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Robert R Maronpot, Yuval Ramot , Mihoko Koyanagi, Nicola Dias, David Cameron, Samuel Eniola, Abraham Nyska, and Shim-mo Hayashi
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Background: Alpha-glycosyl isoquercitrin (AGIQ) is widely used as an anti-oxidative food additive in many products. Nevertheless, information on its safety and toxicity is still very limited. A 90-day toxicity study in rats and comprehensive genotoxic studies have proven AGIQ to be safe.

Aim: The goal of this study was to assess the safety of use of AGIQ in infant formula. To test for potential adverse effects on growth or other safety issues specific for young animals, we performed a 10-day and a 4-week study in preweaning Göttingen minipigs.

Method: Newborn minipigs were treated four times per day with oral AGIQ (0, 100, 300, or 1000 mg/kg/day) for 10 days or 4 weeks.

Results: All animals remained in good health throughout both studies, and there were no treatment-related signs of toxicity or abnormal findings in blood parameters. In the 4-week study, yellow coloration of the bones was seen in all animals in the high-dose group with no related histological findings. Hepatocellular and sinusoidal/Kupffer cell iron deposition was seen in the liver of all animals in both studies, as expected after the routine administration of supplemental iron to newborn animals. There was sufficient evidence of systemic exposure based on plasma levels of AGIQ metabolites.

Conclusion: Taken together, these findings show that oral administration of AGIQ in reconstituted milk supplement to preweaning Göttingen minipigs for 4 weeks at up to 1000 mg/kg/day does not result in any adverse treatment-related effects and further support the safety of AGIQ as a food additive.

Quercetin is a flavonol that is abundant in plants, and together with its glycosides has proven to exert a large number of positive biological functions, such as anti-inflammatory and anti-oxidative effects,1,2 in addition to hepatoprotective, anti-cancer properties and improvement of metabolic disturbances.3–6 Isoquercitrin is a glycoside of quercitrin with a higher bioavailability than quercetin; however, natural amounts of isoquercitrin in plants are low. Large quantities of isoquercitrin can be produced via enzymatic hydrolysis of rutin followed by transglycosylation with dextrin and cyclodextrin glucanotransferase.7–9 The product of this enzymatic process is termed enzymatically modified isoquercitrin or alpha-glycosyl isoquercitrin (AGIQ). AGIQ has been found to possess a higher bioavailability than quercetin or isoquercitrin in humans10 and has proven pharmacological benefits in vivo, including tumor suppressive and anti-oxidative effects.11–19

AGIQ has been used as a natural food additive in Japan since 1987 and is in wide use as an anti-oxidant in a large number of products, ranging from beverages, frozen dairy products and puddings to chewing gums and canned soups.20 Following the Japanese approval in 1996, the Expert Panel of the Flavor and Extract Manufacturers Association granted AGIQ a generally recognized as safe status in 2005, and the same status was granted by the US Food and Drug Administration (FDA) in 2007.21 However, although it is in wide use and sold as a food additive, information on its safety and toxicity is still limited, and based mostly on studies that used AGIQ of low purity or on studies that were not good laboratory practice (GLP)-compliant.21–24 In anticipation of the potential use of AGIQ in infant formula, we conducted this study in preweaning animals to test for potential adverse effects on growth or other safety issues specific for young animals.

According to Constable et al.,25 who recently extensively discussed an integrated approach to the safety assessment of food additives in early life, currently there is neither scientific consensus nor guidance from regulatory bodies on the most suitable animal species to address toxicological data gaps in the early life period. It is suggested that the early life stage of pigs is close to human early life stage.26 Since there is only limited safety information on isoquercitrin and AGIQ, we have performed a comprehensive evaluation process of highly purified AGIQ in a series of GLP-compliant tests in accordance with US FDA, European Food Safety Authority, and the Organisation for Economic Co-operation and Development (OECD) guidelines. As part of this evaluation process, we have performed a 90-day repeat dose toxicity and single dose toxicokinetic (TK) study in Sprague-Dawley rats, which showed no signs of toxicity.27 A genotoxicity assessment revealed that both AGIQ and isoquercitrin caused mutations in several bacterial strains, but no mutagenic potential was evident in any of the several tissues when tested in transgenic mice. Furthermore, although isoquercitrin tested positive in the chromosomal aberration test in Chinese hamster ovary (CHO) cells, all additional in vivo and in vitro assays did not show any numerical or structural chromosomal or DNA damage.28 Taking into consideration the above-mentioned rationale for the suitability of the minipig model, here we report the results of safety and TK studies performed in preweaning Göttingen minipigs. A preliminary 10-day study was performed to aid the selection of the doses for the main 4-week toxicity study.

Animal husbandry and maintenance

Pregnant Göttingen minipigs were obtained from Ellegaard Göttingen Minipigs, Denmark, and were allowed an acclimatization period of at least 4 weeks prior to the expected farrowing date. The test animals were unweaned piglets selected from the litters produced. The piglets were initially housed in litters (with sow) for at least the first 24 h. Thereafter, they were housed with their litter mates (without sow). The first study treatment was given at the age of 2 days. Uniferon® iron III dextran (Pharmacosmos, Denmark) was administered on postnatal day (PND) 1 or 2 (0.5 mL/kg administered intramuscularly into a hind leg).

The animals were housed in indoor pens with farrowing rails, separate piglet creep areas, and infrared heat lamps as appropriate. Temperature was monitored and maintained within the range of 15–24°C, and artificial lighting dictated a 12-h light/12-h dark cycle. For the first approximately 24 h after birth (PND 1/2), the piglets were allowed to suckle from the sow. Afterward, the piglets were given reconstituted milk supplement (Volac Faramate, Volac International Ltd, Devon, UK) mixed at a rate of 150 g/L of mixed milk every 3 h daily. The animals were given free access to potable water from the public supply. The study was conducted in Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, UK, in accordance with the applicable sections of the United Kingdom Animals (Scientific Procedures) Act 1986, Amendment Regulations 2012 (the Act).

Experimental design

As a preliminary assessment of systemic toxicity and TK, a 10-day study consisted of three treated groups without a control group with five animals in each group (Table 1). Daily dosing was from PND 3 to PND 12. A subsequent 4-week study consisted of three treatment and one control groups with 7 animals in each group including 4 (2 male + 2 female) main study animals and 3 animals for TK (Table 1). Daily dosing was from PND 3 to PND 30. AGIQ (consisting of >97% AGIQ and 0.13% quercetin) was supplied by San-Ei Gen F.F.I, Inc. (Osaka, Japan) and was provided orally four times daily (every other feed) with a volume dose of 5 mL/kg body weight. Initially, the first dose was administered via oral gavage in a small amount of Volac Faramate reconstituted milk supplement to facilitate TK sampling. Thereafter, the dose was given in a small amount of reconstituted milk supplement via syringe before offering untreated milk via bottle-feeding. Any dose not taken voluntarily via syringe was administered by oral gavage at the end of the dosing session.

Viability, clinical signs, weight, and food consumption

Animals and their pens were inspected visually at least twice daily for evidence of reaction to treatment or ill-health at least twice in the approximately 24 h after birth, prior to weaning from the sow, and at least eight times daily (at times of feeding) thereafter, and detailed observations were recorded daily during and after each occasion of dosing. The piglets were weighed individually within 24 h after birth, daily from PND 3 and before necropsy in the 10-day study, and daily from PND 3 to 9, twice weekly from PND 9 and before necropsy in the 4-week study. In the 10-day study, the weight of food (milk) supplied to each animal and that remaining was recorded daily from PND 3.

Hematology, biochemistry, coagulation, and urinalysis

Hematology, blood chemistry, coagulation, and urinalysis tests for the 10-day and 4-week studies are listed in Supplemental Table 1. Blood for hematology, biochemistry, and coagulation was collected via the jugular vein pretreatment and at termination in the 10-day study and on day 14 and day 28 in the 4-week study. Urine samples were collected from all animals in the 4-week study before termination.

Necropsy and tissue handling

Piglets were sedated using isoflurane followed by overdose of sodium pentobarbitone solution (200 mg/mL) by intravenous injection followed by exsanguination. All animals were subject to a detailed necropsy, and all organs and tissues were examined for grossly visible lesions. Organs weighed, tissues fixed, and a comprehensive list of tissues examined microscopically are listed in Supplemental Table 2. Tissues were preserved in 10% neutral buffered formalin, except eyes and testes, which were fixed in Davidson’s fluid and modified Davidson’s fluid, respectively. Bone marrow smears were air-dried and subsequently fixed in methanol.

For histology, tissue samples were dehydrated, embedded in paraffin wax, and sectioned at a nominal four- to five-micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required. Sections were stained with hematoxylin and eosin (H&E). Findings were either reported as “present” or assigned a severity grade.29 A single section was prepared from the livers of representative animals and stained with Prussian Blue for the presence of iron deposits.

Toxicokinetics study

In the preliminary10-day study, plasma samples for TK were obtained from all treated animals after the first of four daily doses on PND 3 (the first day of treatment) and after the first dosing on PND 12 (day 10 of dosing). For the main 4-week study, plasma samples were obtained from only one or two minipigs per sex after the first of four daily doses on PND 3 and at study termination on PND 30. The time of sampling after dosing was 0.5 h, 2 h, 6 h (prior to dosing), and 24 h for both studies; all blood samples were collected via the jugular vein. Plasma samples were analyzed for quercetin, isoquercitrin, and quercetin-3-glucuronide by a quantified liquid chromatographic-tandem mass spectrometric (LC-MS/MS, API 4000, Sciex) method with a limit of quantitation of <20.0 ng/mL. All summary data reported in tables are based on rounded numbers. Due to the paucity of quantifiable data, statistical analysis was not considered appropriate. Standard deviations were calculated based on a minimum of three data points.

Survival, clinical observations, hematology and urinalysis parameters, and body and organ weight

Ten-day study

All animals remained in good health throughout the study, and there were no treatment-related signs of toxicity. Body weight gains were similar in all groups, and there was no apparent effect of treatment. Food consumption (milk) was variable between animals with no apparent treatment-related effects. There was some degree of inter-animal variability in organ weights; however, there were no notable differences between the groups and individual organ weights were within normal ranges for this age. There were no apparent effects of treatment on hematological parameters (data not shown).

Four-week study

All animals remained in good health throughout the study, and there were no treatment-related signs of toxicity. Body weight gain in almost all treated groups was slightly less than in controls over the duration of the study (Table 2). Mean daily body weights are provided in Supplemental Table 3.

There was some degree of inter-animal variability in platelet count levels due to the age of the animals that was considered nonadverse, and some degree of inter-animal variability in alkaline phosphatase levels that was attributed to the blood sampl