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Xyloglucan-block-polycaprolactone (XGO-PCL) copolymer nanoparticles have been proposed as nanocarriers for drug delivery. However, the possible harmful effects of exposure to nanoparticles still remain a concern. Therefore, the aim of this study is to evaluate the potential toxicity of XGO-PCL nanoparticles using in vitro and in vivo assays. Cytotoxicity and genotoxicity studies were conducted on MRC-5 human fetal lung fibroblast cells upon exposure to XGO-PCL nanoparticles. No significant reduction in the cell viability and no DNA damage were observed at the different concentrations tested. Erythrocyte toxicity was assessed by the incubation of nanoparticles with human blood. XGO-PCL nanoparticles induced a hemolytic ratio of less than 1%, indicating good blood compatibility. Finally, the subacute toxicity of XGO-PCL nanoparticles (10 mg/kg/day) was evaluated in BALB/c mice when administered orally or intraperitoneally for 14 days. Results of the in vivo toxicity study showed no clinical signs of toxicity, mortality, weight loss, or hematological and biochemical alterations after treatment with nanoparticles. Also, microscopic analysis of the major organs revealed no histopathological abnormalities, corroborating the previous results. Thus, it can be concluded that XGO-PCL nanoparticles induced no effect indicative of toxicity, indicating their potential use as drug delivery systems., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)