Safety Evaluation of Engineered Nanomaterials for Health Risk Assessment: An Experimental Tiered Testing Approach Using Pristine and Functionalized Carbon Nanotubes
Increasing application of engineered nanomaterials within occupational, environmental, and consumer settings has raised the levels of public concern regarding possible adverse effects on human health. We applied a tiered testing strategy including (i) a first in vitro stage to investigate general toxicity endpoints, followed by (ii) a focused in vivo experiment. Cytotoxicity of laboratory-made functionalized multiwalled carbon nanotubes (CNTs) (i.e., MW-COOH and MW-NH2), compared to pristine MWCNTs, carbon black, and silica, has been assessed in human A549 pneumocytes by MTT assay and calcein/propidium iodide (PI) staining. Purity and physicochemical properties of the test nanomaterials were also determined. Subsequently, pulmonary toxic effects were assessed in rats, 16 days after MWCNTs i.t. administration (1？mg/kg b.w.), investigating lung histopathology and monitoring several markers of lung toxicity, inflammation, and fibrosis. In vitro data: calcein/PI test indicated no cell viability loss after all CNTs treatment; MTT assay showed false positive cytotoxic response, occurring not dose dependently at exceedingly low CNT concentrations (1？μg/mL). In vivo results demonstrated a general pulmonary toxicity coupled with inflammatory response, without overt signs of fibrosis and granuloma formation, irrespective of nanotube functionalization. This multitiered approach contributed to clarifying the CNT toxicity mechanisms improving the overall understanding of the possible adverse outcomes resulting from CNT exposure. 1. Introduction Nanotechnology is one of the fastest emerging fields involving development and manipulation of materials ≤100？nm in size. There are numerous potential perspectives for the applications arising from nanotechnology which include their use in a wide range of fields, for example, medicine, environment, occupational setting, chemistry, energy production, information and communication, heavy industry, and consumer goods. Human exposure to engineered nanomaterials (ENMs) can occur at different stages of their life cycle (manufacture, use, and disposal) and several concerns have been expressed about their potential to cause unanticipated adverse effects in humans. The remarkable diversity of engineered nanomaterials (ENMs), together with their unique properties and behaviour, complicates their risk assessment; there are currently about 50,000 different types of carbon nanotubes obtained by different raw materials and production processes. Similar diversity applies to other types of ENMs, rendering ad hoc risk assessment of all of
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