Contents lists available at ScienceDirect Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox Acute and repeated dose (28 days) oral safety studies of phosphatidyl- hydroxytyrosol María-Aránzazu Martínez a,∗ , Irma Ares a , María-Rosa Martínez-Larrañaga a , Arturo Anadón a , Victor Casado b,c , Luis Vazquez b , Diana Martin b,c , Guillermo Reglero b,c,d , Carlos Torres b,c a Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Universidad Complutense de Madrid, 28040, Madrid, Spain b Departamento de Producción y Caracterización de Nuevos Alimentos, Instituto de Investigación en Ciencias de la Alimentación (CIAL) (CSIC-UAM), 28049, Madrid, Spain c Sección Departamental de Ciencias de la Alimentación, Facultad de Ciencias, Universidad Autónoma de Madrid, 28049 Madrid, Spain d Imdea-Food Institute, CEI UAM+CSIC, 28049, Madrid, Spain ARTICLE INFO Keywords: Phosphatidyl-hydroxytyrosol Functional foods Safety evaluation In vivo toxicity study Rats ABSTRACT Phosphatidyl-hydroxytyrosol, a carrier of hydroxytyrosol under the form of phospholipid with high antioxidant capacity, is being actively studied as a potential ingredient of functional foods and supplements. To support the safety, phosphatidyl-hydroxytyrosol has been examined in an acute and in a 28-day repeated dose oral toxicity studies in rats. Phosphatidyl-hydroxytyrosol administered in a single oral gavage dose of 2000 mg/kg of body weight (bw) resulted in no adverse events or mortality. In addition, phosphatidyl-hydroxytyrosol administered as a daily dose of 2000 mg/kg bw for 28 days by gavage resulted in no adverse events or mortality. No evidence or treatment related toxicity was detected during both studies. Data analysis of body weight gain, food con- sumption, clinical observations, blood biochemical, haematology, organ weight ratios and histopathological findings did not show significant differences between control and treated groups. It is concluded that phos- phatidyl-hydroxytirosol orally administered to rats was safe and that no treatment-related toxicity was detected even at the high doses investigated in both acute (2000 mg/kg bw) and repeated dose (28-day) oral (2000 mg/kg bw) toxicity studies. 1. Introduction The popularity of phenolic compounds in general as bioactive nat- ural antioxidants is currently well-known. Within polyphenolic com- pounds, hydroxytyrosol, the major phenolic compound present in virgin olive oil, has been an attractive molecule in the last decades that has shown a great bioactivity and antioxidant power, which has been re- lated to antiatherogenic, antiplatelet aggregation, anti-inflammatory, antimicrobial, and antitumor effects, or aging regulation (Cornwell and Ma, 2008; Granados-Principal et al., 2010; Killeen et al., 2011). Al- though hydroxytyrosol is well absorbed at the gastrointestinal tract, it is rapidly metabolized in enterocyte and liver and it is only found at minor levels in plasma and tissues (D'Angelo et al., 2001; De la Torre, 2008; Rubió et al., 2014). The production of “phenolipids”, namely, lipophilized phenolics resulting from the union of a lipid to the phenolic moiety (Laguerre et al., 2010), has been explored by diverse authors to obtain carriers of hydroxytyrosol. Concerning novel derivatives of phenolic compounds, Casado et al. (2013, 2014) have recently devel- oped a solid to solid reaction system for transphosphatidylation of phosphatidylcholine with two different phenylalkanols, namely tyrosol and hydroxytyrosol, obtaining a new phospholipid, phosphatidyl-hy- droxytyrosol (PHT). Remarkable antioxidant activity of this new mo- lecule for edible oils has been described. It was shown a significant antioxidant activity of PHT in diverse edible oils, which was compar- able or even superior to hydroxytyrosol (Martin et al., 2014a). This new phospholipid (phosphatidyl-hydroxytyrosol, PHT) was proposed as a potential vehicle of hydroxytyrosol considering also the additional re- levance of the phospholipid backbone. Phospholipids are well-known essential molecules for the maintenance of living cells as major con- stituents of cell membranes. Additionally, a positive impact of dietary phospholipids on human health, such as relevant implications in hy- percholesterolemia, atherosclerosis, cardiovascular disease, inflamma- tion and immunity, liver disorders, brain development, as well other chronic diseases has been pointed out (Espinosa-Salinas et al., 2011). https://doi.org/10.1016/j.fct.2018.07.046 Received 19 June 2018; Received in revised form 19 July 2018; Accepted 24 July 2018 ∗ Corresponding author. Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Universidad Complutense de Madrid, 28040, Madrid, Spain. E-mail address: arantxam@vet.ucm.es (M.-A. Martínez). Food and Chemical Toxicology 120 (2018) 462–471 Available online 25 July 2018 0278-6915/ © 2018 Published by Elsevier Ltd. T