Acceptable daily intake

An ADI, which is expressed as mg/kg body weight, has been defined as the amount of a chemical that can be consumed daily over the period of a lifetime with no appreciable risk to health^{( 16 )}. To assign an ADI, long-term, multiple-dose animal studies are typically used to initially establish the no observed adverse effect level by identifying the highest level of exposure which causes no adverse effects in the most sensitive species of animals⁽ Reference Renwick ^{13 –} Reference Logue, Peters and Gallagher ^{15 )}. To account for variation between species and within human subjects, safety factors are then applied (Fig. 1). In the absence of serious adverse effects (e.g. teratogenicity) in animal studies, an overall safety factor of 100 is usually applied to the no observed adverse effect level⁽ Reference Herrman and Younes ^{14 )}, although higher or lower safety factors can also be applied.

Fig. 1. (Colour online) Safety factors applied to the no observed adverse effect level to establish the acceptable daily intake (Source: Logue et al. ⁽ Reference Logue, Peters and Gallagher ^{15 )}). ADI, acceptable daily intake; NOAEL, no observed adverse effect level.

Health effects of low-calorie sweeteners intake

Despite extensive evaluation of the safety of LCS, the potential health effects of consumption has remained topical within the area of nutrition research⁽ Reference Mortensen ^{11 ,} Reference Kroger, Meister and Kava ^{17 ,} Reference Marinovich, Galli and Bosetti ^{18 )}. Moreover, the long-term efficacy of using LCS in place of non-milk extrinsic sugars as a weight management tool has yet to be conclusively established⁽ Reference de la Hunty, Gibson and Ashwell ^{19 –} Reference Swithers ^{25 )}. A recent meta-analysis of randomised controlled trials and prospective cohort studies into the effects of LCS consumption on weight status, reported that in prospective cohort studies, a small positive association was observed between LCS consumption and increased BMI (0·03 kg/m²) but not body weight or fat mass. However in randomised controlled trials, LCS consumption was associated with modest, albeit significant reductions in body weight (−0·80 kg), BMI (−0·24 kg/m²), fat mass (−1·10 kg) and waist circumference (−0·83 cm)⁽ Reference Miller and Perez ^{26 )}. The potential mechanisms by which LCS consumption might influence appetite and food intake were reviewed by Mattes and Popkin⁽ Reference Mattes and Popkin ^{27 )} and they concluded that, although the evidence was lacking for many putative mechanisms (such as cephalic phase stimulation, gut peptide response and increased palatability of products), further research in the free living population via long-term  randomised controlled trials was warranted. In addition to body weight status, LCS consumption in relation to a range of adverse health outcomes including cancer⁽ Reference Weihrauch and Diehl ^{28 ,} Reference Schernhammer, Bertrand and Birmann ^{29 )}, CVD⁽ Reference Duffey, Steffen and Van Horn ^{30 ,} Reference Pereira and Odegaard ^{31 )}, diabetes mellitus⁽ Reference Wiebe, Padwal and Field ^{32 ,} Reference Imamura, O'Connor and Ye ^{33 )} and preterm deliveries⁽ Reference La Vecchia ^{34 )} has also been investigated. No convincing evidence of a risk in the development of any adverse effects as a result of LCS consumption has been presented to date. The French Agency for Food, Environmental and Occupational Health and Safety recently undertook a review of the evidence with regard to many of these outcomes and concluded that, although the data do not demonstrate a risk, further research is required to establish the long-term beneficial effects of LCS consumption on health^{( 35 )}. Furthermore, the French Agency for Food, Environmental and Occupational Health and Safety also recommended that future cohort studies should aim to distinguish the intakes of individual LCS, so that the effects of single and multiple LCS use can be investigated more effectively^{( 35 )}. The ongoing debate around the safety of LCS consumption has served to fuel a somewhat negative perception within the lay media and the population in general. A recent study by Harricharan et al.⁽ Reference Harricharan, Wills and Metzger ^{36 )} investigated the attitudes of dietitians from several European countries towards LCS and highlighted a diversity of opinions ranging from negative, ambivalent to positive; they suggested the provision of guidance similar to that which has been undertaken in the USA^{( 37 )}.

Tiered approaches for assessment of low-calorie sweeteners exposure

An example of a tiered approach for assessing exposure to LCS is illustrated in Fig. 2. Tier 1, which is carried out at European wide level, will usually consist of an initial screening method designed to generate a highly conservative estimate so that potentially at risk groups can be easily identified. Examples of such screening methods include the Sweetener Substitution Method⁽ Reference Renwick ^{41 )} and the Danish Budget Method⁽ Reference Hansen ^{42 )}. In the Danish Budget Method, conservative assumptions are made about the occurrence of LCS in food and beverages and an individual's energy and fluid requirements; this will result in an overestimate of exposure. If, following the initial screening step, the ADI is deemed to be exceeded, further more refined assessments would then be indicated. Tier 2 involves the assessment of actual consumption of foods and beverages known to contain LCS along with the maximum permitted use levels. A Tier 3 assessment would be indicated if the ADI is estimated to be exceeded following the Tier 2 assessment and this will consist of a more refined calculation using actual levels of consumption and actual concentrations in products in order to further elucidate the risk.

Fig. 2. Tiered approach for food additive exposure estimates (adapted from EC^{( 40 )}). MPLs, maximum permitted levels; ADI, acceptable daily intake.

Methods of assessing exposure to low-calorie sweeteners

Dietary intake is traditionally assessed using tools such as food diaries, 24-h recalls and FFQ⁽ Reference Thompson, Subar, Coulston, Boushey and Ferruzzi ^{43 )}. These assessment tools rely on self-reported data and a number of inherent limitations exist which often results in inaccuracies⁽ Reference Livingstone, Prentice and Strain ^{44 –} Reference Kipnis, Midthune and Freedman ^{47 )}. The optimal methodology for assessing habitual LCS exposure is a 1–2-week prospective, brand level diary, including information on portion sizes along with brand-specific information on LCS concentrations in products⁽ Reference Renwick ^{48 )}. Given that obtaining such refined individual level data can be time and resource intensive for investigators, as well as labour intensive for participants, a variety of dietary assessment tools have been utilised in the past including 24-h recalls⁽ Reference Devitt, Daveman and Buccino ^{49 –} Reference Ha, Ha and Choi ^{53 )}, FFQ covering various durations⁽ Reference Toledo and Ioshi ^{54 –} Reference Lino, Costa and Pena ^{57 )} and food diaries lasting 2 d⁽ Reference van Rooij-van den Bos, Konings and Heida ^{58 )}, 5 d⁽ Reference Garnier-Sagne, Leblanc and Verger ^{59 )}, 7 d and 14 d⁽ Reference Leclercq, Berardi and Sorbillo ^{60 )}. Some studies used a combination of retrospective and prospective intake data in order to first identify potential high consumers and then to further investigate these high consumers⁽ Reference Arcella, Le Donne and Piccinelli ^{61 , 62 )}. It is apparent therefore that many of the published studies do not satisfy these criteria, potentially making it difficult to make direct comparisons.

Data on LCS concentrations in products can be obtained from a number of sources; the least resource intensive, yet least accurate, is to use maximum permitted use levels. This source of information is commonly used in initial, conservative estimates of exposure, or when it is not possible to obtain more refined data⁽ Reference Renwick ^{48 )}. However, the actual levels used in products are unlikely to meet these values, particularly when a blend of two or more LCS is used within a particular product, as is frequently done. Therefore using maximum permitted use levels in the calculation will result in an overestimate of exposure. Concentrations in specific products have been obtained from manufacturers⁽ Reference Devitt, Daveman and Buccino ^{49 ,} Reference Serra-Majem, Bassas and Garcia-Glosas ^{56 ,} Reference Leclercq, Berardi and Sorbillo ^{60 – 64 )}, providing a more accurate measure; however, difficulties in obtaining such information in the past have been highlighted^{( 65 , 66 )}. Furthermore, with product innovations and changing tastes among consumers, the concentrations of LCS within products are likely to evolve over time, potentially introducing error into subsequent estimates of exposure unless ongoing data are received from manufacturers. Another method for determining LCS concentrations in products is to directly measure them analytically and to this end, numerous methodologies for the determination of LCS in foods and beverages have been published⁽ Reference Zygler, Wasik and Namiesnik ^{10 )}. Recent studies investigating LCS exposure have adopted this approach⁽ Reference Chung, Suh and Yoo ^{50 –} Reference Ha, Ha and Choi ^{53 ,} Reference Lino, Costa and Pena ^{57 ,} Reference van Rooij-van den Bos, Konings and Heida ^{58 )} and, although it allows for a more accurate and objective measure of LCS concentrations in products, it is also likely to be costly. Furthermore, with the ubiquity of LCS in today's market and the trend towards more widespread use, adopting such an approach as part of future assessments may prove unfeasible.

Recent developments in low-calorie sweeteners exposure assessment

A desire to harmonise food additive exposure assessment across the EU led to guidelines on how Member States should collect intake data for exposure assessments^{( 40 )} and this was further enhanced through the recent implementation of the Flavourings, additives and food contact exposure tool (FACET)^{( 66 )}. As a result of the FACET project, a publicly available exposure software package has been released (available at: http://expofacts.jrc.ec.europa.eu/facet/login.php). One of the strengths of the FACET project is that cooperation was obtained from FoodDrinkEurope, an alliance of national food and drink industries in the EU, for the provision of information on the concentrations of targeted food additives in products, including aspartame and acesulfame-K and this will result in a more sustainable system of monitoring exposure over time.

Alternatively, the Monte Carlo risk assessment software tool, developed as part of the EU-wide ACROPOLIS (Aggregate and cumulative risk of pesticides: an on-line integrated strategy) project, may also be used for the assessment of exposure to chemicals in foods (available at: https://mcra.rivm.nl/Account/Login). This tool is designed to allow a cumulative assessment of exposure to multiple chemicals from multiple sources⁽ Reference van Klaveren, Kennedy and Moretto ^{67 )} and has been successfully applied for the assessment of acute and chronic exposure to a group of pesticides in a number of European countries⁽ Reference Boon, van Donkersgoed and Christodolou ^{68 )}. Like the FACET tool, the ACROPOLIS tool is freely accessible.

Actual low-calorie sweeteners exposure

European based studies carried out over the last 20 years have largely reported that the intakes of LCS fall well within the ADI (Table 2), with only the intake of cyclamate potentially exceeding the ADI in some population sub-groups. Similar results were reported in studies conducted in Korea, Australia and New Zealand and Brazil⁽ Reference Chung, Suh and Yoo ^{50 –} Reference Ha, Ha and Choi ^{52 ,} Reference Leclercq, Berardi and Sorbillo ^{60 )}. A review by Renwick⁽ Reference Renwick ^{48 )} reported that the overall intakes of LCS had not increased significantly during the preceding decade, although it has been reported elsewhere that the numbers of people consuming LCS were increasing⁽ Reference Sylvetsky, Welsh and Brown ^{69 )}.

Table 2. Exposure estimates of low-calorie sweeteners in Europe over the past 20 years

No data reported for other LCS. ‘–’ indicates no data reported.

Ace-k, acesulfame-K; ADI, acceptable daily intake; Asp, aspartame; Cyc, cyclamate; Sac, saccharin; Suc, sucralose; FSA, Food Standards Agency.

* Mean % ADI presented with high consumers (% ADI) where available.

Potential application of a biomarker approach for investigating low-calorie sweeteners intakes

The metabolic fates of LCS are well known (Table 3) and therefore a hypothesis-driven approach would appear to be the most appropriate for the implementation of a biomarker approach for investigating exposure. Following ingestion, aspartame is hydrolysed to aspartic acid, phenylalanine and methanol, each of which commonly occur in a normal diet⁽ Reference Butchko, Stargel and Comer ^{83 ,} Reference Magnuson, Burdock and Doull ^{84 )}, while thaumatin undergoes normal protein digestion^{( 85 )}. Neohesperidine dihydrochalcone, although not known to exist in nature, is structurally similar to naturally occurring flavonoid glycosides and undergoes a similar metabolic fate to these analogues with the same or similar metabolites⁽ Reference Borrego, Montijano and O'Brien Nabors ^{86 )}. This finding would indicate that no obvious specific candidate biomarkers exist for these compounds. Acesulfame-K⁽ Reference Christ and Rupp ^{87 )} and saccharin are almost completely absorbed and excreted unchanged via the urine,⁽ Reference Byard, McChesney and Golberg ^{88 –} Reference Sweatman, Renwick and Burgess ^{90 )} while cyclamate (30–50 %)⁽ Reference Renwick and Williams ^{91 )} and sucralose (10–15 %)⁽ Reference Grice and Goldsmith ^{92 ,} Reference Roberts, Renwick and Sims ^{93 )} undergo partial absorption and the absorbed proportion is excreted unchanged via the urine with the unabsorbed proportions excreted via the faeces. In about 20 % of the population, cyclamate can be converted to cyclohexylamine via bacterial hydrolysis in the gut, which is absorbed and also excreted via the urine⁽ Reference Bopp, Sonders and Kesterson ^{94 )}. Furthermore, the extent of cyclamate conversion to cyclohexylamine can be variable during chronic exposure⁽ Reference Renwick, Thompson and O'Shaughnessy ^{95 )}. Steviol glycosides also undergo bacterial hydrolysis to steviol which is then absorbed and excreted via the urine as steviol glucuronide⁽ Reference Geuns, Buyse and Vankeirsbilck ^{96 –} Reference Wheeler, Boileau and Winkler ^{98 )}. Advantame is converted to advantame acid and a small proportion is absorbed (about 6 %) and excreted via the urine while about 90 % of a dose is excreted via the faeces⁽ Reference Ubukata, Nakayama and Mihara ^{99 )}.

Table 3. Metabolic fates and routes of excretion of low-calorie sweeteners approved in Europe

CAS, Chemical Abstract Service; NHDC, neohesperidine dihydrochalcone; N/A, not applicable as broken down to normal dietary components; JECFA, Joint FAO/WHO Expert Committee on Food Additives.

* No CAS Number.

† Principal route of excretion listed first.

Saccharin, acesulfame-K, cyclamate and sucralose undergo no or limited metabolism following absorption into the body; therefore candidate biomarkers, in the form of the parent compounds, exist for these LCS. For steviol glycosides and advantame, the excretory products may act as suitable biomarkers for intakes. A high level of specificity of the candidate biomarkers exists as they are not found elsewhere in the diet or formed endogenously and given that at least a proportion of each of these compounds is excreted via the urine, a urinary biomarker approach may be feasible. Indeed, such an approach for assessing exposure to acesulfame-K and saccharin was previously described by Wilson et al.⁽ Reference Wilson, Wilkinson and Crews ^{63 )} who measured levels of excretion in 24-h urine samples and found excellent levels of correlation in an intake/excretion study (R ² > 0·99 for both compounds), demonstrating a clear dose–response relationship for both compounds. Slightly lower correlations were observed when validated against an FFQ which is probably more indicative of a limitation with the FFQ rather than the biomarker as the FFQ did not account for non-dietary sources of the LCS. The dose–response relationships for cyclamate, sucralose, steviol glycosides and advantame, however, are less clear so future work will be required to elucidate these relationships before the usefulness of measuring urinary concentrations for investigating intakes is established.

To fully characterise the relationship between urinary excretion and intakes of these LCS, a suitable and validated analytical method is first required⁽ Reference Wild, Andersson and O'Brien ^{76 )}. To this end, a liquid chromatography, tandem-MS/MS method of simultaneously determining urinary levels of acesulfame-K, cyclamate, saccharin, steviol glucuronide and sucralose has recently been developed and validated⁽ Reference Logue, Dowey and Strain ^{100 )} and allows for investigations of the feasibility of implementing a biomarker approach for assessing intakes of these compounds.

Potential limitations of a urinary biomarker approach for assessing low-calorie sweetener intakes

Although a biomarker approach would potentially offer many advantages over more traditional methods of investigating exposure to LCS, potential limitations should also be acknowledged. As urinary biomarkers, only short-term exposure (previous 24–48 h) can be investigated⁽ Reference Kuhnle ^{81 )}. The mode of sampling is also an important factor to consider for the implementation of a biomarker approach and if 24-h urine samples are required, a lack of compliance could also represent a limitation. To mitigate this limitation, however, methods of assessing compliance have been developed such as the paraminobenzoic acid method⁽ Reference Bingham and Cummings ^{101 )}. It must also be acknowledged that a biomarker approach, utilised on its own, will not provide information on the source of exposure. Therefore in the event that the ADI of a particular LCS is being exceeded, more traditional methods would be required to identify the main sources of exposure^{( 39 )}. As such, for the purposes of monitoring exposure in the population, a biomarker approach would likely be most useful when used as an adjunct to more traditional methods.

In summary, knowledge of the intakes of LCS is a legislative requirement in the EU and although the recent development and implementation of the FACET project will help harmonise methods among EU Member States and improve knowledge with regard to exposure to food additives, a successfully implemented biomarker approach for investigating LCS intake would undoubtedly be a useful adjunct to such monitoring activities. Furthermore, with ongoing interest and debate around the efficacy, as well as safety, of long-term LCS use, a biomarker approach would help elucidate the intakes of specific and combinations of LCS, and thereby address a limitation in the evidence to date, as highlighted by the French Agency for Food, Environmental and Occupational Health and Safety.