The Genotoxicity Of Aspartame Biology Essay

In vitro studies

Aspartame was tested for mutagenicity in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 both in the absence and the presence of a rat liver metabolic activation system at dose levels from 10 up to 5000 µg/plate (E97, 1978; E101, 1978) (Annex H). The Panel considered

that, for both studies, the methods implemented appeared to be sufficiently robust to support the results reported. Aspartame was not mutagenic in this test system, either in the absence or in the presence of the metabolic activation system.

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In vivo studies

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Mutagenicity of aspartame was studied using a host-mediated assay in mice (E81, 1974). Mice were treated by gavage with 0 (control), 1000, 2000, 4000, 8000 mg aspartame/kg bw/day (as three separate doses given at two hour intervals for five consecutive days) (E81, 1974). Thirty minutes after the final dose, the animals were inoculated with Salmonella typhimurium, G-46, by intraperitoneal injection. Three hours later the bacteria were recovered and the peritoneal washing was evaluated for the presence of mutants. In mice, the host-mediated assay revealed no evidence for mutagenicity of aspartame. The Panel noted some discrepancies in description of doses in different sections of the report and that the test system employed has not received further validation and is presently considered obsolete and therefore, the results of the study were not included in the assessment.

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Two dominant lethal tests in rats (15 males/group) were reported (E40, 1973; E41, 1973). Aspartame was dosed by gavage to 21-week-old male albino rats of the Charles River CD strain at a dose level of

2000 mg/kg bw given in two equally divided doses administered on the same day. Immediately following treatment, each male was mated with two sexually mature virgin females weekly for eight consecutive weeks. On gestation day (GD) 14, the mated females were sacrificed for ovarian and uterine examinations. The Panel considered that the methods implemented were thought to be sufficiently robust to support the results reported. The following parameters were analysed: paternal growth, maternal pregnancy rate, uterine and ovary examination data and incidence of fetal deaths. None of these parameters was affected by aspartame treatment.

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Aspartame was administered by gavage to five groups of 10 male albino rats for five consecutive days, at dose levels of 0 (control), 500, 1000, 2000 and 4000 mg/kg bw/day (E43, 1972). Twenty-four hours after the last dose, each animal was administered colcemid to arrest mitosis, and sacrificed. Bone marrow cells were prepared and evaluated for chromosome aberrations. Aspartame did not increase the normal aberration frequencies compared to the control rats. The authors concluded that aspartame was not mutagenic. The Panel considered that the methods implemented were sufficiently robust to support the results reported, but considered the study limited since mitotic indexes were not reported (Annex H).

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Aspartame was reported not to induce chromosome aberrations in bone marrow or spermatogonial cells after administration by gavage to rats for five days (E12, 1970). However, the dose levels applied were reported inconsistently. On page 2 of the relevant Study Report (E12, 1970) it is stated that animals received aspartame at dose-levels of 400, 800, 1200 and 1600 mg/kg bw/day. In contrast, it is reported that aspartame was administered at dose-levels of 2000, 4000, 6000, 8000 mg/kg. The Panel considered that the reported results of this study were not supported by the outcome of the methods applied (Annex H).

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Aspartame was tested in a host-mediated assay with rats (E44, 1972). Aspartame was administered by gavage to five groups of 10 male albino rats for five consecutive days, at dose levels of 0 (control),

500, 1000, 2000 and 4000 mg/kg bw/day, given in three equally divided doses. Following the final dose, the animals were inoculated with Salmonella typhimurium, G-46, by intraperitoneal injection. Three hours later the bacteria were recovered, and the peritoneal washing was analysed for the presence of mutants. No statistically significant effects on mutation frequency, as compared to the control, were noted in the treatment groups. The Panel noted that the test system employed has not received further validation and it is presently considered obsolete, and therefore the results of the study were not included in the assessment.

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3.2.3.2. Additional studies on genotoxicity

In vitro studies

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Aspartame was studied in Salmonella mutagenicity tests in the absence and in the presence of metabolic activation (Annex H). No mutagenicity was detected in strains TA98, TA100, TA1535, TA1537 or TA97 for doses up to 10000 μg/plate (NTP, 2005). The Panel considered that the methods implemented were to be sufficiently robust to support the results reported (Annex H). However, the Panel noted a deviation from OECD 471 (i.e. tester strains TA102 or WP2uvrA bearing AT mutation were not used). The authors of the study judged the small increase in mutant colonies with 30% rat liver S9 as equivocal.

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Rencuzogullari et al. (2004) tested aspartame in strains TA98 and TA100 at doses ranging from 50 to

2000 μg/plate in the presence and in the absence of metabolic activation. No mutagenicity was observed. However, the Panel considered that the methods implemented were not sufficiently robust to support the results reported, due to major deviations from OECD guideline (i.e. only two tester strains were used; the highest dose-level employed was lower than 5 mg/ml).

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Bandyopadhyay et al. (2008) reported aspartame to be negative in a test with Salmonella typhimurium

TA97 and TA100 strains (plate incorporation, with and without metabolic activation; 10, 100, 250,

500, 1000 e 10000 µg/plate). The Panel considered that the methods implemented were not sufficiently robust to support the results reported, due to major deviations from OECD guideline (i.e. only two tester strains were used; the concentration intervals were too wide; no replicate experiment were performed).

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Aspartame was tested for DNA damaging activity in the in vitro primary rat hepatocyte/DNA repair assay at concentrations of 5 and 10 mM (corresponding to 1.47 and 2.94 mg/ml, respectively) (Jeffrey and Williams, 2000). The Panel considered that the methods implemented were sufficiently robust to support the results reported. Aspartame was found to be negative in this assay.

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Rencuzogullari et al. (2004) tested aspartame in vitro in a sister chromatid exchange (SCE) assay, a chromosomal aberration test and a micronucleus test on human lymphocytes. Dose-related and statistically significant increases were observed for chromosomal aberration at both 24 and 48 hours and for induction of micronuclei only at the highest dose-levels employed (2000 µg/ml). Negative findings were observed for SCEs. The Panel noted that it cannot be excluded that the positive findings resulted from indirect effects (non-physiological culture conditions) since pH and osmolality were not reported. This conclusion is supported by negative findings obtained for SCEs in parallel cultures; however, these are not usually induced by indirect effects. The possible involvement of an indirect mechanism in the reported clastogenic effect is also supported by the fact that the study was performed in the absence of S9 metabolism: in these experimental conditions, no DNA damaging activity of aspartame is expected, as the molecule does not show any electrophilic centre directly reactive with DNA. The Panel considered that the methods implemented were not sufficiently robust to support the results reported.

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In vivo studies 


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In the study by Durnev et al. (1995) which aimed at evaluating the genotoxicity of five sugar substitutes in mice, aspartame was investigated for induction of chromosomal aberrations in bone marrow cells of C57Bl/6 mice administered aspartame by oral gavage for five days at dose-levels of

40 and 400 mg/kg bw to groups of five animals. A concurrent negative control group was also

included. The animals were sacrificed 6 hours after the last administration of test compound. In the final two hours, colchicine was administered by intraperitoneal injection to accumulate cells in metaphase. A minimum of 100 metaphases per animal were scored. The results obtained indicate that aspartame did not induce any increase in the incidence of chromosomal aberrations compared with negative control values. However, the Panel noted that the study was poorly reported and that a concurrent positive control animal group to show whether the test system was functioning correctly had not been included. Furthermore, the sampling of bone marrow cells 6 hours after the last administration of test compound was not adequate for chromosomal aberration analysis and dose-

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levels administered appear to be very low. On these grounds, the Panel considered that the methods implemented were not sufficiently robust to support the results reported.

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In a study by Mukhopadhyay et al. (2000), male Swiss Albino mice were exposed by oral gavage to a blend of aspartame and acesulfame K (ratio 3.5:1.5) at doses up to 350 mg aspartame/kg bw. The blend of the two sweeteners showed a negative outcome for chromosomal aberrations. The Panel noted that no evaluation of cell cycle progression (e.g. mitotic index) was performed. Given the limitations of the study (blend of sweeteners and no mitotic index determination), the Panel considered it to be of limited relevance for the evaluation of the genotoxicity of aspartame.

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An acute bone marrow micronucleus test was conducted with aspartame administered orally to male Fisher 344 rats at three daily doses of 0 (control), 500, 1000 or 2000 mg/kg bw. No increase in the number of micronucleated polychromatic erythrocytes was observed at any of the tested dose levels (NTP, 2005). The Panel considered that the methods implemented were sufficiently robust to support the results reported.

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Peripheral blood micronucleus tests were conducted in male and female transgenic mice (Tg.AC hemizygous, p53 haploinsufficient or Cdkn2a deficient) after 9 months of exposure to aspartame at doses ranging from 3.1 to 50 g/kg diet. The highest dose tested was equivalent to 7660 and 8180 mg aspartame/kg bw/day in males and in females, respectively (NTP, 2005). The Panel considered that the methods implemented were thought to be sufficiently robust to support the results reported (Annex H; NTP, 2005). Negative results, indicative of an absence of clastogenic activity of aspartame, were obtained in male and female Tg.AC hemizygous and Cdkn2a deficient mice and in male p53 haploinsufficient mice. In female p53 haploinsufficient mice, the results of the test were judged positive by the authors of the study, based on a trend test revealing a statistically significant 2.3 fold increased frequency of micronucleated erythrocytes seen in the 50 g/kg diet group (NTP, 2005). However, the Panel noted that the incidence of micronucleated erythrocytes in female controls was the lowest among the historical control values of the same laboratory; this rendered the outcome of the trend analysis positive. Nevertheless, the observed incidence of micronucleated erythrocytes in the highest dose group fell outside the range of the historical controls. However, the Panel also noted that the reported increase in micronucleated erythrocytes was observed in one gender only. Furthermore, the effect described was observed after 9 months of administration of a daily dose, which exceeded approximately 8-fold, the highest recommended dose level for genotoxic testing according to OECD guideline 474. The Panel concluded that the findings were equivocal in the p53 transgenic strain (positive in female but not in male p53 haploinsufficient mice) but negative in the other two strains, and, overall, did not indicate a genotoxic potential for aspartame.

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Two studies addressing DNA damage as detectable by Comet assay are available, both following administration of aspartame by gavage.

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Sasaki et al. (2002) administered a single dose of aspartame (2000 mg aspartame/kg bw) to mice (four male/group) and analysed the stomach, colon, liver, kidney, bladder, lung, brain, bone marrow. Aspartame did not induce any significant increases in DNA migration. Based on these results, the Panel considered that aspartame was not genotoxic in the organs assayed. However, the Panel noted that for the 24 hour sampling time, a reduction in DNA migration (a marker for DNA cross-linking agents) was observed in all organs analysed, but the reduction was not significant. The Panel considered that the methods implemented were sufficiently robust to support the results reported.

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Bandyopadhyay et al. (2008) administered aspartame as a single dose of 0 (control), 7, 14, 28 and 35 mg aspartame/kg bw to mice (four males/group) by oral gavage, and at the highest dose-level, aspartame was reported to induce DNA damage in bone marrow cells. However, the Panel evaluated the study as poorly reported and noted that the dose levels used were low compared to other studies reporting negative results, and that an insufficient number of cells was scored (total of 50 cells/animal). Therefore, the Panel considered that the methods implemented were not sufficiently robust to support the results reported, and that no conclusion could be drawn from it.

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Kamath et al. (2010) used three endpoints to assess the genotoxic potential of aspartame following administration of 0 (control), 250, 455, 500 and 1000 mg aspartame/kg bw by gavage to mice. The authors concluded that aspartame induced (a) micronuclei in bone marrow erythrocytes, (b) micronuclei in peripheral blood and (c) chromosome aberrations in bone marrow erythrocytes. However, the number of animals and the ratio of polychromatic erythrocytes to normochromatic erythrocytes (PCE/NCE) ratio (the authors only stated that it was changed) are not given. The Panel considered that the methods implemented were thought not to be sufficiently robust to support the results reported, and that no conclusion could be drawn from it.

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AlSuhaibani (2010) tested aspartame for its ability to induce chromosome aberrations (CA), SCE and to affect the mitotic index (MI) in bone marrow cells of mice (five males/group; by gavage) at dose levels of 0 (control), 3.5, 35 and 350 mg aspartame/kg bw. The authors concluded that aspartame induced CA at 35 and 350 mg/kg bw, but neither dose level induced SCE nor decreased the MI. The Panel noted that an insufficient number of cells were scored (total of 50 metaphase cells/animal for CA; 30 metaphases for SCE). Furthermore, no positive control was included in the study and any supplementary information on cytotoxicity relevant for CA and SCE-analysis in the present experiment was lacking. The Panel considered that the methods implemented were not sufficiently robust to support the results reported, and that no conclusion could be drawn from the study.

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Karikas et al. (1998) reported a non-covalent interaction of excess aspartame, aspartic acid and phenylalanine with calf thymus DNA, inferred from the altered chromatographic profile of DNA. This effect was attributed to the electrostatic interaction of amino groups and the negatively charged phosphate in naked DNA. The Panel considered these findings, obtained in an acellular system in presence of excess aspartame, of minimal relevance for the evaluation of the genotoxic potential of aspartame.

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Meier et al. (1990) investigated the kinetic of formation, stability and reactivity of nitrosation products of aspartic acid, aspartame, and glycine ethyl ester. Nitrosation products were obtained in vitro, with incubation of 40 mM substrate and nitrite at pH 2.5 for varying times. The nitrosation products displayed an ‘alkylating’ activity in vitro, as indicated by the reactivity with the nucleophilic scavenger 4-(4-nitrobenzyl)pyridine, measured with a colorimetric method. In the same study, co- administration of glycine ethyl ester and nitrite to rats did not result in the formation of detectable DNA adducts in rat stomach.

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The issue of nitrosation was also addressed by Shephard et al. (1993). Aspartame and several naturally occurring dipeptides were nitrosated in vitro at low pH (3.5) in the presence of 40 mM nitrite and tested for mutagenicity in Salmonella typhimurium TA100. The nitrosation products of some dipeptides (Trp-Trp, Trp-Gly) and aspartame exhibited a direct mutagenic activity, which was related by the study authors to the nitrosation of their primary amino groups.

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Concerning the studies of Meier et al. (1990) and Shephard et al. (1993), the Panel noted the harsh conditions utillised for the in vitro nitrosation of substrates and considered the results of doubtful relevance for the assessment of the genotoxic risk posed by the dietary intake of aspartame or other natural amino acids and dipeptides.

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3.2.3.3. Conclusion on the genotoxicity of aspartame

The Panel concluded that the in vitro genotoxicity data on bacterial reverse mutation exhibited some limitations (e.g. absence of TA102 and WP2 uvrA Escherichia coli). However, the Panel considered the weight-of-evidence was sufficient to conclude that aspartame was not mutagenic in bacterial systems. Concerning mammalian systems in vitro, the Panel concluded that, apart from the valid UDS study that was negative, no conclusion could be drawn at the gene and chromosomal level because no studies dealing with these endpoints were available.

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In vivo, the majority of investigations on systemic genotoxicity reported negative findings. Equivocal positive findings were only described in a NTP study, positive in female but not in male p53 haploinsufficient mice; in two other transgenic mouse strains the results were negative.

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Concerning the possible site of first contact effects in vivo, limited data are available. However, the available in vitro data do not indicate a direct genotoxic activity of aspartame that might predispose to a site of first contact effect in vivo.

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Overall, the Panel concluded that the available data do not indicate a genotoxic concern for aspartame.

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Summary tables on the genotoxicity of aspartame are presented in Annex H.