Vicent ; Yusà

G Quintás

O Pardo

P Martí

A Pastor

Prof Agustín

Food Additives and Contaminants

Keywords: acrylamide, pressurised fluid extraction, LC-MS-MS, design of experiments, food survey

Determination of acrylamide in foods by pressurized fluid extraction and liquid chromatography-tandem mass spectrometry. Survey of Acrylamide in cereal-based foods from Spain

-32,33 0,066 Pressure (psi) 6,67 0,679 a coded units; α = 0.05 b The minimum, central and maximum values of each factor used in the P-B design were: Temperature ºC (35-55-75); time (min) (4-7-10); pressure (psi) (1000-1250-1500). c A solution containing 50 ng mL -1 of Acrylamide and 75 ng mL -1 of [ 13

Table IV

Recovery, repeatability and within-laboratory (W-l) reproducibility of Acrylamide extracted by the ASE method in various food matrices at two incurred levels.

Matrix

Acrylamide a (ng g -1 )

Acrylamide b (ng g -1 )

Repeatability a Mean acrylamide level ± standard deviation (SD)determined by a reference method [START_REF] Roach | Rugged LC-MS/MS Survey Analysis for Acrylamide in Foods[END_REF], (n=5) b Determined by the ASE method; n = 5 c Three-day precision study, n = 5 each day. RSD: relative standard deviation %.

Introduction

The possibility that acrylamide (2-propenamide, CAS Registry No. 79-06-1) may be formed in the heat-processing of foods was first postulated in Sweeden in 2000 [START_REF] Tareke | Acrylamide: A cooking carcinogen?[END_REF]. The same authors published a study showing relatively high levels of acrylamide in many heat-processed foods [START_REF] Tareke | Analysis of Acrylamide, a Carcinogen in Heated Foodstuffs[END_REF]. Acrylamide is formed during the Maillard reaction, and the major reactants leading to the formation of acrylamide are reducing sugars such as glucose and fructose, and the amino acid aspargine [START_REF] Mottram | Acrylamide is formed in the Maillard reaction[END_REF][START_REF] Stadler | Acrylamide from Maillard reaction products[END_REF]. In model experiments, other pathways have been identified as precursors of acrylamide such as acrolein, acrylic acid and other carbonyls (EFSA 2003).

National and international authorities have been concerned, since acrylamide has been classified as "probably carcinogenic to humans" (Group 2A) by the International Agency for Research on cancer (IARC 1994). Consequently, a worldwide monitoring of acrylamide in different food types has started [START_REF] Lineback | Overview of Acrylamide Monitoring Database[END_REF]. Extensive data on levels of acrylamide have been collected by the European Commission (EC 2005) and by the US Food and Drug Administration (FDA 2004). Among the most important matrices included in the European monitoring database are potato chips (median level: 600 µg kg -1 ), crisp bread (median level: 251 µg kg -1 ), breakfast cereals (median level: 60 µg kg -1 ), biscuits (median level: 90 µg kg -1 ) and coffee roasted (median level: 264 µg kg -1 ).

Having sensitive and robust methods that provide reliable data in the different food categories is of crucial importance for intake assessments. During the last four years, different methods have been developed to determine acrylamide in cooked and processed (baked, fried and roasted) foods [START_REF] Wenzl | Analytical methods for the determination of Acrylamide in foods products: a review[END_REF][START_REF] Taeymans | A Review of Acrylamide: An Industry Perspective on Research, Analysis, Formation, and Control[END_REF]). These methods are based either on gas chromatography-mass spectrometry (GC-MS) [START_REF] Pittet | Trace level determination of Acrylamide in cereal foods by gas chromatography-mass spectrometry[END_REF] or on LC-MS-MS [START_REF] Roach | Rugged LC-MS/MS Survey Analysis for Acrylamide in Foods[END_REF][START_REF] Riediker | Analysis of Acrylamide in food by isotope-dilution liquid chromatography coupled with electrospray ionization tandem mass spectrometry[END_REF]. Limits of quantification (LOQ) range from 30-50 µg kg -1 for LC-MS-MS down to 10-30 µg kg -1 for GC-MS. There is no significant difference between results obtained by GC-MS with derivatisation and LC-MS-MS [START_REF] Owen | Acrylamide Analysis: Assessment of Results from Six Rounds of Food Analysis Performance Assessment Scheme (FAPAS ® ) Proficiency Testing[END_REF][START_REF] Klaffke | Results from two Interlaboratory Comparison Tests Organized in Germany at the EU Level for Analysis of Acrylamide in Food[END_REF] [START_REF] Riediker | Analysis of Acrylamide in food by isotope-dilution liquid chromatography coupled with electrospray ionization tandem mass spectrometry[END_REF]), shaking at high speed [START_REF] Pittet | Trace level determination of Acrylamide in cereal foods by gas chromatography-mass spectrometry[END_REF], or in a rotating shaker [START_REF] Roach | Rugged LC-MS/MS Survey Analysis for Acrylamide in Foods[END_REF], using water as extractant. Most clean-up procedures consist of the combination of two different solidphase extractions (SPE) [START_REF] Roach | Rugged LC-MS/MS Survey Analysis for Acrylamide in Foods[END_REF][START_REF] Riediker | Analysis of Acrylamide in food by isotope-dilution liquid chromatography coupled with electrospray ionization tandem mass spectrometry[END_REF]. These procedures are laborious and time consuming. Another more simple approach has been proposed using Carrez I and Carrez II to obtain a clear solution, without further clean-up (Hoenicke et al. 2004[START_REF] Senyuva | Survey of Acrylamide in Turkish foods by an in-house validated LC-MS method[END_REF]. Like the SPE methods, these Carrez-based methods also make use of water as extractant, which difficults the concentration of the extract and, consequently, present problems to reduce the LOD.

Pressurized fluid extraction has been frequently used to extract lipophilic pollutants from environmental matrices and foods [START_REF] Dean | Extraction methods for environmental analysis[END_REF][START_REF] Yusà | Application of accelerated solvent extraction followed by gel performance chromatography and high-performance liquid chromatography for the determination of polycyclic aromatic hydrocarbons in mussel tissue[END_REF]). However, PFE has been scarcely used for the extraction of hydrophilic compounds. The use of PFE for extraction of acrylamide from food has been described by [START_REF] Cavalli | Fast determination of Acrylamide in Food Samples Using Accelerated Solvent Extraction Followed by Ion Chromatography with UV or MS Detection[END_REF]. Hoenicke et al. ( 2004) have reported some problems when extraction was performed using both water and 1% formic acid, as was proposed by Cavalli et al.

This paper presents an automated, simple and fast method for the confirmatory detection of acrylamide. It entails pressurised fluid extraction of acrylamide from food, Carrez precipitation and analysis by LC-MS-MS. The main parameters affecting the performance of the Electrospray Ionisation (ESI) interface and the PFE has been optimized using a chemometric design of experiment (DOE) approach [START_REF] Massart | Handbook of Chemometrics and Qualimetrics[END_REF][START_REF] Riter | Statistical design of experiments as a tool in mass spectrometry[END_REF].

Using the optimised method a survey of acrylamide levels in five cereal-based foods groups (potato chips, breakfast cereals, snacks, crisp bread, and biscuits) from Spanish market was carried out.

F

Sample preparation

Serving size portions of samples were ground using a laboratory mill and sieved through a 1mm-mesh sieve. Sub-samples of the homogenate were stored at 4 ºC in high-density A total of 500 µL of Carrez I (potassium hexacyanoferrate, 150 g L -1 ) and 500 µL of Carrez II (zinc sulphate, 300 g L -1 ) were added to the acetonitrile extracts and the suspension was centrifuged at 5000 g for 15 min. at 5 ºC. Then the precipitate was washed two times with acetonitrile, and the clear supernatant was evaporated in a Turbo Vap 500 (Zymark, Idstein, Germany). Final extracts were reconstituted in 0.5 mL of water and filtered through a 0.22 µm nylon filter prior to LC-MS-MS analysis.

A method based on that reported by Roach et al. (2002) employing water as extractant and a two-step SPE clean up, was used in order to compare the method developed in this work with a more conventional extraction and clean up procedure.

ESI-MS-MS analysis

A TSQ quantum triple stage quadrupole instrument, together with Surveyor LC quaternary pump and Surveyor autosampler, from Thermo Finnigan (San Jose, CA, USA), were used.

Analytical separation was carried out on a porous graphitic carbon column Hypercarb, 50 x 2.1 mm i.d., 5 µm from Thermo Electron Corporation (San Jose, CA, USA),

The mobile phase employed for the isocratic elution of the analyte was a mixture of methanol:water (0.1% HCOOH) (3.75:96.25%, v/v) at a flow rate of 150 µL min -1 . The LC Acrylamide monomer is toxic and can be adsorbed through the skin. Gloves and safety glasses should be worn all time, and standards and sample preparation steps should be carried out in a fume cupboard.

Results and discussion

Optimisation of the ESI-MS-MS interface conditions

Screening design

Considering the literature and previous studies carried out in our laboratory, four factors (k=4) were selected as potentially affecting the ESI efficiency: spray voltage, capillary temperature, sheath gas pressure and auxiliary gas pressure.

A Plackett-Burman (P-B) design [START_REF] Massart | Handbook of Chemometrics and Qualimetrics[END_REF]) was chosen as a screening method to estimate the relative influence of the 4 factors on the analytical response (arbitrary units of peak area of ions m/z 55 and m/z 44). With this screening design the main effects are calculated with a reduced number of experiments (12 runs, 1 block), and the experiment run order was randomized to provide protection against the effect of hidden variables.

The estimated effects of the four factors and their statistical significance at 95% confidence level for responses m/z 55 and m/z 44 are shown in Table I. As can be seen in Table I, only spray voltage had a significant effect on the m/z 55 response (P< 0.05). However, for m/z 44 response, spray voltage and sheath gas pressure were significant. In consequence, both factors were selected for further optimization. For the next stage in the optimization process, the non-significant factors, capillary temperature and auxiliary gas pressure, were fixed at 200 ºC (negative effect) and 5 au (negative effect), respectively.

[ [ [ [Insert Table I about here] ] ] ]

Central composite design

In order to obtain a more accurate optimisation of the two significant ESI parameters (spray voltage and sheath gas pressure), a central composite design (CCD) was used. This type of experimental design permits the response surface to be built and the factor settings or operating conditions that maximise acrylamide response to be found.

The CCD used had a total of 13 randomised chromatographic runs, which included 4 cube points, 5 center points in cube, and 4 axial points, with an Alpha value of 1.4142. The values corresponding to every factor in each experiment and the analytical response are shown in Table II.

[ [ [ [Insert Table II about here] ] ] ]

The responses were fitted by a multiple regression equation, including second-order (curvature) and interaction terms. Three-dimensional response surface display the effect of the two independent variables on the analytical response (arbitrary units of peak area of ions m/z 55, m/z 44). Figure 1 shows the m/z 55 response surface yielded by the model.

[ [ [ [Insert Figure 1 about here] ] ] ]

The following step was to select the factors setting that maximise the acrylamide (m/z 55 and m/z 44) responses. This could be done by the "response optimiser" from response surface design in MINITAB program.

The responses surfaces were similar for the two analytical signals (m/z 55, 44) and therefore there were a factor setting that simultaneously maximises the desirability for each response (the desirability is 0.0 for the lowest values obtained in the CCD; increases as response values increase; and is 1.0 for the highest response obtained in the experiments).

We maximise a composite desirability, which combines the individual desirability of each response into a single measure. The optimised factors settings were time: Spray voltage:

Effect of extraction solvent

Acetonitrile, metanol, acetone, and ethyl acetate were tested as extraction solvents under the following PFE operating conditions: pressure, 1500 psi; temperature, 50 ºC; static time, 5 min; cycles, 2. The experiments were carried out on the reference test material FAPAS T 3002 (potato crisps, 167 µg Kg -1 acrylamide).

Figure 2 shows representative chromatograms obtained for the four extraction solvents.

Methanol, acetone and ethyl acetate extracts contain interfering co-extractives resulting in either strong interferences or ion suppression. On the other hand, extracts produced using acetonitrile were "clear", and higher signal responses were obtained due to less ion suppression effects. For other matrices (breakfast cereals, biscuits, snacks, crisp bread) similar results were obtained. Consequently, acetonitrile was selected extractant.

It was found that the use of extraction thimbles proved to be very useful because it avoids the possible blockage of the PFE system during the extraction of the different matrices.

[ [ [ [Insert Figure 2 about here] ] ] ]

The extraction with acetonitrile followed by Carrez precipitation provides an adequate clean up similar to that provided by SPE clean up for food groups like potato chips, breakfast cereals, biscuits, crisp bread and snacks. Figure 3 compares chromatograms obtained after PSE and Carrez precipitation with other obtained by a reference method that uses water extraction and SPE clean up [START_REF] Roach | Rugged LC-MS/MS Survey Analysis for Acrylamide in Foods[END_REF]. As can be seen, both methods provide "clean" chromatograms for acrylamide, however the Carrez precipitation improves the signal-to-noise ratio of the acrylamide analytical response. III. The temperature and the pressure had both no significant effect on the extraction yield of the PLE (P> 0.05). The time is on the limit of significance (P=0.066), with a negative effect on the recovery of acrylamide.

Consequently, we select the following factor setting: temperature = 35 ºC (negative effect); pressure = 1500 psi (positive effect); time = 4 min (negative effect).

[ [ [ [Insert Table III about here] ] ] ]

Method performance

The acrylamide response was linear over a concentration range of 1-2000 µg L -1 with correlation coefficients better than 0.995.

Assessment of the accuracy was based on the analysis of acrylamide reference test materials FAPAS T3002 (potato crisps, assigned value: 167 µg Kg -1 acrylamide, satisfactory range of 97-237 µg Kg -1 ), FAPAS T3005 (crisp bread, assigned value: 707 µg Kg -1 acrylamide, satisfactory range of 468-945 µg Kg -1 ), and T3011 (potato crisps, assigned value: 1404 g Kg -1 acrylamide). The average value and their standard deviation (SD) for five repetitions were: FAPAS T3002: 170 ± 10; FAPAS T3005: 730 ± 20; FAPAS T3011: 1370 ± 40. These values are within the satisfactory range for each test material. Recovery of the developed method was studied in different incurred matrices, at two levels.

In order to determine the concentration of incurred acrylamide in the different samples, a reference method based on that reported by Roach et al. (2002) was used.

Table IV reports the results of recovery, repeatability and within-laboratory reproducibility (three-day) study carried out on four matrices at two levels each. The relative standard deviation (RSD) values ranged from 3.5 to 6.0%, and from 4.2 to 9.2 % for repeatability and within-laboratory reproducibility, respectively.

A two-sample t-test approach, at 95% confidence level, was used in order to compare both methods. For the different samples and levels P-values>0.05 were obtained. This means that no significant differences exist between both methods.

The limit of quantification (LOQ) (S/N = 10) for the different commodities was set at 5 µg Kg -1 on the basis of compliance with the confirmatory criteria.

[ [ [ [Insert Table IV about here] ] ] ]

Survey data

In this study, 62 food samples from 5 food groups were analysed for their acrylamide content. Different Spanish brands of snacks, potato chips, biscuits, crisp bread and breakfast cereals were selected from local supermarkets in Valencia during June-August

2005.

The mean concentration of acrylamide, the standard deviation (SD) in each group, and the maximum and minimum acrylamide levels found are summarised in Table V.

[ [ [ [Insert Table V about here] ] ] ]

There was a great variation in the acrylamide levels between the brands in each group of foods (see standard deviation in Table V). This high variability is mainly a result of the variability of the raw materials and processing conditions (temperature, time,.

  Response surfaces for Acrylamide (m/z 55) obtained in the optimization of ESI parameters using a central composite design. The fixed parameters were: Capillary temperature, 200 şC; Auxiliary gas pressure, Mass chromatograms of Acrylamide extracted by PFE with A) Acetonitrile, B) Methanol, C) Acetone, and D) Ethyl acetate. 286x199mm (200 x 200 DPI) Mass chromatograms of a crisp bread sample obtained with A) water extraction and SPE clean up, and B) PFE and Carrez precipitation. 286x201mm (200 x 200 DPI)

  plastic screw-capped lids. Then a portion of the sub-sample (1g) was spiked with [ 13 C 3 ] acrylamide as internal standard to achieve a final concentration of 37.5 µg Kg -1 . The sample was ground with diatomaceous earth (DE) with a pestle and mortar and loaded into a cellulose extraction thimble and then placed in a 22-mL stainlesssteel extraction cells containing a cellulose filter in the cell outlet. The sample cells were then closed, to finger tightness, and situated in the carousel of a PFE system, Accelerated Solvent Extraction (ASE ® ) system 200 from Dionex (Sunnyvale, CA, USA).The samples were extracted with acetonitrile, using the following optimized conditions: temperature of 35 ºC with 5 min heat-up time under a pressure of 1500 psi, and three static cycles with a static time of 4 min. The flush volume amounted to 60% of the extraction cell volume. The sample cells were purged using pressurized nitrogen (125-150 psi) for 2 min.

  to the LC-MS interface within the retention time window 0.5-5 min using a diverter valve (Rheodyne, Cotati, USA). The total run time of the chromatograms was 6 min. An injection volume of 10 µL was used. The autosampler and column temperatures were 20 ºC and 26 ºC, respectively.Acrylamide was detected in positive ESI mode. The transitions monitored for acrylamide were m/z 72 > 55 at 12 V and 72 > 44 at 43 V, corresponding to C 2 H 3 CO + and CONH 2  + , respectively. For [13 C 3 ] acrylamide the transition monitored was 75 > 58 at 22 V. The ESI optimized parameters were as follows: spray voltage, 5000 V; capillary temperature, 200ºC; sheath gas pressure, 32 psi; auxiliary gas pressure, 5 arbitrary units (au). Collision gas pressure was 1.5 mTorr. Tube lens offset voltages were optimized for acrylamide using the automated optimization procedure in syringe infusion mode provided by the manufacturer.For quantification, an isotope dilution approach was used. A response curve was plotted of area response ratio for m/z 55/58 versus amount of acrylamide injected with a constant amount of [ 13 C 3 ] acrylamide. Nine-point linear calibration plots with correlation coefficient (R 2 ) > 0.995 in the range 1-2000 ng mL -1 were used. The results were reported without correction for recovery because of the use of [ 13 C 3 ] acrylamide as internal standard made this correction unnecessary.Confirmation of identity of the response is based on three criteria. First, the analyte ions m/z 55, and m/z 44 and the internal standard ion m/z 58 must occur at the same retention time. Second, the relative abundance of the analyte signals for m/z 55 and m/z 44, must be within ±20 % of the observed relative abundance recorded for an acrylamide standard of a similar concentration. Third, the three ions (m/z 55, 44, 58) must have a signal-to-noise (S/N) ratio > 3. These confirmatory criteria fulfil the requirements of the European Community for confirmatory methods(EC 2002), although this Decision is currently only applicable to residues in living animals and animal products.

6

  Analytical quality assurance and safetyEach set of samples were analysed under quality-assurance protocols, including duplicate samples, reagent blanks and three quality control test materials (FAPAS test materials, crisp bread T3005, potato crisps T3002 and potato crisps T3011).During this study, the laboratory participated in one inter-laboratory proficiency testing provided by Central Science Laboratory (CSL) with a satisfactory z-score. The laboratory works under the quality assurance system established by ISO/IEC/EN 17025.

  and sheath gas pressure, 32 psi . These conditions provide a composite desirability of 0.94.

  Optimization of PFE conditionsThe main parameters that have an influence on the performance of the PFE are temperature, pressure and extraction time(Fizpatrich et al. 2000). A P-B design was chosen for screening of the relative influence of these three parameters on the recovery of acrylamide from food samples. The P-B design included 12 plus a triplicate centre point runs. Taking into account the values usually used in PFE technique for pressure and extraction time, and that acrylamide starts to discompose at temperatures above 83 ºC, the minimum and maximum values for the P-B design were: 35-75 ºC for temperature, 1000-1500 psi for pressure, and 4-10 min for static time.The estimated effects of the three factors and their statistical significance at 95% confidence level (P<0.05) are shown in Table

  α = 0.05 b The minimum and maximum values of each factor used in the P-B design were: Spray Voltage (V) (2000-5000); Sheath gas pressure (psi) (10-40); Auxiliary gas pressure (au) (5-20); Capillary Temperature (ºC) (200-350) c A solution containing 50 ng mL -1 of acrylamide and 75 ng mL -1 of [ 13 C 3 analytical response (peak area) a of central composite design used for optimization of the two significant ESI factors in the analysis ; α = 0.05 b The minimum, central and maximum values of each factor used in the P-B design were: Temperature ºC (35-55-75); time (min) (4-7-10); pressure (psi) (1000-1250-1500).c A solution containing 50 ng mL -1 of acrylamide and 75 ng mL -1 of [ 13 C 3 ] acrylamide was used and within-laboratory (W-l) reproducibility of acrylamide extracted by the ASE method in various food matrices at two incurred levels.

Table II

 II Experimental conditions and analytical response (peak area) a of central composite design used for optimization of the two significant ESI factors in the analysis of

	Acrylamide b F Table III Estimated effects a and P-values obtained from Plackett-Burman o design bc used in the optimization of the PLE r Factor Effect P-value
	Factors Spray Voltage (V) Sheath Gas Pressure (psi) 3000 50 3000 15 Temperature (ºC) Run Order 1 2 -5,67 P r Area m/z 44 (au) 64506 72812 0,725 Analytical response Area m/z 55 (au) 1796581 2546153 e e Time (min)
	3 4 5 6 7 8 9 10 11 12	3900 2627 3900 4800 3900 5173 4800 3900 3900 3900	33 33 33 15 33 33 50 8 33 33	86098 66416 98794 86012 84306 107783 80590 53306 80670 83481 R e v i e w	3040198 2230607 3068004 3005588 2926011 3435103 2590584 1931181 2785549 2707467
	13 a A solution containing 50 ng mL -1 of Acrylamide and 75 ng mL -1 of [ 13 C 3 ] Acrylamide 3900 57 56764 1989331 was used b The fixed factors were: capillary temperature, 200 ºC; auxiliary gas pressure, 5 au O n au: arbitrary units. l y

  Germany) and Cambridge Isotope Laboratories Inc. (Andover, MA, USA), respectively. Stock solutions were prepared by dilution in water and stored in capped amber vials at 4 ºC. Working standard solutions for the calibration curve, were prepared by dilutions in water. Concentrations of the calibration standards were 1, 10, 25, 50, 100, 250, 500, 1000, and 2000 ng mL -1 , all with [ 13 C 3 ] acrylamide at 75 ng mL -1 .

	Experimental
	Standards and solvents
	Acrylamide (>99%) and [ 13 C 3 ] acrylamide (99% isotopic purity) were obtained from Sigma
	(Diesenhofen,
	o
	r
	P
	e e r
	R e v i e w
	O n
	l y

Ethyl acetate (SupraSolv ® grade), acetone (SupraSolv ® grade), and formic acid (analyticalreagent grade) were purchased from Merck (Darmstadt, Germany); methanol (gradient HPLC grade) and acetonitrile (Multisolvent ® HPLC, reagent grade) were supplied by Scharlau (Barcelona, Spain). Hyflo super Cell ® diatomaceous earth was supplied by Sigma-Aldrich (Steinheim, Germany). Water was Milli-Q water from a Millipore purification system. acrylamide reference test materials FAPAS T3002 (potato crisps), FAPAS T3005 (crisp bread), and T3011 (potato crisps) were provided by CSL (Central Science Laboratory, York, UK).

Oasis HLB 6 mL, 200 mg, and Oasis MCX 3 mL, 60 mg SPE cartridges were obtained from Waters (Mildford, MA, USA). Statistical data manipulation and numerical analysis of data resulting from experimental design were carried out by means of the statistical package MINITAB for Windows, Release 14 (Minitab Inc., Birmingham, UK).

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

Acknowledgements

We thank Mrs. Clara Coscollà for excellent technical assistance. We also thank the Food Safety Service of Generalitat Valenciana for providing us with the food samples.


Isabel Arranz Hernandez

Joerg Stroka

Michael Neugebauer

Isabel Arranz ¹

Determination of aflatoxin B1 in tiger nut based soft drinks

Keywords: Aflatoxin B 1, tiger nuts, 'chufa', 'horchata', High-performance Liquid-Chromatography

An analytical method for the determination of Aflatoxin B 1 (AfB 1 ) in a tiger nut based soft drinks named 'horchata' is described. The method is based on an immunoaffinity clean-up, followed by HPLC separation and fluorescence detection after electrochemical post-column derivatisation (PCD). The detection limit (S/N = 3) and the quantification limit (S/N = 10) were 0.02 µg/kg and 0.06 µg/kg respectively. The mean recovery at a level of 2 µg/L was 88 % (n = 6) and the coefficient of variation (CV) 9 %. The method was applied to conduct a small market survey for a beverage named 'horchata' that is frequently consumed by all kind of population in Southern Europe. A total of 22 samples from Spanish and Belgian supermarkets were analysed. As a result only one sample was found to contain AfB 1 at the LOQ of the method.

INTRODUCTION

Tiger nut or 'chufa' is a plant that botanically belongs to the genus Cyperus esculentus L.

There are two varieties with different applications: Cyperus esculentus L. var. esculentus and In Europe, the cultivation of tiger nuts is widely spread through the eastern part of Spain (Region of Valencia). Tiger nuts are also grown in Central Africa and in the southern part of United States (Florida).

The Spanish annual consumption of tiger nuts is about 5-7 million of kg/year, and around 3 million kg were imported during 2004. Exporting countries were Nigeria, Niger, Burkina Faso, Benin, Mali and Ghana. The main destination of the tiger nuts in Spain is the production of 'horchata' (40-55 millions litres/year), a soft drink consumed by all kind of population including children (with special importance due to their low body weight). 'Horchata' is either available as liquid, hailstorm or in congealed consistency. It is considered mainly as a refreshing drink, but is also as nutritive due to its caloric contents. After collection of the tiger nuts, they are washed and sorted in order to separate the product from soil, insector otherwise damaged tiger nuts that can be present at the collection. This is an important step for minimizing possible aflatoxin contamination present in the raw material and it is nowadays performed with the help of industrial machines. In the next step the tiger nuts are dried to reduce the water content from 50 to 11 %, this is the last step in the processing and it should be performed carefully since mycotoxin producing micro organisms can still start to grow on not sufficiently dried tiger nuts.

For the preparation of 1 L of 'horchata' approx. 200 g of tiger nuts and 150 g of sugar are used. This product must be distinguished from another different type of 'horchata', that is based on rice and vanilla, is made in Central and South America. [START_REF] Mosquera | Flavor and Stability of "Horchata De Chufas[END_REF].

Tiger nuts are also used for the production of oil for human consumption and they are an important animal feed ingredient in the southern United States (Kelley and Fredricksin 1991) ... [START_REF]methods and the methods of analysis for the official control of the levels of Fusarium toxins in foodstuffs[END_REF] ... [START_REF] Stroka | Immunoaffinity Column Cleanup with Liquid Chromatography Using Post-Column Bromination for Determination of Aflatoxins in Peanut Butter, Pistachio Paste, Fig Paste, and Paprika Powder: Collaborative Study[END_REF] ... [START_REF] Dragacci | Immunoaffinity column cleanup with liquid chromatography for determination of aflatoxin M 1 in liquid milk: collaborative study[END_REF] ... [START_REF] Stroka | Determination of Aflatoxin B 1 in Baby Food (Infant Formula) by Immunoaffinity Column Cleanup Liquid Chromatography Using Post-Column Bromination: Collaborative Study[END_REF] ... [START_REF] Kelley | Chufa biology and management[END_REF] ... [START_REF] Stroka | Comparison of two post column derivatization systems, ultraviolet irradiation and electrochemical determination, for the liquid chromatographic determination of aflatoxins in food[END_REF] ... [START_REF] Adebajo | Survey of aflatoxins and ochratoxin A in stored tubers of Cyperus esculentus[END_REF] ... [16] ... [START_REF] Arranz | Mini-collaborative study for the determination of Aflatoxin B 1 in medical herbs[END_REF] ... [17] ... [START_REF]IARC Monographs on the evaluation of carcinogenic risk to humans[END_REF] ... [START_REF] Bankole | Occurrence of mycoflora and aflatoxin in marketed tiger nut in Nigeria[END_REF] ... [ To our knowledge no method has been reported for the analysis of AfB 1 in 'horchata'. The here-described method has been developed on the basis of a previously described one by [START_REF] Stroka | Immunoaffinity Column Cleanup with Liquid Chromatography Using Post-Column Bromination for Determination of Aflatoxins in Peanut Butter, Pistachio Paste, Fig Paste, and Paprika Powder: Collaborative Study[END_REF]. A small survey on commercially available 'horchata' from Spanish and Belgian supermarkets was conducted after method development.

MATERIALS AND METHOD

Samples

The survey was carried out on 'horchata', from supermarkets in Spain and Belgium (imported product from Spain) with production dates from 2004. The method has been tested for 8

brands and different batches of some of these brands with a total of 22 samples.

Reagents

Solvents and reagents. Solvents used were complying with grade ACS-ISO. Acetonitrile and methanol were HPLC grade from Merck (Darmstadt, Germany). Immunoaffinity columns type EASI-EXTRACT TM aflatoxins were from Rhone-rBiopharm (Glasgow, Scotland).

Water. Purified water was delivered by the Millipore water purification device Milli-Q Gradient A10 ( Bedford, USA).

Phosphate-buffered saline (PBS).

PBS was prepared by adding 0.2 g KCl, 0. The following concentrations were achieved: 0.05, 0.10, 0.20, 0.30, 0.40 in ng/mL.

For recovery experiments, 5 mL of a 0.40 µg/mL AfB 1 standard solution was used to fortify 1 L of blank matrix the day prior to analysis, resulting in a level of fortification of 2 µg/L.

Apparatus.

HPLC system. The HPLC equipment consisted of a combination of Gilson HPLC pump type 309 with a solid phase extraction unit ASPEC XL (Milano, Italy), with a 100 µL injection loop. The fluorescence detector was from Waters type 474 (Milano, Italy). The PCD with KOBRA cell was from Rhone -rBiopharm (Glasgow, Scotland). The column used was a Supelcosil LC-18, 25 cm x 4.6 mm, 5 µm.

Sample preparation and clean-up.

25 mL of PBS and 25 mL of the 'horchata' were mixed and filtered over glass micro fibre filter (Whatman GF/A), 25 mL of this filtrate were passed through an immunoaffinity column

(IAC) at a flow rate of approximately 3 mL/min. The IAC was washed twice with 10 mL of water at the same speed. For elution of AfB 1 , 0.5 mL methanol was applied by gravity on the IAC. The eluate was collected in a volumetric flask of 5.0 mL. After 1 min, a second portion of 0.5 mL methanol was applied. This step was repeated once more after another minute and air was pressed through the IAC to collect most of the applied methanol.

The volumetric flask, containing the combined portions of IAC eluate was filled up to approx.

4.5 mL, shaken and filled up further with water to the mark and shaken again. The clear extracts were directly injected into the HPLC system. The detector was set at 360 nm (excitation wavelength) and 420 nm (emission wavelength).

HPLC equipment and reverse phase conditions

The mobile phase was water/acetonitrile/methanol [62 + 22 + 16 (v+v+v)] with 120 mg of KBr and 350 µL of HNO 3 (4 mol/L) per litre of mobile phase. The flow rate was at 1 mL/min.

Injection volume was 100 µL.

RESULTS AND DISCUSSION

It could be shown, that the clean-up procedure by immunoaffinity chromatography after dilution of the sample with PBS and glass micro fibre filtration is fast, simple and reliable despite a solid residue content of the product of a required minimum of 12 % and a fat content of around 2 % in 'horchata' drinks. Chromatograms were free of interferences in the region of the AfB 1 peak (see Figure 1).

"[Insert Figure 1 here]"

The mean recovery of the method was 88 % (n = 6). These recovery experiments were performed using blank samples spiked at a level of 2 µg/l of AfB 1 in the product. This shows that the method procedure is suitable to purify and concentrate the analyte by immunoaffinity from the matrix and that the matrix has no significant effect on the recovery during clean up.

Additional recovery experiments at lower levels were not carried out as results from collaborative studies and own experience showed that recovery figures for the here used methodology (immunoaffinity clean-up with HPLC-fluorescence detection) and this type of analyte (aflatoxins) are rather stable in terms of fortification levels, while being more influenced by the matrix used for analysis [START_REF] Arranz | Mini-collaborative study for the determination of Aflatoxin B 1 in medical herbs[END_REF][START_REF] Dragacci | Immunoaffinity column cleanup with liquid chromatography for determination of aflatoxin M 1 in liquid milk: collaborative study[END_REF][START_REF] Stroka | Immunoaffinity Column Cleanup with Liquid Chromatography Using Post-Column Bromination for Determination of Aflatoxins in Peanut Butter, Pistachio Paste, Fig Paste, and Paprika Powder: Collaborative Study[END_REF], 2001[START_REF] Stroka | Comparison of two post column derivatization systems, ultraviolet irradiation and electrochemical determination, for the liquid chromatographic determination of aflatoxins in food[END_REF]. A rather simple and fast method for the determination of AfB 1 in 'horchata' was developed and validated under single laboratory conditions. It could be demonstrated that the described method shows good performance characteristics when compared with minimum requirement data calculated from a "fitness for purpose" function that is currently discussed for use in EU legislation on mycotoxin methodology in official food control. The described method therefore is a good candidate for such use.

Based on this small survey, no indication was found for a consumer risk from the 'tiger nut'

based soft drink as sold on the Spanish and Belgian market during the period 2004. However, a survey with more samples would be necessary to obtain a full picture, which was partially difficult as there is a limited amount of producers in Spain.

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Deleted: <#>Agriculture quality policy for the "Protected Designation of Origin" of "Chufa de Valencia".

[internet] Available from: . ¶ Deleted: , The quality of tiger nuts depends on the terrain used for cultivation. Therefore, it has been reported that also the quality and flavour of horchata is influenced by the production area [START_REF] Mosquera | Flavor and Stability of "Horchata De Chufas[END_REF] The end product The process ends with the addition of 1 L of water, the beverage should be served cold.

Deleted: <#>Code of

2

 2 Cyperus esculentus L. var. sativus. The second one is cultivated for human consumption and the rhizomes are used to obtain the tiger nuts. (Fact Sheet HS-583)

  Tiger nuts are also used for the production of oil for human Deleted: For its production Deleted: , collected tiger nuts Deleted: are Deleted: them from rests of sand Deleted: and Deleted: Deleted: other materials Deleted: ,…also mouldy and bitten by insects tiger nuts should be eliminated. Deleted: reducing Deleted: the content of Deleted: s Deleted: in Deleted: ated Deleted: T Deleted: is the Deleted: and Deleted: drying in which the aim is Deleted: should be wash and leave to soak during at least 12 hours, afterwards Deleted: , next is to blend this mixture and to sieve it Deleted: The process ends with the

Formatted

  different type of horchata, based on rice and vanilla, is made in Central and South America. ¶ Deleted: total aflatoxins in Deleted: The EU strategy for consumer protection (Commission Regulation No. 178/2002) Deleted: considers controls throughout the entire food and feed chain. Deleted: aims to provide a methodology for the determination of actions to protect health with the use of three components of risk communication (risk assessment, risk management, and risk communication). Deleted: (Regulation EC/178/2002). Deleted: As a result of this strategy Deleted: , Deleted: , products Deleted: Generally food products Deleted: import Deleted: s that are imported from third countriesto the EU are carried out on the product as it enters the Community Deleted: while Deleted: . In cases Deleted: within the EU these controls are performed throughout the entire food and feed chain Deleted: The determination is performed by HPLC with fluorescent detection and the need of transforming AfB1 in a nonquenchable derivative is Deleted: , Deleted: 2002). Deleted: The market ... [19]

Food

  2 g KH 2 PO 4 , 1.16 g anhydrous Na 2 HPO 4 , and 8.0 g NaCl in 1.0 L water. The pH was adjusted to 7.4 with NaOH c=(0.1 mol/L).Standards. AfB 1 standard solution with a concentration of 10.0 µg/mL dissolved in chloroform was obtained from the Dutch National Institute for Public Health and the Environment, RIVM (Bilthoven, The Netherlands). From this, a dilution with a final concentration of 0.10 µg/mL solution was used for calibration standards purposes. Portions of 125, 250, 500, 750 and 1000 µL from this last standard were evaporated in 250 mL Deleted: a Deleted: ( mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk afterwards re-dissolved with 75 mL of methanol and filled up with water.

Formatted:

  Bullets and NumberingFormatted: English U.K. Formatted: Indent: Before: 0 pt, Hanging: 17.85 pt, Numbered + Level: 1 + Numbering Style: 1, 2, 3, … + Start at: 1 + Alignment: Left + Aligned at: 0 pt + Tab after: 18 pt + Indent at: 0 pt, Tabs: Not at 262.8 pt + 489.6 pt Formatted: Not Highlight Formatted: English U.K., Not Highlight Formatted: Not Highlight Formatted: English U.K. Formatted: Italian Italy Formatted: Bullets and Numbering Formatted: Highlight Formatted: Indent: Before: 0 pt, Hanging: 17.85 pt, Numbered + Level: 1 + Numbering Style: 1, 2, 3, … + Start at: 1 + Alignment: Left + Aligned at: 0 pt + Tab after: 18 pt + Indent at: 0 pt Formatted: Not Highlight Formatted: Not Highlight Formatted: Font: Italic Formatted: Not Highlight Formatted: English U.K.

mainly Page 2 :

 2 [2] Deleted arranis 10/5/2005 9:12:00 AM Tiger nuts are also used for the production of oil for human consumption and they are an important animal feed ingredient in the southern United States (Kelley and Fredricksin 1991) In Spain, the quality of the tiger nuts must fulfil the specifications of the agriculture quality policy of the European Union and the name of the end product "Chufa de Valencia" is a product defined according to the conditions of a Protected Designation of Origin ((ECC) No. 2081/91)

1

 1 Figure 1:

  The Regulating Council of Denomination of Origin "Chufa de Valencia [internet]. Available from: http://www.chufadevalencia.org/. Accessed 2005 June 07. 17. Van Egmond H. 2004. Natural toxins: risks, regulations and the analytical situation in Europe, Review. Analytical and Bioanalytical Chemistry 378: 1152-1160.

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			Stroka (animal feed) ¶
			Stroka (baby food) ¶
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	Practice for the Prevention and Reduction of Mycotoxin Contamination in Cereals, including annexes on Ochratoxin A, Zearalenone, Fumonisins and Tricothecenes. CAC/RCP 51-2003. Available from: http://www.ipfsaph.org/servlet/BinaryDo wnloaderServlet?filename=kopool_data/c odex_0/en_cxc_051e.pdf. Via the INTERNET. Accessed 2005 October 04. ¶ Deleted: <#>Commission Regulation (ECC) No. 178/2002 of 28 January 2002 laying down the general principles and requirements of food law, establishing the ... [20] 9/26/2005 5:22:00 PM n O w v R e P o F r e r e i e l F F Page 2: [1] Deleted arranis Importing o r o r e R e v i e l n y l O n O w w e i v e R r e e r e P P Page 2: [1] Deleted arranis 9/26/2005 5:24:00 PM
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http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

Acknowledgements

The authors are grateful to the RIVM in Bilthoven (The Netherlands) for providing us with the aflatoxin standard and to the Regulating Council of Denomination of Origin "Chufa de Valencia" (CRDO) in Valencia (Spain) for providing us with some technical information about the 'horchata' consumption in Spain.

National Institute for Public Health and the Environment

7

The resulting relative standard deviation was 9 % under repeatability conditions. On the basis of the assumption that the internal reproducibility is linked by a factor of 3/2 with the internal repeatability, the estimate for the internal reproducibility is 14%.

Based on a "fitness for purpose approach" which is characterised by the determination of an acceptable precision parameter that is derived from the function given below, the calculated reproducibility of 14% from this study was compared with the limit calculated from the function below. This approach has recently been implemented in EU legislation on mycotoxin method performance requirements (Commission Directive No. 38/2005) and can be used for those cases where no fully validated method precision (full collaborative trial) is available.

In this formula Uf is the maximum acceptable standard uncertainty in µg/kg, LOD is the Limit of Detection expressed in µg/kg, α is a factor (0.1 -0.2) that depends on the concentration of interest (C in µ g/kg). For concentration below 50 µg/kg α is 0.2. The resulting maximum acceptable standard uncertainty for a target level of 2 µg/L (assuming a liquid density of 1 g/mL for 'horchata') and a LOD of 0.02 µg/L is 0.40 µg/L. Expressed as relative standard deviation this figure is 20 %.

As the internal reproducibility is mostly accepted as standard uncertainty, the calculated figure of 14% shows that the method can be considered as "fit for purpose" according to this scheme.

In none of the tested 22 sample materials AfB 1 could be quantified. Only in one case the presence of AfB 1 could be detected at the level of the estimated LOQ (Figure 1).


Andrew G Renwick

The intake of intense sweeteners -an update review 1 2

Keywords: Intense sweetener, intake, ADI, acesulfame-K, alitame, aspartame, 30 cyclamate, saccharin, sucralose

come

The intake of intense sweeteners -an update review

Intense sweeteners have been subject to scrutiny over the years, both in relation to their 33 safety and the intakes that result from their dietary uses. All approved intense sweeteners 34 have undergone extensive safety testing, and have acceptable daily intakes (ADIs) 35

established by bodies such as the Scientific Committee on Food (SCF) for Europe and the 36 Joint FAO/WHO Expert Committee on Food Additives (JECFA) for international trade. 37

The ADI is an intake "that can be ingested daily over a lifetime without appreciable 38 health risk" [START_REF] Who | Principles for the Safety Assessment of Food Additives and Contaminants in Food[END_REF]. Risk characterization requires comparison of human intakes 39 with the output of hazard characterization [START_REF] Renwick | Risk characterization of chemicals in food and diet[END_REF], which in the case of 40 sweeteners is the relevant ADI. Intakes may vary over time, due to changing patterns of 41 use and food intake, and therefore risk characterization needs to be undertaken at regular 42 intervals, even though the basic safety data and hazard characterization may not have 43 values (in mg/kg body weight) for acesulfame-K are 0-15 (JECFA) and 0-9 (SCF), for 56 alitame is 0-1 (JECFA), for aspartame is 0-40 (JECFA and SCF), for cyclamate are 0-11 57 (JECFA) and 0-7 (SCF), for saccharin is 0-5 (JECFA and SCF), and for sucralose is 0-15 58 (JECFA and SCF). 59 60 Methods used to estimate intakes, and their inherent assumptions 61 Estimation of the intake of an approved food additive is a potentially complex and costly 62 procedure. Because many of the studies on intense sweeteners in recent years were not of 63 optimal design, the different aspects that should be incorporated into an appropriately 64 designed study are given below. 65 66 Study population 67 i.

Should be sufficiently large to define the tail of the distribution of intakes, 68 such as the 90 th , 95 th or 97.5 th percentile of those individuals who consume the 69 additive or sweetener under study (high consumers). 70

ii.

Should include any special groups who would be predicted to have higher 71 than average intakes (such as diabetics for intense sweeteners). 72

iii.

Should include any group that would generally be considered to be of concern 73 irrespective of the subject of the survey, such as pregnant women and 74 children. 75 Should include information on the intakes of those specific products that 77 might contain the additive or sweetener under investigation. Less specific 78 product classifications will result in greater predicted intakes and therefore 79 greater overestimation, conservatism and unreliability in the data generated. 80

ii.

Should give reliable measurements of the amounts of the specific food 81 products consumed daily, which will require information on portion size and 82 frequency of ingestion. Food intake estimates derived from a prospective food 83 diary are more reliable than data derived from a retrospective questionnaire. 84

iii.

Should differentiate between products in the same food classification group 85 that contain the subject of the survey and those that do not. This is particularly 86 important in the case of sweeteners, where different sweeteners can occur in 87 different brands. For example, information on "cola" ingestion represents 88 inadequate data because the product could be sweetened with sugar rather than 89 an intense sweetener, with a single approved sweetener, with a blend of 90 approved sweeteners or with a blend of sugar and sweeteners. Reliable data 91 require analyses to be performed using brand-level intake data. 92

Product composition 93 i.

Ideally the database should include brand-specific data on the concentrations 94 of the additives under study in each product. This information could be 95 obtained from the food producer or by direct measurement, and is particularly 96 important for intense sweeteners since a single food product may contain a 97 blend of different sweeteners. 98 In the absence of product-specific data, it is common to assume that the 99 concentration present is the maximum permitted under the relevant legislation. 100 Because food additives are not always present at their maximum permitted 101 concentrations, this represents another source of conservatism in the final 102 intake estimation. 103 Food additive intake calculation 104 i.

The amounts of each food product consumed and concentrations present are 105 multiplied, and the results for different products are summed and divided by 106 the body weight. The use of individual body weights is of critical importance 107 if the study group covers a wide range of body weights. For example, children 108 aged 1-4years vary widely in body weight but are often reported as a single 109 population group; dividing the highest intake in mg per individual per day by 110 the average body weight could result in a significant overestimation. 111

ii.

There is an added complication in the case of cyclamate because the ADI was 112 calculated using toxicity data on its metabolite, cyclohexylamine, and an 113 assumption that a high % of the ingested cyclamate is converted to this active 114 metabolite [START_REF] Renwick | The metabolism of cyclamate to cyclohexylamine in humans during long-term administration[END_REF]. However, only about 3-4% of the 115 population can metabolize cyclamate to the extent assumed in the ADI 116 calculation, and therefore individual data on the intake of cyclamate does not 117 directly relate to the exposure to cyclohexylamine. 118

Data presentation 119 i.

Information should be provided on the percentage of the population that 120 consumed the additive, the average intake of the additive by those who 121 by "high consumers", such as the 95 th or 97.5 th percentile of the distribution 123 of intakes by consumers. Data for "consumers only" are normally reported 124 because the 95% intake of the additive for the total population would not be 125 representative of high intakes if only a small proportion of the total population 126 consumed foods containing the additive. 127 Time-base for intake estimation 128 i.

It is widely recognized that the correct intake for comparison with the ADI as 129 part of risk characterization would be the average long-term intake by an 130 individual. Because the reliability of intake studies decreases with increasingn 131 duration, reliable data can be obtained for only about 1-2 weeks of diary 132 collection. 133 ii.

Intake data for a single day underestimates the % of a population who will be 134 consumers of an additive, but can grossly overestimate the average intake in 135 those individuals who do report intake of the additive on the day in question. 136

137

The intake studies on intense sweeteners conducted since the previous review (Renwick, 138 1999) Leclercq et al. (1999). 148 This was a specific study on the intakes of intense sweeteners in Italian teenagers using a 149 comprehensive 14-day food diary. 150

Study population -212 teenagers (aged 13-19 years) were recruited from a secondary 151 school in Rome. 152

Country -Italy. 153

Time of intake data collection -September 1996 to December 1996. 154 Nature of intake data -Prospective 14-day food diary with brand information collected 155 and the presence of sweeteners determined from product labels. The amounts of each 156 product consumed on each occasion were defined as small, medium or large. The data 157 were analyzed for acesulfame-K, aspartame, cyclamate and saccharin. 158

Product concentration data -Data were obtained from the product manufacturer. 159

Results -The major sources for acesulfame-K, aspartame and cyclamate were beverages 160 and chewing gum, while for saccharin most of the intake was from table-top products. 161

The means and maximum intakes of all sweeteners were less than 1mg/kg body 162 weight/day and therefore below the corresponding ADI values. 163

Strengths -The results represent a comprehensive assessment of sweetener intakes using 164 the best practical approach. Intakes were calculated using individual body weights and 165 the results were 14-day averages. 166 et al. (1999). 177

F
This study used 24 hour urinary excretions of acesulfame-K and saccharin as biomarkers 178 of intake. The method would only be applicable to these two sweeteners, because they are 179 almost completely absorbed from the intestine and excreted unchanged in urine. The 180 method was validated by giving known amounts of acesulfame-K and saccharin to 181 different volunteers and measuring their urinary excretion over the following 24 hours. 182

The use of the urinary biomarker was then compared with intake estimates derived from a 183 dietary questionnaire specifically on sweetener intake for the same 2-day period, and it is 184 Conclusions -These data provide an interesting approach for the future, rather than 215 giving comprehensive intake data that can be compared with previous studies. The mean 216 intakes of acesulfame-K and saccharin were reported to be below their ADI values. 217

Assuming an average body weight of 60kg for an adult, the maximum intakes of 218 acesulfame-K and saccharin corresponded to less than 1.5mg/kg body weight/day. 219 220 [START_REF] Garnier-Sagne | Calculation of the intake of three intense sweeteners in young insulin-dependent diabetics[END_REF]. 221

This study focused on diabetic children, and used a worst-case analysis to determine the 222 potential for the ADI to be exceeded in this group. 223

Study population -400 subjects aged 2-20 years, who were recruited from the French Aid 224 for Young Diabetics Association, were sent a food intake questionnaire; 227 completed 225 forms were returned. 226

Country -France. 227

Time of intake data collection -June to October 1997. 228 Nature of intake data -The questionnaire included a 5-day prospective food diary which 229 paid particular attention to the types and amounts of sweetened foods that were 230 consumed. The forms were completed by the individual or a parent. 231

Product concentration data -A sweetener concentration database was constructed in 232 which it was assumed that all sugar-free products had been sweetened with the same 233 sweetener and that the concentrations of acesulfame K, aspartame and saccharin used 234 Nature of intake data -The intakes of acesulfame-K, aspartame, cyclamate and saccharin 259 from beverage consumption were measured using a 7-day diary. The volumes of different 260 types or categories of beverage consumed (carbonated, dilutable and powdered drinks, 261 tea/coffee and natural still drinks) were recorded. The report does not state clearly 262 whether the drinks recorded were separated into those sweetened with sugar and low-263 calorie sweeteners or whether brand information was obtained, but it is unclear how the 264 product concentration data could have been used without such detailed information. 265

Product concentration data -Data obtained from the food product manufacturer. 266

Results -The average daily intakes of acesulfame K, aspartame, cyclamate and saccharin 267 by consumers were 0.92, 3.38, 4.46 and 1.16 mg/kg body weight respectively, and the 268 97.5 th percentile intakes were 3.72, 12.01, 14. Weaknesses -The survey was restricted to beverages only, but this is the main source in 274 the age group studied. In some cases, assumptions had to be made about body weight, 275 which can vary widely across the age range 1.5 to 4 years. It is not clear if the data for 276 high consumers related to recorded body weights or to assumptions. It is not clear to what 277 extent brand level information was used because the beverages appear to have been 278 reported as groups rather than brands (the word "brand" does not appear in the report) 279 sweeteners. The individual intake estimates were based on the consumption on a single 297 occasion. Although not stated clearly in the original publication, it appears that the 298 "worst-case" estimates were made by addition of the maximum intake for each product 299 recorded in a single day. This would be highly conservative because the main sources of 300 sweetener intake were fruit syrups, diet sodas and cider and a high consumption of one 301 Product concentration data -The maximum permitted concentrations for each product 304 category were used and it was assumed that the total intake of that product contained a 305 single sweetener. 306

Results -The main sources of intake were tabletop sweeteners and beverages, especially 307 fruit syrups in children . Although it is unclear from the data presentation, it appears that 308 the average intakes for each of the sweeteners in all groups, using these highly 309 conservative assumptions were below the respective ADI values. Estimates of the intakes 310 by high consumers were based on the data for the 10 or 20 individuals in each population 311 subgroup with the highest intakes from all sources. The intakes of aspartame (46mg/kg 312 body weight per day) and saccharin (about 6.3mg/kg body weight per day) slightly 313 exceeded the relevant ADI values for the top 10 children. The intakes for the top 10 314 adults were below the ADI values. The intakes of cyclamate for the 10 children with the 315 highest intakes (about 35mg/kg body weight per day) were about 3 times the JECFA ADI 316 (0-11mg/kg body weight per day) and 5 times the more recent SCF ADI (7mg/kg body 317 weight per day) for cyclamate. The intake for the highest 10 adults slightly exceeded the 318

JEFCA ADI. 319

Strengths -The study focused on diabetics because this was the group expected to have 320 the highest potential intakes. Individual body weights were used. 321

Weaknesses -The authors described the findings as a "worst-case" analysis based on 322 maximum permitted concentrations and maximum intake on a single day. The inclusion 323 Conclusions -The data show that only cyclamate could have an intake significantly 326 above its ADI, and this would only be in a proportion of children. Extrapolation of this 327 observation to non-diabetic children is difficult because the children studied had a high 328 intake of table-top sweeteners, an observation not made for non-diabetic children in other 329 intake studies. The use of highly conservative worst-case assumptions means that this 330 study should be used to identify a possible problem, and should not be interpreted as 331 proving the existence of a real problem. It is unclear why a study which has the power 332 only for hypothesis generation should be performed in 1999, at a time when it was clear 333 that diabetic children consuming cyclamate would represent a group where the ADI for a 334 sweetener might be exceeded. 335 336 Serra- [START_REF] Serra-Majem | Cyclamate intake and cyclohexylamine excretion are not related to male fertility in humans[END_REF]. 337

This was an epidemiological study to investigate the possibility of a relationship between 338 male fertility in humans and the intakes of cyclamate and its metabolism to 339 cyclohexylamine. Cyclohexylamine produces testicular atrophy in experimental animals 340 and this effect was used as the basis for calculation of the ADI of cyclamate. 341

Study population -405 adult males (30-50 years) with clinically defined infertility and 342 379 adult male controls (30-50 years). 343 Strengths -The study employed large group sizes, but this was essential because the 352 majority of individuals do not metabolize cyclamate to cyclohexylamine (Renwick et al., 353 2004). This was the only recent study that has tried to relate cyclamate intake to the 354 excretion of its metabolite cyclohexylamine in urine. 355

Country -Spain. 344

Time of intake data collection

Weaknesses -The population investigated was adult males only, and intake was based on 356 a food frequency questionnaire. 357

Conclusions -The intake data support the findings of previous studies in a population 358 that probably has higher than average cyclamate intakes because of the widespread use of 359 cyclamate in Spain and the use of this sweetener in the popular drink "gaseosa" -a 360 combination of carbonated water containing saccharin and cyclamate taken with wine. 361 362 [START_REF] Arcella | Dietary estimated intake of intense sweeteners by Italian teenagers. Present levels and projections derived from the INRAN-RM-2001 food survey[END_REF]. 363

This was a follow up study to that of [START_REF] Leclercq | Intake of saccharin, aspartame, acesulfame-K and cyclamate in Italian teenagers: present levels and projections[END_REF] 18 362 individuals aged 14-17 years were measured using a food diary as described below. 369

The food diary was completed by 125 males and 108 females selected at random and by 370 139 females who were high consumers of either diet soft drinks or table-top products or 371 both. 372

Country -Italy 373

Time of intake data collection -October 2000 to May 2001 (three 4-day food diaries) 374

Nature of intake data -The randomly selected group and the identified female high 375 consumers from the food frequency questionnaire completed a 4-day food diary on three 376 separate occasions, with different subjects in each group covering all days of the week. 377

Brand information was collected and presence of sweeteners determined from the product 378 label. The amounts of each product consumed on each occasion were defined as small, 379 medium or large. Data were analyzed for saccharin, aspartame, acesulfame-K and 380

cyclamate. 381

Product concentration data -Data were obtained from the product manufacturer. 382

Results -The mean and 95 th percentile intakes of all sweeteners in all individuals who 383 completed the 4-day food diary were well below the corresponding ADI values. The 95 th 384 percentile of cyclamate intake in the selected group of female high consumers of sugar-385 free soft drinks was 0.55mg/kg body weight/day (5% of the JECFA ADI), while the 386 corresponding intakes for acesulfame-K, aspartame and saccharin were 0.25, 0.30 and 387 0.0mg/kg body weight/day (less than 2% of the ADI values). 388

Strengths -This study provides a comprehensive assessment of the intake using the best 389 practical approach. Intakes were calculated using individual body weights. Results were 390 averages of three 4-day diaries collected in different months for all subjects and therefore 391 Weaknesses -The study did not include other groups with high predicted intake, i.e.

children or diabetics 394

Conclusions -The intakes were well below the ADI values. The results of this study are 395 similar to the study of [START_REF] Leclercq | Intake of saccharin, aspartame, acesulfame-K and cyclamate in Italian teenagers: present levels and projections[END_REF] in Italian teenagers. 396 397 [START_REF] Devitt | Assessment of intakes of artificial sweeteners in children with type 1 diabetes mellitus[END_REF]. 398

This study focused on a small number of children treated for Type 1 diabetes mellitus. 399

Study population -A group of 56 children aged 2-6 years were recruited from a total of 400 116 eligible subjects at the Diabetic Clinic at the Hospital for Sick Children in Toronto. 401

Country -Canada. 402

Time of intake data collection -Data were collected over a period of 7 months (dates not 403 given). 404

Nature of intake data -Intake estimates were based on a single interactive 24-hour 405 dietary recall by the parents. Products containing acesulfame-K, aspartame, cyclamate 406 and sucralose were identified by showing product labels to the parents. Saccharin was not 407 used as a food additive in Canada at this time. 408

Product concentration data -Data were obtained from the label or the product 409 manufacturer. 410

Results -The proportion of the group who consumed products containing acesulfame-K, 411 aspartame, cyclamate and sucralose were 25%, 43%, 12% and 2% respectively. The 412 mean and 90 th percentile intakes of cyclamate and sucralose were below 1mg/kg body 413 weight/day, indicating little market penetration by these sweeteners. The mean and 90 th 414 The mean and 90 th percentile intakes of aspartame were 4.1 and 7.8mg/kg body 416 weight/day respectively. 417

Strengths -The study focused in diabetic children. The absence of saccharin in food 418 products and the low use of cyclamate mean that the intake data for aspartame represent a 419 worst-case scenario. 420

Weaknesses -The intake data were based on a single recall assessment and would over-421 estimate average intakes but under-estimate the % consumers. 422

Conclusions -The intakes for all sweeteners were below their ADI values. 423 424 Food Standards Australia and New Zealand (2004). 425

This study was a follow up to the 1994 Australian study which showed that high 426 consumers of saccharin and cyclamate could have intakes that approached or exceeded 427 the ADI. The survey comprised 3 phases; an initial national telephone screen to 428 determine patterns of food intake, a diary survey of potential high consumers of products 429 containing intense sweeteners identified in the screen and a supplementary survey of 430 individuals with diabetes or impaired glucose tolerance. 431

Study population -The initial screen, which was in 3529 individuals aged over 12 years 432 and selected to be representative of the general population, was used to identify 400 433 respondents with high potential intakes of intense sweeteners. The supplementary study 434 in diabetics comprised 111 subjects identified within the group of 400 high consumers of 435 products containing intense sweeteners and these were supplemented by 187 diabetics 436 2.4 and 2.3 mg/kg body weight/day respectively. The mean intakes in the 298 diabetics 460 were 0.6, <0.1, 2.3, 3.3, 0.5 and 0.5 mg/kg body weight/day for acesulfame-K, alitame, 461 aspartame, cyclamate, saccharin and sucralose respectively and the 95 th percentile intakes 462 were 2.0, <0.1, 7.5, 11.6, 1.9 and 1.9 mg/kg body weight/day respectively. 463

Strengths -A major strength of this study is the size of the cohort from which the high 464 consumers and diabetics were selected. It was a recent study in a population where 6 465 different intense sweeteners were available. The study appears to have been conducted in 466

February which corresponds to a potentially high summer intake of beverages containing 467 intense sweeteners. 468

Weaknesses -The participants were not given an individual interview, and the absence of 469 a personal explanation of the protocol may have reduced understanding and compliance 470 with diary completion. 471

Conclusions -The means and 95 th percentile intakes were below the ADI values of the 472 different sweeteners in the selected high consumers and diabetics, with the exception of 473 cyclamate in diabetics, where the 95 th percentile slightly exceeded the JECFA ADI value. 474 475 Van Rooij-Van Den Bos et al. (2004) 476 This study combined data on the concentrations of intense sweeteners in retail food 477 Nature of intake data -Data were collected using a two-day diary. Food consumed at 485 home was recorded in the diaries by the person who usually prepared the meal for the 486 household. Each participant recorded foods consumed out of the house. All products in a 487 single food coding in the food composition database that could contain an intense 488 sweetener were assumed to contain the same sweetener. If the food product could not be 489 related to an existing food coding the intake was assumed to be that of the closest non-490 sweetened coding with correction for the market share of sweetened products in that food 491 coding. For worst-case calculations, the highest consumptions (95 th percentiles) of soft 492 drink, lemonade syrup, yogurt drink and chocolate milk were calculated assuming that all 493 of the products in each category consumed were sweetened with a single sweetener using 494 the average measured concentration. 495

Product concentration data -Concentrations were measured in food products that were 496 purchased in 2003 and which was likely to contain an intense sweetener based on the 497

label. 498

Results -The estimated average and 95 th percentile intakes of acesulfame-K, aspartame, 499 cyclamate and saccharin were all 1mg/kg body weight or less. The highest calculated 95 th 500 percentile intakes were in the 1-4 years age range and the highest value in this age group 501 was for cyclamate (1.1mg/kg body weight/day). The results from the worst-case 502 calculations indicated that for the whole population (1-97years old) the 95 th percentile 503 consumers of soft drinks had intakes of 2, 2, 4 and 0.5mg/kg body weight/day of 504 Weaknesses -The intakes of foods categories are based on national estimates and 508 therefore major assumptions had to be made. Intake and concentration data do not relate 509 to the same time period or to the same products. 510

Conclusions -Despite the conservative assumptions made the mean and 95 th percentile 511 intakes were below the ADI values. The worst-case calculations of the intakes of 512 sweeteners indicated that the only sweetener with the potential to exceed the relevant 513 ADI was cyclamate in 1-4 year olds. 514 515 In addition to the studies outlined above, limited information has been published in 516 summary form for Korea and Japan in which the bases for the intake estimates were not 517 clearly explained and the results were not corrected for body weight. The estimated 518 intakes of acesulfame-K and saccharin in Koreans were 1.3 and 4.1mg/person/day and 519 the theoretical maximum daily intakes were 31 and 106mg/person/day, values that are 520 well below the corresponding ADI values [START_REF] Kim | Estimated dietary intake of sodium saccharin and acesulfame potassium in Koreans[END_REF]). The average daily intakes of 521 acesulfame-K, aspartame, saccharin and sucralose in Japan were reported as 0.8, 7.3, 0.7 522 and 0.4 mg/person (Yomota et al. 2002). 523 524 Estimates of sweetener intakes in the European Union are given in the European 525 Commission Report on Dietary Food Additive Intake. Estimates were made using the 526 "Tier 2" approach, in which the theoretical intake was calculated by combining the mean 527 of the sweetener, and the "Tier 3" approach, in which the theoretical intake was 529 calculated by combining the mean national food consumption data of the whole 530 population with the actual use levels of the sweetener. The results were expressed as the 531 % of the corresponding SCF ADI values (using the old SCF ADI for cyclamate of 0-532 11mg/kg body weight per day). The ranges of intakes in adults were 2-37% for 533 acesulfame-K (Tier 2 data for Denmark, France, Italy, The Netherlands, UK and Norway) 534 and 0-11% for cyclamate (Tier 2 data for Denmark, France, Italy, The Netherlands, UK 535 and Norway). The ranges of intakes in children were 3-107% for acesulfame-K (Tier 3 536 data for France, The Netherlands and UK), 1-40% for aspartame (Tier 2 data for The 537 Netherlands and UK), 1-74% for cyclamate (Tier 2 data for France, The Netherlands and 538 UK) and 2-51% for saccharin (Tier 2 data for France, The Netherlands and UK). 539 540 541 Discussion 542

The recently published studies can be divided into 4 types. 543 i.

Those that were of comprehensive design and capable of producing realistic 544 intake estimates [START_REF] Leclercq | Intake of saccharin, aspartame, acesulfame-K and cyclamate in Italian teenagers: present levels and projections[END_REF], Arcella et al. 2004and Food Standards 545 Australia New Zealand 2004). 546 ii.

Those where the data presentation make it difficult to judge the extent of 547 conservatism in the reported intake estimates (Food Standards Agency UK 548 2001). 549

iii.

Those that included significant weaknesses or conservative assumptions, such 550 that the data obtained can be regarded as "worst-case" estimates only, or 551 of foods and beverages on a body weight basis, and on diabetics (Garnier-Sagne et al. 577 2001[START_REF] Ilback | Estimated intake of the artificial sweeteners acesulfame-K, aspartame, cyclamate and saccharin in a group of Swedish diabetics[END_REF][START_REF] Devitt | Assessment of intakes of artificial sweeteners in children with type 1 diabetes mellitus[END_REF]and Food Standards Australia New Zealand 578 2004) because of their higher potential intakes of intense sweeteners. The studies 579 reported have used a variety of conservative assumptions, and therefore do not provide 580 definitive data, but simply confirm that these groups may have higher than average 581 intakes. 582

583

The only sub-group analyses that have indicated that the 95 th percentile intake of a 584 sweetener may exceed the ADI are for cyclamate in children, particularly those with 585 diabetes. Such a conclusion was apparent from calculations available at the time of the 586 earlier review [START_REF] Renwick | Intake of intense sweeteners[END_REF], and recent publications do not include a specifically 587 designed study producing reliable data for this group, but simply confirm the possibility 588 by further theoretical worst-case calculations. Resolution of this theoretical possibility 589 will require a specifically-designed study using a quantitative prospective 5-day or 7-day 590 diary combined with brand-specific data on intakes and concentrations, in which any 591 dilution of beverages for children is recorded accurately. The recent reduction in the ADI 592 of cyclamate from 0-11mg/kg body weight/day to 0-7 mg/kg body weight/day by the EU-593 SCF [START_REF] Scf | Scientific Committee on Food of the European Commission. Opinion on cyclamic acid and its sodium and calcium salts (expressed on 9th March 2000)[END_REF] has resulted in changes to the uses of cyclamate. Any specifically-594 designed study should be undertaken after these changes have taken effect and the new 595 pattern of uses has stabilized. Interpretation of the intake data on cyclamate is also 596 complex because the ADI is based on the effects of its metabolite cyclohexylamine, and 597 28 only a small percentage of humans, about 3-4% of the population, are able to form 598 significant amounts of this metabolite [START_REF] Renwick | The metabolism of cyclamate to cyclohexylamine in humans during long-term administration[END_REF]. Theoretically, the best study 599 design for estimating exposure to cyclohexylamine following cyclamate ingestion would 600 be based on that of [START_REF] Serra-Majem | Cyclamate intake and cyclohexylamine excretion are not related to male fertility in humans[END_REF], and combine an optimized food-diary, as 601 outlined above, with measurements of the daily urinary excretion of cyclamate and 602 cyclohexylamine. However such a design would not be practicable for studying a group 603 of diabetic children that was large enough to include individuals with high cyclamate 604 metabolizing ability. 605 2 -maximum reported intake 3 -intakes by the 10 children and 10 adults with the highest intakes (values read from published histograms)

4 -the arithmetic mean of the median intakes reported for cases and controls which include non-consumers; the maximum intake was reported to be less than the ADI Ace -Acesulfame-K; Ali -Alitame; Asp -Aspartame; Cyc -Cyclamate; Sac -Saccharin: Suc -Sucralose.

The % ADI values are calculated using the ADIs established by the WHO/FAO Joint Expert Committee on Food Additives of 0-15 (Ace), 0-1 (Ali), 0-40 (Asp), 0-11 (Cyc), 0-5 (Sac) and 0-15 (Suc) mg/kg body weight per day.

  intense sweeteners have been submitted to a previous systematic review, and the Scientific Committee on Food of the European Union (SCF). The ADI 55

  foods and beverages (consumers only) and the intake 122

  this part of the study which is presented below. 185 186 Study population -188 volunteers aged 3-74 years who were family and friends of the 187 laboratory staff. There were 78 adult males, 85 adult females, 19 boys and 6 data collection -Not stated. 190 Nature of intake data -Sweetener intake was measured using a 48 hour intake diary, with 191 information provided on the amounts and brands consumed. The urinary biomarker data 192 were collected only on the second day of the food diary record. The urinary data were 193 accepted only if there was evidence of a complete 24 hour collection based on the urinary 194 recovery of the marker substance p-aminobenzoic acid which was given as three doses 195 with meals. 196 Product concentration data -Data were supplied by the food product manufacturers. 197Results -The mean intakes of acesulfame-K and saccharin determined by the 198 questionnaire were 45mg/day and 33mg/day respectively, and these values were slightly 199 higher than the values derived from the urinary biomarker (35mg/day and 23mg/day 200 respectively). The correlation between the two measurements of intake was analysed in 201 138 subjects submitting complete urine collections, as judged by PABA recovery, was 202 described by the authors as showing generally good agreement (R 2 = 0.6 to 0.7). The 203 highest intakes determined by urinary excretion and questionnaire were 101 and 204 111mg/day respectively for saccharin and 110 and 168mg/day respectively for 205 acesulfame-K. 206Strengths -The intake diaries were comprehensive and could be related to the urinary 207 biomarker data. The questionnaire and concentration data were analyzed down to brand-208 level detail. 209Weaknesses -The study used a small number of subjects for an intake survey, because it 210 was primarily an exercise for the development and validation of biomarkers. The data 211 refer to a 48 hour period only and do not represent long-term average intakes. Detailed 212 intake questionnaire were not given in the publication. The intakes were 213 reported in mg/day. 214

Food

  permitted under European legislation. Such a highly conservative 235 method was used in order to give theoretically maximum daily intakes orTMDIs. 236 Results -The mean TMDIs of acesulfame K, aspartame and saccharin by consumers 237 were 1.1, 2.4 and 0.4mg/kg body weight respectively, and the 97.5 th percentile TMDIs 238 were 4.0, 7.8 and 1.3mg/kg body weight respectively, indicating that intakes by high 239 consumers did not exceed the ADI values. 240Strengths -The study calculated the maximum potential intakes based on 5-day averages. 241Weaknesses -The assumptions about the distribution of sweeteners in food products and 242 the concentrations used are too conservative to allow the data to be taken as realistic 243 estimates, but they do provide an upper bound on the possible intakes of each individual 244 sweetener in the population group predicted to have the highest intake on a body weight 245 basis. Cyclamate intake was not among the sweeteners measured. 246Conclusions -The data appear to be a worst-case analysis of intake in a group of the 247 population with high potential intakes. The study supports the findings of other studies 248 that the intakes of acesulfame K, aspartame and saccharin would not exceed their ADI 249 values, even in the highest consumers. 250 251 Food Standards Agency UK(2003). 252 This study focused on intakes by children, because previous survey data had indicated 253 that this group was likely to have sweetener intakes above the ADI values due to their 254 high intakes of sweetened soft drinks, when expressed per kg of body weight. 255 Study population -1110 children aged 1.5 to 4.5 years across 12 areas of the UK. 256 Country -UK. 257 Page 12 of 35 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk data collection -January to September 2001. 258

  07 and 3.83 mg/kg body weight 269 respectively, indicating that the intakes of cyclamate by high consumers would exceed 270 the ADI values set by the JECFA and the SCF. 271 Strengths -The study focused on a very large group of children with high potential 272 intakes of soft drinks expressed per kg body weight. The results are 7-day averages. 273

  data appear to be a worst-case analysis of intake in a group of the 280 population with high potential intakes. The results support the findings of other studies 281 that it is only cyclamate where the ADI might be exceeded by the highest intakes in 282 young children. 283 284[START_REF] Ilback | Estimated intake of the artificial sweeteners acesulfame-K, aspartame, cyclamate and saccharin in a group of Swedish diabetics[END_REF]. 285This study investigated the intakes of acesulfame-K, aspartame, cyclamate and saccharin 286 in diabetic children and adults. 287 Study population -Subjects were recruited randomly from members of the Association of 288 Diabetics in Stockholm. Data were collected for 243 children (aged 0-15 years), 236 adult 289 males (aged 16-90 years) and 311 adult females (aged 16-90 years). 290 Country -Sweden. 291 Time of intake data collection -January 1999. 292 Nature of intake data -Intakes were estimated from a food-frequency questionnaire 293 (retrospective but of undefined duration) concerning the amounts and frequency of 294 intakes of diet soda, cider, fruit syrup, tabletop sweeteners, light ice cream, chewing 295 gums, sweets, yoghurt, vitamin C supplements, throat lozenges and fluid and dried table 296

  single day would not coincide with high intake of the other two sources on 302 the same day. 303

  at each point in the intake calculation results in an unrealistic 324 intake estimate. 325

  of long term average intakes. 392

  acesulfame-K were 0.6 and 1.9mg/kg body weight/day respectively. 415

  available in 2003 with data from the third Dutch National Food Consumption Survey 478 1997/1998 in order to provide updated intake estimates. 479Study population -Data from the National Food Consumption Survey were used, which 480 was based on 6250 persons in 2774 households with all household members asked to data collection -April 1997 toMarch 1998. 484

  aspartame, cyclamate and saccharin respectively. The corresponding daily 505 intakes for the 1-4 years age range were 6, 8, 14 and 2mg/kg body weight respectively. 506Strengths -The concentrations of sweeteners were measured directly. 507

  data of the population with the maximum permitted use levels 528

  Wilkinson K, Crews HM, Davies AM, Dick CS, Dumsday VL. 1999. Urinary monitoring of saccharin and acesulfame-K as biomarkers of exposure to these additives. Food Additives and Contaminants 16: 227-238. Yomota C, Onishi-abe Y, Tanamoto K, Aizawa H, Osawa T, Miyazaki T, Mouri T, Yamato Y, Tamanaha K. 2002. A study on intake of eight kinds of sweeteners by market basket method in fiscal year 2002. Paper presented at the Japanese Society of Food Chemistry's 9 th General Assembly and Academic Convention. of recent studies on the intakes of intense sweeteners (in chronological order of publication).1 -95 th percentile intakes used for high consumer data except forGarnier-Sagne et al. 2001(97.5 th percentile of the theoretical maximum daily intake), FSA, 2003 (97.5 th percentile) andDevitt et al., 2004 (90 th percentile)

  are outlined below in chronological order of publication, with a description of the 139 findings and the strengths and weaknesses of the study design.

	144	Recent studies are listed in Table 1 in chronological order of publication. They have
	145	investigated different population groups in different countries and represent a significant
	146	body of new data.
	147
		F o
		r
		P
		e e r
		R e v i e w
		O n
	140	l y
	141
	142	Recent intake studies on intense sweeteners.
	143
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not really provide reliable new information(Garnier-Sagne et al. 2001, Ilback