An embryo-fetal survival and development study was conducted to augment the toxicity database for alpha-glycosyl isoquercitrin (AGIQ), a generally recognized as safe (GRAS) additive and flavor in food and beverages. In Phase I, 24 naturally mated New Zealand white (NZW) female rabbits per group were administered AGIQ by oral gavage at 0, 250, 500, or 1000 mg/kg/day once daily during gestation days 6–28, followed by necropsy. There was no evidence of maternal or fetal toxicity except for equivocal findings of unilateral absent kidney and ureter in one and two unrelated fetuses at 500 and 1000 mg/kg/day, respectively. To more thoroughly assess fetal kidney/ureter development, in Phase II groups of time mated NZW rabbits were administered AGIQ at 0, 500, or 1000 mg/kg/day, under the same conditions as Phase I. No occurrences of absent kidney/ureter were noted in the AGIQ-treated Phase II dams or fetuses; although, one control fetus had unilateral missing kidney/ureter. Given the lack of reproducibility following treatment with AGIQ in Phase II using 48 animals per group, the missing kidney/ureter observations in Phase I were considered unrelated to treatment. Since oral gavage administration of AGIQ to pregnant female NZW rabbits at dose levels of 250, 500, or 1000 mg/kg/day was well-tolerated with no adverse treatment-related effects on the maternal animal, pregnancy, or the developing conceptus, the no-observed-adverse-effect-level (NOAEL) for maternal toxicity and embryo-fetal survival, growth, and development was 1000 mg/kg/day.
Introduction
Alpha-glycosyl isoquercitrin (AGIQ) (Figure 1) is an enzymatically modified form of the natural flavonol isoquercitrin (quercetin-3-O-ß D-glucoside), derived from rutin and used in Japan as an additive or flavor ingredient in various beverages and foods. Although quercetin and its glycosides have demonstrated anti-inflammatory, pain-reducing, and cardioprotective properties1–6 and therefore have been promoted as antioxidant dietary supplements to consumers, their poor miscibility in water and limited absorption have hindered their broad application to the food and beverage industry.7 However, the enzymatic modification resulting in AGIQ has been shown to enhance the solubility and bioavailability of isoquercitrin.8,9
Figure 1. Chemical formula of alpha-glycosyl isoquercitrin.
Previous toxicity assessments have shown that AGIQ is safe, non-carcinogenic, and non-genotoxic,5,7,10–13 but some of the earlier studies used incompletely characterized AGIQ and/or did not adhere to current Good Laboratory Practice (GLP).6,10,12,13 Due to the current effort to expand the use of AGIQ in the consumer food and beverage industry, recent GLP-compliant studies of high-purity AGIQ (including comprehensive genotoxic assessment,7 10-day and 4-week studies in preweaning Göttingen minipigs,14 and a 90-day study in rats)5 were conducted to augment the older toxicity database. These studies have generally confirmed the safety profile of AGIQ.
The current study using highly purified AGIQ and New Zealand White (NZW) rabbits was conducted to assess the potential effects of the compound on embryo-fetal development, growth, and survival in a non-rodent species for registration purposes. The objective of the initial dose-response phase of the study (Phase I) was to assess the potential of AGIQ to induce prenatal developmental toxicity after maternal exposure via oral gavage during the critical period of organogenesis and fetal development. A subsequent investigational phase (Phase II) was conducted using 48 animals in each group to verify equivocal Phase I findings of unilateral absent fetal kidney/ureter in one and two (unrelated) fetuses at the mid and high dose levels, respectively. This study provides a base of non-rodent developmental toxicity data obtained in accordance with current developmental toxicity testing guidelines (Phase I)15–17 and GLP (both phases)18–21 for human risk assessment of orally administered AGIQ.
Materials and methods
Test article
AGIQ (>97% pure, 0.13% quercetin, lot no. 170727) was supplied as a yellow to yellow-orange powder by San-Ei Gen F.F.I., Inc., Osaka, Japan.
Dosing formulations were prepared by dissolving AGIQ in purified water at the target dose concentrations of 25, 50, and 100 mg/mL. Representative samples of each formulation concentration prepared for administration during each phase were analyzed on two occasions for achieved concentration of AGIQ. A previous GLP-compliant validation study (Envigo Study No. NL85VW, 21 June 2018; data not presented) found that solutions of AGIQ in purified water at concentrations ranging from 1 to 200 mg/mL were homogeneous and stable for 1 day when stored at ambient temperature (15–25°C) and 15 days when stored refrigerated (2–8°C). Based on these results, the formulations in this study were prepared daily, stored at ambient temperature until use, and administered within 4 h of preparation.
Animal husbandry and mating
Female NZW rabbits (obtained from Envigo RMS UK) were used for this research. For Phase I, 96 sexually mature, virgin females uniquely identified by an ear tag were supplied (88 assigned to study and 8 serving as potential replacements). For Phase II, 144 time-mated females (all assigned to study) were supplied in four deliveries on GD 1 after mating at the supplier. Phase II animals were uniquely identified by a microchip inserted shortly after arrival. Each cage label was color-coded according to group and was numbered uniquely with cage number, study number, and the identity of the occupant.
The rabbits were allowed a 19-day (Phase I) or 5-day (Phase II) period of acclimation to the facility conditions prior to allocation to the study (mating in Phase I [GD 0] or day of arrival [GD 1] in Phase II). Female rabbits were cohabited with stock NZW bucks of established fertility at the performing laboratory (Phase I) or the supplier (Phase II). Males and females were not closely related. Phase I females were assigned to group and cage position in the sequence of observed natural mating (females mating on the same day were evenly distributed among the groups) and Phase II females were randomly assigned to group and cage position, evenly distributed among the groups. After mating, each female was injected intravenously with 25 i.u. luteinizing hormone. The day of observed mating was designated GD 0. Allocation was controlled to prevent any stock male from providing more than one mated female in each treated group and to prevent more than one sibling female in each group, where possible.
The animals were 19–23 (Phase I) or 16–20 (Phase II) weeks of age at the start of the study (GD 0/1). The basal diet, Teklad 2930 Diet, was restricted to 150 g/animal/day during acclimation up to 1 week prior to the onset of mating (Phase I) and 200 g/animal/day thereafter (Phase I and II). In addition to the basal diet, a small supplement of autoclaved hay was given on a daily basis to promote gastric motility and a small amount of chopped fresh vegetables were given twice weekly. Consumption of hay and vegetables were monitored qualitatively but not quantitatively. The animals were allowed unrestricted access to potable drinking water from the public supply, supplied via polycarbonate water bottles equipped with sipper tubes and supplementary water bowls in each cage, throughout the study. Water bottles/bowls were changed at appropriate intervals. All animals were housed individually (except while paired for mating (Phase I) in suspended cages fitted with perforated floor panels and plastic resting platforms. Undertrays lined with absorbent paper were changed at least three times per week. All animals were maintained on a 14-hour daily photoperiod (10 hours dark) at an environmental temperature of 15–21°C and relative humidity of 45–70%. Environmental enrichment for each animal consisted of an Aspen chew block (soft white untreated wood block), a stainless-steel key ring attached to the cage, and nesting paper placed into each cage starting at post-mating GD 20 to allow expression of nesting behavior.
Phase I and II were conducted in an AAALAC accredited facility (Covance Laboratories Limited, Eye, UK (Study JJ43CT)) in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012.22 The number of animals used was the minimum that was consistent with scientific integrity and regulatory acceptability, consideration having been given to the welfare of individual animals in terms of the number and extent of procedures to be carried out on each animal. The NZW rabbit was chosen as the test model based on the availability of Historical Control Data (HCD) in the performing laboratory and the acceptability of this species and strain to regulatory agencies.
Experimental design
This study was conducted in two phases (Table 1): (I) a regulatory guideline-compliant embryo-fetal survival and development study and (II) a follow-up confirmatory investigation of equivocal fetal morphological findings noted in Phase I.
In a preliminary dose range-finding study conducted in the same laboratory, administration of AGIQ to 6 females per group was well-tolerated at 250, 500, and 1000 mg/kg/day, but was not tolerated at 2000 mg/kg/day and this group of animals was terminated early on GD 21/22 due to marked body weight loss and persistently low food consumption in some females. The urine from all treated animals was colored yellow and stained the cage tray paper and animal fur yellow/orange/ brown and the amniotic fluid/sacs in 1/6 animals at 250 mg/kg/day and 1/6 animals at 500 mg/kg/day, but none at 1000 mg/kg/day, were colored yellow. The strong coloration was attributed to the color of AGIQ (yellow to yellow-orange powder). At 1000 mg/kg/day, overall body weight gain was low and food intake was marginally low. Reproductive parameters did not appear to be affected by treatment with AGIQ.
Based on these preliminary results, female rabbits in Phases I (22/group) and II (48/group) were administered AGIQ in purified water once daily during GD 6–28 (inclusive) at approximately the same time each day via oral gavage at a dose volume of 10 mL/kg (calculated from the most recently recorded sch