收集和分析支气管肺泡灌洗液 (BALF) 和组织病理学分析。因此，通过在同一只大鼠中结合肺负荷测量、组织病理学组织制备和 BALF 测定，将减少使用的动物数量并最大化测量的终点数量。