Ninety-day toxicity and single-dose toxicokinetics study of alpha-glycosyl isoquercitrin in Sprague-Dawley rat

Upon ingestion, AGIQ is partially deglycosylated by salivary amylase, partially absorbed in the small intestine, and further metabolized by anaerobic enterobacteria in the large intestine with absorption of quercetin that is rapidly glucuronidated (Hollman et al., 1996,Day et al., 2001 and LePoole, 2003). Other large intestinal bacteria can act on the aromatic rings of quercetin to form short chain fatty acids and phenyl and hydroxyphenylacetic acids that are subsequently absorbed (Valentova et al., 2014).

Isoquercitrin (quercetin-3-O–β-D-glucoside) is a rare natural compound, which has attracted much attention in the food and pharmaceutical industries due to its long list of beneficial properties ( Wang et al., 2015). These include anti-inflammatory, hypotensive, anti-mutagenesis, anti-oxidative, anti-depressant, hypolipidemic and anti-viral effects (Amado et al., 2009, Gasparotto Junior et al., 2011, Kim et al., 2010, Li et al., 2011 and Valentova et al., 2014). Because of its linear glucose moieties, AGIQ is soluble in water, and is absorbed well when given orally (Hara et al., 2014). Similar to isoquercitrin, AGIQ has antioxidative ( Morita et al., 2011, Nishimura et al., 2010 and Shimada et al., 2010) and tumor suppressive properties ( Fujii et al., 2013,Hara et al., 2014 and Kimura et al., 2013) in experimental animals. When tested previously in rats, AGIQ was shown to be safe and non-carcinogenic in a number of toxicological studies ( Salim et al., 2004 and FDA, 2007).

AGIQ was developed in 1987 and approved by the Japanese Ministry of Health and Welfare for use as a food additive in 1996 (MHW, 2009, http://www.ffcr.or.jp/zaidan/FFCRHOME.nsf/pages/list-exst.add). Based on its favorable safety profile, AGIQ has been concluded by the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) (FEMA No. 4225) as a generally recognized as safe (GRAS) compound in 2005 (Smith et al., 2005). The U.S. Food and Drug Administration (US FDA) has granted a GRAS status for AGIQ as an anti-oxidant as well, based on the details given in the GRAS Notice (GRN 000220) (FDA, 2007). Nevertheless, when reviewing the available literature, it is evident that the safety and toxicity information for isoquercitrin and AGIQ is still sparse, and the published safety assessment reports are from older studies that are not Good Laboratory Practice (GLP)-compliant and/or that used AGIQ of low purity (Engen et al., 2015, Valentova et al., 2014 and Salim et al., 2004).

Two previously 13-week rat toxicity studies were conducted using enzymatically modified isoquercitrin (Tamano et al., 2001) and enzymatically decomposed rutin which is principally isoquercitrin (Hasumura et al., 2004). Neither study fully provided details of test agent purity. The Tamano study doses ranged from 0.3 to 2.5% in powdered diet and there was decreased body weight gain in both sexes at the highest dose. Other changes included increased urinary ketones in males at 2.5%, increased reticulocytes in higher doses of both sexes, and yellow pigmentation of bones. The authors suggested the NOAEL at 0.3% for both sexes of F344 rats. The highest dose in the Hasumura study was 5% and this dose was associated with a body weight gain decrement and decreased erythroid parameters in males. The authors concluded a NOAEL of 1% for males and 5% for female Wistar rats. A two-dose two-year study of enzymatically modified isoquercitrin in F344 rats was negative for carcinogenicity at 0.5% and 1.5% in the diet (Salim et al., 2004).

In anticipation of expanded marketing of AGIQ, this report is an initial part of a safety assessment of highly purified AGIQ, and includes a repeated dose 90-day study and a single dose toxicokinetic (TK) study in Sprague-Dawley rats in fully GLP-compliant studies.

2. Methods

2.1. Animal husbandry and maintenance

Previously published isoquercitrin rat studies were conducted in F344 and Wistar rats and diets were varied. We chose to use the Harlan Sprague-Dawley rat and Purina 5002 diet since that is our standard rat strain and diet for food additive studies and because our past use of this combination of rat strain and diet provides us a useful historical control database. Furthermore, in anticipation of our reproductive toxicity studies, we needed a rat strain with high fecundity. It is noted that the NTP recently switched from the F344 rat to the Sprague Dawley rat in part because the low fecundity of the F344 was not ideal for their reproductive toxicity studies.

Hsd: Sprague-Dawley rats, approximately 5 weeks old, were obtained from Harlan Laboratories. A Purina Certified 5002 Meal Diet (Ralston Purina Co., St. Louis, MO) was offered ad libitum throughout the study. The animals were allowed free access to drinking water, supplied to each cage via polycarbonate water bottles. All animals were housed singly in a polycarbonate cage with a micro-isolator top. Absorbent heat-treated hardwood bedding (Northeastern Products Corp., Warrensburg, NY) was provided and changed once per week. The rats were allowed a 7-day acclimation period to the facility conditions prior to inclusion in the study. The study was approved by the Integrated Laboratory Systems (ILS), Inc. (Research Triangle Park, NC, USA) Animal Care and Use Committee, all procedures were in compliance with the Animal Welfare Act Regulations (9 CFR 1–4), and animals were handled and treated according to the Guide for the Care and Use of Laboratory Animals ( ILAR, 2011).

2.2. Experimental design

Ninety-day study was conducted in accordance with OECD Testing Guideline No. 408 (OECD, 1998). Sixty male and 60 female rats were randomized into six exposure groups (Table 1).