Non-nutritive sweeteners (NNS) are often used as alternatives to sugar to reduce calorie and carbohydrate intake. However, despite their approval for use in foods and beverages by authoritative bodies such as the U.S. Food and Drug Administration (FDA) and the European Food Safety Authority (EFSA), controversy surrounding their use remains.

Safety evaluations of food additives such as NNSs by authoritative bodies must include assessments of genotoxicity — the ability of a substance to damage genetic information in cells.^[1]

This narrative review assessed the genotoxicity data on five common NCSs: acesulfame potassium (Ace-K), aspartame, saccharin, steviol glycosides (the compounds responsible for Stevia’s sweet taste), and sucralose.

The results were described based on three categories of endpoints, as discussed above:

• Mutagenicity
• Clastogenicity/aneugenicity
• Other DNA damage

Ace-K:

Mutagenicity: The authors cited two in vitro studies indicating that Ace-K did not induce mutations.

Clastogenicity/aneugenicity: Of the five in vivo studies cited by the authors, two found evidence of clastogenicity/aneugenicity (both in mice).^[4][5]

Other DNA damage: Of the four studies cited by the authors cited (two in vitro and two in-vivo), one reported increased DNA damage in bone marrow of mice.^[6]

Overall, the authors concluded that the weight of evidence indicates that Ace-K doesn’t have genotoxic potential.

Aspartame:

Mutagenicity: Of the five studies cited by the authors (all in vitro), only one found evidence of mutagenicity.^[7] However, the researchers conducting that study performed nitrosation of aspartame before testing, which the authors noted limits the generalizability of the results.

Clastogenicity/aneugenicity: Of the eight studies cited by the authors (two in vitro, six in vivo), six found evidence of clastogenicity/aneugenicity.^{[8][9][10][11][12][13]} However, the authors referenced an EFSA review^[14] that questioned the methods used in several of these studies. Additionally, the authors noted that one of these studies^[13] did not adhere to guidelines set by the Organisation for Economic Cooperation and Developement (OECD) for the in vitro method used.

Other DNA damage: Of the six studies cited by the authors (two in vitro, four in vivo), five found evidence of DNA damage.^{[15][16][17][6][18]} However, they noted that four of these studies did not appear to conform to OECD methodological recommendations, and lacked important information regarding their methods.

Overall, the authors concluded that the weight of evidence supports a lack of genotoxic potential for aspartame.

Sodium saccharin

Mutagenicity: Of the five studies cited by the authors (three in vitro, two in vivo), two showed evidence of mutations.^[19][20] The authors noted that the positive findings in the latter study were likely due to “impurities or contaminants in the test article.”

Clastogenicity/aneugenicity: The authors referenced a 1995 opinion article from the Scientific Committee for Food, noting that, while initial research found some evidence of clastogenicity/aneugenicity, these occurred at very high doses not applicable to human exposure.^[21] They cited four additional studies (all in vivo), two of which found evidence of chromosomal clastogenicity/aneugenicity.^[20][22]

Other DNA damage: The authors cited four studies, two of which found evidence of other DNA damage.^[23][24]

The authors concluded that while recent publications have provided inconsistent findings, the weight of evidence supports conclusions from other authoritative bodies that sodium saccharin is not genotoxic.

Steviol glycosides:

Mutagenicity: Of the five studies (all in vitro) cited by the authors, two demonstrated evidence of mutations.^[25][26] However, these two studies assessed steviol, a metabolite of steviol glycoside, which the authors noted is present at negligible levels in humans.

Clastogenicity/aneugenicity: Of the four studies cited by the authors (two in vitro and two in vivo), one demonstrated evidence of altered chromosome structure.^[27] However, the authors noted that the methods used in this study did not adhere to OECD recommendations.

Other DNA damage: Of the four studies cited by the authors (one in vitro, three in vivo), one study reported evidence of DNA damage.^[28] However, the authors cited the same EFSA review^[14] as mentioned previously, which noted several limitations in this study’s methods, including the use of only a single dose and just five rats.

The authors concluded that recent evidence supports a previous EFSA conclusion that steviol glycosides lack genotoxic potential.

Sucralose:

Mutagenicity: The authors cited one study (a report on numerous genotoxicity studies on sucralose), which found no evidence of mutations from sucralose.

Clastogenicity/aneugenicity: Of the four studies cited by the authors (one in vitro, three in vivo), two reported evidence of chromosomal clastogenicity/aneugenicity.^[29][30]

Other DNA damage: Of the five studies cited by the authors (two in vitro, three in vivo), three reported evidence of other DNA damage.^[24][30][30]

The authors concluded that “recent publications do not provide evidence that would justify a change to the SCF and USFDA conclusions that sucralose lacks genotoxic potential.”

This study was funded by the Calorie Control Council and the American Beverage Association.

When interpreting in vivo data, it’s important to consider the dose of sweetener used in the study. For example, the U.S. FDA has set the acceptable daily intake (ADI) for aspartame to 50 mg per kg of body weight per day (equivalent to about 75 sweetener packets). This figure is based on the no observed adverse effect level (NOAEL, the highest level of exposure that does not produce an adverse effect in test animals) divided by a safety factor (usually 100). Several in vivo studies reporting genotoxic potential used doses exceeding the ADI.

Overview of non-nutritive sweeteners

Sweetener	Brand names	Multiplier of sweetness intensity compared to table sugar	Acceptable Daily Intake (ADI)(mg/kg bw/day)	Number of tabletop sweetener packets equivalent to ADI
	Sweet One®, Sunett®	200x	15	23
	Nutrasweet®, Equal®, Sugar Twin®	200x	50	75
	Sweet and Low®, Sweet Twin®, Sweet’N Low®, Necta Sweet®	200–700x	15	45
	Truvia®, PureVia®, Enliten®	200–400x	4	9
	Splenda®	600x	5	23

It should also be noted that there were numerous instances in which the authors of the current review cited books and review papers when reporting the results of individual studies. This made it difficult to accurately assess the number and type of studies included for each sweetener and endpoint.

Several other reviews have assessed the genotoxicity potential of NNSs:

A 2021 narrative review concluded that “although its genotoxicity is unknown, aspartame has elevated proliferation and slow apoptosis in test cells and could have carcinogenic properties.”^[31]

A 2017 narrative review assessing the safety of sucralose concluded that “the collective evidence supports that sucralose is non-carcinogenic, based on carcinogenicity studies that comply with regulatory standards for appropriate design and conduct and no evidence of genotoxicity.”^[32] The authors reported funding from the Calorie Control Council.

A 2016 systematic review assessing sucralose and carcinogenicity concluded, “The results of in vitro and in vivo assays of sucralose revealed no confirmed genotoxic activity, consistent with the chemical structure and metabolism of sucralose.”^[33] The study was funded by McNeil Nutritionals, a company that has sold and marketed Splenda.

A 2015 narrative review assessing aspartame concluded, “The data available … support the conclusions of EFSA (2013) and of Magnuson et al. (2007) that aspartame does not show any genotoxic potential.”^[34] Funding was provided by the International Sweeteners Association.

A 2012 narrative review concluded, “The recent suggestions that steviol glycosides present a mutagenic — and therefore carcinogenic — risk to consumers are not supported by actual test results.”^[35] The authors received funding from Cargill Incorporated and The Coca-Cola Company.