Consumers are increasingly concerned about the quality and safety of many products present in the diet of industrialized countries, in particular, the use of artificial sweeteners, flavorings, colorings, preservatives, and dietary supplements. General apprehension also exists regarding the possible long-term health effects of the raw materials and technologies used for the packaging, sterilization, and distribution of foods. Of particular concern are the potential carcinogenic effects of these products and processes.
The experimental and epidemiologic data currently available to evaluate the above carcinogenic risks are insufficient and often unreliable because of the inadequate planning and conduct of previous experiments. This inadequacy, combined with the general limited knowledge about the safety and potential carcinogenic effects of substances widely present in the industrialized diet, motivated the design of an integrated project of mega-experiments in 1985 at the Cesare Maltoni Cancer Research Center (CMCRC) of the European Ramazzini Foundation (ERF). The products studied are reported in . The products and agents we selected for this project were those for which committee debate and opinions had often acted as surrogates for good laboratory work. At present, over the course of the project, 32 long-term bioassays have been performed using > 25,000 rodents. Studies have evaluated the carcinogenicity of 12 different products, including the artificial sweetener aspartame (APM).
Beverages and diet products studied at the CMCRC/ERF: status of studies.
In this article we present the results of the mega-experiment on the carcinogenicity of APM in which the sweetener was administered in feed to Sprague-Dawley rats for the life span.
APM, the methyl ester of the dipeptide l
), is a widely used artificial sweetener with a molecular weight of 294.3. Under particular conditions (extreme pH, high temperature, lengthy storage times), APM may be contaminated by the diketopiperazine (DKP) cyclo-aspartylphenylalanine (Butchko et al. 2002a
For more than 30 years, APM has been widely used as a food additive because of its very strong, sweet taste. The sweetening power of APM is estimated to be 200 times that of sucrose, whereas saccharin and cyclamate are 300 and 30 times sweeter, respectively (Mazur 1984
Initial commercial approval of APM in the United States was granted by the Food and Drug Administration (FDA 1974
). The FDA later approved the limited use of APM in solid foods in 1981 and extended this authorization to soft drinks in 1983. APM was eventually approved as a general sweetener in 1996 (FDA 1981
). In the European Union, the safe use of APM was authorized in 1994 (EC Directive 1994
After saccharin, APM is the second most used artificial sweetener in the world. It is estimated that > 8,000 tons of APM are consumed each year in the United States (Hazardous Substances Data Bank 2005
). In terms of world consumption, APM represents 62% of the value of the intense sweetener market (Fry 1999
APM is found in > 6,000 products, including carbonated and powdered soft drinks, hot chocolate, chewing gum, candy, desserts, yogurt, tabletop sweeteners, and some pharmaceutical products, such as vitamins and sugar-free cough drops, and is estimated by the Aspartame Information Center (2005)
to be consumed by > 200 million people worldwide.
Through dietary surveys performed in the United States among APM consumers during the period 1984–1992, the average APM daily intake in the general population has been shown to range from 2 to 3 mg/kg body weight (bw). Consumption by children 2–5 years of age and by females of childbearing age in these surveys ranged from about 2.5 to 5 mg/kg bw/day (Butchko et al. 2002b
). APM intake was also monitored in several other regions, including seven European countries. Although survey methodologies may have differed, the APM intake was remarkably consistent across studies and was well below the acceptable daily intake (ADI) both in the United States (50 mg/kg bw) and in Europe (40 mg/kg bw) (Butchko et al. 2002b
Investigations into the metabolism of APM have shown that, in rodents, nonhuman primates, and humans, it is metabolized in the gastrointestinal tract into three constituents—aspartic acid, phenylalanine, and methanol—which are absorbed into the systemic circulation (Ranney et al. 1976
). For each molecule of APM, one molecule of each constituent is produced. After absorption, they are then used, metabolized, and/or excreted by the body following the same metabolic pathways as when consumed through the ordinary diet: aspartate is transformed into alanine plus oxaloacetate (Stegink 1984
); phenylalanine is transformed mainly into tyrosine and, to a smaller extent, phenylethylamine and phenyl-pyruvate (Harper 1984
); and methanol is transformed into formaldehyde and then to formic acid (Opperman 1984
APM was not genotoxic in the following tests: dominant lethal mutation assay in rats, host-mediated assay in rats and mice, in vivo
cytogenetic assay in rats, and the Ames test (Kotsonis and Hjelle 1996
). Results of an assay to measure induction of unscheduled DNA synthesis in rat hepatocytes treated with APM in vitro
were negative, indicating the absence of APM-induced DNA damage (Jeffrey and Williams 2000
). In a test for the induction of chromosomal aberration in bone marrow cells of male Swiss mice, Mukhopadhyay et al. (2000)
reported that a mixture of APM (up to 350 mg/kg) and a second sweetener, acesul-fame potassium (up to 150 mg/kg) administered by gavage was negative. However, a dose-related increase in the percentage of cells with chromosomal aberrations was noted with increasing doses of the two sweeteners, even though the increase was not statistically significant (Mukhopadhyay et al. 2000
Two long-term feeding carcinogenicity bioassays on APM were performed on rats and one on mice in the early 1970s by the producer Searle & Co. Results were reviewed by the FDA and then summarized in the Federal Register
). To date, the details of the experiments have not been published.
In the first study, groups of 40 male and 40 female Sprague-Dawley rats were treated with 1, 2, 4, or 6–8 g/kg bw/day of APM in the diet. The treatment started at 4 weeks of age and lasted for a period of 104 weeks. A control group of 60 rats per sex was fed the same diet without APM. At the end of the treatment, all surviving animals were sacrificed and their brains, as well as other organs (not specified in the report), were examined histologically. Brain tumors were observed in 7 of 155 (4.5%) exposed males versus 1 of 59 (1.7%) controls, and in 5 of 158 (3.2%) exposed females versus 0 of 59 (0%) controls. Overall, the FDA considered the study to be negative with regard to the carcinogenicity of APM (FDA 1981
In the second study, groups of 40 male and 40 female Sprague-Dawley rats were exposed to APM, at doses of 2 and 4 g/kg bw/day, through their mothers’ diet both in utero
and during lactation, and then for 104 weeks with APM in their own diets. A control group of 60 rats per sex was fed the same diet without APM. The animals were necropsied at the time of death or at 104 weeks after weaning. Three brain tumors were observed among control males and one among control females. Brain tumors were also observed in two males and one female in the 2 g/kg bw group, and in one male and one female in the 4 g/kg bw group. Again, the FDA considered the study to be negative with regard to the carcinogenicity of APM (FDA 1981
Regarding the third chronic APM study, in this case performed on mice, the FDA reported that the results did not show any treatment-related carcinogenic effect. In this experiment, as reported by Molinary (1984)
, groups of 36 male and 36 female mice were fed 1, 2, or 4 g/kg bw/day until 110 weeks of age. A group of 72 males and 72 females served as the control. There were no treatment-related effects on survival and behavior, nor were any lesions recorded during macroscopic or microscopic analysis.
An APM carcinogenicity study was also conducted in Japan during this period (Ishii 1981
; Ishii et al. 1981
). Groups of 86 male and 86 female Wistar rats were treated with APM in feed at doses of 0, 1, 2, or 4 g/kg bw/day from 6 to 110 weeks of age. No increase in the incidence of brain tumors was observed in the treated groups compared with the controls. Exhaustive experimental details of this study were not published.
Epidemiologic studies to evaluate the relationship between APM intake and cancer development in humans are not currently available.
Although all of the aforementioned studies were considered negative with respect to the carcinogenicity of APM, in our opinion, these studies did not comply with today’s basic requirements for testing the carcinogenic potential of a physical or chemical agent, in particular concerning the number of animals for each experimental group and the duration of the experiment until 110 weeks of age of the animals.
For these reasons, and in light of the ever-increasing diffusion of APM in the diet of industrialized countries (particularly in products consumed by young children and pregnant women), we considered it important to perform a mega-experiment following today’s internationally recognized good laboratory practices for carcinogenicity bioassays and, more specifically, the life-span carcinogenicity bioassay design followed for many years at the CMCRC and described in previous publications (Soffritti et al. 1999