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The applications of magnetic nanoparticles (MNPs) as biocatalysts in different biomedical areas have been evolved very recently. One of the main challenges in this field is to design affective MNPs surfaces with catalytically active atomic centres, while producing minimal toxicological side effects on the hosting cell or tissues. MNPs of vanadium spinel ferrite (VFe 2 O 4 ) are a promising material for mimicking the action of natural enzymes in degrading harmful substrates due to the presence of active V 5+ centres. However, the toxicity of this material has not been yet studied in detail enough to grant biomedical safety. In this work, we have extensively measured the structural, compositional, and magnetic properties of a series of V x Fe 3-x O 4 spinel ferrite MNPs to assess the surface composition and oxidation state of V atoms, and also performed systematic and extensive in vitro cytotoxicity and genotoxicity testing required to assess their safety in potential clinical applications. We could establish the presence of V 5+ at the particle surface even in water-based colloidal samples at pH 7, as well as different amounts of V 2+ and V 3+ substitution at the A and B sites of the spinel structure. All samples showed large heating efficiency with Specific Loss Power values up to 400 W/g (H 0  = 30 kA/m; f = 700 kHz). Samples analysed for safety in human hepatocellular carcinoma (HepG2) cell line with up to 24h of exposure showed that these MNPs did not induce major genomic abnormalities such as micronuclei, nuclear buds, or nucleoplasmic bridges (MNIs, NBUDs, and NPBs), nor did they cause DNA double-strand breaks (DSBs) or aneugenic effects-types of damage considered most harmful to cellular genetic material. The present study is an essential step towards the use of these type of nanomaterials in any biomedical or clinical application., Competing Interests: Declaration of competing interest The authors declare that they do not have any affiliations that would lead to conflicts of interest. There were no financial or non-financial forms of assistance provided by third parties specifically for the conduct of the research presented in this manuscript. The authors have had no financial interests or relationships within the past three years that are related to the subject matter of the manuscript, including advisory roles, consulting, equity ownership, or receipt of non-financial support. None of the co-authors hold any patents or copyrights relevant to the work in this manuscript. There are no additional activities or relationships that the authors need to disclose, which could be perceived as influencing the research or its interpretation., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)