Research on the uses and manufacturing of nano graphene has increased dramatically in the past decade. Thus, worker inhalation of graphene nanopowders is likely. The goal of this study was to evaluate the lung toxicity of three non-oxidized graphene (Gr) samples of different sizes [20 um lateral x 7-10 nm thick (Gr20), 5 um lateral x 7-10 nm thick (Gr5), and <2 um lateral x 1-2 nm thick (Gr1)]. Gr samples were diluted in physiological dispersion medium (DM) and characterized for size, surface reactivity, and free radical generation in vitro. Male C57BL/6J mice received 4 or 40 ug of Gr1, Gr5, or Gr20, or 40 ug of carbon black (CB; particle control), or DM (vehicle control) by aspiration. Mice were sacrificed at 4 hr (day 0), 1, 7, and 28 days post-exposure. Lung lavage was performed, the fluid and cells were retained, and indices of lung injury and inflammation were examined. Particle/aggregate size ranged from ~ 5-300 um, 0.5-60 um, and 0.2-5um for Gr20, Gr5, and Gr1, respectively, with CB being similar to Gr1. Electron spin resonance (ESR) indicated that all Gr samples and CB had low to no surface reactivity as compared to a positive control (á-quartz). In vitro, ESR showed all Gr samples induced free radical production by mouse monocytes with significantly greater response in Gr20- and Gr5-treated cells compared to Gr1- and CB-treated cells. Indices of lung injury in lavage fluid were increased for the 40 ug doses of Gr20 and Gr5 on days 0, 1, and 7 when compared to control. Gr1 (40 ug) produced an increase only at day 7. Increased lung injury in the CB group was comparable to Gr20 and Gr5 on days 1 and 7. Injury decreased in all groups by day 28. Inflammation was elevated in the 40 ug Gr20, Gr5, Gr1 and CB groups on day 1, but only in the 40 ug Gr20 and Gr5 groups on days 0 and 7. In summary, the larger Gr particles appeared to produce more toxicity at the early time points post-exposure when compared to controls.
The Toxicologist. Society of Toxicology 52nd Annual Meeting and ToxExpo, March 10-14, 2013, San Antonio, Texas