Safety of docosahexaenoic acid (DHA) administered as DHA ethyl ester in a 9-month toxicity study in dogs.

DHA Ethyl Ester (DHA-EE) is a 90% concentrated ethyl ester of docosahexaenoic acid manufactured from the microalgal oil. The objective of the 9-month study was to evaluate safety of DHA-EE administered to beagle dogs at dose levels 150, 1000 and 2000 mg/kg bw/day by oral gavage and to determine reversibility of any findings after a 2-month recovery period. DHA-EE was well tolerated at all doses. There were observations of dry flaky skin with occasional reddened areas at doses ≥1000 mg/kg bw/day. These findings lacked any microscopic correlate and were no longer present after the recovery period. There were no toxicologically relevant findings in body weights, body weight gains, food consumption, ophthalmological examinations, and ECG measurements. Test article-related changes in hematology parameters were limited to decreases in reticulocyte count in the high-dose males and considered non-adverse. In clinical chemistry parameters, dose-related decreases in cholesterol and triglycerides levels were observed at all doses in males and females and attributed to the known lipid-lowering effects of DHA. There were no effects on other clinical chemistry, urinalysis or coagulation parameters. There were no abnormal histopathology findings attributed to test article. The No-Observable-Adverse-Effect Level of DHA-EE was established at 2000 mg/kg bw/day for both genders.

Toxicological evaluation of an olive extract, H35: Subchronic toxicity in the rat.

The safety of olive extract H35 containing 35% hydroxytyrosol (HT) was tested in a 90-day oral gavage study in Wistar rats. H35 was administered at 0, 345, 691 and 1381 mg/kg bw/day, equivalent to 0, 125, 250 and 500 mg HT/kg bw/day. Reductions in terminal body weight (9%), and a statistically significant reduction in body weight gain (17%, P < 0.05) at week 13 were observed in high dose males, as well as a statistically significant increase in relative weights of the liver, heart, and kidneys of high dose males and females. These changes were not accompanied by pathological or clinical observations and a trend towards reversal was observed in the recovery phase. H35 was well-tolerated and no toxicologically significant treatment-related changes were observed in condition and appearance of rats, neurobehavioral outcomes, motor activity assessments, functional observational battery (FOB), food intake, ophthalmoscopic examinations, hematology, clinical chemistry, urinalysis, necropsy findings, sperm parameters or estrus cycle. The lowest observed adverse effect level (LOAEL) was the 500 mg HT/kg bw/day based on statistically significant reductions in body weight gain and decreased body weight in males. The no observed adverse effect level (NOAEL) was 250 mg HT/kg bw/day, equivalent to 691 mg/kg bw/day of H35 extract.

Investigations into the genotoxic potential of olive extracts.

The phenolic anti-oxidant 3-hydroxytyrosol (HT) is a major constituent of olives and olive oil. Published data showed it was negative in the Ames test at concentrations up to 5 µL per plate, but did induce chromosomal aberrations in human lymphocytes. HIDROX, an olive extract containing approximately 2.4% HT, was reported as both positive and equivocal in an Ames test in different papers from the same laboratory. Negative results for micronucleus induction in vivo in both an acute study and as part of a 90-day rat toxicity study were also reported for HIDROX. Given the widespread use and consumption of olives, olive oil and olive extracts, it was important to obtain more data. Here we confirm that pure HT, and an olive extract containing 15% HT, both induced micronuclei in cultured cells in vitro, but show that these responses were either due to high levels of cytotoxicity or to reaction of HT with culture medium components to produce hydrogen peroxide. Another extract (H40) containing 40% HT also induced micronuclei in vitro, probably via the same mechanism. However, both extracts were negative in robust Ames tests. The 15% HT formulated extract did not induce micronuclei in rat bone marrow after 4 weeks of dosing up to 561 mg HT/kg/day. H40 produced increased rat bone marrow micronucleus frequencies at 250 and 500 mg HT/kg/day in a 90-day toxicity study, but the results were questionable for various reasons. However, when two different batches of this extract were tested in acute micronucleus studies at doses up to 2000 mg HT/kg, giving plasma exposures that exceeded those in the 90-day study, negative results were obtained. Based on weight of evidence it is concluded that the olive extracts tested are not genotoxic at high doses in vivo, and any genotoxic risks for human consumers are negligible.