A subchronic inhalation toxicity study of inhaled vapor grown carbon nanofibers (CNF) (VGCF-H) was conducted in male and female Sprague Dawley rats. The CNF test sample was composed of > 99.5% carbon with virtually no catalyst metals; Brunauer, Emmett, and Teller (BET) surface area measurements of 13.8 m2/g; and mean lengths and diameters of 5.8 µm and 158 nm, respectively.Four groups of rats per sex were exposed nose-only, 6 h/day, for 5 days/week to target concentrations of 0, 0.50, 2.5, or 25 mg/m3 VGCF-H over a 90-day period and evaluated 1 day later. Assessments included conventional clinical and histopathological methods, bronchoalveolar lavage fluid (BALF) analysis, and cell proliferation (CP) studies of the terminal bronchiole (TB), alveolar duct (AD), and subpleural regions of the respiratory tract. In addition, groups of 0 and 25 mg/m3 exposed rats were evaluated at 3 months postexposure (PE). Aerosol exposures of rats to 0.54 (4.9 f/cc), 2.5 (56 f/cc), and 25 (252 f/cc) mg/m(3) of VGCF-H CNFs produced concentration-related small, detectable accumulation of extrapulmonary fibers with no adverse tissue effects. At the two highest concentrations, inflammation of the TB and AD regions of the respiratory tract was noted wherein fiber-laden alveolar macrophages had accumulated. This finding was characterized by minimal infiltrates of inflammatory cells in rats exposed to 2.5mg/m(3) CNF, inflammation along with some thickening of interstitial walls, and hypertrophy/hyperplasia of type II epithelial cells, graded as slight for the 25mg/m(3) concentration. At 3 months PE, the inflammation in the high dose was reduced. No adverse effects were observed at 0.54mg/m(3). BALF and CP endpoint increases versus controls were noted at 25mg/m(3) VGCF-H but not different from control values at 0.54 or 2.5mg/m(3). After 90 days PE, BALF biomarkers were still increased at 25mg/m(3), indicating that the inflammatory response was not fully resolved. Greater than 90% of CNF-exposed, BALF-recovered alveolar macrophages from the 25 and 2.5mg/m(3) exposure groups contained nanofibers (> 60% for 0.5mg/m(3)). A nonspecific inflammatory response was also noted in the nasal passages. The no-observed-adverse-effect level for VGCF-H nanofibers was considered to be 0.54mg/m(3) (4.9 fibers/cc) for male and female rats, based on the minimal inflammation in the terminal bronchiole and alveolar duct areas of the lungs at 2.5mg/m(3) exposures. It is noteworthy that the histopathology observations at the 2.5mg/m(3) exposure level did not correlate with the CP or BALF data at that exposure concentration. In addition, the results with CNF are compared with published findings of 90-day inhalation studies in rats with carbon nanotubes, and hypotheses are presented for potency differences based on CNT physicochemical characteristics. Finally, the (lack of) relevance of CNF for the high aspect ratio nanomaterials/fiber paradigm is discussed.

Exposure to particulate matter (PM) may exacerbate preexisting respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), bronchitis, and pneumonia. However, few experimental studies have addressed the effects of PM on lower respiratory tract (LRT) viral infection. Respiratory syncytial virus (RSV) is a major etiological agent for LRT infections in infants, the elderly, and the immunocompromised and may lead to chronic wheezing and the development of asthma in children. In this study, we examined the effects of carbon black (CB) on RSV-induced pulmonary inflammation, chemokine and cytokine expression, and airway hyperresponsiveness in a mouse model of RSV. Female BALB/c mice were instilled via the trachea (i.t.) with 1 x 106 plaque forming units (pfu) RSV or with uninfected culture media. On day 3 of infection, mice were i.t. instilled with either 40 micro g ultrafine CB particles or with saline. End points were examined on days 4, 5, 7, and 14 of RSV infection. Viral titer and clearance in the lung were unaffected by CB exposure. Neutrophil numbers were elevated on days 4 and 7, and lymphocyte numbers were higher on days 4 and 14 of infection in CB-exposed, RSV-infected mice. CB exposure also enhanced RSV-induced airway hyperresponsiveness to methacholine, bronchoalveolar lavage (BAL) total protein, and virus-associated chemokines monocyte chemoattractant protein (MCP-1), macrophage inflammatory protein (MIP-1 alpha), and regulated upon activation, normal T cell expressed and secreted (RANTES). MIP-1 alpha mRNA expression was increased in the alveolar epithelium, where ultrafine particles deposit in the lung. These data demonstrate a synergistic effect of ultrafine CB particles on RSV infection, and suggest a potential mechanism for increased respiratory infections in human populations after PM exposure.

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New approaches are needed to assess the effects of inhaled substances on human health. These approaches will be based on mechanisms of toxicity, an understanding of dosimetry, and the use of in silico modeling and in vitro test methods. In order to accelerate wider implementation of such approaches, development of adverse outcome pathways (AOPs) can help identify and address gaps in our understanding of relevant parameters for model input and mechanisms, and optimize non-animal approaches that can be used to investigate key events of toxicity. This paper describes the AOPs and the toolbox of in vitro and in silico models that can be used to assess the key events leading to toxicity following inhalation exposure. Because the optimal testing strategy will vary depending on the substance of interest, here we present a decision tree approach to identify an appropriate non-animal integrated testing strategy that incorporates consideration of a substance's physicochemical properties, relevant mechanisms of toxicity, and available in silico models and in vitro test methods. This decision tree can facilitate standardization of the testing approaches. Case study examples are presented to provide a basis for proof-of-concept testing to illustrate the utility of non-animal approaches to inform hazard identification and risk assessment of humans exposed to inhaled substances.

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