Applicability of in silico genotoxicity models on food and feed ingredients.

Evaluation of the genotoxic potential of food and feed ingredients is required in the development of new substances and for their registration. In addition to in vitro and in vivo assays, in silico tools such as expert alert-based and statistical models can be used for data generation. These in silico models are commonly used among the pharmaceutical industry, whereas the food industry has not widely adopted them. In this study, the applicability of in silico tools for predicting genotoxicity was evaluated, with a focus on bacterial mutagenicity, in vitro and in vivo chromosome damage assays. For this purpose, a test set of 27 food and feed ingredients including vitamins, carotenoids, and nutraceuticals with experimental genotoxicity data was constructed from proprietary data. This dataset was run through multiple models and the model applicability was analyzed. The compounds were generally within the applicability domain of the models and the models predicted the compounds correctly in most of the cases. Although the regulatory acceptance of in silico tools as single data source is still limited, the models are applicable and can be used in the safety evaluation of food and feed ingredients.

Slight hypercalcemia is not associated with positive responses in the Comet Assay in male rat liver.

Maintenance of physiological levels of intracellular and extracellular calcium is essential for life. Increased intracellular calcium levels are involved in cell death (apoptosis and necrosis) and are associated with positive responses in the Comet assay in vitro. In addition, high calcium and vitamin D intakes were reported to induce apoptosis in adipose tissue in obese mice and to increase DNA-migration in the Comet assay. To investigate increased serum concentration of calcium as a potential confounding factor in the regulatory Comet assay in vivo, we induced mild hypercalcemia in male Wistar rats by 3-day continuous intravenous infusion of calcium gluconate and performed the Comet assay in the liver in line with regulatory guidelines. The results of the study showed that mild increases in serum calcium concentration (up to 1.4 times above the concurrent control) and increased urinary calcium concentration (up to 27.8 times above the concurrent control) results in clinical signs like mild tremor, faster respiration rate and decreased activity in a few animals. However, under the conditions of the study, no increase in the %Tail DNA in the Comet assay and no indication of liver damage as determined by histopathological means were observed. Thus, mild increases in plasma calcium did not lead to positive results in a genotoxicity assessment by the Comet assay in the rat liver. This result is important as it confirms the reliability of this assay for regulatory evaluation of safety.

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.

Intra- and inter-laboratory reproducibility and accuracy of the LuSens assay: A reporter gene-cell line to detect keratinocyte activation by skin sensitizers.

Several non-animal methods are now available to address the key events leading to skin sensitization as defined by the adverse outcome pathway. The KeratinoSens assay addresses the cellular event of keratinocyte activation and is a method accepted under OECD TG 442D. In this study, the results of an inter-laboratory evaluation of the "me-too" LuSens assay, a bioassay that uses a human keratinocyte cell line harboring a reporter gene construct composed of the rat antioxidant response element (ARE) of the NADPH:quinone oxidoreductase 1 gene and the luciferase gene, are described. Earlier in-house validation with 74 substances showed an accuracy of 82% in comparison to human data. When used in a battery of non-animal methods, even higher predictivity is achieved. To meet European validation criteria, a multicenter study was conducted in 5 laboratories. The study was divided into two phases, to assess 1) transferability of the method, and 2) reproducibility and accuracy. Phase I was performed by testing 8 non-coded test substances; the results showed a good transferability to naïve laboratories even without on-site training. Phase II was performed with 20 coded test substances (performance standards recommended by OECD, 2015). In this phase, the intra- and inter-laboratory reproducibility as well as accuracy of the method was evaluated. The data demonstrate a remarkable reproducibility of 100% and an accuracy of over 80% in identifying skin sensitizers, indicating a good concordance with in vivo data. These results demonstrate good transferability, reliability and accuracy of the method thereby achieving the standards necessary for use in a regulatory setting to detect skin sensitizers.

Review of genotoxicity and rat carcinogenicity investigations with astaxanthin.

Synthetic astaxanthin has been extensively tested for safety. Genotoxicity studies including Ames and in vitro Micronucleus Tests show absence of genotoxic potential. Although a long-term mouse study showed no carcinogenicity potential, the rat carcinogenicity study with dietary dosages of 0 (control), 0 (placebo beadlet), 40, 200 and 1000 mg astaxanthin/kg bw/day showed an increased incidence of benign, hepatocellular adenoma in females only, at 200 mg/kg bw/day and above. There was no clear evidence of toxicity during the in-life phase. Discoloration of feces was observed and a reduction in body weight gain in all groups receiving beadlets, probably reflecting a nutritional influence. Blood sampling confirmed systemic exposure and some minor clinical chemistry differences in females at 200 and 1000 mg/kg bw/day. There was no effect on adjusted liver weight. Histopathological examination showed hepatic changes indicative of slight hepatotoxicity and hepatocyte regeneration in females at 200 and 1000 mg/kg bw/day, in addition to the adenoma. Taking into account this pathological background in the female rat, and a wide variety of other supporting information, it is concluded that the hepatocellular adenoma in female rats was secondary to hepatotoxicity and regeneration, and is most probably a species-specific phenomenon of doubtful human relevance.

Subchronic (13-week) toxicity and prenatal developmental toxicity studies of dietary astaxanthin in rats.

Two studies examined the effects of dietary astaxanthin on Hanlbm Wistar (SPF) rats. Male and female rats receiving astaxanthin concentrations up to 1.52% of the feed for 13 weeks showed no evidence of toxicity; no effects were noted in the offspring of female rats exposed to astaxanthin at up to 1.39% of the feed during the period of organogenesis (GD 7-16). Discoloration of the feces and yellow pigmentation of adipose tissue was seen in the 13-week study, an intrinsic property of the substance, and not a sign of toxicity. Differences between the control and astaxanthin groups, some of which reached statistical significance, were generally sporadic (i.e., transient and/or not related to astaxanthin concentration) and not considered of biological or toxicological significance. Blood cholesterol levels, for example, were greater in animals receiving astaxanthin for 13 weeks, but remained within the normal range. The highest dietary concentration of astaxanthin in each of the studies is proposed as a no-observable-adverse-effect level (NOAEL). Specifically, 1.52% for the 13-week study, corresponding to a mean intake of 1033 mg/kg bw/day (range: 880-1240 mg/kg bw/day), and 1.39% for the developmental toxicity study, corresponding to a mean intake of approximately 830 mg/kg bw/day (range: 457-957 mg/kg bw/day).

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.

Black pepper constituent piperine: genotoxicity studies in vitro and in vivo.

Piperine is responsible for the hot taste of black pepper. Publications on genotoxicity of piperine are reported: negative Ames Tests and one in vitro micronucleus test (MNT). In vivo tests were mainly negative. In the majority of the data the administered dose levels did not follow the dose selection requirements of regulatory guidelines of having dose levels up to the maximum tolerated dose (MTD). The only oral high dose studies were a positive in vivo MNT in mice in contrast to a negative in vivo chromosome aberration test in rats. Thus, conflicting results in genotoxicity testing are published. To investigate this further, we administered piperine to mice up to the MTD and determined micronuclei-frequency. Piperine reduces core body temperature and interferes with blood cells both being known to result in irrelevant positive in vivo MNTs. Therefore we added mechanistic endpoints: core body temperature, haematology, erythropoietin level, and organ weights. Additionally an in vitro MNT in Chinese hamster ovary cells was performed. Piperine was negative in the in vitro MNT. It caused significant reduction of core body temperature, decrease of white blood cells and spleen weights but no increase in the micronucleus-frequency. Thus, in our studies piperine was not genotoxic.