Advantame--an overview of the toxicity data.

Advantame is an N-substituted (aspartic acid portion) derivative of aspartame that is similar in structure to neotame, another N-substituted aspartame. An extensive series of studies, were conducted on advantame to define the pharmacokinetics and metabolism in various species, subchronic and chronic toxicity in the rat and dog, carcinogenicity in the rat and mouse, genotoxicity, reproductive, and developmental toxicity, and human tolerability studies. The results of these studies, presented in overview in the present publication, and in greater detail in the accompanying publications, show that advantame is well tolerated by both animals and humans and does not possess systemic toxicity. The metabolic data demonstrate that the animal species used in the toxicity testing are relevant to the evaluation of human safety. The no-observed-adverse-effect levels (NOAELs) identified in the animal studies in which advantame was administered in the diet were generally the highest doses tested. Under the anticipated conditions of use, the predicted intakes of advantame are about 20,000- to 70,000-fold lower than the identified animal study NOAEL values. The results of the animal toxicology and human trial data support the safety of use of advantame in food.

In vitro and in vivo assessment of the mutagenic activity of N-[N-[3-(3-hydroxy-4-methoxyphenyl) propyl]-α-aspartyl]-L-phenylalanine 1-methyl ester, monohydrate (advantame).

Advantame (N-[N-[3-(3-hydroxy-4-methoxyphenyl) propyl]-α-aspartyl]-L-phenylalanine 1-methyl ester, monohydrate), an N-substituted analogue of aspartame, has been developed as a high-intensity sweetener. It is approximately 100 and 20,000 times sweeter than aspartame and sucrose, respectively. In this study the safety of advantame has been evaluated using a series of in vitro and in vivo genotoxicity assays including, bacterial mutation, mammalian cell mutation, and mouse micronucleus tests. Advantame did not induce reverse mutations in Salmonella typhimurium and Escherichia coli at concentrations of up to 5000 µg/plate. In the mammalian cell mutation assay, advantame did not induce mutation at the Hprt locus of L5178Y mouse lymphoma cells in two independent experiments, either in the absence or presence of S9. In vivo, there was no effect on the incidence of micronucleated immature or mature erythrocytes in bone marrow after oral administration of the test substance at any dose level (up to 2000 mg/kg body weight) or sampling time (24 and 48 h). The results of these studies demonstrate that advantame is without genotoxic potential.

Chronic toxicity and carcinogenicity of N-[N-[3-(3-hydroxy-4-methoxyphenyl) propyl]-α-aspartyl]-L-phenylalanine 1-methyl ester, monohydrate (advantame) in the rat.

Groups of 55 male and 55 female Han Wistar rats were administered advantame (98.9-99.8% purity) in the diet at concentrations of 0, 2000, 10,000, or 50,000 ppm for 104 weeks, following parental exposure to the same levels from prior to mating and throughout gestation. Additional groups of 20 rats/sex and 10 rats/sex were dosed for a period of 52 weeks and constituted the toxicity and reversibility phases of the study. Achieved doses of advantame over the carcinogenicity study were 0, 97, 488, and 2621 mg/kg body weight/day in males and 0, 125, 630, and 3454 mg/kg body weight/day in females, respectively. A high incidence of a pale and swollen anus and changes in fecal composition were observed in the high-dose groups. There was no effect of treatment on mortality. Body weight gain in the high-dose males (50,000 ppm) was slightly reduced compared to controls after 52 and 104 weeks of treatment; the decrease was not considered to be of toxicological significance, but due to the non-nutritive nature of the high dietary concentration of advantame. During the toxicity phase, food conversion efficiency was slightly decreased in both sexes, at the 50,000 ppm dose level. Given the non-nutritive content of the diet, this finding was not considered biologically significant. There were no relationships between treatment and the results of hematological or urinalysis investigations. Clinical chemistry evaluations showed consistently lower plasma urea concentrations in both sexes treated at 50,000 ppm, which was reversed during the 6-week recovery phase following 52 weeks of treatment, indicating a lack of permanent effects. Terminal investigations at both the 52 and 104-week revealed a number of intergroup differences in absolute and/or relative organ weights; however, the differences did not show dose-response relationships, were minor in nature, and/or occurred only in one sex, and were not associated with any pathological findings, and they were considered not to be treatment-related. Evaluation of the histopathology of the carcinogenicity phase animals revealed an increased incidence of pancreatic islet cell carcinomas in males (incidence rates of 0/55, 1/55, 2/55, and 3/55 in the 0, 2000, 10,000, or 50,000 ppm groups, respectively) and of mammary gland adenomas in the high-dose females (incidence rates of 0 in the control through 10,000 ppm dose groups and 4/41 in the 50,000 ppm dose group). The incidence rates of these tumors did not attain statistical significance and/or remained within background historical control values, and they were considered to be unrelated to advantame treatment. The no-observed-adverse-effect level was considered to be 50,000 ppm in the diet, the highest concentration tested, equivalent to 2621 and 3454 mg/kg body weight/day in males and females, respectively. Advantame was concluded to be without carcinogenic activity.

Chronic oral toxicity of N-[N-[3-(3-hydroxy-4-methoxyphenyl) propyl]-α-aspartyl]-L-phenylalanine 1-methyl ester, monohydrate (advantame) in the dog.

Advantame (N-[N-[3-(3-hydroxy-4-methoxyphenyl) propyl]-α-aspartyl]-L-phenylalanine 1-methyl ester, monohydrate), an N-substituted analog of aspartame, has been developed as a high-intensity sweetener. Groups of 4 dogs of each sex were treated at 0, 2000, 10,000, or 50,000 ppm of advantame in the diet for 52 weeks. Additional groups of 2 dogs/sex at the control, and mid- and high-dose groups were treated for 52 weeks followed by a 6-week recovery period. There was no effect of treatment on mortality, body weight, organ weights, food consumption, or the results of ophthalmological, electrocardiographic, haematological, clinical chemistry or urinalysis examinations. No histopathological changes were associated with advantame treatment. The NOAEL was considered to be 50,000 ppm, the highest concentration tested, which was equivalent to 2057 and 2139 mg/kg body weight/day in males, and females, respectively. The results of the study support the safety of advantame for use as a high-intensity sweetener.

Evaluation of the teratogenic potential of N-[N-[3-(3-hydroxy-4-methoxyphenyl) propyl]-α-aspartyl]-L-phenylalanine 1-methyl ester, monohydrate (advantame) in the rat and rabbit.

To assess its teratogenic potential, advantame (N-[N-[3-(3-hydroxy-4-methoxyphenyl) propyl]-α-aspartyl]-L-phenylalanine 1-methyl ester, monohydrate) was administered to mated rats (22/group) in the diet at 0, 5000, 15,000, and 50,000 ppm (providing approximately 465, 1418, and 4828 mg/kg body weight/day), and to mated rabbits (24/group) via oral gavage at 0, 500, 1000, and 2000 mg/kg body weight/day throughout gestation. Shortly before delivery (rats: day 20; rabbits: day 29), animals were killed and subjected to a detailed necropsy. Fetuses were examined for external, visceral, and skeletal alterations. Atypical coloration of the feces and cage liners seen with test diets in both rats and rabbits was attributed to excretion of test material/metabolites in the feces and urine. Advantame had no adverse effect on rat offspring survival or development. The no-observed-adverse-effect level (NOAEL) for both maternal and developmental toxicity in rats was 50,000 ppm, the highest dietary concentration tested. Due to adverse effects associated with reduced food intake and fecal output, approximately 20% of mated rabbits receiving 200 0mg/kg body weight/day and 1 animal at 1000 mg/kg body weight/day had to be terminated before scheduled necropsy. A NOAEL of 500 mg/kg body weight/day was established for maternal toxicity in rabbits. No teratogenic effects were observed in any animals, and based on a slightly increased incidence of fetal deaths at 2000 mg/kg body weight/day, a finding that was considered to be indirectly related to advantame treatment, 1000 mg/kg body weight/day was considered the NOAEL for developmental toxicity.

Acute and multiple-dose studies to determine the safety, tolerability, and pharmacokinetic profile of advantame in healthy volunteers.

Advantame, a new, high-intensity sweetener that is thought not to be absorbed from the human gastrointestinal tract in appreciable quantities, was evaluated for safety and tolerability in a total of 48 healthy adult volunteers in 2 studies. In the first study 24 subjects were randomized to receive escalating single doses of 0.1, 0.25, or 0.5mg/kg of body weight, and pharmacokinetic and safety parameters were assessed subsequently for 8 days. In the second study, 24 subjects were randomized to receive over 4 weeks either 30 mg advantame/day (split into 3 doses per day), or placebo. All subjects completed both studies and no significant treatment-related adverse effects were observed in any subjects in either study. There were no clinically relevant changes in laboratory parameters, vital signs, electrocardiogram, or physical examination findings. Plasma concentrations of advantame were mostly below the limit of quantification in all samples taken after a single dose or prior to the dose in the repeat-dose study. The concentrations of the hydrolysis product, advantame-acid, were also below the limit of quantification at 12, 36, and 48 h after a single dose of 0.1, 0.25, and 0.5mg/kg body weight, respectively. These studies demonstrate the safety and tolerability of advantame in healthy subjects at doses far exceeding those likely to be encountered in food and beverage use.

A two-generation reproductive toxicity study of the high-intensity sweetener advantame in CD rats.

Rats received diets containing 0, 2000, 10,000, or 50,000 ppm advantame (N-[N-[3-(3-hydroxy-4-methoxyphenyl) propyl]-α-aspartyl]-L-phenylalanine 1-methyl ester, monohydrate) for 2 generations. F(0) animals (30/sex/group) were treated from 10 weeks before pairing. Males continued until week 16; females through gestation and lactation. Once weaned, F(1) animals (25/sex/group) continued receiving the same diet until F(2) pups were weaned. Mean advantame intakes from each of the diets were 164, 833, and 4410 mg/kg bw/day among F(0) males, and 204, 1036, and 5431 mg/kg bw/day among F(1) males. F(0) and F(1) females had comparable intakes up to lactation, when intakes increased (up to 8447 mg/kg bw/day from 50,000 ppm diet). No treatment-related effects on mortality, body weights, reproduction, litter observations, or postnatal offspring development were noted. Atypical coloration of the feces and cage liners seen with test diets was attributed to excretion of test material/metabolites in the feces and urine. Slightly higher food consumption was seen in F(0) and F(1) animals, especially males, receiving 50,000 ppm. However, these differences were considered to be a secondary response to the high levels of non-nutritive material in the diet. The no-observed-adverse-effect level for reproductive and developmental toxicity was considered to be 50,000 ppm, the highest dietary concentration tested.