The NPCB (Printex 90) was a gift from Evonik (Frankfurt, Germany). Particle preparation, characterization, and instillation procedures were described in detail previously [Jacobsen et al., 2007 (link); Saber et al., 2009 (link); Bourdon et al., 2012 (link); Boisen et al., 2013 (link)]. NPCB has a primary particle size of 14nm and the particles were composed of 99% C, 0.8% N, and 0.01% H2 [Jacobsen et al., 2007 (link)]. The total content of polycyclic aromatic hydrocarbons in Printex 90 was 0.0742 mg/g, that is, about 3000-fold less than diesel exhaust particles [Jacobsen et al., 2007 (link)]. The endotoxin level was 0.142 EU/mg Printex 90 [Jackson et al., 2011b (link)]. The specific surface area was 295–338 m2/g, corresponding to a theoretical average spherical particle size 8.1–9.5 nm [Saber et al., 2005 (link)]. NPCB particles were suspended in 0.2 µm filtered, -irradiated Nanopure Diamond UV water (Pyrogens: < 0,001 EU/ml, total organic carbon: < 3.0 ppb [Jackson et al., 2012a (link), b (link)]), and sonicated on ice, for 16 min without pause using a Branson Sonifier S-450D (Branson Ultrasonics Corp., Danbury, CT) equipped with a disruptor horn (model number 101–147-037). Two suspensions were prepared at concentrations 0.12 mg/ml (6 µg/instillation) and 3.24 mg/ml (162 µg/instillation). A suspension with a final concentration of 0.12 mg/ml was subsequently diluted 1:3 to obtain 0.04 mg/ml (2 µg/instillation) and diluted further 1:3 for the lowest dose of 0.00134 mg/ml (0.67 µg/instillation). After each dilution, suspensions were sonicated for 2 minutes. Particle suspensions were instilled in mice directly after sonication up to 1 hr to assure their homogeneity. Vehicle solution was prepared by sonication of Nanopure Diamond water in the same conditions as described above.