Metabolism and Toxicity Studies Supporting the Safety of Rebaudioside D

Abstract

Rebaudioside D (Reb D) is one of the several glycosides found in the leaves of Stevia rebaudiana (Bertoni) Bertoni (Compositae) which has been identified as a potential sweetener. The metabolism of Reb A and Reb D was evaluated in various in vitro matrices (simulated gastrointestinal fluids, rat liver microsomes, and rat cecal contents) and through analysis of plasma collected from rats in a dietary toxicity study. Reb A and Reb D showed similar stability when exposed to simulated stomach and small intestine fluids, with susceptibility to hydrolytic degradation by enteric bacteria collected from the cecum. Incubations with rat liver microsomes indicated that neither compound is expected to be metabolized by the liver enzymes. Plasma concentrations of Reb D, Reb A, and/or the final hydrolysis product of each compound, free/conjugated steviol, were consistent between animals administered either Reb D or Reb A in the diet. A repeated exposure dietary toxicity study was conducted to compare the safety of Reb D, when administered at target exposure levels of 500, 1000, and 2000 mg/kg body weight (bw)/d to Sprague-Dawley rats for 28 days, to that of Reb A administered at a target exposure level of 2000 mg/kg bw/d. There were no treatment-related effects on the general condition and behavior of the animals and no toxicologically relevant, treatment-related effects on hematology, serum chemistry, or urinalysis. Macroscopic and microscopic findings revealed no treatment-related effects on any organ evaluated. Results were comparable between the group administered 2000 mg/kg/d Reb D and the group administered 2000 mg/kg/d Reb A.

Chemical-specific adjustment factors (inter-species toxicokinetics) to establish the ADI for steviol glycosides

“…Although steviol glycosides share a common metabolic fate in both rats and humans (Wingard et al, 1980;Hutapea et al, 1997;Gardana et al, 2003;Koyama et al, 2003a;Geuns et al, 2007;Roberts and Renwick, 2008;Wheeler et al, 2008;Nikiforov et al, 2013;Purkayastha et al, 2014Purkayastha et al, , 2015Purkayastha et al, , 2016, some differences in pharmacokinetics have been noted. As evaluated by JECFA at their sixty-ninth meeting (JECFA, 2009), studies comparing the pharmacokinetics of steviol and steviol glucuronide following oral administration of steviol glycosides demonstrated substantial differences in the concentration of circulating plasma steviol and steviol glucuronide between rats and humans (Roberts and Renwick, 2008;Wheeler et al, 2008).…”

Scientific opinion on the safety of the proposed amendment of the specifications for steviol glycosides (E 960) as a food additive

“…The authors of both these papers attribute the apparent decrease in the rate and extent of metabolism of rebaudioside D to the difference in the substituent at R1. Nikiforov et al (2013) also demonstrated that in contrast to rebaudioside A, there was no detectable rebaudioside D in the plasma of rats following oral administration at 2000 mg/kg bw per day for 28 days. These data suggest that increasing size of the side chain at R1 may also decrease the absorption of rebaudiosides into the systemic circulation.…”

“…Therefore, steviol glycosides entered the colon intact, where they were subjected to microbial degradation by members of the Bacteroidaceae family, resulting in the release of the aglycone steviol (Renwick and Tarka, 2008). Several in vitro studies mimicking the anaerobic conditions of the colon demonstrated the ability of the gut microbiota from mice, rats, hamsters and humans to hydrolyse steviol glycosides completely to steviol (Wingard et al, 1980;Hutapea et al, 1997;Gardana et al, 2003;Koyama et al, 2003b;Nikiforov et al, 2013;Purkayastha et al, 2014).…”

“…Although there was evidence that rebaudioside A can be available systemically this was only detectable following high doses due to the extensive cleavage to steviol and the much more rapid absorption of steviol (2-300 times faster in an in vitro model system). There were data on rebaudioside D and a comparison of its metabolism in the recent paper by Nikiforov et al (2013). Rebaudioside D can be cleaved to steviol by the gastrointestinal microflora but it appears that the extent and rate of this cleavage may be lower than that seen with rebaudioside A.…”

“…Two new ADME studies have been recently published: metabolisms of rebaudioside A and D (Nikiforov et al, 2013) were compared in various in vitro matrices (simulated gastrointestinal fluids, rat liver microsomes, and rat cecal contents) and through analysis of plasma collected from rats in a dietary toxicity study. This repeated exposure dietary toxicity study was conducted to compare the safety of rebaudioside D administered at levels of 500, 1000 and 2000 mg/kg bw/day to Sprague-Dawley rats for 28 days, to that of rebaudioside A administered at a target exposure level of 2000 mg/kg bw/day.…”